U.S. patent application number 14/131174 was filed with the patent office on 2014-08-14 for biological methods for preparing a fatty dicarboxylic acid.
The applicant listed for this patent is Tom Beardslee, Tom Fahland, Alex Hutagalung, Stephen Picataggio. Invention is credited to Tom Beardslee, Tom Fahland, Alex Hutagalung, Stephen Picataggio.
Application Number | 20140228587 14/131174 |
Document ID | / |
Family ID | 46514834 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140228587 |
Kind Code |
A1 |
Beardslee; Tom ; et
al. |
August 14, 2014 |
BIOLOGICAL METHODS FOR PREPARING A FATTY DICARBOXYLIC ACID
Abstract
The technology relates in part to biological methods for
producing a fatty dicarboxylic acid and engineered microorganisms
capable of such production.
Inventors: |
Beardslee; Tom; (Carlsbad,
CA) ; Picataggio; Stephen; (Carlsbad, CA) ;
Hutagalung; Alex; (Carlsbad, CA) ; Fahland; Tom;
(Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Beardslee; Tom
Picataggio; Stephen
Hutagalung; Alex
Fahland; Tom |
Carlsbad
Carlsbad
Carlsbad
Carlsbad |
CA
CA
CA
CA |
US
US
US
US |
|
|
Family ID: |
46514834 |
Appl. No.: |
14/131174 |
Filed: |
July 5, 2012 |
PCT Filed: |
July 5, 2012 |
PCT NO: |
PCT/US12/45622 |
371 Date: |
April 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61505092 |
Jul 6, 2011 |
|
|
|
61523216 |
Aug 12, 2011 |
|
|
|
Current U.S.
Class: |
554/121 ;
435/134; 435/254.2 |
Current CPC
Class: |
C12N 15/815 20130101;
C12N 9/001 20130101; C12N 1/16 20130101; C12N 9/0071 20130101; C12N
15/81 20130101; C12P 7/6409 20130101; C12Y 103/03006 20130101; C12P
7/44 20130101; C12N 9/0042 20130101; C12N 1/28 20130101 |
Class at
Publication: |
554/121 ;
435/254.2; 435/134 |
International
Class: |
C12P 7/64 20060101
C12P007/64 |
Claims
1-159. (canceled)
160. A genetically modified yeast, comprising an active, modified
endogenous acyl-coA Oxidase polypeptide, which yeast is capable of
producing a diacid from a feedstock comprising one or more
components from a vegetable oil.
161. (canceled)
162. The genetically modified yeast of claim 160, wherein the
acyl-coA Oxidase polypeptide comprises an amino acid modification
at one or more amino acid positions chosen from 81, 82, 83, 84, 85,
86, 88, 93, 94, 95, 96, 98, 102, 284, 287, 290, 291, 292, 294, 295,
436, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 and 463.
163. The genetically modified yeast of claim 160, wherein said
modified acyl-coA Oxidase polypeptide comprises at least one amino
acid substitution.
164-167. (canceled)
168. The genetically modified yeast of claim 162, which comprises a
genetic modification that reduces the activity of an enoyl coA
isomerase polypeptide.
169. The genetically modified yeast of claim 168, wherein the
genetic modification disrupts a polynucleotide that encodes the
enoyl coA isomerase polypeptide.
170. (canceled)
171. The genetically modifies yeast of claim 168, wherein the enoyl
coA isomerase polypeptide is a polypeptide native to a yeast,
wherein the yeast is a Candida spp. yeast.
172. (canceled)
173. The genetically modified yeast of claim 168, wherein the
diacid is a C4 to C24 diacid.
174. The genetically modified yeast of claim 173, wherein the
diacid is a C10, C12, C14, C16, C18 or C20 diacid.
175. (canceled)
176. (canceled)
177. The genetically modified yeast of claim 174, wherein the
diacid is the predominant diacid in a mixture of diacids.
178. (canceled)
179. The genetically modified yeast of claim 177, wherein the
feedstock comprises a plurality of fatty acids.
180-182. (canceled)
183. The genetically modified yeast of claim 168, wherein the
vegetable oil is from a plant chosen from palm, palm kernel,
coconut, soy, safflower, canola, palm, palm kernel or combination
thereof.
184. A method for producing a diacid, comprising: contacting a
genetically modified yeast of claim 162 with a feedstock comprising
one or more components from a vegetable oil capable of being
converted by yeast to a diacid; and culturing the yeast under
conditions in which the diacid is produced from the feedstock.
185. A diacid produced by the method of claim 184.
186. An expression product comprising a desired diacid, said
expression product obtained by contacting a genetically modified
yeast of claim 168 with a feedstock capable of being converted by
the yeast to said desired diacid, and culturing the yeast under
conditions in which the expression product comprising said diacid
is produced from the feedstock.
187. An expression product comprising at least one of sebacic acid
and dodecanedioic acid, said product obtained by contacting a
genetically modified yeast comprising an active, modified
endogenous acyl-coA oxidase polypeptide with a feedstock capable of
being converted by the yeast to at least one of sebacic acid and
dodecanedioic acid; and culturing the yeast under conditions in
which said expression product is produced.
Description
RELATED PATENT APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/505,092 filed on Jul. 6,
2011, entitled "BIOLOGICAL METHODS FOR PREPARING SEBACIC ACID"
naming Stephen Picataggio and Tom Beardslee as inventors, and
designated by Attorney Docket No. VRD-1005-PV. This patent
application also claims the benefit of U.S. provisional patent
application No. 61/523,216 filed Aug. 12, 2011, entitled
"BIOLOGICAL METHODS FOR PREPARING DODECANEDIOIC ACID" naming
Stephen Picataggio and Tom Beardslee as inventors, and designated
by Attorney Docket No. VRD-1006-PV. The entire content of each of
the foregoing provisional patent applications is incorporated
herein by reference, including all text, tables and drawings.
SEQUENCE LISTING
[0002] The instant application contains a "lengthy" Sequence
Listing which has been submitted via CD-R in lieu of a printed
paper copy, and is hereby incorporated by reference in its
entirety. Said CD-R, recorded on Jul. 2, 2012, are labeled "CRF,"
"Copy 1--SEQUENCE LISTING PART," "Copy 2--SEQUENCE LISTING PART,"
and "Copy 3--SEQUENCE LISTING PART," respectively, and each
contains only one identical 19,578,880 byte file
(VRD1006P.TXT).
FIELD
[0003] The technology relates in part to biological methods for
producing a fatty dicarboxylic acid and engineered microorganisms
capable of such production.
BACKGROUND
[0004] Microorganisms employ various enzyme-driven biological
pathways to support their own metabolism and growth. A cell
synthesizes native proteins, including enzymes, in vivo from
deoxyribonucleic acid (DNA). DNA first is transcribed into a
complementary ribonucleic acid (RNA) that comprises a
ribonucleotide sequence encoding the protein. RNA then directs
translation of the encoded protein by interaction with various
cellular components, such as ribosomes. The resulting enzymes
participate as biological catalysts in pathways involved in
production of molecules by the organism.
[0005] These pathways can be exploited for the harvesting of the
naturally produced products. The pathways also can be altered to
increase production or to produce different products that may be
commercially valuable. Advances in recombinant molecular biology
methodology allow researchers to isolate DNA from one organism and
insert it into another organism, thus altering the cellular
synthesis of enzymes or other proteins. Advances in recombinant
molecular biology methodology also allow endogenous genes, carried
in the genomic DNA of a microorganism, to be increased in copy
number, thus altering the cellular synthesis of enzymes or other
proteins. Such genetic engineering can change the biological
pathways within the host organism, causing it to produce a desired
product. Microorganic industrial production can minimize the use of
caustic chemicals and the production of toxic byproducts, thus
providing a "clean" source for certain compounds. The use of
appropriate plant derived feedstocks allows production of "green"
compounds while further minimizing the need for and use of
petroleum derived compounds.
SUMMARY
[0006] Provided in certain aspects is a genetically modified yeast
that includes an active, modified endogenous acyl-coA oxidase
polypeptide or an active, modified endogenous acyl-coA
dehydrogenase polypeptide, which yeast is capable of producing a
diacid from a feedstock comprising one or more components from a
vegetable oil.
[0007] In certain instances a modified endogenous acyl-coA oxidase
polypeptide comprises one or more amino acid modifications in one
or more structures chosen from the N-terminal loop, D alpha helix,
loop between the D alpha helix and the E' alpha helix, an amino
acid in effective contact with carbons 6 to 9 in a feedstock
component, an amino acid in effective contact with carbons 10 to 12
in a feedstock component, L alpha helix, loop C-terminal to the L
alpha helix, and loop between the L alpha helix and the M alpha
helix.
[0008] In some instances a modified endogenous acyl-coA oxidase
polypeptide is a modified POX4 or POX5 polypeptide from a Candida
spp. yeast (e.g., strain ATCC20336 or ATCC20962). In some cases a
modified POX4 polypeptide comprises a modified amino acid sequence
of SEQ ID NO: 30. Sometimes the POX4 polypeptide comprises an amino
acid modification at one or more amino acid positions chosen from
88, 90, 96, 98, 99, 100, 102, 103, 302, 309, 310, 473, 474, 475,
476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,
489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,
502, 503, 504 and 505. A modified endogenous acyl-coA oxidase
polypeptide that is not a modified POX4 polypeptide can include an
amino acid modification at one or more positions corresponding to
one or more of the foregoing positions in the POX4 polypeptide.
[0009] In some instances a modified POX5 polypeptide comprises a
modified amino acid sequence of SEQ ID NO: 32. Sometimes the POX5
polypeptide comprises an amino acid modification at one or more
amino acid positions chosen from 81, 82, 83, 84, 85, 86, 88, 93,
94, 95, 96, 98, 102, 284, 287, 290, 291, 292, 294, 295, 436, 453,
454, 455, 456, 457, 458, 459, 460, 461, 462 and 463. A modified
endogenous acyl-coA oxidase polypeptide that is not a modified POX5
polypeptide can include an amino acid modification at one or more
positions corresponding to one or more of the foregoing positions
in the POX5 polypeptide.
[0010] In certain instances a modified endogenous acyl-coA
dehydrogenase polypeptide is chosen from a modified ACAD, VLCAD,
LCAD, MCAD and SCAD polypeptide. In some cases the acyl-coA
dehydrogenase polypeptide comprises an amino acid modification
corresponding to position 461 of a VLCAD polypeptide.
[0011] Provided in certain aspects is a genetically modified yeast
that includes a heterologous acyl-coA oxidase polypeptide or a
heterologous acyl-coA dehydrogenase polypeptide, which yeast is
capable of producing a diacid from a feedstock comprising one or
more components from a vegetable oil. The heterologous acyl-coA
oxidase polypeptide sometimes is a native polypeptide and sometimes
is an active, modified polypeptide. In some embodiments, the
heterologous acyl-coA dehydrogenase polypeptide sometimes is a
native polypeptide and sometimes is an active, modified
polypeptide. In certain instances, a heterologous acyl-coA oxidase
polypeptide is chosen from a polypeptide having an amino acid
sequence set forth in SEQ ID NO: 51 to SEQ ID NO: 3673. In some
cases a heterologous acyl-coA dehydrogenase polypeptide is chosen
from SEQ ID NOs: 3679 to 3683, 3686, 3689, 3691, 3693, 3695, 3697,
3699, 3701 and 3703.
[0012] A genetically modified yeast sometimes is chosen from a
Candida spp. yeast, Yarrowia spp. yeast, Pichia spp. yeast,
Saccharomyces spp. yeast and Kluyveromyces spp. yeast. In certain
cases a genetically modified yeast includes one or more genetic
modifications that reduce the activity of one or more native
endogenous acyl-coA oxidase polypeptides. In some instances a
genetically modified yeast includes a genetic modification that
reduces the activity of an enoyl coA isomerase polypeptide (e.g.,
ECI polypeptide (e.g., ECI1, ECI2)). In certain cases, a
genetically modified yeast includes a genetic modification that
reduces the activity of an acyl-CoA synthetase (ACS) polypeptide
(e.g., a genetic modification reduces the activity of a cytopasmic
and/or peroxisomal ACS polypeptide). An ACS polypeptide sometimes
is an ACS1 and/or FAT1 polypeptide. In certain instances, a
genetically modified yeast includes a genetic modification that
reduces the activity of a polypeptide that transports long-chain
fatty acyl-CoA molecules from the cytoplasm into the peroxisome
(e.g., the peroxisomal matrix). Such a polypeptide sometimes is a
PXA polypeptide. In some cases, a genetic modification that reduces
the activity of a certain polypeptide disrupts a polynucleotide
that encodes the polypeptide.
[0013] In some aspects provided is a method for producing a diacid,
which includes contacting a genetically modified yeast described
herein with a feedstock comprising one or more components from a
vegetable oil capable of being converted by the yeast to a diacid;
and culturing the yeast under conditions in which the diacid is
produced from the feedstock. The diacid sometimes is a C4 to C24
diacid, and sometimes is chosen from one or more of a C10, C12,
C14, C16, C18 or C20 diacid. A diacid sometimes contains no
unsaturation (e.g., double bond) and sometimes contains one or more
unsaturations. A particular diacid sometimes is the predominant
diacid in a mixture of diacids. In some instances the feedstock
includes a substantially pure oil. Sometimes the feedstock includes
a plurality of fatty acids, and sometimes the feedstock includes a
soapstock and/or fatty acid distillate. In certain cases the
vegetable oil is from a plant chosen from palm, palm kernel,
coconut, soy, safflower, canola, palm, palm kernel or combination
thereof.
[0014] In certain aspects, provided herein are isolated nucleic
acids described herein.
[0015] Certain embodiments are described further in the following
description, examples, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The drawings illustrate embodiments of the technology and
are not limiting. For clarity and ease of illustration, the
drawings are not made to scale and, in some instances, various
aspects may be shown exaggerated or enlarged to facilitate an
understanding of particular embodiments.
[0017] FIG. 1 is a schematic representation of the conversion of
decane to sebacic acid in a beta-oxidation blocked microorgansim.
Capric acid is formed as an intermediate during omega
oxidation.
[0018] FIG. 2 is a schematic representation of the conversion of
dodecane to dodecanedioic acid in a beta-oxidation blocked
microorganism. Lauric acid is formed as an intermediate during
omega oxidation.
[0019] FIG. 3 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes to mixed diacids
products, including sebacic acid in a beta-oxidation blocked
microorganism. Mixed chain-length fatty acids are formed as
intermediates during omega oxidation. Sebacic acid can be separated
from other diacid products by the use of appropriate separation
techniques.
[0020] FIG. 4 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes to mixed diacids
products, including dodecanedioic acid in a beta-oxidation blocked
microorganism. Mixed chain-length fatty acids are formed as
intermediates during omega oxidation. Dodecanedioic acid can be
separated from other diacid products by the use of appropriate
separation techniques.
[0021] FIG. 5 is a schematic representation of the conversion of a
long-chain alkane into sebacic acid in a partially beta-oxidation
blocked microorganism. The long-chain alkane is first converted
into a long-chain fatty acid and then into a long-chain diacid by
activities in the omega-oxidation pathway. The long-chain diacid
can be converted to sebacic acid by activities in the
beta-oxidation pathway, with the simultaneous generation of
acetyl-CoA.
[0022] FIG. 6 is a schematic representation of the conversion of a
long-chain alkane into dodecanedioic acid in a partially
beta-oxidation blocked microorganism. The long-chain alkane is
first converted into a long-chain fatty acid and then into a
long-chain diacid by activities in the omega-oxidation pathway. The
long-chain diacid can be converted to dodecanedioic acid by
activities in the beta-oxidation pathway, with the simultaneous
generation of acetyl-CoA.
[0023] FIG. 7 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes into sebacic acid
in a partially beta-oxidation blocked microorganism. The mixed
chain-length alkanes are first converted into mixed chain-length
fatty acids and then mixed diacids by activities in the
omega-oxidation pathway. Mixed diacids can be converted to sebacic
acid by activities in the beta-oxidation pathway, with the
simultaneous generation of acetyl-CoA.
[0024] FIG. 8 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes into dodecanedioic
acid in a partially beta-oxidation blocked microorganism. The mixed
chain-length alkanes are first converted into mixed chain-length
fatty acids and then mixed diacids by activities in the
omega-oxidation pathway. Mixed diacids can be converted to
dodecanedioic acid by activities in the beta-oxidation pathway,
with the simultaneous generation of acetyl-CoA.
[0025] FIG. 9 graphically illustrates the conversion of decane to
sebacic acid in a fully beta-oxidation blocked Candida yeast
strain. After incubation for the times shown in the graph, the
media was subjected to gas chromatography. The results indicate
that greater than 99% of the decane was converted into sebacic
acid, with a minimal amount of capric acid also detected by gas
chromatography. No significant accumulation of any other monoacid
or diacid was detected by gas chromatography. Experimental details
and results are given in Example 1.
[0026] FIG. 10 graphically illustrates the conversion of capric
acid to sebacic acid in a Candida yeast strain. GC analysis was
performed after a predetermined period of growth. Nearly all the
capric acid added was converted to sebacic acid using a starting
concentration of capric acid. Experimental details and results are
given in Example 2.
[0027] FIG. 11 graphically illustrates the distribution of diacids
produced during the conversion of long-chain fatty acids to mixed
diacids under fermentation conditions using a partially
beta-oxidation blocked Candida tropicalis strain (e.g., sAA106).
Experimental details and results are given in Example 5.
[0028] FIG. 12 graphically illustrates the conversion of decane to
sebacic acid in a fully beta-oxidation blocked Candida yeast strain
having additional genetic modifications. Strain sAA003 is the fully
beta-oxidation blocked control strain. +CPR indicates the fully
beta-oxidation blocked strain also includes an increased number of
copies of cytochrome P450 reductase. +CPR+A12 indicates starting
strain sAA003 includes the addition genetic modifications of an
increased number of copies of cytochrome P450 reductase and also
includes an increased number of copies of cytochrome P450 A12
(e.g., CYP52A12). +CPR+A18 indicates starting strain sAA003
includes the addition genetic modifications of an increased number
of copies of cytochrome P450 reductase and also includes an
increased number of copies of cytochrome P450 A18 (e.g., CYP52A18).
+CPR+A19 indicates starting strain sAA003 includes the addition
genetic modifications of an increased number of copies of
cytochrome P450 reductase and also includes an increased number of
copies of cytochrome P450 A19 (e.g., CYP52A19). +CPR+A20 indicates
starting strain sAA003 includes the addition genetic modifications
of an increased number of copies of cytochrome P450 reductase and
also includes an increased number of copies of cytochrome P450 A20
(e.g., CYP52A20). The y-axis of FIG. 12 is percent of theoretical
maximum yield. Experimental details and results are given in
Example 7.
[0029] FIG. 13 graphically illustrates the results of conversion of
methyl laurate to dodecanedioic acid in a fully beta-oxidation
blocked Candida yeast strain also contain genetic alterations to a
monooxygenase reductase activity, a monooxygenase activity, or a
monooxygenase reductase activity and a monooxygenase activity.
After 48 hours of incubation the media was subjected to gas
chromatography. The results indicate that Candida strains
containing an increased number of copies of a CYP52A18
monooxygenase activity and an increased number of copies of a
monooxygenase reductase activity (e.g., CPR750) gave the highest
yield of dodecanedioic acid (e.g., DDDA), in shake flask
fermentation experiments. Experimental details and results are
given in Example 8.
[0030] FIG. 14 and FIG. 15 schematically illustrate a screening
and/or selection method for identifying acyl-CoA oxidase activities
with specific substrate specificities. The method can be utilized
in conjunction with generating and/or identifying acyl-CoA oxidase
activities with altered chain-length substrate specificities.
Screening/selection method details are given in Example 9.
[0031] FIG. 16 graphically illustrates the results of engineered
microorganisms described herein converting decane to sebacic acid
under fermentation conditions using different amounts of decane as
the feedstock. Experimental details and results are given in
Example 3.
[0032] FIG. 17 graphically illustrates the results of engineered
microorganisms described herein converting a mixed fatty acid
feedstock (e.g., mixed chain-length fatty acids) to sebacic acid
under fermentation conditions. Experimental details and results are
given in Example 4.
[0033] FIG. 18 shows a diagram of a plasmid designated pAA073
containing a POX4 promoter and a POX4 terminator.
[0034] FIG. 19 illustrates the generation of a full-length deletion
cassette for ECI1 using PCR overlap extension.
[0035] FIG. 20 illustrates the generation of a full-length deletion
cassette for the second allele of ECI1 using PCR overlap
extension.
[0036] FIG. 21 shows an acyl CoA oxidase activity profile for Pox5
isolated from a Candida strain.
[0037] FIG. 22 illustrates a PCR overlap extension method for
introducing site-directed point mutations into Acyl-CoA Oxidase
genes.
[0038] FIG. 23--shows a sequence alignment of the N-terminal 180
amino acids of AcoI and AcoII. The amino acids highlighted in grey
are located within alpha helices and those in bold are located
within beta sheets. The center sequence represents the consensus
sequence showing conserved residues.
[0039] FIG. 24--illustrates a HotSpot Wizard analysis of Pox4 (FIG.
24A) and Pox5 (FIG. 24B) from Candida strain ATCC20336. Residues
highlighted in dark grey or light grey are mutagenic "hot spots".
Dark grey residues show greater variability at that position than
light grey residues. Residues in bold are found within or close to
the substrate binding pocket.
[0040] FIG. 25--illustrates a HotSpot Wizard analysis of RnAcoII.
Residues highlighted in dark grey or light grey are mutagenic "hot
spots". Dark grey residues show greater variability at that
position than light grey residues. Residues in bold are found
within or close to the substrate binding pocket.
[0041] FIG. 26 shows a multiple sequence alignment of all three
proteins. The underlined portion of RnAcoII (AcoII from R.
norvegicus) represents the alternatively spliced exon 3.
[0042] FIG. 27 shows the acyl CoA activity profile associated with
Pox5 mutants. The substrate referred to as "C18" is shortened and
pertains to a C18:1 substrate.
[0043] FIG. 28 shows the acyl CoA activity profile associated with
Pox4 mutants.
[0044] FIG. 29 shows a diagram of a plasmid designated pAA298.
DETAILED DESCRIPTION
[0045] Certain fatty dicarboxylic acids (i.e., diacids, e.g.,
dodecanedioic acid or sebacic acid) are chemical intermediates in
manufacturing processes used to make certain polyamides,
polyurethanes and plasticizers, all of which have wide applications
in producing items such as antiseptics, top-grade coatings,
hot-melt coating and adhesives, painting materials, corrosion
inhibitor, surfactant, engineering plastics and can also be used as
a starting material in the manufacture of fragrances, for example.
For example dodecanedioic acid, also known as 1,12 dodecanedioic
acid, and DDDA, is a 12 carbon organic molecule that is a fatty
dicarboxylic acid. In another example, sebacic acid, also known as
1,10 decanedioic acid, and 1,8 octanedicarboxylic acid, is a 10
carbon organic molecule that is a fatty dicarboxylic acid.
[0046] Provided herein are methods for producing a fatty
dicarboxylic acid (also referred to herein as a diacid). Any
suitable diacid can be produced, and a diacid produced often
includes acid moieties at each terminus of the molecule (e.g.,
alpha omega diacids). A diacid sometimes is a C4 to a C24 diacid
(i.e., a diacid containing 4 carbons to 24 carbons) and sometimes
is a C8, C10, C12, C14, C16, C18, or C20 diacid. Yeast and
processes herein are capable of producing a diacid containing an
odd number of carbons, and sometimes a product contains one or more
diacids chosen from a C5, C7, C9, C11, C13, C15, C17, C19, C21 and
C23 diacid. A hydrocarbon portion of a diacid sometimes is fully
saturated and sometimes a diacid includes one or more unsaturations
(e.g., double bonds).
[0047] Non-limiting examples of diacids include octadecanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) and
other organic intermediates using biological systems. Non-limiting
examples of fatty dicarboxylic acids include suberic acid (i.e.,
octanedioic acid, 1,8-octanedioic acid, octanedioic acid,
octane-1,8-dioic acid, 1,6-hexanedicarboxylic acid, capryllic
diacids), sebacic acid (i.e., 1,10-decanedioic acid, decanedioic
acid, decane-1,10-dioic acid, 1,8-octanedicarboxylic acid, capric
diacid), dodecanedioic acid (i.e., DDDA, 1,12-dodecanedioic acid,
dodecanedioic acid, dodecane-1,12-dioic acid,
1,10-decanedicarboxylic acid, decamethylenedicarboxylic acid,
1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e.,
TDDA, 1,14-tetradecanedioic acid, tetradecanedioic acid,
tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid,
myristic diacid), thapsic acid (i.e., hexadecanedioic acid,
1,16-hexadecanedioic acid, hexadecanedioic acid,
hexadecane-1,16-dioic acid, 1,14-tetradecanedicarboxylic acid,
palmitic diacid), cis-9-hexadecenedioic acid (i.e., palmitoleic
diacids), octanedioic acid (i.e., 1,18-octadecanedioic acid,
octadecanedioic acid, octadecane-1,18-dioic acid,
1,16-hexadecanedicarboxylic acid, stearic diacid),
cis-9-octadecenedioic acid (i.e., oleic diacids),
cis-9,12-octadecenedioic acid (i.e., linoleic diacids),
cis-9,12,15-octadecenedioic acid (i.e., linolenic diacids),
arachidic diacid (i.e., eicosanoic diacid, icosanoic diacid),
11-eicosenoic diacid (i.e., cis-11-eicosenedioic acid),
13-eicosenoic diacids (i.e., cis-13-eicosenedioic acid),
arachidonic diacid (i.e., cis-5,8,11,14-eicosatetraenedioic
acid).
[0048] A genetically modified yeast can be provided with a
feedstock to produce a diacid, and the feedstock sometimes includes
a substantially pure aliphatic molecule from which the diacid is
produced. In certain embodiments, the feedstock contains a mixed
set of aliphatic molecules from which diacids may be produced. In
some embodiments, an aliphatic molecule in the feedstock is the
predominant aliphatic species and sometimes a particular diacid
produced from that aliphatic molecule is the predominant diacid
species produced. A predominant species generally is 51% or more by
weight of aliphatic molecule species in a feedstock or 51% or more
by weight of diacid species in a product (e.g., about 55% or more,
60% or more, 65% or more, 70% or more, 75% or more, 80% or more,
85% or more, 90% or more or 95% or more).
[0049] Such production systems may have significantly less
environmental impact and could be economically competitive with
current manufacturing systems. Thus, provided in part herein are
methods for manufacturing a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) by engineered microorganisms. In some
embodiments microorganisms are engineered to contain at least one
heterologous gene encoding an enzyme, where the enzyme is a member
of a novel and/or altered pathway engineered into the
microorganism. In certain embodiments, an organism may be selected
for elevated activity of a native enzyme.
Microorganisms
[0050] A microorganism selected often is suitable for genetic
manipulation and often can be cultured at cell densities useful for
industrial production of a target fatty dicarboxylic acid product.
A microorganism selected often can be maintained in a fermentation
device.
[0051] The term "engineered microorganism" as used herein refers to
a modified microorganism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point (hereafter a "host microorganism"). An engineered
microorganism includes a heterologous polynucleotide in some
embodiments, and in certain embodiments, an engineered organism has
been subjected to selective conditions that alter an activity, or
introduce an activity, relative to the host microorganism. Thus, an
engineered microorganism has been altered directly or indirectly by
a human being. A host microorganism sometimes is a native
microorganism, and at times is a microorganism that has been
engineered to a certain point.
[0052] In some embodiments an engineered microorganism is a single
cell organism, often capable of dividing and proliferating. A
microorganism can include one or more of the following features:
aerobe, anaerobe, filamentous, non-filamentous, monoploid, dipoid,
auxotrophic and/or non-auxotrophic. In certain embodiments, an
engineered microorganism is a prokaryotic microorganism (e.g.,
bacterium), and in certain embodiments, an engineered microorganism
is a non-prokaryotic microorganism. In some embodiments, an
engineered microorganism is a eukaryotic microorganism (e.g.,
yeast, fungi, amoeba). In some embodiments, an engineered
microorganism is a fungus. In some embodiments, an engineered
organism is a yeast.
[0053] Any suitable yeast may be selected as a host microorganism,
engineered microorganism, genetically modified organism or source
for a heterologous or modified polynucleotide. Yeast include, but
are not limited to, Yarrowia yeast (e.g., Y. lipolytica (formerly
classified as Candida lipolytica)), Candida yeast (e.g., C.
revkaufi, C. viswanathii, C. pulcherrima, C. tropicalis, C.
utilis), Rhodotorula yeast (e.g., R. glutinus, R. graminis),
Rhodosporidium yeast (e.g., R. toruloides), Saccharomyces yeast
(e.g., S. cerevisiae, S. bayanus, S. pastorianus, S.
carlsbergensis), Cryptococcus yeast, Trichosporon yeast (e.g., T.
pullans, T. cutaneum), Pichia yeast (e.g., P. pastoris) and
Lipomyces yeast (e.g., L. starkeyii, L. lipoferus). In some
embodiments, a suitable yeast is of the genus Arachniotus,
Aspergillus, Aureobasidium, Auxarthron, Blastomyces, Candida,
Chrysosporuim, Chrysosporuim Debaryomyces, Coccidiodes,
Cryptococcus, Gymnoascus, Hansenula, Histoplasma, Issatchenkia,
Kluyveromyces, Lipomyces, Lssatchenkia, Microsporum, Myxotrichum,
Myxozyma, Oidiodendron, Pachysolen, Penicillium, Pichia,
Rhodosporidium, Rhodotorula, Rhodotorula, Saccharomyces,
Schizosaccharomyces, Scopulariopsis, Sepedonium, Trichosporon, or
Yarrowia. In some embodiments, a suitable yeast is of the species
Arachniotus flavoluteus, Aspergillus flavus, Aspergillus fumigatus,
Aspergillus niger, Aureobasidium pullulans, Auxarthron thaxteri,
Blastomyces dermatitidis, Candida albicans, Candida dubliniensis,
Candida famata, Candida glabrata, Candida guilliermondii, Candida
kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida
lustitaniae, Candida parapsilosis, Candida pulcherrima, Candida
revkaufi, Candida rugosa, Candida tropicalis, Candida utilis,
Candida viswanathii, Candida xestobii, Chrysosporuim
keratinophilum, Coccidiodes immitis, Cryptococcus albidus var.
diffluens, Cryptococcus laurentii, Cryptococcus neofomans,
Debaryomyces hansenii, Gymnoascus dugwayensis, Hansenula anomala,
Histoplasma capsulatum, Issatchenkia occidentalis, Isstachenkia
orientalis, Kluyveromyces lactis, Kluyveromyces marxianus,
Kluyveromyces thermotolerans, Kluyveromyces waltii, Lipomyces
lipoferus, Lipomyces starkeyii, Microsporum gypseum, Myxotrichum
deflexum, Oidiodendron echinulatum, Pachysolen tannophilis,
Penicillium notatum, Pichia anomala, Pichia pastoris, Pichia
stipitis, Rhodosporidium toruloides, Rhodotorula glutinus,
Rhodotorula graminis, Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe, Scopulariopsis acremonium,
Sepedonium chrysospermum, Trichosporon cutaneum, Trichosporon
pullans, Yarrowia lipolytica, or Yarrowia lipolytica (formerly
classified as Candida lipolytica). In some embodiments, a yeast is
a Y. lipolytica strain that includes, but is not limited to,
ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 and LGAM S(7)1
strains (Papanikolaou S., and Aggelis G., Bioresour. Technol.
82(1):43-9 (2002)). In certain embodiments, a yeast is a Candida
species (i.e., Candida spp.) yeast. Any suitable Candida species
can be used and/or genetically modified for production of a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid). In some embodiments,
suitable Candida species include, but are not limited to Candida
albicans, Candida dubliniensis, Candida famata, Candida glabrata,
Candida guilliermondii, Candida kefyr, Candida krusei, Candida
lambica, Candida lipolytica, Candida lustitaniae, Candida
parapsilosis, Candida pulcherrima, Candida revkaufi, Candida
rugosa, Candida tropicalis, Candida utilis, Candida viswanathii,
Candida xestobii and any other Candida spp. yeast described herein.
Non-limiting examples of Candida spp. strains include, but are not
limited to, sAA001 (ATCC20336), sAA002 (ATCC20913), sAA003
(ATCC20962), sAA496 (US2012/0077252), sAA106 (US2012/0077252), SU-2
(ura3-/ura3-), H5343 (beta oxidation blocked; U.S. Pat. No.
5,648,247) strains. Any suitable strains from Candida spp. yeast
may be utilized as parental strains for genetic modification.
[0054] Yeast genera, species and strains are often so closely
related in genetic content that they can be difficult to
distinguish, classify and/or name. In some cases strains of C.
lipolytica and Y. lipolytica can be difficult to distinguish,
classify and/or name and can be, in some cases, considered the same
organism. In some cases, various strains of C. tropicalis and C.
viswanathii can be difficult to distinguish, classify and/or name
(for example see Arie et. al., J. Gen. Appl. Microbiol., 46,
257-262 (2000). Some C. tropicalis and C. viswanathii strains
obtained from ATCC as well as from other commercial or academic
sources can be considered equivalent and equally suitable for the
embodiments described herein. In some embodiments, some parental
strains of C. tropicalis and C. viswanathii are considered to
differ in name only.
[0055] Any suitable fungus may be selected as a host microorganism,
engineered microorganism or source for a heterologous
polynucleotide. Non-limiting examples of fungi include, but are not
limited to, Aspergillus fungi (e.g., A. parasiticus, A. nidulans),
Thraustochytrium fungi, Schizochytrium fungi and Rhizopus fungi
(e.g., R. arrhizus, R. oryzae, R. nigricans). In some embodiments,
a fungus is an A. parasiticus strain that includes, but is not
limited to, strain ATCC24690, and in certain embodiments, a fungus
is an A. nidulans strain that includes, but is not limited to,
strain ATCC38163.
[0056] Any suitable prokaryote may be selected as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. A Gram negative or Gram positive
bacteria may be selected. Examples of bacteria include, but are not
limited to, Bacillus bacteria (e.g., B. subtilis, B. megaterium),
Acinetobacter bacteria, Norcardia bacteria, Xanthobacter bacteria,
Escherichia bacteria (e.g., E. coli (e.g., strains DH10B, Stb12,
DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 and ccdA-over (e.g., U.S.
application Ser. No. 09/518,188))), Streptomyces bacteria, Erwinia
bacteria, Klebsiella bacteria, Serratia bacteria (e.g., S.
marcessans), Pseudomonas bacteria (e.g., P. aeruginosa), Salmonella
bacteria (e.g., S. typhimurium, S. typhi), Megasphaera bacteria
(e.g., Megasphaera elsdenii). Bacteria also include, but are not
limited to, photosynthetic bacteria (e.g., green non-sulfur
bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus),
Chloronema bacteria (e.g., C. gigateum)), green sulfur bacteria
(e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon
bacteria (e.g., P. luteolum), purple sulfur bacteria (e.g.,
Chromatium bacteria (e.g., C. okenii)), and purple non-sulfur
bacteria (e.g., Rhodospirillum bacteria (e.g., R. rubrum),
Rhodobacter bacteria (e.g., R. sphaeroides, R. capsulatus), and
Rhodomicrobium bacteria (e.g., R. vanellii)).
[0057] Cells from non-microbial organisms can be utilized as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. Examples of such cells, include, but
are not limited to, insect cells (e.g., Drosophila (e.g., D.
melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells)
and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C.
elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis
cells); reptilian cells; mammalian cells (e.g., NIH3T3, 293, CHO,
COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells); and
plant cells (e.g., Arabidopsis thaliana, Nicotania tabacum, Cuphea
acinifolia, Cuphea aequipetala, Cuphea angustifolia, Cuphea
appendiculata, Cuphea avigera, Cuphea avigera var. pulcherrima,
Cuphea axilliflora, Cuphea bahiensis, Cuphea baillonis, Cuphea
brachypoda, Cuphea bustamanta, Cuphea calcarata, Cuphea calophylla,
Cuphea calophylla subsp. mesostemon, Cuphea carthagenensis, Cuphea
circaeoides, Cuphea confertiflora, Cuphea cordata, Cuphea
crassiflora, Cuphea cyanea, Cuphea decandra, Cuphea denticulata,
Cuphea disperma, Cuphea epilobiifolia, Cuphea ericoides, Cuphea
flava, Cuphea flavisetula, Cuphea fuchsiifolia, Cuphea gaumeri,
Cuphea glutinosa, Cuphea heterophylla, Cuphea hookeriana, Cuphea
hyssopifolia (Mexican-heather), Cuphea hyssopoides, Cuphea ignea,
Cuphea ingrata, Cuphea jorullensis, Cuphea lanceolata, Cuphea
linarioides, Cuphea llavea, Cuphea lophostoma, Cuphea lutea, Cuphea
lutescens, Cuphea melanium, Cuphea melvilla, Cuphea micrantha,
Cuphea micropetala, Cuphea mimuloides, Cuphea nitidula, Cuphea
palustris, Cuphea parsonsia, Cuphea pascuorum, Cuphea paucipetala,
Cuphea procumbens, Cuphea pseudosilene, Cuphea pseudovaccinium,
Cuphea pulchra, Cuphea racemosa, Cuphea repens, Cuphea salicifolia,
Cuphea salvadorensis, Cuphea schumannii, Cuphea sessiliflora,
Cuphea sessilifolia, Cuphea setosa, Cuphea spectabilis, Cuphea
spermacoce, Cuphea splendida, Cuphea splendida var. viridiflava,
Cuphea strigulosa, Cuphea subuligera, Cuphea teleandra, Cuphea
thymoides, Cuphea tolucana, Cuphea urens, Cuphea utriculosa, Cuphea
viscosissima, Cuphea watsoniana, Cuphea wrightii, Cuphea
lanceolata)
[0058] Microorganisms or cells used as host organisms or source for
a heterologous polynucleotide are commercially available.
Microorganisms and cells described herein, and other suitable
microorganisms and cells are available, for example, from
Invitrogen Corporation, (Carlsbad, Calif.), American Type Culture
Collection (Manassas, Va.), and Agricultural Research Culture
Collection (NRRL; Peoria, Ill.).
[0059] Host microorganisms and engineered microorganisms may be
provided in any suitable form. For example, such microorganisms may
be provided in liquid culture or solid culture (e.g., agar-based
medium), which may be a primary culture or may have been passaged
(e.g., diluted and cultured) one or more times. Microorganisms also
may be provided in frozen form or dry form (e.g., lyophilized).
Microorganisms may be provided at any suitable concentration.
[0060] Carbon Processing Pathways and Activities
[0061] FIGS. 1-8 schematically illustrate non-limiting embodiments
of engineered pathways that can be used to produce a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) from various starting
carbon sources or feedstocks. FIG. 1 depicts an embodiment of a
non-limiting engineered biological pathway for producing sebacic
acid in microorganisms having a fully blocked beta-oxidation
pathway, using decane as the carbon source starting material. FIG.
2 depicts an embodiment of a non-limiting engineered biological
pathway for producing dodecanedioic acid in microorganisms having a
fully blocked beta-oxidation pathway, using dodecane as the carbon
source starting material. FIGS. 3 and 4 depict an embodiment of a
non-limiting engineered biological pathway for producing mixed
chain-length diacids in a microorganism having a fully blocked
beta-oxidation pathway, using mixed chain-length alkanes as the
carbon source starting material. Sebacic acid (FIG. 3) and
dodecanedioic acid (FIG. 4) can be separated and/or purified away
from other diacid products using a suitable combination of
centrifugation, organic solvent extraction, chromatography, and/or
other purification/separation techniques. FIGS. 5 and 6 depict an
embodiment of a non-limiting engineered biological pathway for
producing sebacic acid (FIG. 5) and dodecanedioic acid (FIG. 6) in
microorganisms having a partially blocked beta oxidation pathway,
using long-chain alkanes as the carbon source starting material.
FIGS. 7 and 8 depict an embodiment of a non-limiting engineered
biological pathway for producing sebacic acid (FIG. 7) and
dodecanedioic acid (FIG. 8) in microorganisms having a partially
blocked beta oxidation pathway, using mixed-chain length alkanes as
the carbon source starting material. The alkane carbon source
starting materials are initially metabolized using naturally
occurring and/or engineered activities in naturally occurring
and/or engineered pathways to yield an intermediate alcohol which
can then be converted to a carboxylic acid (e.g., fatty acid) by
the action of other naturally occurring and/or engineered
activities in the omega-oxidation pathway depicted in FIGS.
1-8.
[0062] Alkanes are omega-hydroxylated by the activity of cytochrome
P450 enzymes, thereby generating the equivalent chain-length
alcohol derivative of the starting alkane carbon source material.
In certain embodiments, a cytochrome P450 activity can be increased
by increasing the number of copies of a cytochrome P450 gene (e.g.,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more copies of the gene),
by increasing the activity of a promoter that regulates
transcription of a cytochrome P450 gene, or by increasing the
number of copies of a cytochrome P450 gene and increasing the
activity of a promoter that regulates transcription of a cytochrome
P450 gene, thereby increasing the production of target product
(e.g., sebacic or dodecanedioic acid) via increased activity of one
or more cytochrome P450 enzymes. In some embodiments, a cytochrome
P450 enzyme is endogenous to the host microorganism. One or more
cytochrome P450 activities can be added and/or increased dependent
on the carbon source starting material, in certain embodiments.
Cytochrome P450's sometimes exhibit increased activities in
response to stimulation by certain feedstocks or carbon source
starting materials. In some embodiments, an engineered
microorganism includes an increased number of copies of one or more
cytochrome P450s that are stimulated by a chosen carbon source
starting material or feedstock. Cytochrome P450 responsiveness to a
chosen starting carbon source or feedstock can be determined using
any suitable assay. Non-limiting examples of assays suitable for
identification of cytochrome P450 responsiveness to a starting
carbon source or feedstock include RT-PCR or qRT-PCR after the host
microorganism has been exposed to the chosen carbon source or
feedstock for varying amounts of time.
[0063] Cytochrome P450 is reduced by the activity of cytochrome
P450 reductase (CPR), thereby recycling cytochrome P450 to allow
further enzymatic activity. In certain embodiments, the CPR enzyme
is endogenous to the host microorganism. In some embodiments, host
CPR activity can be increased by increasing the number of copies of
a CPR gene (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more
copies of the gene), by increasing the activity of a promoter that
regulates transcription of a CPR gene, or by increasing the number
of copies of a CPR gene and increasing the activity of a promoter
that regulates transcription of a CPR gene, thereby increasing the
production of target product (e.g., sebacic or dodecanedioic acid)
via increased recycling of cytochrome P450. In certain embodiments,
the promoter can be a heterologous promoter (e.g., endogenous or
exogenous promoter). In some embodiments, the CPR gene is
heterologous and exogenous and can be isolated from any suitable
organism. Non-limiting examples of organisms from which a CPR gene
can be isolated include C. tropicalis, S. cerevisiae and Bacillus
megaterium.
[0064] Oxidation of the alcohol to an aldehyde may be performed by
an enzyme in the fatty alcohol oxidase family (e.g., long-chain
fatty alcohol oxidase EC 1.1.3.20), or an enzyme in the alcohol
dehydrogenase family (e.g., fatty alcohol dehydrogenase; EC
1.1.1.1). The aldehyde may be oxidized to a carboxylic acid (e.g.,
sebacic or dodecanedioic acid) by the activity of the enzyme
aldehyde dehydrogenase (e.g., long-chain-aldehyde dehydrogenase or
fatty aldehyde dehydrogenase; EC 1.2.1.48). In some embodiments,
the long chain fatty alcohol oxidase, fatty alcohol dehydrogenase
and/or the long-chain-aldehyde dehydrogenase exist in a host
organism. Flux through these two steps may sometimes be augmented
by increasing the copy number of the enzymes, or by increasing the
activity of the promoter transcribing the genes. In some
embodiments alcohol and aldehyde dehydrogenases specific for 10, 12
or 14 carbon substrates may be isolated from another organism, and
inserted into the host organism.
[0065] FIG. 1 depicts a non-limiting embodiment of an engineered
biological pathway for making sebacic acid using decane (e.g., a
C10 alkane) as the carbon source starting material. Due to the
carbon chain length of decane, no chain shortening is necessary to
arrive at the 10 carbon diacid, sebacic acid. Thus a fully beta
oxidation blocked microorganism can be utilized to minimize
conversion of the desired 10 carbon diacid into diacids having
shorter chain lengths.
[0066] FIG. 2 depicts a non-limiting embodiment of an engineered
biological pathway for making dodecanedioic acid using dodecane
(e.g., a C12 alkane) as the carbon source starting material. Due to
the carbon chain length of dodecane, no chain shortening is
necessary to arrive at the 12 carbon diacid, dodecanedioic acid.
Thus a fully beta oxidation blocked microorganism can be utilized
to minimize conversion of the desired 12 carbon diacid into diacids
having shorter chain lengths.
[0067] FIGS. 3 and 4 depict a non-limiting embodiment of an
engineered biological pathway for generating a mixed population of
diacid (fatty dicarboxylic acid) products, including sebacic acid
(FIG. 3) and dodecanedioic acid (FIG. 4), using a carbon source or
feedstock that contains mixed-chain-length alkanes as the carbon
source starting material. Any suitable mixed-chain-length alkane,
fatty alcohol, mixed chain length fatty alcohol feedstock, fatty
acid, mixed fatty acid feedstock, paraffin, fat or oil can be used.
In some embodiments, the distribution of carbon chain lengths in
the starting material is substantially similar to the desired
carbon chain length distribution in the mixed diacid product. In
certain embodiments, the feedstock is enriched for a desired chain
length. In some embodiments, the enriched fraction is enriched for
carbon chain lengths of about 10 carbons. In some embodiments, the
enriched fraction is enriched for carbon chain lengths of about 12
carbons. Because, in some embodiments, the diacids generated have
substantially the same chain lengths as the chain lengths found in
the carbon source starting material, a fully beta-oxidation blocked
microorganism can be utilized to minimize conversion of the diacids
of desired chain length into diacids of shorter chain lengths. The
lower part of the pathways in FIG. 3 AND FIG. 4 show the separation
of sebacic acid and dodecanedioic acid, respectively, away from the
mixed diacid products by the use of separation techniques described
herein, or those known in the art.
[0068] In certain embodiments involving genetically modified
organisms having partially blocked beta-oxidation pathways (see
FIGS. 5-8), feedstocks suitable for use include, but are not
limited to, fatty acid distillates or soapstocks of renewable oils
(palm oil fatty acid distillate, soybean oil soapstock, coconut oil
soapstock), renewable oils (coconut oil, palm oil, palm kernel oil,
soybean oil, corn oil, etc.), fatty acids of chain length equal to
or greater than C10 (in substantially single form (e.g., in
substantially pure form) or in mixture form, alkanes of chain
length equal to or greater than C10 in substantially single form
(e.g., substantially pure form) or in mixture form.
[0069] Carbon sources with longer chain lengths (e.g., 12 carbons
or greater in length) can be metabolized using naturally occurring
and/or engineered pathways to yield molecules that can be further
metabolized using the beta oxidation pathway shown in the lower
portion of FIGS. 5 through 8. In some embodiments, beta-oxidation
activities in the pathways shown in FIGS. 5 through 8 also can be
engineered (e.g., as described herein) to enhance metabolism and
target product formation. In some embodiments, one acyl-CoA oxidase
activity of the beta-oxidation pathway is engineered to be
enhanced, and in certain embodiments, the other acyl-CoA oxidase
activity in the beta-oxidation pathway is altered to reduce or
eliminate the activity, thereby optimizing the production of a
diacid of a desired chain-length or diacids with a distribution of
desired chain lengths. In some embodiments, an acyl-CoA oxidase is
selected and/or engineered to alter the substrate specificity of
the enzyme. In certain embodiments, the substrate specificity of a
heterologous and/or engineered acyl-CoA oxidase is for carbon chain
lengths of between about 12 carbons and about 18 carbons, and in
some embodiments a heterologous and/or engineered acyl-CoA oxidase
exhibits no activity on substrates below 12 carbons in length. In
certain embodiments, a heterologous acyl-CoA oxidase with a desired
chain length specificity can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for acyl-CoA oxidase enzymes include yeast (e.g.,
Candida, Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0070] In certain embodiments, a carbon source starting material
(e.g., alkane, fatty acid, fatty alcohol, dicarboxylic acid) of
intermediate or long chain length (e.g., between about 10 carbons
and 22 carbons) is converted into an acyl-CoA derivative for entry
into the beta-oxidation pathway. The acyl-CoA derivative can be
generated by the activity of an acyl-CoA ligase enzyme, in some
embodiments. The acyl-CoA derivative is subsequently oxidized by
the activity of an acyl-CoA oxidase enzyme (e.g., also known as
acyl-CoA oxidoreductase and fatty acyl-coenzyme A oxidase) of
natural or altered substrate specificity, in certain embodiments.
The trans-2,3-dehydroacyl-CoA derivative long chain fatty alcohol,
fatty acid or dicarboxylic acid may be further converted to
3-hydroxyacyl-CoA by the activity of enoyl-CoA hydratase.
3-hydroxyacyl-CoA can be converted to 3-oxoacyl-CoA by the activity
of 3-hydroxyacyl-CoA dehydrogenase. 3-oxoacyl-CoA may be converted
to an acyl-CoA molecule, shortened by 2 carbons and an acetyl-CoA,
by the activity of Acetyl-CoA C-acyltransferase (e.g., also known
as beta-ketothiolase and beta-ketothiolase). In some embodiments,
acyl-CoA molecules may be repeatedly shortened by beta oxidation
until a desired carbon chain length is generated (e.g., 10 or 12
carbons, sebacic acid or dodecanedioic acid, respectively). A
shortened fatty acid can be further processed using omega oxidation
to yield a dicarboxylic acid (e.g., dodecanedioic acid).
[0071] Beta-Oxidation Activities
[0072] The term "beta oxidation pathway" as used herein, refers to
a series of enzymatic activities utilized to metabolize fatty
alcohols, fatty acids, or dicarboxylic acids. The activities
utilized to metabolize fatty alcohols, fatty acids, or dicarboxylic
acids include, but are not limited to, acyl-CoA ligase activity,
acyl-CoA oxidase activity, acyl-CoA hydrolase activity, acyl-CoA
thioesterase activity, enoyl-CoA hydratase activity,
3-hydroxyacyl-CoA dehydrogenase activity and acetyl-CoA
C-acyltransferase activity. The term "beta oxidation activity"
refers to any of the activities in the beta oxidation pathway
utilized to metabolize fatty alcohols, fatty acids or dicarboxylic
acids.
[0073] Beta-Oxidation--Acyl-CoA Ligase
[0074] An acyl-CoA ligase enzyme sometimes is encoded by the host
organism and can be added to generate an engineered organism. In
some embodiments, host acyl-CoA ligase activity can be increased by
increasing the number of copies of an acyl-CoA ligase gene, by
increasing the activity of a promoter that regulates transcription
of an acyl-CoA ligase gene, or by increasing the number copies of
the gene and by increasing the activity of a promoter that
regulates transcription of the gene, thereby increasing production
of target product (e.g., sebacic or dodecanedioic acid) due to
increased carbon flux through the pathway. In certain embodiments,
the acyl-CoA ligase gene can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for, acyl-CoA ligase enzymes include Candida,
Saccharomyces, or Yarrowia.
[0075] Beta-Oxidation--Enoyl-CoA Hydratase
[0076] An enoyl-CoA hydratase enzyme catalyzes the addition of a
hydroxyl group and a proton to the unsaturated .beta.-carbon on a
fatty-acyl CoA and sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the enoyl-CoA hydratase activity is unchanged
in a host or engineered organism. In some embodiments, the host
enoyl-CoA hydratase activity can be increased by increasing the
number of copies of an enoyl-CoA hydratase gene, by increasing the
activity of a promoter that regulates transcription of an enoyl-CoA
hydratase gene, or by increasing the number copies of the gene and
by increasing the activity of a promoter that regulates
transcription of the gene, thereby increasing the production of
target product (e.g., sebacic or dodecanedioic acid) due to
increased carbon flux through the pathway. In certain embodiments,
the enoyl-CoA hydratase gene can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for, enoyl-CoA hydratase enzymes include Candida,
Saccharomyces, or Yarrowia.
[0077] Beta-Oxidation--3-Hydroxyacyl-CoA Dehydrogenase
[0078] 3-hydroxyacyl-CoA dehydrogenase enzyme catalyzes the
formation of a 3-ketoacyl-CoA by removal of a hydrogen from the
newly formed hydroxyl group created by the activity of enoyl-CoA
hydratase. In some embodiments, the activity is encoded by the host
organism and sometimes can be added or increased to generate an
engineered organism. In certain embodiments, the 3-hydroxyacyl-CoA
activity is unchanged in a host or engineered organism. In some
embodiments, the host 3-hydroxyacyl-CoA dehydrogenase activity can
be increased by increasing the number of copies of a
3-hydroxyacyl-CoA dehydrogenase gene, by increasing the activity of
a promoter that regulates transcription of a 3-hydroxyacyl-CoA
dehydrogenase gene, or by increasing the number copies of the gene
and by increasing the activity of a promoter that regulates
transcription of the gene, thereby increasing production of target
product (e.g., sebacic or dodecanedioic acid) due to increased
carbon flux through the pathway. In certain embodiments, the
3-hydroxyacyl-CoA dehydrogenase gene can be isolated from any
suitable organism. Non-limiting examples of organisms that include,
or can be used as donors for, 3-hydroxyacyl-CoA dehydrogenase
enzymes include Candida, Saccharomyces, or Yarrowia.
[0079] Beta-Oxidation--Acetyl-CoA C-Acyltransferase
[0080] An Acetyl-CoA C-acyltransferase (e.g., beta-ketothiolase)
enzyme catalyzes the formation of a fatty acyl-CoA shortened by 2
carbons by cleavage of the 3-ketoacyl-CoA by the thiol group of
another molecule of CoA. The thiol is inserted between C-2 and C-3,
which yields an acetyl CoA molecule and an acyl CoA molecule that
is two carbons shorter. An Acetyl-CoA C-acyltransferase sometimes
is encoded by the host organism and sometimes can be added to
generate an engineered organism. In certain embodiments, the
acetyl-CoA C-acyltransferase activity is unchanged in a host or
engineered organism. In some embodiments, the host acetyl-CoA
C-acyltransferase activity can be increased by increasing the
number of copies of an acetyl-CoA C-acyltransferase gene, or by
increasing the activity of a promoter that regulates transcription
of an acetyl-CoA C-acyltransferase gene, thereby increasing the
production of target product (e.g., sebacic or dodecanedioic acid)
due to increased carbon flux through the pathway. In certain
embodiments, the acetyl-CoA C-acyltransferase gene can be isolated
from any suitable organism. Non-limiting examples of organisms that
include, or can be used as donors for, acetyl-CoA C-acyltransferase
enzymes include Candida, Saccharomyces, or Yarrowia.
[0081] Beta-Oxidation--Enoyl CoA Isomerase
[0082] Feedstocks, such as fatty acid distillates and soapstocks
can comprise unsaturated fatty acids, for example, such as oleic
acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). In
some embodiments, unsaturated fatty acids are converted to
dicarboxylic acids that maintain the position and orientation of
the double bonds. Cells can employ additional enzymes to allow the
oxidation (e.g., beta oxidation) of these types of unsaturated
fatty acids or diacids. In some instances, an enzyme enoyl-CoA
isomerase (ECI) is required for the beta-oxidation of substrates
with double bonds at odd numbered positions. In some instances, the
enzyme dienoyl-CoA reductase (DCR) is required for the
beta-oxidation of substrates with double bonds at even numbered
positions).
[0083] Enoyl CoA Isomerase (ECI) can also be known as enoyl-CoA
delta isomerase 1, dodecenoyl-CoA isomerase, 3,2 trans-enoyl-CoA
isomerase, acetylene-allene isomerase, delta3, delta2-enoyl-CoA
isomerase, dodecenoyl-CoA delta isomerase, and EC 5.3.3.8 (in human
for example). Several alternatively spliced transcript variants are
also known. ECI is a member of the hydratase/isomerase superfamily.
ECI can be a key mitochondrial enzyme involved in beta-oxidation of
unsaturated fatty acids. This enzyme can isomerize both 3-cis and
3-trans double bonds into the 2-trans form in a range of ECI
enzymes from different species. ECI can catalyze the transformation
of 3-cis and 3-trans-enoyl-CoA esters arising during the stepwise
degradation of cis-, mono-, and polyunsaturated fatty acids to the
2-trans-enoyl-CoA intermediates.
[0084] In some embodiments, ECI is a critical enzyme because of its
activity and the normal position of double bonds in some feedstocks
(e.g., soapstocks and fatty acid distillates). Many unsaturated
fatty acids have a cis-.DELTA.9 double bond. During the
beta-oxidation of an 18-carbon diacid with a cis-A9 double bond,
the double bond is encountered when it has been chain shortened to
12 carbons. At this stage the 12-carbon molecule can have a
cis-.DELTA.3 double bond that is not a substrate for an acyl-CoA
oxidase. Therefore, in some embodiments, ECI is required to convert
the cis-.DELTA.3 double bond to a trans-.DELTA.2 double bond. In
some instances, the product of the ECI reaction is a substrate for
the second step in beta-oxidation, so ECI can effectively bypasses
acyl-CoA oxidase in this particular round of beta-oxidation. In
some instances, this is important because even if a strain
comprises an acyl-CoA oxidase that is not active on feedstocks of
.ltoreq.C12 (i.e., 12 carbons), an active ECI can effect the
shortening of one more round of beta-oxidation which can produce a
10-carbon product for substrates with a cis-.DELTA.9 double bond.
Therefore, in some embodiments the ECI gene is disrupted (e.g.,
knocked out or deleted) in a yeast (e.g., in a Candida strain) to
prevent chain shortening past a desired chain-length (e.g., in this
instance, 12 carbons). In some embodiments, disrupting the
expression (e.g. knocking out the expression) of an ECI gene can
result in an increase in the production of a fatty dicarboxylic
acid comprising 10 to 18 carbons. In some embodiments, disrupting
the expression (e.g. knocking out the expression) of an ECI gene
can result in an increase in the production of a fatty dicarboxylic
acid comprising 10, 12, 14, 16 or 18 carbons. In some embodiments,
disrupting the expression of an enoyl CoA isomerase can increase
the production of fatty dicarboxylic acid comprising 10, 12, 14, 16
or 18 carbons when using certain feedstocks (e.g., certain
soapstocks or fatty acid distillates).
[0085] In some embodiments, an ECI knock out (i.e. eci.DELTA.)
strain is able to produce DDDA from oleic acid even in the presence
of acyl-CoA oxidase with activity on substrates of chain-length
less than 12 carbons.
[0086] In some embodiments, a 12 carbon dicarboxylic acids produced
from fatty acid feedstocks comprising unsaturated fatty acids
require hydrogenation to arrive at the fully saturated DDDA
product.
[0087] Omega Oxidation Activities
[0088] The term "omega oxidation activity" refers to any of the
activities in the omega oxidation pathway utilized to metabolize
alkanes, fatty alcohols, fatty acids, dicarboxylic acids, or
sugars. The activities utilized to metabolize fatty alcohols, fatty
acids, or dicarboxylic acids include, but are not limited to,
monooxygenase activity (e.g., cytochrome P450 activity),
monooxygenase reductase activity (e.g., cytochrome P450 reductase
activity), alcohol dehydrogenase activity (e.g., fatty alcohol
dehydrogenase activity, or long-chain alcohol dehydrogenase
activity), fatty alcohol oxidase activity, fatty aldehyde
dehydrogenase activity, and thioesterase activity.
[0089] Omega Oxidation--Monooxygenases
[0090] A cytochrome P450 enzyme (e.g., monooxygenase activity)
often catalyzes the insertion of one atom of oxygen into an organic
substrate (RH) while the other oxygen atom is reduced to water.
Insertion of the oxygen atom near the omega carbon of a substrate
yields an alcohol derivative of the original starting substrate
(e.g., yields a fatty alcohol). A cytochrome P450 sometimes is
encoded by the host organism and sometimes can be added to generate
an engineered organism.
[0091] In certain embodiments, the monooxygenase activity is
unchanged in a host or engineered organism. In some embodiments,
the host monooxygenase activity can be increased by increasing the
number of copies of a cytochrome P450 gene, or by increasing the
activity of a promoter that regulates transcription of a cytochrome
P450 gene, thereby increasing the production of target product
(e.g., sebacic or dodecanedioic acid) due to increased carbon flux
through the pathway. In certain embodiments, the cytochrome P450
gene can be isolated from any suitable organism. Non-limiting
examples of organisms that include, or can be used as donors for,
cytochrome P450 enzymes include yeast (e.g., Candida,
Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0092] Omega Oxidation--Monooxygenase Reductases
[0093] A cytochrome P450 reductase (e.g., monooxygenase reductase
activity) catalyzes the reduction of the heme-thiolate moiety in
cytochrome P450 by transferring an electron to the cytochrome P450.
A cytochrome P450 reductase sometimes is encoded by the host
organism and sometimes can be added to generate an engineered
organism. In certain embodiments, the monooxygenase reductase
activity is unchanged in a host or engineered organism. In some
embodiments, the host monooxygenase reductase activity can be
increased by increasing the number of copies of a cytochrome P450
reductase gene, or by increasing the activity of a promoter that
regulates transcription of a cytochrome P450 reductase gene,
thereby increasing the production of target product (e.g., sebacic
or dodecanedioic acid) due to increased carbon flux through the
pathway. In certain embodiments, the cytochrome P450 reductase gene
can be isolated from any suitable organism. Non-limiting examples
of organisms that include, or can be used as donors for, cytochrome
P450 reductase enzymes include yeast (e.g., Candida, Saccharomyces,
Debaryomyces, Meyerozyma, Lodderomyces, Scheffersomyces,
Clavispora, Yarrowia, Pichia, Kluyveromyces, Eremothecium,
Zygosaccharomyces, Lachancea, Nakaseomyces), animals (e.g., Homo,
Rattus), bacteria (e.g., Escherichia, Pseudomonas, Bacillus), or
plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0094] Omega Oxidation--Alcohol Dehydrogenases
[0095] An alcohol dehydrogenase (e.g., fatty alcohol dehydrogenase,
long-chain alcohol dehydrogenase) catalyzes the removal of a
hydrogen from an alcohol to yield an aldehyde or ketone and a
hydrogen atom and NADH, in the endoplasmic reticulum of a cell. An
alcohol dehydrogenase sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the alcohol dehydrogenase activity is
unchanged in a host or engineered organism. In some embodiments,
the host alcohol dehydrogenase activity can be increased by
increasing the number of copies of an alcohol dehydrogenase gene,
or by increasing the activity of a promoter that regulates
transcription of an alcohol dehydrogenase gene, thereby increasing
the production of target product (e.g., sebacic or dodecanedioic
acid) due to increased carbon flux through the pathway. In certain
embodiments, the alcohol dehydrogenase gene can be isolated from
any suitable organism. Non-limiting examples of organisms that
include, or can be used as donors for, alcohol dehydrogenase
enzymes include yeast (e.g., Candida, Saccharomyces, Debaryomyces,
Meyerozyma, Lodderomyces, Scheffersomyces, Clavispora, Yarrowia,
Pichia, Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,
Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,
Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,
Nictotania, Cuphea).
[0096] Omega Oxidation--Fatty Alcohol Oxidases
[0097] A fatty alcohol oxidase (e.g., long-chain alcohol oxidase,
EC 1.1.3.20) enzyme catalyzes the addition of oxygen to two
molecules of a long-chain alcohol to yield 2 long chain aldehydes
and 2 molecules of water, in the peroxisome of a cell. A fatty
alcohol oxidase sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the fatty alcohol oxidase activity is
unchanged in a host or engineered organism. In some embodiments,
the host fatty alcohol oxidase activity can be increased by
increasing the number of copies of a fatty alcohol oxidase gene, or
by increasing the activity of a promoter that regulates
transcription of a fatty alcohol oxidase gene, thereby increasing
the production of target product (e.g., sebacic or dodecanedioic
acid) due to increased carbon flux through the pathway. In certain
embodiments, the fatty alcohol oxidase gene can be isolated from
any suitable organism. Non-limiting examples of organisms that
include, or can be used as donors for, fatty alcohol oxidase
enzymes include yeast (e.g., Candida, Saccharomyces, Debaryomyces,
Meyerozyma, Lodderomyces, Scheffersomyces, Clavispora, Yarrowia,
Pichia, Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,
Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,
Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,
Nictotania, Cuphea).
[0098] Omega Oxidation--Aldehyde Dehydrogenases
[0099] A fatty aldehyde dehydrogenase (e.g., long chain aldehyde
dehydrogenase) enzyme catalyzes the oxidation of long chain
aldehydes to a long chain carboxylic acid, NADH and H.sup.+. A
fatty aldehyde dehydrogenase sometimes is encoded by the host
organism and sometimes can be added to generate an engineered
organism. In certain embodiments, the fatty aldehyde dehydrogenase
activity is unchanged in a host or engineered organism. In some
embodiments, the host fatty aldehyde dehydrogenase activity can be
increased by increasing the number of copies of a fatty aldehyde
dehydrogenase gene, or by increasing the activity of a promoter
that regulates transcription of a fatty aldehyde dehydrogenase
gene, thereby increasing the production of target product (e.g.,
sebacic or dodecanedioic acid) due to increased carbon flux through
the pathway. In certain embodiments, the fatty aldehyde
dehydrogenase gene can be isolated from any suitable organism.
Non-limiting examples of organisms that include, or can be used as
donors for, fatty aldehyde dehydrogenase enzymes include yeast
(e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,
Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,
Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,
Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,
Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,
Nictotania, Cuphea).
[0100] Omega Oxidation--Thioesterases
[0101] A thioesterase enzyme (e.g., acyl-CoA thioesterase activity,
acyl-ACP thioesterase activity) catalyzes the removal of Coenzyme A
or acyl carrier protein (e.g., ACP) from a fatty acid including
acyl-CoA or acyl carrier protein (e.g., esterified fatty acid) to
yield a fatty acid and an alcohol. The reaction occurs in the
presence of water and Coenzyme A or acyl carrier protein is
specifically removed at a thiol group. A thioesterase sometimes is
encoded by the host organism and sometimes can be added to generate
an engineered organism. In certain embodiments, the thioesterase
activity is unchanged in a host or engineered organism. In some
embodiments, the host thioesterase activity can be increased by
increasing the number of copies of a thioesterase gene, or by
increasing the activity of a promoter that regulates transcription
of a thioesterase gene, thereby increasing the production of target
product (e.g., sebacic or dodecanedioic acid) due to increased
carbon flux through the pathway. In certain embodiments, a
thioesterase gene can be isolated from any suitable organism.
Non-limiting examples of organisms that include, or can be used as
donors for, thioesterase enzymes include yeast (e.g., Candida,
Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0102] Engineered Pathways
[0103] FIGS. 1-8 depict embodiments of biological pathways for
making sebacic acid and dodecanedioic acid, using various alkanes,
fatty acids, fatty alcohols or combinations thereof. Any suitable
alkane, fatty acid, fatty alcohol, plant based oil, seed based oil,
non-petroleum derived soap stock or the like can be used as the
feedstock for the organism (e.g., dodecane, methyl laurate, lauric
acid, carbon sources having 10 or greater carbons (e.g. for sebacic
acid production) or carbon sources having 12 or greater carbons
(e.g. for dodecanedioic acid production). In some embodiments,
carbon sources with greater than 12 carbons can be metabolized
using naturally occurring and/or engineered pathways to yield
molecules that can be further metabolized using the beta oxidation
pathway shown in the lower portion of FIGS. 5-8. In some
embodiments, the activities in the pathways depicted in FIGS. 1-8
can be engineered, as described herein, to enhance metabolism and
target product formation.
[0104] In certain embodiments, one or more activities in one or
more metabolic pathways can be engineered to increase carbon flux
through the engineered pathways to produce a desired product (e.g.,
sebacic or dodecanedioic acid). The engineered activities can be
chosen to allow increased production of metabolic intermediates
that can be utilized in one or more other engineered pathways to
achieve increased production of a desired product with respect to
the unmodified host organism. The engineered activities also can be
chosen to allow decreased activity of enzymes that reduce
production of a desired intermediate or end product (e.g., reverse
activities). This "carbon flux management" can be optimized for any
chosen feedstock, by engineering the appropriate activities in the
appropriate pathways. Non-limiting examples are given herein using
pure alkanes (e.g., single chain length alkanes, dodecane or
example), mixed chain-length alkanes, long-chain alkanes, pure
fatty acids (e.g., single chain length fatty acids, capric acid for
example) and mixed chain length fatty acids (see FIGS. 1-8). The
process of "carbon flux management" through engineered pathways
produces a dicarboxylic acid (e.g. sebacic acid or dodecanedioic
acid) at a level and rate closer to the calculated maximum
theoretical yield for any given feedstock, in certain embodiments.
The terms "theoretical yield" or "maximum theoretical yield" as
used herein refer to the yield of product of a chemical or
biological reaction that can be formed if the reaction went to
completion. Theoretical yield is based on the stoichiometry of the
reaction and ideal conditions in which starting material is
completely consumed, undesired side reactions do not occur, the
reverse reaction does not occur, and there no losses in the work-up
procedure.
[0105] A microorganism may be modified and engineered to include or
regulate one or more activities in a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) pathway. The term "activity" as used herein
refers to the functioning of a microorganism's natural or
engineered biological pathways to yield various products including
a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid) and its precursors.
A fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid) producing activity
can be provided by any non-mammalian source in certain embodiments.
Such sources include, without limitation, eukaryotes such as yeast
and fungi and prokaryotes such as bacteria. In some embodiments, a
reverse activity in a pathway described herein can be altered
(e.g., disrupted, reduced) to increase carbon flux through a beta
oxidation pathway, an omega oxidation pathway, or a beta oxidation
and omega oxidation pathway, towards the production of target
product (e.g., sebacic or dodecanedioic acid). In some embodiments,
a genetic modification disrupts an activity in the beta oxidation
pathway, or disrupts a polynucleotide that encodes a polypeptide
that carries out a forward reaction in the beta oxidation pathway,
which renders beta oxidation activity undetectable. The term
"undetectable" as used herein refers to an amount of an analyte
that is below the limits of detection, using detection methods or
assays known (e.g., described herein). In certain embodiments, the
genetic modification partially reduces beta oxidation activity. The
term "partially reduces beta oxidation activity" as used here
refers to a level of activity in an engineered organism that is
lower than the level of activity found in the host or starting
organism.
[0106] In some embodiments, a beta-oxidation activity can be
modified to alter the catalytic specificity of the chosen activity.
In certain embodiments, an acyl-CoA oxidase activity can be altered
by modifying a catalytic domain associated with carbon chain length
preference and/or specificity. In some embodiments, the altered
catalytic specificity can be found by screening naturally occurring
variant or mutant populations of a host organism. In certain
embodiments, the altered catalytic can be generated by various
mutagenesis techniques in conjunction with selection and/or
screening for the desired activity. In some embodiments, the
altered catalytic activity can be generated by generating chimeric
acyl-CoA oxidases using a mix and match approach, followed by
selection and/or screening for the desired catalytic activity.
Examples of experiments performed to generate acyl-CoA oxidases
with altered catalytic activity are described herein.
[0107] An activity within an engineered microorganism provided
herein can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or all) of the following activities:
6-oxohexanoic acid dehydrogenase activity; 6-hydroxyhexanoic acid
dehydrogenase activity; cytochrome P450 activity; cytochrome P450
reductase activity; fatty alcohol oxidase activity; acyl-CoA ligase
activity, acyl-CoA oxidase activity; enoyl-CoA hydratase activity,
3-hydroxyacyl-CoA dehydrogenase activity, fatty acid synthase
activity, lipase activity, acetyl-CoA carboxylase activity,
acyltransferase activity (diacylglycerol acyl transferase,
lecithin-cholesterol acyltransferase, phospholipid:diacylglycerol
acyltransferase) and thioesterase activity (e.g., acyl-CoA
hydrolase, acyl-CoA thioesterase, acyl-ACP thioesterase, acetyl-CoA
C-acyltransferase, beta-ketothiolase, and the like). In certain
embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
all) of the foregoing activities is altered by way of a genetic
modification. In some embodiments, one or more (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the foregoing
activities is altered by way of (i) adding a heterologous
polynucleotide that encodes a polypeptide having the activity,
and/or (ii) altering or adding a regulatory sequence that regulates
the expression of a polypeptide having the activity. In certain
embodiments, one or more of the foregoing activities is altered by
way of (i) disrupting an endogenous polynucleotide that encodes a
polypeptide having the activity (e.g., insertional mutagenesis),
(ii) deleting a regulatory sequence that regulates the expression
of a polypeptide having the activity, and/or (iii) deleting the
coding sequence that encodes a polypeptide having the activity
(e.g., knock out mutagenesis).
[0108] The term "omega hydroxyl fatty acid dehydrogenase activity"
as used herein refers to conversion of an omega hydroxyl fatty acid
to an omega oxo fatty acid. The omega hydroxyl fatty acid
dehydrogenase activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. In certain
embodiments, an endogenous polypeptide having the omega hydroxyl
fatty acid dehydrogenase activity is identified in the host
microorganism, and the host microorganism is genetically altered to
increase the amount of the polypeptide produced (e.g., a
heterologous promoter is introduced in operable linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the polypeptide is increased (e.g., by
introducing a plasmid that includes the polynucleotide)). Nucleic
acid sequences conferring omega hydroxyl fatty acid dehydrogenase
activity can be obtained from a number of sources, including
Actinobacter, Norcardia, Pseudomonas and Xanthobacter bacteria.
Examples of an amino acid sequence of a polypeptide having omega
hydroxyl fatty acid dehydrogenase activity and a nucleotide
sequence of a polynucleotide that encodes the polypeptide, are
presented herein. Presence, absence or amount of omega hydroxyl
fatty acid dehydrogenase activity can be detected by any suitable
method known in the art. In some embodiments, omega hydroxyl fatty
acid dehydrogenase activity is not altered in a host microorganism,
and in certain embodiments, the activity is added or increased in
the engineered microorganism relative to the host
microorganism.
[0109] The term "monooxygenase activity" as used herein refers to
inserting one atom of oxygen from O.sub.2 into an organic substrate
(RH) and reducing the other oxygen atom to water. In some
embodiments, monooxygenase activity refers to incorporation of an
oxygen atom onto a six-carbon organic substrate. In certain
embodiments, monooxygenase activity refers to conversion of
hexanoate to 6-hydroxyhexanoic acid. Monooxygenase activity can be
provided by any suitable polypeptide, such as a cytochrome P450
polypeptide (hereafter "CYP450") in certain embodiments. Nucleic
acid sequences conferring CYP450 activity can be obtained from a
number of sources, including Bacillus megaterium and may be induced
in organisms including but not limited to Candida tropicalis,
Yarrowia lipolytica, Aspergillus nidulans, and Aspergillus
parasiticus. Examples of oligonucleotide sequences utilized to
isolate a polynucleotide sequence encoding a polypeptide having
CYP450 activity (e.g., CYP52A12 polynucleotide, a CYP52A13
polynucleotide, a CYP52A14 polynucleotide, a CYP52A15
polynucleotide, a CYP52A16 polynucleotide, a CYP52A17
polynucleotide, a CYP52A18 polynucleotide, a CYP52A19
polynucleotide, a CYP52A20 polynucleotide, a CYP52D2
polynucleotide, and/or a BM3 polynucleotide) are presented herein.
In some embodiments, monooxygenase activity is not altered in a
host microorganism, and in certain embodiments, the activity is
added or increased in the engineered microorganism relative to the
host microorganism. In some embodiments, the altered monooxygenase
activity is an endogenous activity, and in certain embodiments, the
altered monooxygenase activity is an exogenous activity. In some
embodiments, the exogenous activity is a single polypeptide with
both monooxygenase and monooxygenase reductase activities (e.g., B.
megaterium cytochrome P450:NADPH P450 reductase).
[0110] Presence, absence or amount of cytochrome P450 activity can
be detected by any suitable method known in the art. For example,
detection can be performed by assaying a reaction containing
cytochrome P450 (CYP52A family) and NADPH-cytochrome P450 reductase
(see Appl Environ Microbiol 69: 5983 and 5992). Briefly, cells are
grown under standard conditions and harvested for production of
microsomes, which are used to detect CYP activity. Microsomes are
prepared by lysing cells in Tris-buffered sucrose (10 mM Tris-HCl
pH 7.5, 1 mM EDTA, 0.25M sucrose). Differential centrifugation is
performed first at 25,000.times.g then at 100,000.times.g to pellet
cell debris then microsomes, respectively. The microsome pellet is
resuspended in 0.1 M phosphate buffer (pH 7.5), 1 mM EDTA to a
final concentration of approximately 10 mg protein/mL. A reaction
mixture containing approximately 0.3 mg microsomes, 0.1 mM sodium
hexanoate, 0.7 mM NADPH, 50 mM Tris-HCl pH 7.5 in 1 mL is initiated
by the addition of NADPH and incubated at 37.degree. C. for 10
minutes. The reaction is terminated by addition of 0.25 mL 5M HCl
and 0.25 mL 2.5 ug/mL 10-hydroxydecanoic acid is added as an
internal standard (3.3 nmol). The mixture is extracted with 4.5 mL
diethyl ether under NaCl-saturated conditions. The organic phase is
transferred to a new tube and evaporated to dryness. The residue is
dissolved in acetonitrile containing 10 mM
3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1 mL of
15 mg/mL 18-crown-6 in acetonitrile saturated with K.sub.2CO.sub.3.
The solution is incubated at 40.degree. C. for 30 minutes before
addition of 0.05 mL 2% acetic acid. The fluorescently labeled
omega-hydroxy fatty acids are resolved via HPLC with detection at
430 nm and excitation at 355 nm (Yamada et al., 1991, AnalBiochem
199: 132-136). Optionally, specifically induced CYP gene(s) may be
detected by Northern blotting and/or quantitative RT-PCR. (Craft et
al., 2003, AppEnvironMicro 69: 5983-5991).
[0111] The term "monooxygenase reductase activity" as used herein
refers to the transfer of an electron from NAD(P)H, FMN, or FAD by
way of an electron transfer chain, reducing the ferric heme iron of
cytochrome P450 to the ferrous state. The term "monooxygenase
reductase activity" as used herein also can refer to the transfer
of a second electron via the electron transport system, reducing a
dioxygen adduct to a negatively charged peroxo group. In some
embodiments, a monooxygenase activity can donate electrons from the
two-electron donor NAD(P)H to the heme of cytochrome P450 (e.g.,
monooxygenase activity) in a coupled two-step reaction in which
NAD(P)H can bind to the NAD(P)H-binding domain of the polypeptide
having the monooxygenase reductase activity and electrons are
shuttled from NAD(P)H through FAD and FMN to the heme of the
monooxygenase activity, thereby regenerating an active
monooxygenase activity (e.g., cytochrome P450). Monooxygenase
reductase activity can be provided by any suitable polypeptide,
such as a cytochrome P450 reductase polypeptide (hereafter "CPR")
in certain embodiments. Nucleic acid sequences conferring CPR
activity can be obtained from and/or induced in a number of
sources, including but not limited to Bacillus megaterium, Candida
tropicalis, Yarrowia lipolytica, Aspergillus nidulans, and
Aspergillus parasiticus. Examples of oligonucleotide sequences
utilized to isolate a polynucleotide sequence encoding a
polypeptide having CPR activity are presented herein. In some
embodiments, monooxygenase reductase activity is not altered in a
host microorganism, and in certain embodiments, the activity is
added or increased in the engineered microorganism relative to the
host microorganism. In some embodiments, the altered monooxygenase
reductase activity is an endogenous activity, and in certain
embodiments, the altered monooxygenase reductase activity is an
exogenous activity. In some embodiments, the exogenous activity is
a single polypeptide with both monooxygenase and monooxygenase
reductase activities (e.g., B. megaterium cytochrome P450:NADPH
P450 reductase).
[0112] Presence, absence or amount of CPR activity can be detected
by any suitable method known in the art. For example, an engineered
microorganism having an increased number of genes encoding a CPR
activity, relative to the host microorganism, could be detected
using quantitative nucleic acid detection methods (e.g., southern
blotting, PCR, primer extension, the like and combinations
thereof). An engineered microorganism having increased expression
of genes encoding a CPR activity, relative to the host
microorganism, could be detected using quantitative expression
based analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like and combinations thereof). Alternately, an
enzymatic assay can be used to detect Cytochrome P450 reductase
activity, where the enzyme activity alters the optical absorbance
at 550 nanometers of a substrate solution (Masters, B. S. S.,
Williams, C. H., Kamin, H. (1967) Methods in Enzymology, X,
565-573).
[0113] Acyl-CoA Oxidases
[0114] The term "acyl-CoA oxidase activity" as used herein refers
to the oxidation of a long chain fatty-acyl-CoA to a
trans-2,3-dehydroacyl-CoA fatty alcohol. In some embodiments, the
acyl-CoA activity is from a peroxisome. In certain embodiments, the
acyl-CoA oxidase activity is a peroxisomal acyl-CoA oxidase (PDX)
activity, carried out by a PDX polypeptide. In some embodiments the
acyl-CoA oxidase activity is encoded by the host organism and
sometimes can be altered to generate an engineered organism.
Acyl-CoA oxidase activity is encoded by the POX4 and POX5 genes of
Candida strain ATCC20336. In certain embodiments, endogenous
acyl-CoA oxidase activity can be increased. In some embodiments,
acyl-CoA oxidase activity of the POX4 polypeptide or the POX5
polypeptide can be altered independently of each other (e.g.,
increase activity of POX4 alone, POX5 alone, increase one and
disrupt the other, and the like). Increasing the activity of one
PDX activity, while disrupting the activity of another PDX
activity, may alter the specific activity of acyl-CoA oxidase with
respect to carbon chain length, while maintaining or increasing
overall flux through the beta oxidation pathway, in certain
embodiments.
[0115] In certain embodiments, host acyl-CoA oxidase activity of
one of the PDX genes can be increased by genetically altering
(e.g., increasing) the amount of the polypeptide produced (e.g., a
strongly transcribed or constitutively expressed heterologous
promoter is introduced in operable linkage with a polynucleotide
that encodes the polypeptide; the copy number of a polynucleotide
that encodes the polypeptide is increased (e.g., by introducing a
plasmid that includes the polynucleotide, integration of additional
copies in the host genome)). In some embodiments, the host acyl-CoA
oxidase activity can be decreased by disruption (e.g., knockout,
insertion mutagenesis, the like and combinations thereof) of an
acyl-CoA oxidase gene, or by decreasing the activity of the
promoter (e.g., addition of repressor sequences to the promoter or
5'UTR) which transcribes an acyl-CoA oxidase gene.
[0116] As noted above, disruption of nucleotide sequences encoding
POX4, PDX 5, or POX4 and POX5 sometimes can alter pathway
efficiency, specificity and/or specific activity with respect to
metabolism of carbon chains of different lengths (e.g., carbon
chains including fatty alcohols, fatty acids, paraffins,
dicarboxylic acids of between about 1 and about 60 carbons in
length). In some embodiments, the nucleotide sequence of POX4,
POX5, or POX4 and POX5 is disrupted with a URA3 nucleotide sequence
encoding a selectable marker, and introduced to a host
microorganism, thereby generating an engineered organism deficient
in POX4, POX5 or POX4 and POX5 activity. Nucleic acid sequences
encoding POX4 and POX5 can be obtained from a number of sources,
including Candida tropicalis, for example. Examples of POX4 and
POX5 amino acid sequences and nucleotide sequences of
polynucleotides that encode the polypeptides, are presented herein.
Described in the examples are experiments conducted to amplify the
activity encoded by the POX5 gene.
[0117] Also as noted above, catalytic specificity of acyl-CoA
oxidases (e.g., POX4, POX5) can be altered by a variety of methods.
Altering the binding and/or catalytic specificity of acyl-CoA
oxidases may prove advantageous for generating novel acyl-CoA
oxidases with altered chain length recognition, altered chain
length catalytic activity, and/or generation of an acyl-CoA oxidase
activity with a narrow or specific chain length specificity,
thereby allowing further increases in pathway efficiency,
specificity and/or specific activity with respect to metabolism of
carbon chains of different lengths or metabolism of carbon chain
distributions found in a particular chosen feedstock. In some
embodiments the altered acyl-CoA oxidase sequences are identified
and/or generated by; (i) screening naturally occurring variant
populations; (ii) mutagenesis of endogenous sequences; (iii)
introduction of heterologous sequences having a desired
specificity; (iv) generation of chimeric sequences having a portion
of the coding sequence from one polynucleotide source (e.g., gene,
organism) and a portion of the coding sequence from another source
and/or (v) intelligent design using nucleotide sequences and three
dimensional structure analysis from an acyl-CoA oxidase having a
desired specificity to remodel an endogenous acyl-CoA oxidase,
thereby generating a novel specificity enzyme. In some embodiments
a chimeric acyl-CoA oxidase sequence can have polynucleotide
sequence contributions from two or more sources. In some
embodiments, a chimeric acyl-CoA oxidase sequence comprises a
portion of the coding sequences from an endogenous polynucleotide
and a portion of the coding sequence from a heterologous
polynucleotide. Described in the examples are methods utilized to
identify and/or generate acyl-CoA oxidases with novel catalytic and
binding specificities.
[0118] Introduction of Heterologous Acyl CoA Oxidase Sequences
Having a Desired Specificity
[0119] Thousands of Acyl CoA Oxidases and Acyl CoA-like Oxidases
have been cloned, sequenced and isolated from a variety of
organisms (SEQ ID NO. 51 through SEQ ID NO. 3673 and SEQ ID NO.
3810 through SEQ ID NO. 3882). Many of these enzymes have reported
catalytic activity with selective substrate specificity. For
example, some Acyl CoA Oxidases (e.g., Pox5p from a Candida strain)
display optimal activity on substrates of 12 to 18 carbons (FIG.
21). In some embodiments, an organism (e.g., a yeast) or a
genetically modified organism (e.g., a genetically modified yeast,
e.g., a yeast in which .beta.-oxidation activity is blocked) is
engineered to express a heterologous Acyl-CoA Oxidase with
selective substrate specificity. In some embodiments, an organism
(e.g., a yeast) or a genetically modified organism (e.g., a
genetically modified yeast, e.g., a yeast in which .beta.-oxidation
activity is blocked) is engineered to express an Acyl-CoA Oxidase
or Acyl CoA-like Oxidase selected from SEQ ID NO. 51 to SEQ ID NO.
3673. In some embodiments, an organism (e.g., a yeast) or a
genetically modified organism (e.g., a genetically modified yeast,
e.g., a yeast in which .beta.-oxidation activity is blocked) is
engineered to express an Acyl-CoA Oxidase or Acyl CoA-like Oxidase
selected from SEQ ID NO. 3810 through SEQ ID NO. 3882.
[0120] Presence, absence or amount of acyl-coA oxidase activity can
be detected by any suitable method known in the art. For example,
using enzymatic assays as described in Shimizu et al, 1979, and as
described herein in the Examples, POX4, POX5 and other acyl-coA
oxidase activities can be assessed. Alternatively, nucleic acid
sequences representing native and/or disrupted POX4 and POX5
sequences also can be detected using nucleic acid detection methods
(e.g., PCR, primer extension, nucleic acid hybridization, the like
and combinations thereof), or quantitative expression based
analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like and combinations thereof), where the engineered
organism exhibits decreased RNA and/or polypeptide levels as
compared to the host organism.
Genetic Modification of Acyl-CoA Oxidases
[0121] A rate-limiting step for .beta.-oxidation is the first step
in the pathway carried out by the enzyme acyl CoA oxidase.
Different Acyl-CoA oxidases can display different chain-length
substrate specificities. Some acyl CoA oxidases display broad
chain-length specificity and can accept any fatty acyl CoA (or
diacyl-CoA) as a substrate. However, some acyl CoA oxidases can
display narrow chain-length specificity.
[0122] For example the Pox5 enzyme from Candida strain ATCC20336
displays a decrease in activity on substrates below C10 (FIG. 21)
and has low activity on C6 and C8 substrates. In a cell with Pox5
as the only functional acyl CoA oxidase, long chain fatty acyl-CoA
or diacyl-CoA substrates can be shortened to about 8 carbons and do
not typically enter another cycle of 3-oxidation. The shorter
substrates (e.g., a C8 fatty dicarboxylic acid) are not typically
recognized as a substrate by Pox5, the CoA is removed by
peroxisomal thioesterases and the fatty dicarboxylic acid (e.g., an
.alpha.,.omega.-dicarboxylic acid) product is secreted from the
cell. In this embodiment, the acyl CoA oxidase chain-length
substrate specificity effectively controls the chain length of a
diacid produced.
[0123] In some embodiments, a .beta.-oxidation pathway in a yeast
is active and includes a genetically modified acyl CoA oxidase. In
some embodiments, an acyl CoA oxidase is genetically modified to
prevent complete oxidation of fatty acyl-CoA or diacyl-CoA
substrates. Genetic modification of an acyl CoA oxidase can
increase the production yield of a desired fatty acid or fatty
dicarboxylic acid product. Therefore, in some embodiments,
metabolic degradation of a fatty acid of a specified chain length
(e.g., the chain length of a desired or target fatty acid or fatty
dicarboxylic acid product) is reduced significantly, when an acyl
CoA oxidase is genetically modified. In some embodiments, metabolic
degradation of a fatty dicarboxylic acid product (e.g., DDDA) by
beta-oxidation is reduced significantly, when an acyl CoA oxidase
is genetically modified. This can be accomplished by modifying the
substrate specificity of an acyl CoA oxidase such that the enzyme
has low activity (e.g., enzymatic activity) with chain lengths less
than that of a desired product.
[0124] In some embodiments, the substrate specificity of an acyl
CoA oxidase is modified such that the enzyme has low activity for
aliphatic molecules with chain lengths less than C24 (i.e., 24
carbons). In some embodiments, the substrate specificity of an acyl
CoA oxidase is modified such that the enzyme has very low activity
with chain lengths less than 24, 22, 20, 18, 16, 14, 12, 10, 8, 6
or 4 carbons. In some embodiments, the substrate specificity of an
acyl CoA oxidase is modified such that the enzyme has very low
activity with chain lengths less than 18, 16, 14, 12, 10 or 8
carbons. In some embodiments, the substrate specificity of an acyl
CoA oxidase is modified such that the enzyme has very low activity
with chain lengths less than C12. In some embodiments, the
substrate specificity of an acyl CoA oxidase is modified such that
the enzyme has very low activity with chain lengths less than
C10.
[0125] In some embodiments, genes encoding a genetically modified
acyl CoA oxidase are engineered and expressed in a suitable
organism (e.g., a bacteria (e.g., E. coli) or a yeast) to test the
substrate specificity of the modified enzyme in vitro. In some
embodiments, genes encoding a genetically modified acyl CoA oxidase
are engineered and expressed in a suitable yeast and the substrate
specificity is tested. In some embodiments, yeast that express a
modified acyl CoA oxidase are tested for production of the desired
fatty acid or fatty dicarboxylic acid product. A modified acyl CoA
oxidase can be generated in any suitable manner, non limiting
examples of which are provided hereafter.
Random Mutagenesis of Acyl-CoA Oxidase
[0126] A library of genetically modified acyl CoA oxidases can be
generated using several methods known in the art (e.g.,
site-directed mutagenesis). Genetically modified acyl CoA oxidase
genes can then be transformed into a .beta.-oxidation blocked
strain of a suitable yeast strain (e.g., Candida spp. (e.g.,
Candida viswanathii or Candida tropicalis)). In some embodiments, a
genetically modified acyl CoA oxidase is expressed under the
control of the POX4 promoter or another strong constitutive or
inducible promoter in a pox4.DELTA./pox4.DELTA.
pox5.DELTA./pox5.DELTA. (e.g., an organism that lacks endogenouse
acyl CoA oxidase activity) background. In some embodiments, the
genetically modified acyl CoA oxidase is expressed under the
control of endogenous promoter. In some embodiments, the
genetically modified acyl CoA oxidase is expressed under the
control of a heterologous promoter. The transformants can be
selected by growth in a fatty acid or methyl-derivate fatty acid
containing fatty acids with two more carbons than the diacid
product of interest. For example, for adipic acid, the
transformants can be grown in caprylic acid or methyl-caprylate.
For example, for dodecanedioic acid, the transformants can be grown
in tetradecanedioic acid. The group of transformants can then be
moved to a medium with a carbon source of a fatty acid of interest
(for example dodecanedioic acid) in the presence of an agent that
kills growing cells (e.g., Nystatin) and cells that cannot
metabolize the carbon source (e.g., dodecanedioic acid in this
example) can be selected. The resulting modified strains can then
be further characterized for acyl CoA oxidase activity. This method
can be used to select for any modified acyl CoA oxidase (e.g.,
those listed and/or described in TABLES 9 through 26). In addition,
this method can be used to select for any heterologous acyl CoA
oxidase (e.g., those listed in SEQ ID NO. 51 through 3273 and SEQ
ID NO. 3728 through 3810) expressed in a suitable organism.
[0127] Rational Mutagenesis of Acyl-CoA Oxidase
[0128] Structural and sequence information and experimental data
can be combined to determine specific mutations to be tested in a
acyl-CoA oxidase for altered specificity. For example, primary
sequences of acyl-CoA oxidases tested can be compared and
correlated with substrate specificity. Based on such an analysis,
single amino-acids, small numbers of contiguous amino acids and/or
domains can be proposed for providing a desired substrate
specificity. Those amino acids positions can be targeted for
specific or random mutations for improve specificity.
[0129] Acyl CoA oxidase structure also can be modeled against a
known tertiary structure using modeling methods known in the art.
The models can be used to propose amino acids and regions
pertaining to substrate selectivity. For example, biochemical,
structure and sequence data suggest that the N-terminus of acyl CoA
oxidases often, in part, determines substrate specificity.
Mutations or region replacements can be introduced based on such
analyses and the specificity of the new acyl CoA oxidase tested as
described before. The resulting information can be used to go back
to the models to postulate new potential mutations. As for random
mutagenesis, any suitable acyl CoA oxidase can be modified to alter
substrate specificity (e.g., those listed in SEQ ID NO. 51 through
3273 and SEQ ID NO. 3728 through 3810)
[0130] The term "acyl CoA oxidase activity" as used herein refers
to the enzymatic activity (e.g., catalytic activity) of a acyl CoA
oxidase. An acyl CoA oxidase can catalyze the following chemical
reaction:
acyl-CoA+O.sub.2trans-2,3-dehydroacyl-CoA+H.sub.2O.sub.2
[0131] In some embodiments, acyl CoA oxidase activity refers to
oxidation of a long chain fatty-acyl-CoA to a
trans-2,3-dehydroacyl-CoA fatty alcohol. In some embodiments, acyl
CoA oxidase activity refers to its enzyme activity (or lack
thereof) on a selective set of substrates. The activity of an acyl
CoA oxidase can be affected by its ability to bind a substrate,
oxidize a substrate and/or release a product. In some embodiments,
an acyl CoA oxidase is active in one compartment of a cell and not
in another compartment of the cell. In some embodiments, the acyl
CoA oxidase activity is from a peroxisome. In certain embodiments,
the acyl CoA oxidase activity is a peroxisomal acyl CoA oxidase
(PDX) activity, carried out by a PDX polypeptide. In some
embodiments the acyl CoA oxidase activity is encoded by the host
organism and sometimes can be altered to generate an engineered
organism. Acyl-CoA oxidase activity can be encoded by the POX4 and
POX5 genes of Candida spp. In certain embodiments, endogenous acyl
CoA oxidase activity can be increased. In some embodiments, acyl
CoA oxidases in an organism, containing one or more acyl CoA
oxidases, can be independently modified (e.g., one or more acyl CoA
oxidases can be modified). In some embodiments, acyl CoA oxidase
activity of a POX4 polypeptide or a POX5 polypeptide can be altered
independently of each other (e.g., increase activity of POX4 alone,
POX5 alone, increase one and disrupt the other, and the like).
Increasing the activity of one PDX activity, while disrupting the
activity of another PDX activity, may alter the specific activity
of acyl CoA oxidase with respect to carbon chain length, while
maintaining or increasing overall flux through the beta oxidation
pathway, in certain embodiments.
[0132] In certain embodiments, host activity of one or more acyl
CoA oxidase genes can be increased by genetically altering (e.g.,
increasing) the amount of a polypeptide produced (e.g., a strongly
transcribed or constitutively expressed heterologous promoter is
introduced in operable linkage with a polynucleotide that encodes a
polypeptide; the copy number of a polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that
includes the polynucleotide, integration of additional copies in
the host genome)). In some embodiments, host activity of one or
more acyl CoA oxidases can be decreased by disruption (e.g.,
knockout, insertion mutagenesis, the like and combinations thereof)
of an acyl CoA oxidase gene, or by decreasing the activity of the
promoter (e.g., addition of repressor sequences to the promoter or
5'UTR) which transcribes an acyl CoA oxidase gene.
[0133] As noted above, disruption of nucleotide sequences encoding
one or more acyl CoA oxidases (e.g., POX4, PDX 5, or POX4 and POX5)
sometimes can alter pathway efficiency, specificity and/or specific
activity with respect to metabolism of carbon chains of different
lengths (e.g., carbon chains including fatty alcohols, fatty acids,
paraffins, dicarboxylic acids, aliphatic molecules of between about
1 and about 60 carbons in length). In some embodiments, the
nucleotide sequence of one or more acyl CoA oxidases (e.g., POX4,
PDX 5, or POX4 and POX5) is disrupted with a URA3 nucleotide
sequence encoding a selectable marker, and introduced to a host
microorganism, thereby generating an engineered organism deficient
in an acyl CoA oxidase activity.
[0134] Also as noted above, catalytic specificity of acyl CoA
oxidases (e.g., POX4, POX5) can be altered by a variety of methods.
Altering the binding and/or catalytic specificity of acyl CoA
oxidases may prove advantageous for generating novel acyl CoA
oxidases with altered chain length recognition, altered chain
length catalytic activity, and/or generation of an acyl CoA oxidase
activity with a narrow or specific chain length specificity,
thereby allowing further increases in pathway efficiency,
specificity and/or specific activity with respect to metabolism of
carbon chains of different lengths or metabolism of carbon chain
distributions found in a particular chosen feedstock. In some
embodiments the altered acyl CoA oxidase sequences are identified
and/or generated by: (i) screening naturally occurring variant
populations; (ii) mutagenesis of endogenous sequences; (iii)
introduction of heterologous sequences having a desired specificity
(e.g., introduction of one or more unmodified or modified acyl CoA
oxidases from another organism into a host organism in which one or
more endogenous acyl-CoA oxidases are optionally disrupted); (iv)
generation of chimeric sequences having a portion of the coding
sequence from one polynucleotide source (e.g., gene, organism) and
a portion of the coding sequence from another source and/or (v)
intelligent design using nucleotide sequences and three dimensional
structure analysis from an acyl CoA oxidase having a desired
specificity to remodel an endogenous acyl CoA oxidase, thereby
generating a novel specificity enzyme. In some embodiments a
chimeric acyl CoA oxidase sequence can have polynucleotide sequence
contributions from two or more sources. In some embodiments, a
chimeric acyl CoA oxidase sequence comprises a portion of the
coding sequences from an endogenous polynucleotide and a portion of
the coding sequence from a heterologous polynucleotide. Described
in the examples are methods utilized to identify and/or generate
acyl CoA oxidases with novel catalytic and binding
specificities.
[0135] Nucleic acid sequences encoding acyl CoA oxidases (e.g.,
POX4 and POX5) can be obtained from any suitable source, including
any animal (e.g., mammals, fish, reptiles, amphibians, etc.), any
plant, fungus, yeast, protozoan, bacteria, virus, phage, and the
like). Non-limiting examples of suitable yeast sources include
Yarrowia yeast (e.g., Y. lipolytica (formerly classified as Candida
lipolytica)), Candida yeast (e.g., C. revkaufi, C. viswanathii, C.
pulcherrima, C. tropicalis, C. utilis), Rhodotorula yeast (e.g., R.
glutinus, R. graminis), Rhodosporidium yeast (e.g., R. toruloides),
Saccharomyces yeast (e.g., S. cerevisiae, S. bayanus, S.
pastorianus, S. carlsbergensis), Cryptococcus yeast, Trichosporon
yeast (e.g., T. pullans, T. cutaneum), Pichia yeast (e.g., P.
pastoris) and Lipomyces yeast (e.g., L. starkeyii, L. lipoferus).
In some embodiments, a suitable yeast is of the genus Arachniotus,
Aspergillus, Aureobasidium, Auxarthron, Blastomyces, Candida,
Chrysosporuim, Chrysosporuim Debaryomyces, Coccidiodes,
Cryptococcus, Gymnoascus, Hansenula, Histoplasma, Issatchenkia,
Kluyveromyces, Lipomyces, Lssatchenkia, Microsporum, Myxotrichum,
Myxozyma, Oidiodendron, Pachysolen, Penicillium, Pichia,
Rhodosporidium, Rhodotorula, Rhodotorula, Saccharomyces,
Schizosaccharomyces, Scopulariopsis, Sepedonium, Trichosporon, or
Yarrowia. In some embodiments, a suitable yeast is of the species
Arachniotus flavoluteus, Aspergillus flavus, Aspergillus fumigatus,
Aspergillus niger, Aureobasidium pullulans, Auxarthron thaxteri,
Blastomyces dermatitidis, Candida albicans, Candida dubliniensis,
Candida famata, Candida glabrata, Candida guilliermondii, Candida
kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida
lustitaniae, Candida parapsilosis, Candida pulcherrima, Candida
revkaufi, Candida rugosa, Candida tropicalis, Candida utilis,
Candida viswanathii, Candida xestobii, Chrysosporuim
keratinophilum, Coccidiodes immitis, Cryptococcus albidus var.
diffluens, Cryptococcus laurentii, Cryptococcus neofomans,
Debaryomyces hansenii, Gymnoascus dugwayensis, Hansenula anomala,
Histoplasma capsulatum, Issatchenkia occidentalis, Isstachenkia
orientalis, Kluyveromyces lactis, Kluyveromyces marxianus,
Kluyveromyces thermotolerans, Kluyveromyces waltii, Lipomyces
lipoferus, Lipomyces starkeyii, Microsporum gypseum, Myxotrichum
deflexum, Oidiodendron echinulatum, Pachysolen tannophilis,
Penicillium notatum, Pichia anomala, Pichia pastoris, Pichia
stipitis, Rhodosporidium toruloides, Rhodotorula glutinus,
Rhodotorula graminis, Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe, Scopulariopsis acremonium,
Sepedonium chrysospermum, Trichosporon cutaneum, Trichosporon
pullans, Yarrowia lipolytica, or Yarrowia lipolytica (formerly
classified as Candida lipolytica). In some embodiments, a suitable
yeast is a Y. lipolytica strain that includes, but is not limited
to, ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 and LGAM
S(7)1 strains (Papanikolaou S., and Aggelis G., Bioresour. Technol.
82(1):43-9 (2002)). In certain embodiments, a suitable yeast is a
Candida species (i.e., Candida spp.) yeast. Any nucleic acid
sequence encoding an acyl CoA oxidase, acyl CoA oxidase-like
activity or acyl-CoA dehyrogenase activity can be used to alter the
substrate specificity of a yeast as described herein. Non-limiting
examples of acyl CoA oxidase, acyl CoA oxidase-like and acyl CoA
dehydrogenase amino acid sequences and nucleotide sequences are
provided herein and in SEQ ID NO. 51 through 3810. Described in the
examples are experiments conducted to modify and amplify the
activity of an acyl CoA oxidase gene (e.g., the POX5 gene).
[0136] Presence, absence or amount of acyl CoA oxidase activity can
be detected by any suitable method known in the art. For example,
enzymatic assays as described in Shimizu et al, 1979, and as
described herein in the Examples can be used to assess acyl CoA
oxidase activity. Nucleic acid sequences representing native and/or
disrupted acyl CoA oxidase sequences also can be detected using
nucleic acid detection methods (e.g., PCR, primer extension,
nucleic acid hybridization, the like and combinations thereof), or
quantitative expression based analysis (e.g., RT-PCR, western blot
analysis, northern blot analysis, the like and combinations
thereof), where the engineered organism exhibits decreased RNA
and/or polypeptide levels as compared to the host organism.
[0137] Acyl CoA Dehydrogenase
[0138] Acyl-CoA dehydrogenases (ACADs) are a class of enzymes that
can function to catalyze the initial step in each cycle of fatty
acid .beta.-oxidation in the mitochondria of cells. They can be
very similar in structure and function to Acyl CoA oxidases. Their
action results in the introduction of a trans double-bond between
C2 and C3 of an acyl-CoA thioester substrate. FAD is a required
co-factor in the mechanism in order for the enzyme to bind to its
appropriate substrate.
[0139] Acyl-CoA dehydrogenases can be categorized into four
distinct groups based on their specificity for short-, medium-, or
long-chain fatty acid, and very long-chain fatty acid acyl-CoA
substrates. While different dehydrogenases target fatty acids of
varying chain length, all types of acyl-CoA dehydrogenases can be
mechanistically similar. Differences in ACADs can occur based on
the location of the active site along the amino acid sequence.
[0140] The medium chain acyl-CoA dehydrogenase is a homotetramer
with each subunit containing roughly 400 amino acids and one
equivalent of FAD. The tetramer is classified as a "dimer of
dimers".
[0141] The interface between the two monomers of a single dimer of
an acyl-CoA dehydrogenase contains the FAD binding sites and has
extensive bonding interactions. In contrast, the interface between
the two dimers has few interactions. There are a total of 4 active
sites within the tetramer, each of which contains a single FAD
molecule and an acyl-CoA substrate. This gives a total of four FAD
molecules and four acyl-CoA substrates per enzymatic molecule.
[0142] FAD is bound between the three domains of the monomer, where
only the nucleotide portion is accessible. FAD binding contributes
significantly to overall enzyme stability. The acyl-CoA substrate
is bound completely within each monomer of the enzyme. In some
ACADs, the active site is lined with the residues F252, T255, V259,
T96, T99, A100, L103, Y375, Y375, and E376. The area of interest
within the substrate can become wedged between Glu 376 and FAD,
lining up the molecules into an ideal position for the
reaction.
[0143] Some ACAD sequences are presented in SEQ ID NO.s 3728
through 3810.
[0144] Thioesterase
[0145] The term "thioesterase activity" as used herein refers to
removal of Coenzyme A from hexanoate.
[0146] The term "thioesterase activity" as used herein also refers
to the removal of Coenzyme A from an activated fatty acid (e.g.,
fatty-acyl-CoA). A Non-limiting example of an enzyme with
thioesterase activity includes acyl-CoA hydrolase (e.g., EC
3.1.2.20; also referred to as acyl coenzyme A thioesterase,
acyl-CoA thioesterase, acyl coenzyme A hydrolase, thioesterase B,
thioesterase II, lecithinase B, lysophopholipase L1, acyl-CoA
thioesterase 1, and acyl-CoA thioesterase). Thioesterases that
remove Coenzyme A from fatty-acyl-CoA molecules catalyze the
reaction,
acyl-CoA+H2O--.fwdarw.CoA+a carboxylate,
where the carboxylate often is a fatty acid. The released Coenzyme
A can then be reused for other cellular activities.
[0147] The thioesterase activity can be provided by a polypeptide.
In certain embodiments, the polypeptide is an endogenous nucleotide
sequence that is increased in copy number, operably linked to a
heterologous and/or endogenous promoter, or increased in copy
number and operably linked to a heterologous and/or endogenous
promoter. In some embodiments, the polypeptide is encoded by a
heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring thioesterase
activity can be obtained from a number of sources, including Cuphea
lanceolata, C. tropicalis (e.g., see SEQ ID NOS: 33 and 35), and E.
coli (e.g., see SEQ ID NO: 37). Additional organisms that can be
used as thioesterase polynucleotide sequence donors are given
herein. Examples of such polypeptides include, without limitation,
acyl-(ACP) thioesterase type B from Cuphea lanceolata (see SEQ ID
NO: 1), acyl-CoA hydrolase (e.g., ACHA and ACHB, see SEQ ID NOS: 34
and 36)) from C. tropicalis, acyl-CoA thioesterase (e.g., TESA, see
SEQ ID NO: 38) from E. coli. A non-limiting example of a
thioesterase polynucleotide sequences is referenced by accession
number CAB60830 at the World Wide Web Uniform Resource Locator
(URL) ncbi.nlm.nih.gov of the National Center for Biotechnology
Information (NCBI).
[0148] Presence, absence or amount of thioesterase activity can be
detected by any suitable method known in the art. An example of
such a method is described Chemistry and Biology 9: 981-988. In
some embodiments, thioesterase activity is not altered in a host
microorganism, and in certain embodiments, the activity is added or
increased in the engineered microorganism relative to the host
microorganism. In some embodiments, a polypeptide having
thioesterase activity is linked to another polypeptide (e.g., a
hexanoate synthase A or hexanoate synthase B polypeptide).
Non-limiting examples of polynucleotide sequences encoding
thioesterase activities and polypeptides having thioesterase
activity are provided in Example 33.
[0149] Reducing Omega Fatty Acid Conversion--General
[0150] The term "a genetic modification that reduces omega hydroxyl
fatty acid conversion" as used herein refer to genetic alterations
of a host microorganism that reduce an endogenous activity that
converts an omega hydroxyl fatty acid to another product. In some
embodiments, an endogenous omega hydroxyl fatty acid dehydrogenase
activity is reduced. Such alterations can advantageously increase
the amount of a dicarboxylic acid, which can be purified and
further processed.
[0151] Reducing Beta Oxidaiton--General
[0152] The term "a genetic modification that reduces beta-oxidation
activity" as used herein refers to a genetic alteration of a host
microorganism that reduces an endogenous activity that oxidizes a
beta carbon of carboxylic acid containing organic molecules. In
certain embodiments, the organic molecule is a ten or twelve carbon
molecule, and sometimes contains one or two carboxylic acid
moieties located at a terminus of the molecule (e.g., sebacic or
dodecanedioic acid). Such alterations can advantageously increase
yields of end products, such as a fatty dicarboxylic acid
[0153] (e.g., octanedioic acid, decanedioic acid, dodecanedioic
acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic
acid, eicosanedioic acid).
[0154] Increasing Fatty Acid Synthesis--General
[0155] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts fatty acids into fatty-acyl-CoA
intermediates. In some embodiments, an endogenous activity that
converts fatty acids into fatty-acyl-CoA intermediates is reduced.
In certain embodiments, an acyl-CoA synthetase activity is reduced.
Such alterations can advantageously increase yields of end
products, such as a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0156] Acyl-CoA Synthetase
[0157] Fatty acids can be converted into fatty-acyl-CoA
intermediates by the activity of an acyl-CoA synthetase (e.g.,
ACS1, ACS2; EC 6.2.1.3; also referred to as acyl-CoA synthetase,
acyl-CoA ligase), in many organisms. Acyl-CoA synthetase has six
isoforms encoded by ACS1, FAT1, ACS2A, ACS2B, ACS2C and ACS2D,
respectively, in Candida spp. (e.g., homologous to FAA1, FAT1, and
FAA2 in S. cerevisiae). Acyl-CoA synthetase is a member of the
ligase class of enzymes and catalyzes the reaction,
ATP+Fatty Acid+CoA<=>AMP+Pyrophosphate+Fatty-Acyl-CoA.
[0158] Fatty acids and Coenzyme A often are utilized in the
activation of fatty acids to fatty-acyl-CoA intermediates for entry
into various cellular processes. Without being limited by theory,
it is believed that reduction in the amount of fatty-acyl-CoA
available for various cellular processes can increase the amount of
fatty acids available for conversion into a fatty dicarboxylic acid
(e.g., a sebacic or dodecanedioic acid) by other engineered
pathways in the same host organism (e.g., omega oxidation pathway,
beta oxidation pathway, omega oxidation pathway and beta oxidation
pathway). Acyl-CoA synthetase can be inactivated by any suitable
means. Described herein are gene knockout methods suitable for use
to disrupt the nucleotide sequence that encodes a polypeptide
having ACS1 activity. A nucleotide sequence of ACS1 is provided in
Example 33, SEQ ID NO: 39. An example of an integration/disruption
construct, configured to generate a deletion mutant for ACS1 is
also provided in the Examples.
[0159] The presence, absence or amount of acyl-CoA synthetase
activity can be detected by any suitable method known in the art.
Non-limiting examples of suitable detection methods include
enzymatic assays (e.g., Lageweg et al "A Fluorimetric Assay for
Acyl-CoA Synthetase Activity", Analytical Biochemistry,
197(2):384-388 (1991)), PCR based assays (e.g., qPCR, RT-PCR),
immunological detection methods (e.g., antibodies specific for
acyl-CoA synthetase), the like and combinations thereof.
[0160] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts long chain and very long chain fatty acids
into activated fatty-acyl-CoA intermediates. In some embodiments,
an endogenous activity that converts long chain and very long chain
fatty acids into activated fatty-acyl-CoA intermediates is reduced.
In certain embodiments, a long chain acyl-CoA synthetase activity
is reduced. Such alterations can advantageously increase yields of
end products, such as a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0161] Long chain fatty acids (e.g., C12-C18 chain lengths) and
very long chain fatty acids (e.g., C20-C26) often are activated
and/or transported by the thioesterification activity of a
long-chain acyl-CoA synthetase (e.g., FAT1; EC 6.2.1.3; also
referred to as long-chain fatty acid-CoA ligase, acyl-CoA
synthetase; fatty acid thiokinase (long chain); acyl-activating
enzyme; palmitoyl-CoA synthase; lignoceroyl-CoA synthase;
arachidonyl-CoA synthetase; acyl coenzyme A synthetase; acyl-CoA
ligase; palmitoyl coenzyme A synthetase; thiokinase; palmitoyl-CoA
ligase; acyl-coenzyme A ligase; fatty acid CoA ligase; long-chain
fatty acyl coenzyme A synthetase; oleoyl-CoA synthetase;
stearoyl-CoA synthetase; long chain fatty acyl-CoA synthetase;
long-chain acyl CoA synthetase; fatty acid elongase (ELO); LCFA
synthetase; pristanoyl-CoA synthetase; ACS3; long-chain acyl-CoA
synthetase I; long-chain acyl-CoA synthetase II; fatty
acyl-coenzyme A synthetase; long-chain acyl-coenzyme A synthetase;
and acid:CoA ligase (AMP-forming)), in some organisms. Fatty acids
also can be transported into the host organism from feedstocks by
the activity of long chain acyl-CoA synthetase.
[0162] Long-chain acyl-CoA synthetase catalyzes the reaction,
ATP+a long-chain carboxylic acid+CoA=AMP+diphosphate+an
acyl-CoA,
where "an acyl-CoA" refers to a fatty-acyl-CoA molecule. As noted
herein, activation of fatty acids is often necessary for entry of
fatty acids into various cellular processes (e.g., as an energy
source, as a component for membrane formation and/or remodeling, as
carbon storage molecules). Deletion mutants of FAT1 have been shown
to accumulate very long chain fatty acids and exhibit decreased
activation of these fatty acids. Without being limited by theory,
it is believed that reduction in the activity of long-chain
acyl-CoA synthetase may reduce the amount of long chain fatty acids
converted into fatty-acyl-CoA intermediates, thereby increasing the
amount of fatty acids available for conversion into a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) by other engineered
pathways in the same host organism (e.g., omega oxidation pathway,
beta oxidation pathway, omega oxidation pathway and beta oxidation
pathway). Long-chain-acyl-CoA synthetase activity can be reduced or
inactivated by any suitable means. Described herein are gene
knockout methods suitable for disrupting the nucleotide sequence
that encodes the polypeptide having FAT1 activity. The nucleotide
sequence of FAT1 is provided in Example 33, SEQ ID NO: 41. DNA
vectors suitable for use in constructing "knockout" constructs are
described herein.
[0163] The presence, absence or amount of long-chain-acyl-CoA
synthetase activity can be detected by any suitable method known in
the art. Non-limiting examples of suitable detection methods
include enzymatic assays, binding assays (e.g., Erland et al,
Analytical Biochemistry 295(1):38-44 (2001)), PCR based assays
(e.g., qPCR, RTPCR), immunological detection methods (e.g.,
antibodies specific for long-chain-acyl-CoA synthetase), the like
and combinations thereof.
[0164] Selective Modification of ACS Activity
[0165] In some embodiments, a beta-oxidation pathway is functional
and is modified for selective substrate specificity. In some
embodiments a beta-oxidation pathway is selective for only
diacyl-CoA thioesters and in some embodiments only on diacyl-CoA's
of a chain length greater than 6, 8, 10, 12, 14, 16, 18 or 20
carbons. Beta-oxidation selectivity can be achieved by: 1)
utilizing the difference in transport of acyl-CoA's and diacids
across the peroxisomal membrane, 2) selectively knocking out
acyl-CoA synthetase (ACS) activity in the cytosolic compartment, 3)
knocking out ACS activity in the peroxisomal compartment for
isozymes with substrate specificity for short chain substrates,
and/or 4) engineering a beta-oxidation pathway that will work only
on substrates longer than 6, 8, 10, 12, 14, 16, 18 or 20
carbons.
[0166] In S. cerevisiae, cytoplasmic ACS activity is encoded by
FAA1, FAA3, FAA4 and FAT1, while peroxisomal activity is encoded by
FAA2. Homologs for FAA1 and FAT1 were identified in Candida strains
however there were no identified homologs for FAA3 or FAA4. As many
as five homologs for the S. cerevisiae peroxisomal FAA2 were
identified in Candida strains. Two of the five homologs display 95%
identity to one another and are most likely alleles of the same
gene. Four FAA2 homologs were identified in Candida strain
ATCC20336 (e.g., ACS2A through ACS2D).
[0167] In some embodiments, one strategy is to control the
subcellular location of ACS enzyme activity so that it is only
present in the peroxisome. FAA1 and FAT1 mutants, faa1.DELTA. and
fat1.DELTA. were constructed and should have very little ACS
activity targeted to the cytoplasm. In these mutant strains,
exogenously supplied long-chain free fatty acids accumulate in the
cytoplasm since they cannot be transported into the peroxisome
unless they are activated to the acyl-CoA thioester. High
concentrations of free fatty acid can be toxic, so the cell acts to
detoxify itself by oxidizing the free fatty acids to dicarboxylic
acids that are much less toxic. Unlike long-chain fatty acids,
long-chain dicarboxylic acids are able to diffuse into the
peroxisomal compartment where they can then be activated to
diacyl-CoA thioesters, which is required for entry into the
beta-oxidation pathway. With multiple peroxisomal ACS isozymes it
may be that each isozyme has different substrate specificity. In
some embodiments, it is desired to retain those peroxisomal ACS
enzymes with substrate specificity matching the chain-length of the
fatty acid feedstock but without activity (or low activity) on
diacids of chain-length.ltoreq.6, 8, 10, 12, 14, 16, 18 or 20
carbons. With this strategy any long-chain dicarboxyl-CoA that is
chain-shortened by beta-oxidation to 12 carbons, for example, that
is subsequently hydrolyzed to a dicarboxylic acid and free CoA
cannot be reactivated to a dicarboxyl-CoA for re-entry into
beta-oxidation for further chain shortening. In some embodiments,
in combination with controlling the substrate chain-length
specificity of the peroxisomal ACS, a peroxisomal thioesterase
activity is amplified with maximum activity at the desired
chain-length of our product. This strategy can control the
chain-length of the dicarboxylic acid produced by
beta-oxidation.
[0168] In some embodiments, the flow of fatty acids into the
peroxisome is controlled by knocking out the genes PXA1 and PXA2.
These genes encode subunits of an ATP binding cassette transporter
that is responsible for transporting long-chain fatty acyl-CoA's
from the cytoplasm across the peroxisomal membrane into the
peroxisomal matrix. Even though, in some embodiments, the genes
encoding the cytoplasmic ACS's are knocked out, there may still be
some residual ACS activity in the cytoplasm from the peroxisomal
ACS's. The ACS isozymes destined for the peroxisome are translated
in the cytoplasm and fully folded prior to import into the
peroxisome. Therefore the peroxisomal ACS's may contribute to a
small amount of cytoplasmic ACS activity. Deletion of the
Pxa1p/Pxa2p transporter can prevent any of the long-chain fatty
acids activated to acyl-CoA thioesters from being transported into
the peroxisome for degradation.
[0169] Acyl-CoA Sterol Acyltransferase
[0170] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts fatty acids into cholesterol esters. In some
embodiments, an endogenous activity that converts fatty acids into
cholesterol esters is reduced. In certain embodiments, an acyl-CoA
sterol acyltransferase activity is reduced. Such alterations can
advantageously increase yields of end products, such as a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid).
[0171] Fatty acids can be converted into a cholesterol-ester by the
activity of acyl-CoA sterol acyltransferase (e.g., ARE1, ARE2, EC
2.3.1.26; also referred to as sterol O-acyltransferase; cholesterol
acyltransferase; sterol-ester synthase; sterol-ester synthetase;
sterol-ester synthase; acyl coenzyme
A-cholesterol-O-acyltransferase; acyl-CoA:cholesterol
acyltransferase; ACAT; acylcoenzyme A:cholesterol
O-acyltransferase; cholesterol ester synthase; cholesterol ester
synthetase; and cholesteryl ester synthetase), in many organisms.
Without being limited by any theory, cholesterol esterification may
be involved in directing fatty acids away from incorporation into
cell membranes and towards storage forms of lipids. Acyl-CoA sterol
acyltransferase catalyzes the reaction,
acyl-CoA+cholesterol=CoA+cholesterol ester.
[0172] The esterification of cholesterol is believed to limit its
solubility in cell membrane lipids and thus promotes accumulation
of cholesterol ester in the fat droplets (e.g., a form of carbon
storage molecule) within cytoplasm. Therefore, without being
limited by any theory esterification of cholesterol may cause the
accumulation of lipid storage molecules, and disruption of the
activity of acyl-CoA sterol acyltransferase may cause an increase
in acyl-CoA levels that can be converted into a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) by other engineered pathways in the same host
organism (e.g., omega oxidation pathway, beta oxidation pathway,
omega oxidation pathway and beta oxidation pathway). Acyl-CoA
sterol acyltransferase can be inactivated by any suitable means.
Described herein are gene knockout methods suitable for disrupting
nucleotide sequences that encode polypeptides having ARE1 activity,
ARE2 activity or ARE1 activity and ARE2 activity. The nucleotide
sequences of ARE1 and ARE2 are provided in Example 33, SEQ ID NOS:
43 and 45. DNA vectors suitable for use in constructing "knockout"
constructs are described herein.
[0173] The presence, absence or amount of acyl-CoA sterol
acyltransferase activity can be detected by any suitable method
known in the art. Non-limiting examples of suitable detection
methods include enzymatic assays (e.g., Chen et al, Plant
Physiology 145:974-984 (2007)), binding assays, PCR based assays
(e.g., qPCR, RT-PCR), immunological detection methods (e.g.,
antibodies specific for long-chain-acyl-CoA synthetase), the like
and combinations thereof.
[0174] Diacylglycerol Acyltransferase & Acyltransferases
[0175] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that catalyzes diacylglycerol esterification (e.g.,
addition of acyl group to a diacylglycerol to form a
triacylglycerol). In some embodiments, an endogenous activity that
converts diacylglycerol into triacylglycerol is reduced. In certain
embodiments, an acyltransferase activity is reduced. In some
embodiments a diacylglycerol acyltransferase activity is reduced.
In some embodiments a diacylglycerol acyltransferase (e.g., DGA1,
EC 2.3.1.20) activity and an acyltransferase (e.g., LRO1) activity
are reduced. Such alterations can advantageously increase yields of
end products, such as a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0176] Diacylglycerol can be converted into triacylglycerol by the
activity of diacylglycerol acyltransferase (e.g., DGA1; EC
2.3.1.20; also referred to as diglyceride acyltransferase;
1,2-diacylglycerol acyltransferase; diacylglycerol acyltransferase;
diglyceride O-acyltransferase; palmitoyl-CoA-sn-1,2-diacylglycerol
acyltransferase; acyl-CoA:1,2-diacylglycerol O-acyltransferase and
acyl-CoA:1,2-diacyl-sn-glycerol O-acyltransferase), in many
organisms. Diacylglycerol acyltransferase catalyzes the
reaction,
Acyl-CoA+1,2-diacyl-sn-glycerol=CoA+triacylglycerol,
and is generally considered the terminal and only committed step in
triglyceride synthesis. The product of the DGA1 gene in yeast
normally is localized to lipid particles.
[0177] In addition to the diacylglycerol esterification activity
described for DGA1, many organisms also can generate triglycerides
by the activity of other acyltransferase activities, non-limiting
examples of which include lecithin-cholesterol acyltransferase
activity (e.g., LRO1; EC 2.3.1.43; also referred to as
phosphatidylcholine-sterol O-acyltransferase activity;
lecithin-cholesterol acyltransferase activity;
phospholipid-cholesterol acyltransferase activity; LCAT
(lecithin-cholesterol acyltransferase) activity;
lecithin:cholesterol acyltransferase activity; and lysolecithin
acyltransferase activity) and phospholipid:diacylglycerol
acyltransferase (e.g., EC 2.3.1.158; also referred to as PDAT
activity and phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase
activity). Acyltransferases of the families EC 2.3.1.43 and EC
2.3.1.58 catalyze the general reaction,
phospholipid+1,2-diacylglycerol=lysophospholipid+triacylglycerol.
[0178] Triacylglycerides often are utilized as carbon (e.g., fatty
acid or lipid) storage molecules. Without being limited by any
theory, it is believe that reducing the activity of acyltransferase
may reduce the conversion of diacylglycerol to triacylglycerol,
which may cause increased accumulation of fatty acid, in
conjunction with additional genetic modifications (e.g., lipase to
further remove fatty acids from the glycerol backbone) that can be
converted into a fatty dicarboxylic acid (e.g., octanedioic acid,
decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) by
other engineered pathways in the same host organism (e.g., omega
oxidation pathway, beta oxidation pathway, omega oxidation pathway
and beta oxidation pathway). Acyltransferases can be inactivated by
any suitable means. Described herein are gene knockout methods
suitable for disrupting nucleotide sequences that encode
polypeptides having DGA1 activity, LRO1 activity or DGA1 activity
and LRO1 activity. The nucleotide sequence of DGA1 is provided in
Example 33, SEQ ID NO: 47 The nucleotide sequence of LRO1 is
provided in Example 33, SEQ ID NO: 49. DNA vectors suitable for use
in constructing "knockout" constructs are described herein.
[0179] The presence, absence or amount of acyltransferase activity
can be detected by any suitable method known in the art.
Non-limiting examples of suitable detection methods include
enzymatic assays (e.g., Geelen, Analytical Biochemistry
322(2):264-268 (2003), Dahlqvist et al, PNAS 97(12):6487-6492
(2000)), binding assays, PCR based assays (e.g., qPCR, RTPCR),
immunological detection methods (e.g., antibodies specific for a
DGA1 or LRO1 acyltransferase), the like and combinations
thereof.
[0180] Polynucleotides and Polypeptides
[0181] A nucleic acid (e.g., also referred to herein as nucleic
acid reagent, target nucleic acid, target nucleotide sequence,
nucleic acid sequence of interest or nucleic acid region of
interest) can be from any source or composition, such as DNA, cDNA,
gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA
or mRNA, for example, and can be in any form (e.g., linear,
circular, supercoiled, single-stranded, double-stranded, and the
like). A nucleic acid can also comprise DNA or RNA analogs (e.g.,
containing base analogs, sugar analogs and/or a non-native backbone
and the like). It is understood that the term "nucleic acid" does
not refer to or infer a specific length of the polynucleotide
chain, thus polynucleotides and oligonucleotides are also included
in the definition. Deoxyribonucleotides include deoxyadenosine,
deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the
uracil base is uridine.
[0182] A nucleic acid sometimes is a plasmid, phage, autonomously
replicating sequence (ARS), centromere, artificial chromosome,
yeast artificial chromosome (e.g., YAC) or other nucleic acid able
to replicate or be replicated in a host cell. In certain
embodiments a nucleic acid can be from a library or can be obtained
from enzymatically digested, sheared or sonicated genomic DNA
(e.g., fragmented) from an organism of interest. In some
embodiments, nucleic acid subjected to fragmentation or cleavage
may have a nominal, average or mean length of about 5 to about
10,000 base pairs, about 100 to about 1,000 base pairs, about 100
to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000 or 10000 base pairs.
[0183] Fragments can be generated by any suitable method in the
art, and the average, mean or nominal length of nucleic acid
fragments can be controlled by selecting an appropriate
fragment-generating procedure by the person of ordinary skill. In
some embodiments, the fragmented DNA can be size selected to obtain
nucleic acid fragments of a particular size range.
[0184] Nucleic acid can be fragmented by various methods known to
the person of ordinary skill, which include without limitation,
physical, chemical and enzymic processes. Examples of such
processes are described in U.S. Patent Application Publication No.
20050112590 (published on May 26, 2005, entitled
"Fragmentation-based methods and systems for sequence variation
detection and discovery," naming Van Den Boom et al.). Certain
processes can be selected by the person of ordinary skill to
generate non-specifically cleaved fragments or specifically cleaved
fragments. Examples of processes that can generate non-specifically
cleaved fragment sample nucleic acid include, without limitation,
contacting sample nucleic acid with apparatus that expose nucleic
acid to shearing force (e.g., passing nucleic acid through a
syringe needle; use of a French press); exposing sample nucleic
acid to irradiation (e.g., gamma, x-ray, UV irradiation; fragment
sizes can be controlled by irradiation intensity); boiling nucleic
acid in water (e.g., yields about 500 base pair fragments) and
exposing nucleic acid to an acid and base hydrolysis process.
[0185] Nucleic acid may be specifically cleaved by contacting the
nucleic acid with one or more specific cleavage agents. The term
"specific cleavage agent" as used herein refers to an agent,
sometimes a chemical or an enzyme that can cleave a nucleic acid at
one or more specific sites. Specific cleavage agents often will
cleave specifically according to a particular nucleotide sequence
at a particular site. Examples of enzymic specific cleavage agents
include without limitation endonucleases (e.g., DNase (e.g., DNase
I, II); RNase (e.g., RNase E, F, H, P); Cleavase.TM. enzyme; Taq
DNA polymerase; E. coli DNA polymerase I and eukaryotic
structure-specific endonucleases; murine FEN-1 endonucleases; type
I, II or III restriction endonucleases such as Acc I, Afl III, Alu
I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl
I. Bgl II, Bln I, Bsm I, BssH II, BstE II, Cfo I, Cla I, Dde I, Dpn
I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hae II, Hae II,
Hind III, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, Msp
I, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I,
Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I,
Sma I, Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho
I); glycosylases (e.g., uracil-DNA glycolsylase (UDG),
3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase
II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase,
thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase,
5-Hydroxymethyluracil DNA glycosylase (HmUDG),
5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA
glycosylase); exonucleases (e.g., exonuclease III); ribozymes, and
DNAzymes. Sample nucleic acid may be treated with a chemical agent,
or synthesized using modified nucleotides, and the modified nucleic
acid may be cleaved. In non-limiting examples, sample nucleic acid
may be treated with (i) alkylating agents such as methylnitrosourea
that generate several alkylated bases, including N3-methyladenine
and N3-methylguanine, which are recognized and cleaved by alkyl
purine DNA-glycosylase; (ii) sodium bisulfite, which causes
deamination of cytosine residues in DNA to form uracil residues
that can be cleaved by uracil N-glycosylase; and (iii) a chemical
agent that converts guanine to its oxidized form, 8-hydroxyguanine,
which can be cleaved by formamidopyrimidine DNA N-glycosylase.
Examples of chemical cleavage processes include without limitation
alkylation, (e.g., alkylation of phosphorothioate-modified nucleic
acid); cleavage of acid lability of
P3'-N5'-phosphoroamidate-containing nucleic acid; and osmium
tetroxide and piperidine treatment of nucleic acid.
[0186] As used herein, the term "complementary cleavage reactions"
refers to cleavage reactions that are carried out on the same
nucleic acid using different cleavage reagents or by altering the
cleavage specificity of the same cleavage reagent such that
alternate cleavage patterns of the same target or reference nucleic
acid or protein are generated. In certain embodiments, nucleic
acids of interest may be treated with one or more specific cleavage
agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific
cleavage agents) in one or more reaction vessels (e.g., nucleic
acid of interest is treated with each specific cleavage agent in a
separate vessel).
[0187] A nucleic acid suitable for use in the embodiments described
herein sometimes is amplified by any amplification process known in
the art (e.g., PCR, RT-PCR and the like). Nucleic acid
amplification may be particularly beneficial when using organisms
that are typically difficult to culture (e.g., slow growing,
require specialize culture conditions and the like). The terms
"amplify", "amplification", "amplification reaction", or
"amplifying" as used herein refer to any in vitro processes for
multiplying the copies of a target sequence of nucleic acid.
Amplification sometimes refers to an "exponential" increase in
target nucleic acid. However, "amplifying" as used herein can also
refer to linear increases in the numbers of a select target
sequence of nucleic acid, but is different than a one-time, single
primer extension step. In some embodiments, a limited amplification
reaction, also known as pre-amplification, can be performed.
Pre-amplification is a method in which a limited amount of
amplification occurs due to a small number of cycles, for example
10 cycles, being performed. Pre-amplification can allow some
amplification, but stops amplification prior to the exponential
phase, and typically produces about 500 copies of the desired
nucleotide sequence(s).
[0188] Use of pre-amplification may also limit inaccuracies
associated with depleted reactants in standard PCR reactions.
[0189] In some embodiments, a nucleic acid reagent sometimes is
stably integrated into the chromosome of the host organism, or a
nucleic acid reagent can be a deletion of a portion of the host
chromosome, in certain embodiments (e.g., genetically modified
organisms, where alteration of the host genome confers the ability
to selectively or preferentially maintain the desired organism
carrying the genetic modification). Such nucleic acid reagents
(e.g., nucleic acids or genetically modified organisms whose
altered genome confers a selectable trait to the organism) can be
selected for their ability to guide production of a desired protein
or nucleic acid molecule. When desired, the nucleic acid reagent
can be altered such that codons encode for (i) the same amino acid,
using a different tRNA than that specified in the native sequence,
or (ii) a different amino acid than is normal, including
unconventional or unnatural amino acids (including detectably
labeled amino acids). As described herein, the term "native
sequence" refers to an unmodified nucleotide sequence as found in
its natural setting (e.g., a nucleotide sequence as found in an
organism).
[0190] A nucleic acid or nucleic acid reagent can comprise certain
elements often selected according to the intended use of the
nucleic acid. Any of the following elements can be included in or
excluded from a nucleic acid reagent. A nucleic acid reagent, for
example, may include one or more or all of the following nucleotide
elements: one or more promoter elements, one or more 5'
untranslated regions (5'UTRs), one or more regions into which a
target nucleotide sequence may be inserted (an "insertion
element"), one or more target nucleotide sequences, one or more 3'
untranslated regions (3'UTRs), and one or more selection elements.
A nucleic acid reagent can be provided with one or more of such
elements and other elements may be inserted into the nucleic acid
before the nucleic acid is introduced into the desired organism. In
some embodiments, a provided nucleic acid reagent comprises a
promoter, 5'UTR, optional 3'UTR and insertion element(s) by which a
target nucleotide sequence is inserted (i.e., cloned) into the
nucleotide acid reagent. In certain embodiments, a provided nucleic
acid reagent comprises a promoter, insertion element(s) and
optional 3'UTR, and a 5' UTR/target nucleotide sequence is inserted
with an optional 3'UTR. The elements can be arranged in any order
suitable for expression in the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example), and in some embodiments a nucleic acid
reagent comprises the following elements in the 5' to 3' direction:
(1) promoter element, 5'UTR, and insertion element(s); (2) promoter
element, 5'UTR, and target nucleotide sequence; (3) promoter
element, 5'UTR, insertion element(s) and 3'UTR; and (4) promoter
element, 5'UTR, target nucleotide sequence and 3'UTR.
[0191] Promoters
[0192] A promoter element typically is required for DNA synthesis
and/or RNA synthesis. A promoter element often comprises a region
of DNA that can facilitate the transcription of a particular gene,
by providing a start site for the synthesis of RNA corresponding to
a gene. Promoters generally are located near the genes they
regulate, are located upstream of the gene (e.g., 5' of the gene),
and are on the same strand of DNA as the sense strand of the gene,
in some embodiments. In some embodiments, a promoter element can be
isolated from a gene or organism and inserted in functional
connection with a polynucleotide sequence to allow altered and/or
regulated expression. A non-native promoter (e.g., promoter not
normally associated with a given nucleic acid sequence) used for
expression of a nucleic acid often is referred to as a heterologous
promoter. In certain embodiments, a heterologous promoter and/or a
5'UTR can be inserted in functional connection with a
polynucleotide that encodes a polypeptide having a desired activity
as described herein. The terms "operably linked" and "in functional
connection with" as used herein with respect to promoters, refer to
a relationship between a coding sequence and a promoter element.
The promoter is operably linked or in functional connection with
the coding sequence when expression from the coding sequence via
transcription is regulated, or controlled by, the promoter element.
The terms "operably linked" and "in functional connection with" are
utilized interchangeably herein with respect to promoter
elements.
[0193] A promoter often interacts with a RNA polymerase. A
polymerase is an enzyme that catalyses synthesis of nucleic acids
using a preexisting nucleic acid reagent. When the template is a
DNA template, an RNA molecule is transcribed before protein is
synthesized. Enzymes having polymerase activity suitable for use in
the present methods include any polymerase that is active in the
chosen system with the chosen template to synthesize protein. In
some embodiments, a promoter (e.g., a heterologous promoter) also
referred to herein as a promoter element, can be operably linked to
a nucleotide sequence or an open reading frame (ORF). Transcription
from the promoter element can catalyze the synthesis of an RNA
corresponding to the nucleotide sequence or ORF sequence operably
linked to the promoter, which in turn leads to synthesis of a
desired peptide, polypeptide or protein.
[0194] Promoter elements sometimes exhibit responsiveness to
regulatory control. Promoter elements also sometimes can be
regulated by a selective agent. That is, transcription from
promoter elements sometimes can be turned on, turned off,
up-regulated or down-regulated, in response to a change in
environmental, nutritional or internal conditions or signals (e.g.,
heat inducible promoters, light regulated promoters, feedback
regulated promoters, hormone influenced promoters, tissue specific
promoters, oxygen and pH influenced promoters, promoters that are
responsive to selective agents (e.g., kanamycin) and the like, for
example). Promoters influenced by environmental, nutritional or
internal signals frequently are influenced by a signal (direct or
indirect) that binds at or near the promoter and increases or
decreases expression of the target sequence under certain
conditions.
[0195] Non-limiting examples of selective or regulatory agents that
can influence transcription from a promoter element used in
embodiments described herein include, without limitation, (1)
nucleic acid segments that encode products that provide resistance
against otherwise toxic compounds (e.g., antibiotics); (2) nucleic
acid segments that encode products that are otherwise lacking in
the recipient cell (e.g., essential products, tRNA genes,
auxotrophic markers); (3) nucleic acid segments that encode
products that suppress the activity of a gene product; (4) nucleic
acid segments that encode products that can be readily identified
(e.g., phenotypic markers such as antibiotics (e.g.,
.beta.-lactamase), .beta.-galactosidase, green fluorescent protein
(GFP), yellow fluorescent protein (YFP), red fluorescent protein
(RFP), cyan fluorescent protein (CFP), and cell surface proteins);
(5) nucleic acid segments that bind products that are otherwise
detrimental to cell survival and/or function; (6) nucleic acid
segments that otherwise inhibit the activity of any of the nucleic
acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products
that modify a substrate (e.g., restriction endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g., specific protein binding sites); (9)
nucleic acid segments that encode a specific nucleotide sequence
that can be otherwise non-functional (e.g., for PCR amplification
of subpopulations of molecules); (10) nucleic acid segments that,
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; (11) nucleic acid segments
that encode products that either are toxic or convert a relatively
non-toxic compound to a toxic compound (e.g., Herpes simplex
thymidine kinase, cytosine deaminase) in recipient cells; (12)
nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like). In some embodiments, the
regulatory or selective agent can be added to change the existing
growth conditions to which the organism is subjected (e.g., growth
in liquid culture, growth in a fermentor, growth on solid nutrient
plates and the like for example).
[0196] In some embodiments, regulation of a promoter element can be
used to alter (e.g., increase, add, decrease or substantially
eliminate) the activity of a peptide, polypeptide or protein (e.g.,
enzyme activity for example). For example, a microorganism can be
engineered by genetic modification to express a nucleic acid
reagent that can add a novel activity (e.g., an activity not
normally found in the host organism) or increase the expression of
an existing activity by increasing transcription from a homologous
or heterologous promoter operably linked to a nucleotide sequence
of interest (e.g., homologous or heterologous nucleotide sequence
of interest), in certain embodiments. In some embodiments, a
microorganism can be engineered by genetic modification to express
a nucleic acid reagent that can decrease expression of an activity
by decreasing or substantially eliminating transcription from a
homologous or heterologous promoter operably linked to a nucleotide
sequence of interest, in certain embodiments.
[0197] In some embodiments the activity can be altered using
recombinant DNA and genetic techniques known to the artisan.
Methods for engineering microorganisms are further described
herein. Tables herein provide non-limiting lists of yeast promoters
that are up-regulated by oxygen, yeast promoters that are
down-regulated by oxygen, yeast transcriptional repressors and
their associated genes, DNA binding motifs as determined using the
MEME sequence analysis software. Potential regulator binding motifs
can be identified using the program MEME to search intergenic
regions bound by regulators for overrepresented sequences. For each
regulator, the sequences of intergenic regions bound with p-values
less than 0.001 were extracted to use as input for motif discovery.
The MEME software was run using the following settings: a motif
width ranging from 6 to 18 bases, the "zoops" distribution model, a
6.sup.th order Markov background model and a discovery limit of 20
motifs. The discovered sequence motifs were scored for significance
by two criteria: an E-value calculated by MEME and a specificity
score. The motif with the best score using each metric is shown for
each regulator. All motifs presented are derived from datasets
generated in rich growth conditions with the exception of a
previously published dataset for epitope-tagged Gal4 grown in
galactose.
[0198] In some embodiments, the altered activity can be found by
screening the organism under conditions that select for the desired
change in activity. For example, certain microorganisms can be
adapted to increase or decrease an activity by selecting or
screening the organism in question on a media containing substances
that are poorly metabolized or even toxic. An increase in the
ability of an organism to grow a substance that is normally poorly
metabolized may result in an increase in the growth rate on that
substance, for example. A decrease in the sensitivity to a toxic
substance might be manifested by growth on higher concentrations of
the toxic substance, for example. Genetic modifications that are
identified in this manner sometimes are referred to as naturally
occurring mutations or the organisms that carry them can sometimes
be referred to as naturally occurring mutants. Modifications
obtained in this manner are not limited to alterations in promoter
sequences. That is, screening microorganisms by selective pressure,
as described above, can yield genetic alterations that can occur in
non-promoter sequences, and sometimes also can occur in sequences
that are not in the nucleotide sequence of interest, but in a
related nucleotide sequences (e.g., a gene involved in a different
step of the same pathway, a transport gene, and the like).
Naturally occurring mutants sometimes can be found by isolating
naturally occurring variants from unique environments, in some
embodiments.
[0199] Homology and Identity
[0200] In addition to the regulated promoter sequences, regulatory
sequences, and coding polynucleotides provided herein, a nucleic
acid reagent may include a polynucleotide sequence 80% or more
identical to the foregoing (or to the complementary sequences).
That is, a nucleotide sequence that is at least 80% or more, 81% or
more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or
more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or
more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or
more, 97% or more, 98% or more, or 99% or more identical to a
nucleotide sequence described herein can be utilized. The term
"identical" as used herein refers to two or more nucleotide
sequences having substantially the same nucleotide sequence when
compared to each other. One test for determining whether two
nucleotide sequences or amino acids sequences are substantially
identical is to determine the percent of identical nucleotide
sequences or amino acid sequences shared.
[0201] Calculations of sequence identity can be performed as
follows. Sequences are aligned for optimal comparison purposes
(e.g., gaps can be introduced in one or both of a first and a
second amino acid or nucleic acid sequence for optimal alignment
and non-homologous sequences can be disregarded for comparison
purposes). The length of a reference sequence aligned for
comparison purposes is sometimes 30% or more, 40% or more, 50% or
more, often 60% or more, and more often 70% or more, 80% or more,
90% or more, or 100% of the length of the reference sequence. The
nucleotides or amino acids at corresponding nucleotide or
polypeptide positions, respectively, are then compared among the
two sequences. When a position in the first sequence is occupied by
the same nucleotide or amino acid as the corresponding position in
the second sequence, the nucleotides or amino acids are deemed to
be identical at that position. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, introduced for optimal alignment of the two
sequences.
[0202] Comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. Percent identity between two amino acid or
nucleotide sequences can be determined using the algorithm of
Meyers & Miller, CABIOS 4: 11-17 (1989), which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. Also, percent identity between two amino acid sequences can
be determined using the Needleman & Wunsch, J. Mol. Biol. 48:
444-453 (1970) algorithm which has been incorporated into the GAP
program in the GCG software package (available at the http address
www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight
of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide
sequences can be determined using the GAP program in the GCG
software package (available at http address www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often
used is a Blossum 62 scoring matrix with a gap open penalty of 12,
a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0203] Sequence identity can also be determined by hybridization
assays conducted under stringent conditions. As use herein, the
term "stringent conditions" refers to conditions for hybridization
and washing. Stringent conditions are known to those skilled in the
art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and
non-aqueous methods are described in that reference and either can
be used. An example of stringent hybridization conditions is
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C. Another example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
55.degree. C. A further example of stringent hybridization
conditions is hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C. Often, stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C. More
often, stringency conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C.
[0204] UTRs
[0205] As noted above, nucleic acid reagents may also comprise one
or more 5' UTR's, and one or more 3'UTR's. A 5' UTR may comprise
one or more elements endogenous to the nucleotide sequence from
which it originates, and sometimes includes one or more exogenous
elements. A 5' UTR can originate from any suitable nucleic acid,
such as genomic DNA, plasmid DNA, RNA or mRNA, for example, from
any suitable organism (e.g., virus, bacterium, yeast, fungi, plant,
insect or mammal). The artisan may select appropriate elements for
the 5' UTR based upon the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example). A 5' UTR sometimes comprises one or more of
the following elements known to the artisan: enhancer sequences
(e.g., transcriptional or translational), transcription initiation
site, transcription factor binding site, translation regulation
site, translation initiation site, translation factor binding site,
accessory protein binding site, feedback regulation agent binding
sites, Pribnow box, TATA box, -35 element, E-box (helix-loop-helix
binding element), ribosome binding site, replicon, internal
ribosome entry site (IRES), silencer element and the like. In some
embodiments, a promoter element may be isolated such that all 5'
UTR elements necessary for proper conditional regulation are
contained in the promoter element fragment, or within a functional
subsequence of a promoter element fragment.
[0206] A 5' UTR in the nucleic acid reagent can comprise a
translational enhancer nucleotide sequence. A translational
enhancer nucleotide sequence often is located between the promoter
and the target nucleotide sequence in a nucleic acid reagent. A
translational enhancer sequence often binds to a ribosome,
sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a
40S ribosome binding sequence) and sometimes is an internal
ribosome entry sequence (IRES). An IRES generally forms an RNA
scaffold with precisely placed RNA tertiary structures that contact
a 40S ribosomal subunit via a number of specific intermolecular
interactions. Examples of ribosomal enhancer sequences are known
and can be identified by the artisan (e.g., Mignone et al., Nucleic
Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids
Research 31: 722-733 (2003); Akbergenov et al., Nucleic Acids
Research 32: 239-247 (2004); Mignone et al., Genome Biology 3(3):
reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:
3401-3411 (2002); Shaloiko et al., http address
www.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et
al., Nucleic Acids Research 15: 3257-3273 (1987)).
[0207] A translational enhancer sequence sometimes is a eukaryotic
sequence, such as a Kozak consensus sequence or other sequence
(e.g., hydroid polyp sequence, GenBank accession no. U07128). A
translational enhancer sequence sometimes is a prokaryotic
sequence, such as a Shine-Dalgarno consensus sequence. In certain
embodiments, the translational enhancer sequence is a viral
nucleotide sequence. A translational enhancer sequence sometimes is
from a 5' UTR of a plant virus, such as Tobacco Mosaic Virus (TMV),
Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus (ETV); Potato Virus
Y (PVY); Turnip Mosaic (poty) Virus and Pea Seed Borne Mosaic
Virus, for example. In certain embodiments, an omega sequence about
67 bases in length from TMV is included in the nucleic acid reagent
as a translational enhancer sequence (e.g., devoid of guanosine
nucleotides and includes a 25 nucleotide long poly (CAA) central
region).
[0208] A 3' UTR may comprise one or more elements endogenous to the
nucleotide sequence from which it originates and sometimes includes
one or more exogenous elements. A 3' UTR may originate from any
suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or
mRNA, for example, from any suitable organism (e.g., a virus,
bacterium, yeast, fungi, plant, insect or mammal). The artisan can
select appropriate elements for the 3' UTR based upon the chosen
expression system (e.g., expression in a chosen organism, for
example). A 3' UTR sometimes comprises one or more of the following
elements known to the artisan: transcription regulation site,
transcription initiation site, transcription termination site,
transcription factor binding site, translation regulation site,
translation termination site, translation initiation site,
translation factor binding site, ribosome binding site, replicon,
enhancer element, silencer element and polyadenosine tail. A 3' UTR
often includes a polyadenosine tail and sometimes does not, and if
a polyadenosine tail is present, one or more adenosine moieties may
be added or deleted from it (e.g., about 5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45 or about
50 adenosine moieties may be added or subtracted).
[0209] In some embodiments, modification of a 5' UTR and/or a 3'
UTR can be used to alter (e.g., increase, add, decrease or
substantially eliminate) the activity of a promoter. Alteration of
the promoter activity can in turn alter the activity of a peptide,
polypeptide or protein (e.g., enzyme activity for example), by a
change in transcription of the nucleotide sequence(s) of interest
from an operably linked promoter element comprising the modified 5'
or 3' UTR. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can add a novel activity (e.g., an
activity not normally found in the host organism) or increase the
expression of an existing activity by increasing transcription from
a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest (e.g., homologous or heterologous
nucleotide sequence of interest), in certain embodiments. In some
embodiments, a microorganism can be engineered by genetic
modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can decrease the expression of an
activity by decreasing or substantially eliminating transcription
from a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest, in certain embodiments.
[0210] Target Nucleotide Sequence
[0211] A nucleotide reagent sometimes can comprise a target
nucleotide sequence. A "target nucleotide sequence" as used herein
encodes a nucleic acid, peptide, polypeptide or protein of
interest, and may be a ribonucleotide sequence or a
deoxyribonucleotide sequence. A target nucleic acid sometimes is an
untranslated ribonucleic acid and sometimes is a translated
ribonucleic acid. An untranslated ribonucleic acid may include, but
is not limited to, a small interfering ribonucleic acid (siRNA), a
short hairpin ribonucleic acid (shRNA), other ribonucleic acid
capable of RNA interference (RNAi), an antisense ribonucleic acid,
or a ribozyme. A translatable target nucleotide sequence (e.g., a
target ribonucleotide sequence) sometimes encodes a peptide,
polypeptide or protein, which are sometimes referred to herein as
"target peptides," "target polypeptides" or "target proteins."
[0212] Any peptides, polypeptides or proteins, or an activity
catalyzed by one or more peptides, polypeptides or proteins may be
encoded by a target nucleotide sequence and may be selected by a
user. Representative proteins include enzymes (e.g., acetyl-CoA
carboxylase, acyl-CoA oxidase, thioesterase, monooxygenase,
monooxygenase reductase, fatty alcohol oxidase, acyltransferase and
the like, for example), antibodies, serum proteins (e.g., albumin),
membrane bound proteins, hormones (e.g., growth hormone,
erythropoietin, insulin, etc.), cytokines, etc., and include both
naturally occurring and exogenously expressed polypeptides.
Representative activities (e.g., enzymes or combinations of enzymes
which are functionally associated to provide an activity) include
thioesterase activity, monooxygenase activity, monooxygenase
reductase activity, acyltransferase activity, omega hydroxyl fatty
acid dehydrogenase activity, beta-oxidation activity,
omega-oxidation activity and the like, for example. The term
"enzyme" as used herein refers to a protein which can act as a
catalyst to induce a chemical change in other compounds, thereby
producing one or more products from one or more substrates.
[0213] Specific polypeptides (e.g., enzymes) useful for embodiments
described herein are listed herein. The term "protein" as used
herein refers to a molecule having a sequence of amino acids linked
by peptide bonds. This term includes fusion proteins,
oligopeptides, peptides, cyclic peptides, polypeptides and
polypeptide derivatives, whether native or recombinant, and also
includes fragments, derivatives, homologs, and variants thereof. A
protein or polypeptide sometimes is of intracellular origin (e.g.,
located in the nucleus, cytosol, or interstitial space of host
cells in vivo) and sometimes is a cell membrane protein in vivo. In
some embodiments (described above, and in further detail hereafter
in Engineering and Alteration Methods), a genetic modification can
result in a modification (e.g., increase, substantially increase,
decrease or substantially decrease) of a target activity.
[0214] A translatable nucleotide sequence generally is located
between a start codon (AUG in ribonucleic acids and ATG in
deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre), UAG
(amber) or UGA (opal) in ribonucleic acids and TAA, TAG or TGA in
deoxyribonucleic acids), and sometimes is referred to herein as an
"open reading frame" (ORF). A translatable nucleotide sequence
(e.g., ORF) sometimes is encoded differently in one organism (e.g.,
most organisms encode CTG as leucine) than in another organism
(e.g., C. tropicalis encodes CTG as serine). In some embodiments, a
translatable nucleotide sequence is altered to correct alternate
genetic code (e.g., codon usage) differences between a nucleotide
donor organism and an nucleotide recipient organism (e.g.,
engineered organism). In certain embodiments, a translatable
nucleotide sequence is altered to improve; (i) codon usage, (ii)
transcriptional efficiency, (iii) translational efficiency, (iv)
the like, and combinations thereof.
[0215] Nucleic Acid Reagents & Tools
[0216] A nucleic acid reagent sometimes comprises one or more ORFs.
An ORF may be from any suitable source, sometimes from genomic DNA,
mRNA, reverse transcribed RNA or complementary DNA (cDNA) or a
nucleic acid library comprising one or more of the foregoing, and
is from any organism species that contains a nucleic acid sequence
of interest, protein of interest, or activity of interest.
Non-limiting examples of organisms from which an ORF can be
obtained include bacteria, yeast, fungi, human, insect, nematode,
bovine, equine, canine, feline, rat or mouse, for example.
[0217] A nucleic acid reagent sometimes comprises a nucleotide
sequence adjacent to an ORF that is translated in conjunction with
the ORF and encodes an amino acid tag. The tag-encoding nucleotide
sequence is located 3' and/or 5' of an ORF in the nucleic acid
reagent, thereby encoding a tag at the C-terminus or N-terminus of
the protein or peptide encoded by the ORF. Any tag that does not
abrogate in vitro transcription and/or translation may be utilized
and may be appropriately selected by the artisan. Tags may
facilitate isolation and/or purification of the desired ORF product
from culture or fermentation media.
[0218] A tag sometimes specifically binds a molecule or moiety of a
solid phase or a detectable label, for example, thereby having
utility for isolating, purifying and/or detecting a protein or
peptide encoded by the ORF. In some embodiments, a tag comprises
one or more of the following elements: FLAG (e.g., DYKDDDDKG), V5
(e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV (e.g.,
QPELAPEDPED), influenza hemaglutinin, HA (e.g., YPYDVPDYA), VSV-G
(e.g., YTDIEMNRLGK), bacterial glutathione-S-transferase, maltose
binding protein, a streptavidin- or avidin-binding tag (e.g.,
pcDNA.TM. 6 BioEase.TM. Gateway.RTM. Biotinylation System
(Invitrogen)), thioredoxin, R-galactosidase, VSV-glycoprotein, a
fluorescent protein (e.g., green fluorescent protein or one of its
many color variants (e.g., yellow, red, blue)), a polylysine or
polyarginine sequence, a polyhistidine sequence (e.g., His6) or
other sequence that chelates a metal (e.g., cobalt, zinc, copper),
and/or a cysteine-rich sequence that binds to an arsenic-containing
molecule. In certain embodiments, a cysteine-rich tag comprises the
amino acid sequence CC-Xn-CC, wherein X is any amino acid and n is
1 to 3, and the cysteine-rich sequence sometimes is CCPGCC. In
certain embodiments, the tag comprises a cysteine-rich element and
a polyhistidine element (e.g., CCPGCC and His6).
[0219] A tag often conveniently binds to a binding partner. For
example, some tags bind to an antibody (e.g., FLAG) and sometimes
specifically bind to a small molecule. For example, a polyhistidine
tag specifically chelates a bivalent metal, such as copper, zinc
and cobalt; a polylysine or polyarginine tag specifically binds to
a zinc finger; a glutathione S-transferase tag binds to
glutathione; and a cysteine-rich tag specifically binds to an
arsenic-containing molecule. Arsenic-containing molecules include
LUMIO.TM. agents (Invitrogen, California), such as FlAsH.TM.
(EDT2[4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethan-
edithiol)2]) and ReAsH reagents (e.g., U.S. Pat. No. 5,932,474 to
Tsien et al., entitled "Target Sequences for Synthetic Molecules;"
U.S. Pat. No. 6,054,271 to Tsien et al., entitled "Methods of Using
Synthetic Molecules and Target Sequences;" U.S. Pat. Nos. 6,451,569
and 6,008,378; published U.S. Patent Application 2003/0083373, and
published PCT Patent Application WO 99/21013, all to Tsien et al.
and all entitled "Synthetic Molecules that Specifically React with
Target Sequences"). Such antibodies and small molecules sometimes
are linked to a solid phase for convenient isolation of the target
protein or target peptide.
[0220] A tag sometimes comprises a sequence that localizes a
translated protein or peptide to a component in a system, which is
referred to as a "signal sequence" or "localization signal
sequence" herein. A signal sequence often is incorporated at the
N-terminus of a target protein or target peptide, and sometimes is
incorporated at the C-terminus. Examples of signal sequences are
known to the artisan, are readily incorporated into a nucleic acid
reagent, and often are selected according to the organism in which
expression of the nucleic acid reagent is performed. A signal
sequence in some embodiments localizes a translated protein or
peptide to a cell membrane. Examples of signal sequences include,
but are not limited to, a nucleus targeting signal (e.g., steroid
receptor sequence and N-terminal sequence of SV40 virus large T
antigen); mitochondrial targeting signal (e.g., amino acid sequence
that forms an amphipathic helix); peroxisome targeting signal
(e.g., C-terminal sequence in YFG from S. cerevisiae); and a
secretion signal (e.g., N-terminal sequences from invertase, mating
factor alpha, PHO5 and SUC2 in S. cerevisiae; multiple N-terminal
sequences of B. subtilis proteins (e.g., Tjalsma et al., Microbiol.
Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylase signal
sequence (e.g., U.S. Pat. No. 6,288,302); pectate lyase signal
sequence (e.g., U.S. Pat. No. 5,846,818); precollagen signal
sequence (e.g., U.S. Pat. No. 5,712,114); OmpA signal sequence
(e.g., U.S. Pat. No. 5,470,719); lam beta signal sequence (e.g.,
U.S. Pat. No. 5,389,529); B. brevis signal sequence (e.g., U.S.
Pat. No. 5,232,841); and P. pastoris signal sequence (e.g., U.S.
Pat. No. 5,268,273)).
[0221] A tag sometimes is directly adjacent to the amino acid
sequence encoded by an ORF (i.e., there is no intervening sequence)
and sometimes a tag is substantially adjacent to an ORF encoded
amino acid sequence (e.g., an intervening sequence is present). An
intervening sequence sometimes includes a recognition site for a
protease, which is useful for cleaving a tag from a target protein
or peptide. In some embodiments, the intervening sequence is
cleaved by Factor Xa (e.g., recognition site I (E/D)GR), thrombin
(e.g., recognition site LVPRGS), enterokinase (e.g., recognition
site DDDDK), TEV protease (e.g., recognition site ENLYFQG) or
PreScission.TM. protease (e.g., recognition site LEVLFQGP), for
example.
[0222] An intervening sequence sometimes is referred to herein as a
"linker sequence," and may be of any suitable length selected by
the artisan. A linker sequence sometimes is about 1 to about 20
amino acids in length, and sometimes about 5 to about 10 amino
acids in length. The artisan may select the linker length to
substantially preserve target protein or peptide function (e.g., a
tag may reduce target protein or peptide function unless separated
by a linker), to enhance disassociation of a tag from a target
protein or peptide when a protease cleavage site is present (e.g.,
cleavage may be enhanced when a linker is present), and to enhance
interaction of a tag/target protein product with a solid phase. A
linker can be of any suitable amino acid content, and often
comprises a higher proportion of amino acids having relatively
short side chains (e.g., glycine, alanine, serine and
threonine).
[0223] A nucleic acid reagent sometimes includes a stop codon
between a tag element and an insertion element or ORF, which can be
useful for translating an ORF with or without the tag. Mutant tRNA
molecules that recognize stop codons (described above) suppress
translation termination and thereby are designated "suppressor
tRNAs." Suppressor tRNAs can result in the insertion of amino acids
and continuation of translation past stop codons (e.g., U.S. Patent
Application No. 60/587,583, filed Jul. 14, 2004, entitled
"Production of Fusion Proteins by Cell-Free Protein Synthesis,";
Eggertsson, et al., (1988) Microbiological Review 52(3):354-374,
and Engleerg-Kukla, et al. (1996) in Escherichia coli and
Salmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,
Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number of
suppressor tRNAs are known, including but not limited to, supE,
supP, supD, supF and supZ suppressors, which suppress the
termination of translation of the amber stop codon; supB, gIT,
supL, supN, supC and supM suppressors, which suppress the function
of the ochre stop codon and glyT, trpT and Su-9 suppressors, which
suppress the function of the opal stop codon. In general,
suppressor tRNAs contain one or more mutations in the anti-codon
loop of the tRNA that allows the tRNA to base pair with a codon
that ordinarily functions as a stop codon. The mutant tRNA is
charged with its cognate amino acid residue and the cognate amino
acid residue is inserted into the translating polypeptide when the
stop codon is encountered. Mutations that enhance the efficiency of
termination suppressors (i.e., increase stop codon read-through)
have been identified. These include, but are not limited to,
mutations in the uar gene (also known as the prfA gene), mutations
in the ups gene, mutations in the sueA, sueB and sueC genes,
mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in
the rplL gene.
[0224] Thus, a nucleic acid reagent comprising a stop codon located
between an ORF and a tag can yield a translated ORF alone when no
suppressor tRNA is present in the translation system, and can yield
a translated ORF-tag fusion when a suppressor tRNA is present in
the system. Suppressor tRNA can be generated in cells transfected
with a nucleic acid encoding the tRNA (e.g., a replication
incompetent adenovirus containing the human tRNA-Ser suppressor
gene can be transfected into cells, or a YAC containing a yeast or
bacterial tRNA suppressor gene can be transfected into yeast cells,
for example). Vectors for synthesizing suppressor tRNA and for
translating ORFs with or without a tag are available to the artisan
(e.g., Tag-On-Demand.TM. kit (Invitrogen Corporation, California);
Tag-On-Demand.TM. Suppressor Supernatant Instruction Manual,
Version B, 6 Jun. 2003, at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;
Tag-On-Demand.TM. Gateway.RTM. Vector Instruction Manual, Version
B, 20 Jun., 2003 at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_vectors_man.pdf;
and Capone et al., Amber, ochre and opal suppressor tRNA genes
derived from a human serine tRNA gene. EMBO J. 4:213, 1985).
[0225] Any convenient cloning strategy known in the art may be
utilized to incorporate an element, such as an ORF, into a nucleic
acid reagent. Known methods can be utilized to insert an element
into the template independent of an insertion element, such as (1)
cleaving the template at one or more existing restriction enzyme
sites and ligating an element of interest and (2) adding
restriction enzyme sites to the template by hybridizing
oligonucleotide primers that include one or more suitable
restriction enzyme sites and amplifying by polymerase chain
reaction (described in greater detail herein). Other cloning
strategies take advantage of one or more insertion sites present or
inserted into the nucleic acid reagent, such as an oligonucleotide
primer hybridization site for PCR, for example, and others
described herein. In some embodiments, a cloning strategy can be
combined with genetic manipulation such as recombination (e.g.,
recombination of a nucleic acid reagent with a nucleic acid
sequence of interest into the genome of the organism to be
modified, as described further herein). In some embodiments, the
cloned ORF(s) can produce (directly or indirectly) a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid), by engineering a
microorganism with one or more ORFs of interest, which
microorganism comprises one or more altered activities selected
from the group consisting of omega hydroxyl fatty acid
dehydrogenase activity, acyl-CoA oxidase activity, acyltransferase
activity, thioesterase activity, monooxygenase activity and
monooxygenase reductase activity.
[0226] In some embodiments, the nucleic acid reagent includes one
or more recombinase insertion sites. A recombinase insertion site
is a recognition sequence on a nucleic acid molecule that
participates in an integration/recombination reaction by
recombination proteins. For example, the recombination site for Cre
recombinase is loxP, which is a 34 base pair sequence comprised of
two 13 base pair inverted repeats (serving as the recombinase
binding sites) flanking an 8 base pair core sequence (e.g., FIG. 1
of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Other
examples of recombination sites include attB, attP, attL, and attR
sequences, and mutants, fragments, variants and derivatives
thereof, which are recognized by the recombination protein A Int
and by the auxiliary proteins integration host factor (IHF), FIS
and excisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557;
6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S. patent
application Ser. No. 09/517,466, filed Mar. 2, 2000, and
09/732,914, filed Aug. 14, 2003, and in U.S. patent publication no.
2002-0007051-A1; Landy, Curr. Opin. Biotech. 3:699-707 (1993)).
[0227] Examples of recombinase cloning nucleic acids are in
Gateway.RTM. systems (Invitrogen, California), which include at
least one recombination site for cloning a desired nucleic acid
molecules in vivo or in vitro. In some embodiments, the system
utilizes vectors that contain at least two different site-specific
recombination sites, often based on the bacteriophage lambda system
(e.g., attl and att2), and are mutated from the wild-type (att0)
sites. Each mutated site has a unique specificity for its cognate
partner att site (i.e., its binding partner recombination site) of
the same type (for example attB1 with attP1, or attL1 with attR1)
and will not cross-react with recombination sites of the other
mutant type or with the wild-type att0 site. Different site
specificities allow directional cloning or linkage of desired
molecules thus providing desired orientation of the cloned
molecules. Nucleic acid fragments flanked by recombination sites
are cloned and subcloned using the Gateway.RTM. system by replacing
a selectable marker (for example, ccdB) flanked by att sites on the
recipient plasmid molecule, sometimes termed the Destination
Vector. Desired clones are then selected by transformation of a
ccdB sensitive host strain and positive selection for a marker on
the recipient molecule. Similar strategies for negative selection
(e.g., use of toxic genes) can be used in other organisms such as
thymidine kinase (TK) in mammals and insects.
[0228] A recombination system useful for engineering yeast is
outlined briefly. The system makes use of the URA3 gene (e.g., for
S. cerevisiae and C. albicans, for example) or URA4 and URA5 genes
(e.g., for S. pombe, for example) and toxicity of the nucleotide
analogue 5-Fluoroorotic acid (5-FOA). The URA3 or URA4 and URA5
genes encode orotine-5'-monophosphate (OMP) dicarboxylase. Yeast
with an active URA3 or URA4 and URA5 gene (phenotypically Ura+)
convert 5-FOA to fluorodeoxyuridine, which is toxic to yeast cells.
Yeast carrying a mutation in the appropriate gene(s) or having a
knock out of the appropriate gene(s) can grow in the presence of
5-FOA, if the media is also supplemented with uracil.
[0229] A nucleic acid engineering construct can be made which may
comprise the URA3 gene or cassette (for S. cerevisiae), flanked on
either side by the same nucleotide sequence in the same
orientation. The URA3 cassette comprises a promoter, the URA3 gene
and a functional transcription terminator. Target sequences which
direct the construct to a particular nucleic acid region of
interest in the organism to be engineered are added such that the
target sequences are adjacent to and abut the flanking sequences on
either side of the URA3 cassette. Yeast can be transformed with the
engineering construct and plated on minimal media without uracil.
Colonies can be screened by PCR to determine those transformants
that have the engineering construct inserted in the proper location
in the genome. Checking insertion location prior to selecting for
recombination of the ura3 cassette may reduce the number of
incorrect clones carried through to later stages of the procedure.
Correctly inserted transformants can then be replica plated on
minimal media containing 5-FOA to select for recombination of the
URA3 cassette out of the construct, leaving a disrupted gene and an
identifiable footprint (e.g., nucleic acid sequence) that can be
use to verify the presence of the disrupted gene. The technique
described is useful for disrupting or "knocking out" gene function,
but also can be used to insert genes or constructs into a host
organisms genome in a targeted, sequence specific manner.
[0230] In certain embodiments, a nucleic acid reagent includes one
or more topoisomerase insertion sites.
[0231] A topoisomerase insertion site is a defined nucleotide
sequence recognized and bound by a site-specific topoisomerase. For
example, the nucleotide sequence 5'-(C/T)CCTT-3' is a topoisomerase
recognition site bound specifically by most poxvirus
topoisomerases, including vaccinia virus DNA topoisomerase I. After
binding to the recognition sequence, the topoisomerase cleaves the
strand at the 3'-most thymidine of the recognition site to produce
a nucleotide sequence comprising 5'-(C/T)CCIT-PO4-TOPO, a complex
of the topoisomerase covalently bound to the 3' phosphate via a
tyrosine in the topoisomerase (e.g., Shuman, J. Biol. Chem.
266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res.
22:5360-5365, 1994; U.S. Pat. No. 5,766,891; PCT/US95/16099; and
PCT/US98/12372). In comparison, the nucleotide sequence
5'-GCAACTT-3' is a topoisomerase recognition site for type IA E.
coli topoisomerase III. An element to be inserted often is combined
with topoisomerase-reacted template and thereby incorporated into
the nucleic acid reagent (e.g., World Wide Web URL
invitrogen.com/downloads/F-13512_Topo_Flyer.pdf; World Wide Web URL
invitrogen.com/content/sfs/brochures/710.sub.--021849%20_B_TOPOCloning_br-
o.pdf; TOPO TA Cloning.RTM. Kit and Zero Blunt.RTM. TOPO.RTM.
Cloning Kit product information).
[0232] A nucleic acid reagent sometimes contains one or more origin
of replication (ORI) elements. In some embodiments, a template
comprises two or more ORIs, where one functions efficiently in one
organism (e.g., a bacterium) and another functions efficiently in
another organism (e.g., a eukaryote, like yeast for example). In
some embodiments, an ORI may function efficiently in one species
(e.g., S. cerevisiae, for example) and another ORI may function
efficiently in a different species (e.g., S. pombe, for example). A
nucleic acid reagent also sometimes includes one or more
transcription regulation sites.
[0233] A nucleic acid reagent can include one or more selection
elements (e.g., elements for selection of the presence of the
nucleic acid reagent, and not for activation of a promoter element
which can be selectively regulated). Selection elements often are
utilized using known processes to determine whether a nucleic acid
reagent is included in a cell. In some embodiments, a nucleic acid
reagent includes two or more selection elements, where one
functions efficiently in one organism and another functions
efficiently in another organism. Examples of selection elements
include, but are not limited to, (1) nucleic acid segments that
encode products that provide resistance against otherwise toxic
compounds (e.g., antibiotics); (2) nucleic acid segments that
encode products that are otherwise lacking in the recipient cell
(e.g., essential products, tRNA genes, auxotrophic markers); (3)
nucleic acid segments that encode products that suppress the
activity of a gene product; (4) nucleic acid segments that encode
products that can be readily identified (e.g., phenotypic markers
such as antibiotics (e.g., .beta.-lactamase), .beta.-galactosidase,
green fluorescent protein (GFP), yellow fluorescent protein (YFP),
red fluorescent protein (RFP), cyan fluorescent protein (CFP), and
cell surface proteins); (5) nucleic acid segments that bind
products that are otherwise detrimental to cell survival and/or
function; (6) nucleic acid segments that otherwise inhibit the
activity of any of the nucleic acid segments described in Nos. 1-5
above (e.g., antisense oligonucleotides); (7) nucleic acid segments
that bind products that modify a substrate (e.g., restriction
endonucleases); (8) nucleic acid segments that can be used to
isolate or identify a desired molecule (e.g., specific protein
binding sites); (9) nucleic acid segments that encode a specific
nucleotide sequence that can be otherwise non-functional (e.g., for
PCR amplification of subpopulations of molecules); (10) nucleic
acid segments that, when absent, directly or indirectly confer
resistance or sensitivity to particular compounds; (11) nucleic
acid segments that encode products that either are toxic or convert
a relatively non-toxic compound to a toxic compound (e.g., Herpes
simplex thymidine kinase, cytosine deaminase) in recipient cells;
(12) nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like).
[0234] A nucleic acid reagent is of any form useful for in vivo
transcription and/or translation. A nucleic acid sometimes is a
plasmid, such as a supercoiled plasmid, sometimes is a yeast
artificial chromosome (e.g., YAC), sometimes is a linear nucleic
acid (e.g., a linear nucleic acid produced by PCR or by restriction
digest), sometimes is single-stranded and sometimes is
double-stranded. A nucleic acid reagent sometimes is prepared by an
amplification process, such as a polymerase chain reaction (PCR)
process or transcription-mediated amplification process (TMA). In
TMA, two enzymes are used in an isothermal reaction to produce
amplification products detected by light emission (see, e.g.,
Biochemistry 1996 Jun. 25; 35(25):8429-38 and http address
www.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR
processes are known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195;
4,965,188; and 5,656,493), and generally are performed in cycles.
Each cycle includes heat denaturation, in which hybrid nucleic
acids dissociate; cooling, in which primer oligonucleotides
hybridize; and extension of the oligonucleotides by a polymerase
(i.e., Taq polymerase). An example of a PCR cyclical process is
treating the sample at 95 C for 5 minutes; repeating forty-five
cycles of 95 C for 1 minute, 59 C for 1 minute, 10 seconds, and 72
C for 1 minute 30 seconds; and then treating the sample at 72 C for
5 minutes. Multiple cycles frequently are performed using a
commercially available thermal cycler. PCR amplification products
sometimes are stored for a time at a lower temperature (e.g., at 4
C) and sometimes are frozen (e.g., at -20 C) before analysis.
[0235] In some embodiments, a nucleic acid reagent, protein
reagent, protein fragment reagent or other reagent described herein
is isolated or purified. The term "isolated" as used herein refers
to material removed from its original environment (e.g., the
natural environment if it is naturally occurring, or a host cell if
expressed exogenously), and thus is altered "by the hand of man"
from its original environment. The term "purified" as used herein
with reference to molecules does not refer to absolute purity.
Rather, "purified" refers to a substance in a composition that
contains fewer substance species in the same class (e.g., nucleic
acid or protein species) other than the substance of interest in
comparison to the sample from which it originated. "Purified," if a
nucleic acid or protein for example, refers to a substance in a
composition that contains fewer nucleic acid species or protein
species other than the nucleic acid or protein of interest in
comparison to the sample from which it originated. Sometimes, a
protein or nucleic acid is "substantially pure," indicating that
the protein or nucleic acid represents at least 50% of protein or
nucleic acid on a mass basis of the composition. Often, a
substantially pure protein or nucleic acid is at least 75% on a
mass basis of the composition, and sometimes at least 95% on a mass
basis of the composition.
[0236] Engineering and Alteration Methods
[0237] Methods and compositions (e.g., nucleic acid reagents)
described herein can be used to generate engineered microorganisms.
As noted above, the term "engineered microorganism" as used herein
refers to a modified organism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point for modification (e.g., host microorganism or
unmodified organism). Engineered microorganisms typically arise as
a result of a genetic modification, usually introduced or selected
for, by one of skill in the art using readily available techniques.
Non-limiting examples of methods useful for generating an altered
activity include, introducing a heterologous polynucleotide (e.g.,
nucleic acid or gene integration, also referred to as "knock in"),
removing an endogenous polynucleotide, altering the sequence of an
existing endogenous nucleic acid sequence (e.g., site-directed
mutagenesis), disruption of an existing endogenous nucleic acid
sequence (e.g., knock outs and transposon or insertion element
mediated mutagenesis), selection for an altered activity where the
selection causes a change in a naturally occurring activity that
can be stably inherited (e.g., causes a change in a nucleic acid
sequence in the genome of the organism or in an epigenetic nucleic
acid that is replicated and passed on to daughter cells), PCR-based
mutagenesis, and the like. The term "mutagenesis" as used herein
refers to any modification to a nucleic acid (e.g., nucleic acid
reagent, or host chromosome, for example) that is subsequently used
to generate a product in a host or modified organism. Non-limiting
examples of mutagenesis include, deletion, insertion, substitution,
rearrangement, point mutations, suppressor mutations and the like.
Mutagenesis methods are known in the art and are readily available
to the artisan. Non-limiting examples of mutagenesis methods are
described herein and can also be found in Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Another non-limiting example of mutagenesis can be conducted using
a Stratagene (San Diego, Calif.) "QuickChange" kit according to the
manufacturer's instructions.
[0238] The term "genetic modification" as used herein refers to any
suitable nucleic acid addition, removal or alteration that
facilitates production of a target fatty dicarboxylic acid product
(e.g., sebacic or dodecanedioic acid) in an engineered
microorganism. Genetic modifications include, without limitation,
insertion of one or more nucleotides in a native nucleic acid of a
host organism in one or more locations, deletion of one or more
nucleotides in a native nucleic acid of a host organism in one or
more locations, modification or substitution of one or more
nucleotides in a native nucleic acid of a host organism in one or
more locations, insertion of a non-native nucleic acid into a host
organism (e.g., insertion of an autonomously replicating vector),
and removal of a non-native nucleic acid in a host organism (e.g.,
removal of a vector).
[0239] The term "heterologous polynucleotide" as used herein refers
to a nucleotide sequence not present in a host microorganism in
some embodiments. In certain embodiments, a heterologous
polynucleotide is present in a different amount (e.g., different
copy number) than in a host microorganism, which can be
accomplished, for example, by introducing more copies of a
particular nucleotide sequence to a host microorganism (e.g., the
particular nucleotide sequence may be in a nucleic acid autonomous
of the host chromosome or may be inserted into a chromosome). A
heterologous polynucleotide is from a different organism in some
embodiments, and in certain embodiments, is from the same type of
organism but from an outside source (e.g., a recombinant
source).
[0240] In some embodiments, an organism engineered using the
methods and nucleic acid reagents described herein can produce a
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid). In certain embodiments,
an engineered microorganism described herein that produces a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) may comprise one or more
altered activities selected from the group consisting of omega oxo
fatty acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity, acyl-CoA oxidase activity, monooxygenase
activity and monooxygenase reductase activity. In some embodiments,
an engineered microorganism as described herein may comprise a
genetic modification that adds or increases the omega oxo fatty
acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity, acyl-CoA oxidase activity, monooxygenase
activity and monooxygenase reductase activity.
[0241] In certain embodiments, an engineered microorganism
described herein can comprise an altered thioesterase activity. In
some embodiments, the engineered microorganism may comprise a
genetic alteration that adds or increases a thioesterase activity.
In some embodiments, the engineered microorganism comprising a
genetic alteration that adds or increases a thioesterase activity,
may further comprise a heterologous polynucleotide encoding a
polypeptide having thioesterase activity.
[0242] The term "altered activity" as used herein refers to an
activity in an engineered microorganism that is added or modified
relative to the host microorganism (e.g., added, increased,
reduced, inhibited or removed activity). An activity can be altered
by introducing a genetic modification to a host microorganism that
yields an engineered microorganism having added, increased,
reduced, inhibited or removed activity.
[0243] An added activity often is an activity not detectable in a
host microorganism. An increased activity generally is an activity
detectable in a host microorganism that has been increased in an
engineered microorganism. An activity can be increased to any
suitable level for production of a target fatty dicarboxylic acid
product (e.g., sebacic or dodecanedioic acid), including but not
limited to less than 2-fold (e.g., about 10% increase to about 99%
increase; about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% increase),
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of
10-fold increase, or greater than about 10-fold increase. A reduced
or inhibited activity generally is an activity detectable in a host
microorganism that has been reduced or inhibited in an engineered
microorganism. An activity can be reduced to undetectable levels in
some embodiments, or detectable levels in certain embodiments. An
activity can be decreased to any suitable level for production of a
target fatty dicarboxylic acid product (e.g., sebacic or
dodecanedioic acid), including but not limited to less than 2-fold
(e.g., about 10% decrease to about 99% decrease; about 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% decrease), 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of 10-fold decrease, or
greater than about 10-fold decrease.
[0244] An altered activity sometimes is an activity not detectable
in a host organism and is added to an engineered organism. An
altered activity also may be an activity detectable in a host
organism and is increased in an engineered organism. An activity
may be added or increased by increasing the number of copies of a
polynucleotide that encodes a polypeptide having a target activity,
in some embodiments. In some embodiments, the activity of a native
polypeptide can be increased by increasing in the modified organism
the number of copies of a polynucleotide that encodes the
polypeptide (e.g., introducing 1 to about 100 additional copies of
the polynucleotide (e.g., introducing 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 or
more additional copies of the polynucleotide). In certain
embodiments an activity can be added or increased by inserting into
a host microorganism a polynucleotide that encodes a heterologous
polypeptide having the added activity or encodes a modified
endogenous polypeptide. In such embodiments, 1 to about 100 copies
of the polynucleotide can be introduced (e.g., introducing 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,
24, 26, 28, 30 copies). A "modified endogenous polypeptide" often
has an activity different than an activity of a native polypeptide
counterpart (e.g., different catalytic activity and/or different
substrate specificity), and often is active (e.g., an activity
(e.g., substrate turnover) is detectable). In certain embodiments,
an activity can be added or increased by inserting into a host
microorganism a heterologous polynucleotide that is (i) operably
linked to another polynucleotide that encodes a polypeptide having
the added activity, and (ii) up regulates production of the
polynucleotide. Thus, an activity can be added or increased by
inserting or modifying a regulatory polynucleotide operably linked
to another polynucleotide that encodes a polypeptide having the
target activity. In certain embodiments, an activity can be added
or increased by subjecting a host microorganism to a selective
environment and screening for microorganisms that have a detectable
level of the target activity. Examples of a selective environment
include, without limitation, a medium containing a substrate that a
host organism can process and a medium lacking a substrate that a
host organism can process.
[0245] An altered activity sometimes is an activity detectable in a
host organism and is reduced, inhibited or removed (i.e., not
detectable) in an engineered organism. An activity may be reduced
or removed by decreasing the number of copies of a polynucleotide
that encodes a polypeptide having a target activity, in some
embodiments. In some embodiments, an activity can be reduced or
removed by (i) inserting a polynucleotide within a polynucleotide
that encodes a polypeptide having the target activity (disruptive
insertion), and/or (ii) removing a portion of or all of a
polynucleotide that encodes a polypeptide having the target
activity (deletion or knock out, respectively). In certain
embodiments, an activity can be reduced or removed by inserting
into a host microorganism a heterologous polynucleotide that is (i)
operably linked to another polynucleotide that encodes a
polypeptide having the target activity, and (ii) down regulates
production of the polynucleotide. Thus, an activity can be reduced
or removed by inserting or modifying a regulatory polynucleotide
operably linked to another polynucleotide that encodes a
polypeptide having the target activity.
[0246] An activity also can be reduced or removed by (i) inhibiting
a polynucleotide that encodes a polypeptide having the activity or
(ii) inhibiting a polynucleotide operably linked to another
polynucleotide that encodes a polypeptide having the activity. A
polynucleotide can be inhibited by a suitable technique known in
the art, such as by contacting an RNA encoded by the polynucleotide
with a specific inhibitory RNA (e.g., RNAi, siRNA, ribozyme). An
activity also can be reduced or removed by contacting a polypeptide
having the activity with a molecule that specifically inhibits the
activity (e.g., enzyme inhibitor, antibody). In certain
embodiments, an activity can be reduced or removed by subjecting a
host microorganism to a selective environment and screening for
microorganisms that have a reduced level or removal of the target
activity.
[0247] In some embodiments, an untranslated ribonucleic acid, or a
cDNA can be used to reduce the expression of a particular activity
or enzyme. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent that reduces
the expression of an activity by producing an RNA molecule that is
partially or substantially homologous to a nucleic acid sequence of
interest which encodes the activity of interest. The RNA molecule
can bind to the nucleic acid sequence of interest and inhibit the
nucleic acid sequence from performing its natural function, in
certain embodiments. In some embodiments, the RNA may alter the
nucleic acid sequence of interest which encodes the activity of
interest in a manner that the nucleic acid sequence of interest is
no longer capable of performing its natural function (e.g., the
action of a ribozyme for example).
[0248] In certain embodiments, nucleotide sequences sometimes are
added to, modified or removed from one or more of the nucleic acid
reagent elements, such as the promoter, 5'UTR, target sequence, or
3'UTR elements, to enhance, potentially enhance, reduce, or
potentially reduce transcription and/or translation before or after
such elements are incorporated in a nucleic acid reagent. In some
embodiments, one or more of the following sequences may be modified
or removed if they are present in a 5'UTR: a sequence that forms a
stable secondary structure (e.g., quadruplex structure or stem loop
stem structure (e.g., EMBL sequences X12949, AF274954, AF139980,
AF152961, S95936, U194144, AF116649 or substantially identical
sequences that form such stem loop stem structures)); a translation
initiation codon upstream of the target nucleotide sequence start
codon; a stop codon upstream of the target nucleotide sequence
translation initiation codon; an ORF upstream of the target
nucleotide sequence translation initiation codon; an iron
responsive element (IRE) or like sequence; and a 5' terminal
oligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidines
adjacent to the cap). A translational enhancer sequence and/or an
internal ribosome entry site (IRES) sometimes is inserted into a
5'UTR (e.g., EMBL nucleotide sequences J04513, X87949, M95825,
M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427,
D14838 and M17446 and substantially identical nucleotide
sequences).
[0249] An AU-rich element (ARE, e.g., AUUUA repeats) and/or
splicing junction that follows a non-sense codon sometimes is
removed from or modified in a 3'UTR. A polyadenosine tail sometimes
is inserted into a 3'UTR if none is present, sometimes is removed
if it is present, and adenosine moieties sometimes are added to or
removed from a polyadenosine tail present in a 3'UTR. Thus, some
embodiments are directed to a process comprising: determining
whether any nucleotide sequences that increase, potentially
increase, reduce or potentially reduce translation efficiency are
present in the elements, and adding, removing or modifying one or
more of such sequences if they are identified. Certain embodiments
are directed to a process comprising: determining whether any
nucleotide sequences that increase or potentially increase
translation efficiency are not present in the elements, and
incorporating such sequences into the nucleic acid reagent.
[0250] In some embodiments, an activity can be altered by modifying
the nucleotide sequence of an ORF. An ORF sometimes is mutated or
modified (for example, by point mutation, deletion mutation,
insertion mutation, PCR based mutagenesis and the like) to alter,
enhance or increase, reduce, substantially reduce or eliminate the
activity of the encoded protein or peptide. The protein or peptide
encoded by a modified ORF sometimes is produced in a lower amount
or may not be produced at detectable levels, and in other
embodiments, the product or protein encoded by the modified ORF is
produced at a higher level (e.g., codons sometimes are modified so
they are compatible with tRNA's preferentially used in the host
organism or engineered organism). To determine the relative
activity, the activity from the product of the mutated ORF (or cell
containing it) can be compared to the activity of the product or
protein encoded by the unmodified ORF (or cell containing it).
[0251] In some embodiments, an ORF nucleotide sequence sometimes is
mutated or modified to alter the triplet nucleotide sequences used
to encode amino acids (e.g., amino acid codon triplets, for
example). Modification of the nucleotide sequence of an ORF to
alter codon triplets sometimes is used to change the codon found in
the original sequence to better match the preferred codon usage of
the organism in which the ORF or nucleic acid reagent will be
expressed. The codon usage, and therefore the codon triplets
encoded by a nucleic acid sequence, in bacteria may be different
from the preferred codon usage in eukaryotes, like yeast or plants
for example. Preferred codon usage also may be different between
bacterial species. In certain embodiments an ORF nucleotide
sequences sometimes is modified to eliminate codon pairs and/or
eliminate mRNA secondary structures that can cause pauses during
translation of the mRNA encoded by the ORF nucleotide sequence.
Translational pausing sometimes occurs when nucleic acid secondary
structures exist in an mRNA, and sometimes occurs due to the
presence of codon pairs that slow the rate of translation by
causing ribosomes to pause. In some embodiments, the use of lower
abundance codon triplets can reduce translational pausing due to a
decrease in the pause time needed to load a charged tRNA into the
ribosome translation machinery. Therefore, to increase
transcriptional and translational efficiency in bacteria (e.g.,
where transcription and translation are concurrent, for example) or
to increase translational efficiency in eukaryotes (e.g., where
transcription and translation are functionally separated), the
nucleotide sequence of a nucleotide sequence of interest can be
altered to better suit the transcription and/or translational
machinery of the host and/or genetically modified microorganism. In
certain embodiments, slowing the rate of translation by the use of
lower abundance codons, which slow or pause the ribosome, can lead
to higher yields of the desired product due to an increase in
correctly folded proteins and a reduction in the formation of
inclusion bodies.
[0252] Codons can be altered and optimized according to the
preferred usage by a given organism by determining the codon
distribution of the nucleotide sequence donor organism and
comparing the distribution of codons to the distribution of codons
in the recipient or host organism. Techniques described herein
(e.g., site directed mutagenesis and the like) can then be used to
alter the codons accordingly. Comparisons of codon usage can be
done by hand, or using nucleic acid analysis software commercially
available to the artisan.
[0253] Modification of the nucleotide sequence of an ORF also can
be used to correct codon triplet sequences that have diverged in
different organisms. For example, certain yeast (e.g., C.
tropicalis and C. maltosa) use the amino acid triplet CUG (e.g.,
CTG in the DNA sequence) to encode serine. CUG typically encodes
leucine in most organisms. In order to maintain the correct amino
acid in the resultant polypeptide or protein, the CUG codon must be
altered to reflect the organism in which the nucleic acid reagent
will be expressed. Thus, if an ORF from a bacterial donor is to be
expressed in either Candida yeast strain mentioned above, the
heterologous nucleotide sequence must first be altered or modified
to the appropriate leucine codon. Therefore, in some embodiments,
the nucleotide sequence of an ORF sometimes is altered or modified
to correct for differences that have occurred in the evolution of
the amino acid codon triplets between different organisms. In some
embodiments, the nucleotide sequence can be left unchanged at a
particular amino acid codon, if the amino acid encoded is a
conservative or neutral change in amino acid when compared to the
originally encoded amino acid.
[0254] In some embodiments, an activity can be altered by modifying
translational regulation signals, like a stop codon for example. A
stop codon at the end of an ORF sometimes is modified to another
stop codon, such as an amber stop codon described above. In some
embodiments, a stop codon is introduced within an ORF, sometimes by
insertion or mutation of an existing codon. An ORF comprising a
modified terminal stop codon and/or internal stop codon often is
translated in a system comprising a suppressor tRNA that recognizes
the stop codon. An ORF comprising a stop codon sometimes is
translated in a system comprising a suppressor tRNA that
incorporates an unnatural amino acid during translation of the
target protein or target peptide. Methods for incorporating
unnatural amino acids into a target protein or peptide are known,
which include, for example, processes utilizing a heterologous
tRNA/synthetase pair, where the tRNA recognizes an amber stop codon
and is loaded with an unnatural amino acid (e.g., World Wide Web
URL iupac.org/news/prize/2003/wang.pdf).
[0255] Depending on the portion of a nucleic acid reagent (e.g.,
Promoter, 5' or 3' UTR, ORI, ORF, and the like) chosen for
alteration (e.g., by mutagenesis, introduction or deletion, for
example) the modifications described above can alter a given
activity by (i) increasing or decreasing feedback inhibition
mechanisms, (ii) increasing or decreasing promoter initiation,
(iii) increasing or decreasing translation initiation, (iv)
increasing or decreasing translational efficiency, (v) modifying
localization of peptides or products expressed from nucleic acid
reagents described herein, or (vi) increasing or decreasing the
copy number of a nucleotide sequence of interest, (vii) expression
of an anti-sense RNA, RNAi, siRNA, ribozyme and the like. In some
embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter a region involved in feedback inhibition (e.g.,
5' UTR, promoter and the like). A modification sometimes is made
that can add or enhance binding of a feedback regulator and
sometimes a modification is made that can reduce, inhibit or
eliminate binding of a feedback regulator.
[0256] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in
transcription initiation (e.g., promoters, 5' UTR, and the like). A
modification sometimes can be made that can enhance or increase
initiation from an endogenous or heterologous promoter element. A
modification sometimes can be made that removes or disrupts
sequences that increase or enhance transcription initiation,
resulting in a decrease or elimination of transcription from an
endogenous or heterologous promoter element.
[0257] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in translational
initiation or translational efficiency (e.g., 5' UTR, 3' UTR, codon
triplets of higher or lower abundance, translational terminator
sequences and the like, for example). A modification sometimes can
be made that can increase or decrease translational initiation,
modifying a ribosome binding site for example. A modification
sometimes can be made that can increase or decrease translational
efficiency. Removing or adding sequences that form hairpins and
changing codon triplets to a more or less preferred codon are
non-limiting examples of genetic modifications that can be made to
alter translation initiation and translation efficiency.
[0258] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in localization
of peptides, proteins or other desired products (e.g., a sebacic
acid or dodecanedioic acid, for example). A modification sometimes
can be made that can alter, add or remove sequences responsible for
targeting a polypeptide, protein or product to an intracellular
organelle, the periplasm, cellular membranes, or extracellularly.
Transport of a heterologous product to a different intracellular
space or extracellularly sometimes can reduce or eliminate the
formation of inclusion bodies (e.g., insoluble aggregates of the
desired product).
[0259] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in increasing or
decreasing the copy number of a nucleotide sequence of interest. A
modification sometimes can be made that increases or decreases the
number of copies of an ORF stably integrated into the genome of an
organism or on an epigenetic nucleic acid reagent. Non-limiting
examples of alterations that can increase the number of copies of a
sequence of interest include, adding copies of the sequence of
interest by duplication of regions in the genome (e.g., adding
additional copies by recombination or by causing gene amplification
of the host genome, for example), cloning additional copies of a
sequence onto a nucleic acid reagent, or altering an ORI to
increase the number of copies of an epigenetic nucleic acid
reagent. Non-limiting examples of alterations that can decrease the
number of copies of a sequence of interest include, removing copies
of the sequence of interest by deletion or disruption of regions in
the genome, removing additional copies of the sequence from
epigenetic nucleic acid reagents, or altering an ORI to decrease
the number of copies of an epigenetic nucleic acid reagent.
[0260] In certain embodiments, increasing or decreasing the
expression of a nucleotide sequence of interest can also be
accomplished by altering, adding or removing sequences involved in
the expression of an anti-sense RNA, RNAi, siRNA, ribozyme and the
like. The methods described above can be used to modify expression
of anti-sense RNA, RNAi, siRNA, ribozyme and the like.
[0261] The methods and nucleic acid reagents described herein can
be used to generate genetically modified microorganisms with
altered activities in cellular processes involved in a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) synthesis. In some
embodiments, an engineered microorganism described herein may
comprise an increased number of copies of an endogenous
polynucleotide encoding a polypeptide having omega oxo fatty acid
dehydrogenase activity. In certain embodiments, an engineered
microorganism described herein may comprise an increased number of
copies of an endogenous polynucleotide encoding a polypeptide
having omega hydroxyl fatty acid dehydrogenase activity. In some
embodiments, an engineered microorganism described herein may
comprise a heterologous polynucleotide encoding a polypeptide
having omega oxo fatty acid dehydrogenase activity. In some
embodiments, an engineered microorganism described herein may
comprise a heterologous polynucleotide encoding a polypeptide
having omega hydroxyl fatty acid dehydrogenase activity. In some
embodiments, the heterologous polynucleotide can be from a
bacterium. In some embodiments, the bacterium can be an
Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium.
[0262] In some embodiments, an engineered microorganism described
herein may comprise a heterologous polynucleotide encoding a
polypeptide having monooxygenase activity. In certain embodiments,
the heterologous polynucleotide can be from a bacterium. In some
embodiments, the bacterium can be a Bacillus bacterium. In certain
embodiments, the Bacillus bacterium is B. megaterium.
[0263] In certain embodiments, an engineered microorganism
described herein may comprise a genetic modification that reduces
omega hydroxyl fatty acid conversion. In some embodiments, the
genetic modification can reduce omega hydroxyl fatty acid
dehydrogenase activity. In certain embodiments, an engineered
microorganism described herein may comprise a genetic modification
that reduces beta-oxidation activity. In some embodiments, the
genetic modification can reduce a target activity described
herein.
[0264] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
monooxygenase activity, in certain embodiments. In some
embodiments, the engineered microorganism described herein may
comprise a genetic modification that alters the monooxygenase
activity. In certain embodiments, the engineered microorganism
described herein can comprise an increase number of copies of an
endogenous polynucleotide encoding a polypeptide having
monooxygenase activity. In some embodiments, the engineered
microorganism described herein can comprise a heterologous
polynucleotide encoding a polypeptide having monooxygenase
activity. In certain embodiments, the heterologous polynucleotide
can be from a bacterium. In some embodiments, the bacterium can be
a Bacillus bacterium. In certain embodiments, the Bacillus
bacterium is B. megaterium. In some embodiments, the genetic
modification can reduce a polyketide synthase activity.
[0265] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
thioesterase activity, in certain embodiments. In some embodiments,
the engineered microorganism may comprise a genetic modification
that adds or increases the thioesterase activity. In certain
embodiments, the engineered microorganism may comprise a
heterologous polynucleotide encoding a polypeptide having
thioesterase activity.
[0266] In some embodiments, the engineered microorganism with an
altered thioesterase activity may comprise an altered omega oxo
fatty acid dehydrogenase activity. In certain embodiments, the
engineered microorganism with an altered thioesterase activity may
comprise a genetic modification that adds or increases omega oxo
fatty acid dehydrogenase activity. In some embodiments, the
engineered microorganism may comprise a heterologous polynucleotide
encoding a polypeptide having altered omega oxo fatty acid
dehydrogenase activity. In certain embodiments, the heterologous
polynucleotide can be from a bacterium. In some embodiments, the
bacterium can be an Acinetobacter, Nocardia, Pseudomonas or
Xanthobacter bacterium.
[0267] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
omega hydroxyl fatty acid dehydrogenase activity. In certain
embodiments, the engineered microorganism may comprise a genetic
modification that adds or increases the omega hydroxyl fatty acid
dehydrogenase activity. In certain embodiments, the engineered
microorganism may comprise a heterologous polynucleotide encoding a
polypeptide having altered omega hydroxyl fatty acid dehydrogenase
activity. In some embodiments, the heterologous polynucleotide is
from a bacterium. In certain embodiments, the bacterium can be an
Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium. In
some embodiments, the engineered microorganism can be a eukaryote.
In certain embodiments, the eukaryote can be a yeast. In some
embodiments, the eukaryote may be a fungus. In certain embodiments,
the yeast can be a Candida yeast. In some embodiments, the Candida
yeast may be C. troplicalis. In certain embodiments, the fungus can
be a Yarrowia fungus. In some embodiments the Yarrowia fungus may
be Y. lipolytica. In certain embodiments, the fungus can be an
Aspergillus fungus. In some embodiments, the Aspergillus fungus may
be A. parasiticus or A. nidulans. In some embodiments, an
engineered microorganism as described above may comprise a genetic
modification that reduces omega hydroxyl fatty acid conversion. In
certain embodiments, the genetic modification can reduce omega
hydroxyl fatty acid dehydrogenase activity. In some embodiments the
genetic may reduce beta-oxidation activity. In certain embodiments,
the genetic modification may reduce a target activity described
herein.
[0268] Engineered microorganisms can be prepared by altering,
introducing or removing nucleotide sequences in the host genome or
in stably maintained epigenetic nucleic acid reagents, as noted
above. The nucleic acid reagents use to alter, introduce or remove
nucleotide sequences in the host genome or epigenetic nucleic acids
can be prepared using the methods described herein or available to
the artisan.
[0269] Nucleic acid sequences having a desired activity can be
isolated from cells of a suitable organism using lysis and nucleic
acid purification procedures described in a known reference manual
(e.g., Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.) or using commercially available cell lysis and
DNA purification reagents and kits. In some embodiments, nucleic
acids used to engineer microorganisms can be provided for
conducting methods described herein after processing of the
organism containing the nucleic acid. For example, the nucleic acid
of interest may be extracted, isolated, purified or amplified from
a sample (e.g., from an organism of interest or culture containing
a plurality of organisms of interest, like yeast or bacteria for
example). The term "isolated" as used herein refers to nucleic acid
removed from its original environment (e.g., the natural
environment if it is naturally occurring, or a host cell if
expressed exogenously), and thus is altered "by the hand of man"
from its original environment. An isolated nucleic acid generally
is provided with fewer non-nucleic acid components (e.g., protein,
lipid) than the amount of components present in a source sample. A
composition comprising isolated sample nucleic acid can be
substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid
components). The term "purified" as used herein refers to sample
nucleic acid provided that contains fewer nucleic acid species than
in the sample source from which the sample nucleic acid is derived.
A composition comprising sample nucleic acid may be substantially
purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or greater than 99% free of other nucleic acid species). The
term "amplified" as used herein refers to subjecting nucleic acid
of a cell, organism or sample to a process that linearly or
exponentially generates amplicon nucleic acids having the same or
substantially the same nucleotide sequence as the nucleotide
sequence of the nucleic acid in the sample, or portion thereof. As
noted above, the nucleic acids used to prepare nucleic acid
reagents as described herein can be subjected to fragmentation or
cleavage.
[0270] Amplification of nucleic acids is sometimes necessary when
dealing with organisms that are difficult to culture. Where
amplification may be desired, any suitable amplification technique
can be utilized. Non-limiting examples of methods for amplification
of polynucleotides include, polymerase chain reaction (PCR);
ligation amplification (or ligase chain reaction (LCR));
amplification methods based on the use of O-beta replicase or
template-dependent polymerase (see US Patent Publication Number
US20050287592); helicase-dependant isothermal amplification
(Vincent et al., "Helicase-dependent isothermal DNA amplification".
EMBO reports 5 (8): 795-800 (2004)); strand displacement
amplification (SDA); thermophilic SDA nucleic acid sequence based
amplification (3SR or NASBA) and transcription-associated
amplification (TAA). Non-limiting examples of PCR amplification
methods include standard PCR, AFLP-PCR, Allele-specific PCR,
Alu-PCR, Asymmetric PCR, Colony PCR, Hot start PCR, Inverse PCR
(IPCR), In situ PCR (ISH), Intersequence-specific PCR (ISSR-PCR),
Long PCR, Multiplex PCR, Nested PCR, Quantitative PCR, Reverse
Transcriptase PCR (RT-PCR), Real Time PCR, Single cell PCR, Solid
phase PCR, combinations thereof, and the like. Reagents and
hardware for conducting PCR are commercially available.
[0271] Protocols for conducting the various type of PCR listed
above are readily available to the artisan. PCR conditions can be
dependent upon primer sequences, target abundance, and the desired
amount of amplification, and therefore, one of skill in the art may
choose from a number of PCR protocols available (see, e.g., U.S.
Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to
Methods and Applications, Innis et al., eds, 1990. PCR often is
carried out as an automated process with a thermostable enzyme. In
this process, the temperature of the reaction mixture is cycled
through a denaturing region, a primer-annealing region, and an
extension reaction region automatically. Machines specifically
adapted for this purpose are commercially available. A non-limiting
example of a PCR protocol that may be suitable for embodiments
described herein is, treating the sample at 95.degree. C. for 5
minutes; repeating forty-five cycles of 95.degree. C. for 1 minute,
59.degree. C. for 1 minute, 10 seconds, and 72.degree. C. for 1
minute 30 seconds; and then treating the sample at 72.degree. C.
for 5 minutes. Additional PCR protocols are described in the
example section. Multiple cycles frequently are performed using a
commercially available thermal cycler. Suitable isothermal
amplification processes known and selected by the person of
ordinary skill in the art also may be applied, in certain
embodiments. In some embodiments, nucleic acids encoding
polypeptides with a desired activity can be isolated by amplifying
the desired sequence from an organism having the desired activity
using oligonucleotides or primers designed based on sequences
described herein.
[0272] Amplified, isolated and/or purified nucleic acids can be
cloned into the recombinant DNA vectors described in Figures herein
or into suitable commercially available recombinant DNA vectors.
Cloning of nucleic acid sequences of interest into recombinant DNA
vectors can facilitate further manipulations of the nucleic acids
for preparation of nucleic acid reagents, (e.g., alteration of
nucleotide sequences by mutagenesis, homologous recombination,
amplification and the like, for example). Standard cloning
procedures (e.g., enzymic digestion, ligation, and the like) are
known (e.g., described in Maniatis, T., E. F. Fritsch and J.
Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0273] In some embodiments, nucleic acid sequences prepared by
isolation or amplification can be used, without any further
modification, to add an activity to a microorganism and thereby
create a genetically modified or engineered microorganism. In
certain embodiments, nucleic acid sequences prepared by isolation
or amplification can be genetically modified to alter (e.g.,
increase or decrease, for example) a desired activity. In some
embodiments, nucleic acids, used to add an activity to an organism,
sometimes are genetically modified to optimize the heterologous
polynucleotide sequence encoding the desired activity (e.g.,
polypeptide or protein, for example). The term "optimize" as used
herein can refer to alteration to increase or enhance expression by
preferred codon usage. The term optimize can also refer to
modifications to the amino acid sequence to increase the activity
of a polypeptide or protein, such that the activity exhibits a
higher catalytic activity as compared to the "natural" version of
the polypeptide or protein.
[0274] Nucleic acid sequences of interest can be genetically
modified using methods known in the art. Mutagenesis techniques are
particularly useful for small scale (e.g., 1, 2, 5, 10 or more
nucleotides) or large scale (e.g., 50, 100, 150, 200, 500, or more
nucleotides) genetic modification. Mutagenesis allows the artisan
to alter the genetic information of an organism in a stable manner,
either naturally (e.g., isolation using selection and screening) or
experimentally by the use of chemicals, radiation or inaccurate DNA
replication (e.g., PCR mutagenesis). In some embodiments, genetic
modification can be performed by whole scale synthetic synthesis of
nucleic acids, using a native nucleotide sequence as the reference
sequence, and modifying nucleotides that can result in the desired
alteration of activity. Mutagenesis methods sometimes are specific
or targeted to specific regions or nucleotides (e.g., site-directed
mutagenesis, PCR-based site-directed mutagenesis, and in vitro
mutagenesis techniques such as transplacement and in vivo
oligonucleotide site-directed mutagenesis, for example).
Mutagenesis methods sometimes are non-specific or random with
respect to the placement of genetic modifications (e.g., chemical
mutagenesis, insertion element (e.g., insertion or transposon
elements) and inaccurate PCR based methods, for example).
[0275] Site directed mutagenesis is a procedure in which a specific
nucleotide or specific nucleotides in a DNA molecule are mutated or
altered. Site directed mutagenesis typically is performed using a
nucleic acid sequence of interest cloned into a circular plasmid
vector. Site-directed mutagenesis requires that the wild type
sequence be known and used a platform for the genetic alteration.
Site-directed mutagenesis sometimes is referred to as
oligonucleotide-directed mutagenesis because the technique can be
performed using oligonucleotides which have the desired genetic
modification incorporated into the complement a nucleotide sequence
of interest. The wild type sequence and the altered nucleotide are
allowed to hybridize and the hybridized nucleic acids are extended
and replicated using a DNA polymerase. The double stranded nucleic
acids are introduced into a host (e.g., E. coli, for example) and
further rounds of replication are carried out in vivo. The
transformed cells carrying the mutated nucleic acid sequence are
then selected and/or screened for those cells carrying the
correctly mutagenized sequence. Cassette mutagenesis and PCR-based
site-directed mutagenesis are further modifications of the
site-directed mutagenesis technique. Site-directed mutagenesis can
also be performed in vivo (e.g., transplacement "pop-in pop-out",
In vivo site-directed mutagenesis with synthetic oligonucleotides
and the like, for example).
[0276] PCR-based mutagenesis can be performed using PCR with
oligonucleotide primers that contain the desired mutation or
mutations. The technique functions in a manner similar to standard
site-directed mutagenesis, with the exception that a thermocycler
and PCR conditions are used to replace replication and selection of
the clones in a microorganism host. As PCR-based mutagenesis also
uses a circular plasmid vector, the amplified fragment (e.g.,
linear nucleic acid molecule) containing the incorporated genetic
modifications can be separated from the plasmid containing the
template sequence after a sufficient number of rounds of
thermocycler amplification, using standard electrophorectic
procedures. A modification of this method uses linear amplification
methods and a pair of mutagenic primers that amplify the entire
plasmid. The procedure takes advantage of the E. coli Dam methylase
system which causes DNA replicated in vivo to be sensitive to the
restriction endonucleases DpnI. PCR synthesized DNA is not
methylated and is therefore resistant to DpnI. This approach allows
the template plasmid to be digested, leaving the genetically
modified, PCR synthesized plasmids to be isolated and transformed
into a host bacteria for DNA repair and replication, thereby
facilitating subsequent cloning and identification steps. A certain
amount of randomness can be added to PCR-based sited directed
mutagenesis by using partially degenerate primers.
[0277] Recombination sometimes can be used as a tool for
mutagenesis. Homologous recombination allows the artisan to
specifically target regions of known sequence for insertion of
heterologous nucleotide sequences using the host organisms natural
DNA replication and repair enzymes. Homologous recombination
methods sometimes are referred to as "pop in pop out" mutagenesis,
transplacement, knock out mutagenesis or knock in mutagenesis.
Integration of a nucleic acid sequence into a host genome is a
single cross over event, which inserts the entire nucleic acid
reagent (e.g., pop in). A second cross over event excises all but a
portion of the nucleic acid reagent, leaving behind a heterologous
sequence, often referred to as a "footprint" (e.g., pop out).
Mutagenesis by insertion (e.g., knock in) or by double
recombination leaving behind a disrupting heterologous nucleic acid
(e.g., knock out) both server to disrupt or "knock out" the
function of the gene or nucleic acid sequence in which insertion
occurs. By combining selectable markers and/or auxotrophic markers
with nucleic acid reagents designed to provide the appropriate
nucleic acid target sequences, the artisan can target a selectable
nucleic acid reagent to a specific region, and then select for
recombination events that "pop out" a portion of the inserted
(e.g., "pop in") nucleic acid reagent.
[0278] Such methods take advantage of nucleic acid reagents that
have been specifically designed with known target nucleic acid
sequences at or near a nucleic acid or genomic region of interest.
Popping out typically leaves a "foot print" of left over sequences
that remain after the recombination event. The left over sequence
can disrupt a gene and thereby reduce or eliminate expression of
that gene. In some embodiments, the method can be used to insert
sequences, upstream or downstream of genes that can result in an
enhancement or reduction in expression of the gene. In certain
embodiments, new genes can be introduced into the genome of a host
organism using similar recombination or "pop in" methods. An
example of a yeast recombination system using the ura3 gene and
5-FOA were described briefly above and further detail is presented
herein.
[0279] A method for modification is described in Alani et al., "A
method for gene disruption that allows repeated use of URA3
selection in the construction of multiply disrupted yeast strains",
Genetics 116(4):541-545 August 1987. The original method uses a
Ura3 cassette with 1000 base pairs (bp) of the same nucleotide
sequence cloned in the same orientation on either side of the URA3
cassette. Targeting sequences of about 50 bp are added to each side
of the construct. The double stranded targeting sequences are
complementary to sequences in the genome of the host organism. The
targeting sequences allow site-specific recombination in a region
of interest. The modification of the original technique replaces
the two 1000 bp sequence direct repeats with two 200 bp direct
repeats. The modified method also uses 50 bp targeting sequences.
The modification reduces or eliminates recombination of a second
knock out into the 1000 bp repeat left behind in a first
mutagenesis, therefore allowing multiply knocked out yeast.
Additionally, the 200 bp sequences used herein are uniquely
designed, self-assembling sequences that leave behind identifiable
footprints. The technique used to design the sequences incorporate
design features such as low identity to the yeast genome, and low
identity to each other. Therefore a library of the self-assembling
sequences can be generated to allow multiple knockouts in the same
organism, while reducing or eliminating the potential for
integration into a previous knockout.
[0280] As noted above, the URA3 cassette makes use of the toxicity
of 5-FOA in yeast carrying a functional URA3 gene. Uracil synthesis
deficient yeast are transformed with the modified URA3 cassette,
using standard yeast transformation protocols, and the transformed
cells are plated on minimal media minus uracil. In some
embodiments, PCR can be used to verify correct insertion into the
region of interest in the host genome, and certain embodiments the
PCR step can be omitted. Inclusion of the PCR step can reduce the
number of transformants that need to be counter selected to "pop
out" the URA3 cassette. The transformants (e.g., all or the ones
determined to be correct by PCR, for example) can then be
counter-selected on media containing 5-FOA, which will select for
recombination out (e.g., popping out) of the URA3 cassette, thus
rendering the yeast ura3 deficient again, and resistant to 5-FOA
toxicity. Targeting sequences used to direct recombination events
to specific regions are presented herein. A modification of the
method described above can be used to integrate genes in to the
chromosome, where after recombination a functional gene is left in
the chromosome next to the 200 bp footprint.
[0281] In some embodiments, other auxotrophic or dominant selection
markers can be used in place of URA3 (e.g., an auxotrophic
selectable marker), with the appropriate change in selection media
and selection agents. Auxotrophic selectable markers are used in
strains deficient for synthesis of a required biological molecule
(e.g., amino acid or nucleoside, for example). Non-limiting
examples of additional auxotrophic markers include; HIS3, TRP1,
LEU2, LEU2-d, and LYS2. Certain auxotrophic markers (e.g., URA3 and
LYS2) allow counter selection to select for the second
recombination event that pops out all but one of the direct repeats
of the recombination construct. HIS3 encodes an activity involved
in histidine synthesis. TRP1 encodes an activity involved in
tryptophan synthesis. LEU2 encodes an activity involved in leucine
synthesis. LEU2-d is a low expression version of LEU2 that selects
for increased copy number (e.g., gene or plasmid copy number, for
example) to allow survival on minimal media without leucine. LYS2
encodes an activity involved in lysine synthesis, and allows
counter selection for recombination out of the LYS2 gene using
alpha-amino adipate (-amino adipate).
[0282] Dominant selectable markers are useful because they also
allow industrial and/or prototrophic strains to be used for genetic
manipulations. Additionally, dominant selectable markers provide
the advantage that rich medium can be used for plating and culture
growth, and thus growth rates are markedly increased. Non-limiting
examples of dominant selectable markers include; Tn903 kan.sup.r,
Cm.sup.r, Hyg.sup.r, CUP1, and DHFR. Tn903 kan.sup.r encodes an
activity involved in kanamycin antibiotic resistance (e.g.,
typically neomycin phosphotransferase II or NPTII, for example).
Cm.sup.r encodes an activity involved in chloramphenicol antibiotic
resistance (e.g., typically chloramphenicol acetyl transferase or
CAT, for example). Hyg.sup.r encodes an activity involved in
hygromycin resistance by phosphorylation of hygromycin B (e.g.,
hygromycin phosphotransferase, or HPT). CUP1 encodes an activity
involved in resistance to heavy metal (e.g., copper, for example)
toxicity. DHFR encodes a dihydrofolate reductase activity which
confers resistance to methotrexate and sulfanilamde compounds.
[0283] In contrast to site-directed or specific mutagenesis, random
mutagenesis does not require any sequence information and can be
accomplished by a number of widely different methods. Random
mutagenesis often is used to generate mutant libraries that can be
used to screen for the desired genotype or phenotype. Non-limiting
examples of random mutagenesis include; chemical mutagenesis,
UV-induced mutagenesis, insertion element or transposon-mediated
mutagenesis, DNA shuffling, error-prone PCR mutagenesis, and the
like.
[0284] Chemical mutagenesis often involves chemicals like ethyl
methanesulfonate (EMS), nitrous acid, mitomycin C,
N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1, 2, 7,
8-diepoxyoctane (DEO), methyl methane sulfonate (MMS),
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline
1-oxide (4-NQO),
2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridine-
dihydrochloride (ICR-170), 2-amino purine (2AP), and hydroxylamine
(HA), provided herein as non-limiting examples. These chemicals can
cause base-pair substitutions, frameshift mutations, deletions,
transversion mutations, transition mutations, incorrect
replication, and the like. In some embodiments, the mutagenesis can
be carried out in vivo. Sometimes the mutagenic process involves
the use of the host organisms DNA replication and repair mechanisms
to incorporate and replicate the mutagenized base or bases.
[0285] Another type of chemical mutagenesis involves the use of
base-analogs. The use of base-analogs cause incorrect base pairing
which in the following round of replication is corrected to a
mismatched nucleotide when compared to the starting sequence. Base
analog mutagenesis introduces a small amount of non-randomness to
random mutagenesis, because specific base analogs can be chose
which can be incorporated at certain nucleotides in the starting
sequence. Correction of the mispairing typically yields a known
substitution. For example, Bromo-deoxyuridine (BrdU) can be
incorporated into DNA and replaces T in the sequence. The host DNA
repair and replication machinery can sometime correct the defect,
but sometimes will mispair the BrdU with a G. The next round of
replication then causes a G-C transversion from the original A-T in
the native sequence.
[0286] Ultra violet (UV) induced mutagenesis is caused by the
formation of thymidine dimers when UV light irradiates chemical
bonds between two adjacent thymine residues. Excision repair
mechanism of the host organism correct the lesion in the DNA, but
occasionally the lesion is incorrectly repaired typically resulting
in a C to T transition.
[0287] Insertion element or transposon-mediated mutagenesis makes
use of naturally occurring or modified naturally occurring mobile
genetic elements. Transposons often encode accessory activities in
addition to the activities necessary for transposition (e.g.,
movement using a transposase activity, for example). In many
examples, transposon accessory activities are antibiotic resistance
markers (e.g., see Tn903 kan.sup.r described above, for example).
Insertion elements typically only encode the activities necessary
for movement of the nucleic acid sequence. Insertion element and
transposon mediated mutagenesis often can occur randomly, however
specific target sequences are known for some transposons. Mobile
genetic elements like IS elements or Transposons (Tn) often have
inverted repeats, direct repeats or both inverted and direct
repeats flanking the region coding for the transposition genes.
Recombination events catalyzed by the transposase cause the element
to remove itself from the genome and move to a new location,
leaving behind a portion of an inverted or direct repeat. Classic
examples of transposons are the "mobile genetic elements"
discovered in maize. Transposon mutagenesis kits are commercially
available which are designed to leave behind a 5 codon insert
(e.g., Mutation Generation System kit, Finnzymes, World Wide Web
URL finnzymes.us, for example). This allows the artisan to identify
the insertion site, without fully disrupting the function of most
genes.
[0288] DNA shuffling is a method which uses DNA fragments from
members of a mutant library and reshuffles the fragments randomly
to generate new mutant sequence combinations. The fragments are
typically generated using DNaseI, followed by random annealing and
re-joining using self priming PCR. The DNA overhanging ends, from
annealing of random fragments, provide "primer" sequences for the
PCR process. Shuffling can be applied to libraries generated by any
of the above mutagenesis methods.
[0289] Error prone PCR and its derivative rolling circle error
prone PCR uses increased magnesium and manganese concentrations in
conjunction with limiting amounts of one or two nucleotides to
reduce the fidelity of the Taq polymerase. The error rate can be as
high as 2% under appropriate conditions, when the resultant mutant
sequence is compared to the wild type starting sequence. After
amplification, the library of mutant coding sequences must be
cloned into a suitable plasmid.
[0290] Although point mutations are the most common types of
mutation in error prone PCR, deletions and frameshift mutations are
also possible. There are a number of commercial error-prone PCR
kits available, including those from Stratagene and Clontech (e.g.,
World Wide Web URL strategene.com and World Wide Web URL
clontech.com, respectively, for example). Rolling circle
error-prone PCR is a variant of error-prone PCR in which wild-type
sequence is first cloned into a plasmid, then the whole plasmid is
amplified under error-prone conditions.
[0291] As noted above, organisms with altered activities can also
be isolated using genetic selection and screening of organisms
challenged on selective media or by identifying naturally occurring
variants from unique environments. For example, 2-Deoxy-D-glucose
is a toxic glucose analog. Growth of yeast on this substance yields
mutants that are glucose-deregulated. A number of mutants have been
isolated using 2-Deoxy-D-glucose including transport mutants, and
mutants that ferment glucose and galactose simultaneously instead
of glucose first then galactose when glucose is depleted. Similar
techniques have been used to isolate mutant microorganisms that can
metabolize plastics (e.g., from landfills), petrochemicals (e.g.,
from oil spills), and the like, either in a laboratory setting or
from unique environments.
[0292] Similar methods can be used to isolate naturally occurring
mutations in a desired activity when the activity exists at a
relatively low or nearly undetectable level in the organism of
choice, in some embodiments. The method generally consists of
growing the organism to a specific density in liquid culture,
concentrating the cells, and plating the cells on various
concentrations of the substance to which an increase in metabolic
activity is desired. The cells are incubated at a moderate growth
temperature, for 5 to 10 days. To enhance the selection process,
the plates can be stored for another 5 to 10 days at a low
temperature. The low temperature sometimes can allow strains that
have gained or increased an activity to continue growing while
other strains are inhibited for growth at the low temperature.
Following the initial selection and secondary growth at low
temperature, the plates can be replica plated on higher or lower
concentrations of the selection substance to further select for the
desired activity.
[0293] A native, heterologous or mutagenized polynucleotide can be
introduced into a nucleic acid reagent for introduction into a host
organism, thereby generating an engineered microorganism. Standard
recombinant DNA techniques (restriction enzyme digests, ligation,
and the like) can be used by the artisan to combine the mutagenized
nucleic acid of interest into a suitable nucleic acid reagent
capable of (i) being stably maintained by selection in the host
organism, or (ii) being integrating into the genome of the host
organism. As noted above, sometimes nucleic acid reagents comprise
two replication origins to allow the same nucleic acid reagent to
be manipulated in bacterial before final introduction of the final
product into the host organism (e.g., yeast or fungus for example).
Standard molecular biology and recombinant DNA methods are known
(e.g., described in Maniatis, T., E. F. Fritsch and J. Sambrook
(1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0294] Nucleic acid reagents can be introduced into microorganisms
using various techniques. Non-limiting examples of methods used to
introduce heterologous nucleic acids into various organisms
include; transformation, transfection, transduction,
electroporation, ultrasound-mediated transformation, particle
bombardment and the like. In some instances the addition of carrier
molecules (e.g., bis-benzimdazolyl compounds, for example, see U.S.
Pat. No. 5,595,899) can increase the uptake of DNA in cells
typically though to be difficult to transform by conventional
methods. Conventional methods of transformation are known (e.g.,
described in Maniatis, T., E. F. Fritsch and J. Sambrook (1982)
Molecular Cloning: a Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
Modified Activities
[0295] Certain activities in a genetically modified organism can be
modified by techniques known in the art. An acyl-coA oxidase
activity or acyl-coA dehydrogenase activity, or acyl-coA oxidase
activity and acyl-coA dehydrogenase activity, can be modified in an
organism in certain embodiments. In some embodiments, a modified
endogenous acyl-coA oxidase polypeptide, modified endogenous
acyl-coA dehydrogenase polypeptide, modified heterologous acyl-coA
oxidase polypeptide, and/or modified heterologous acyl-coA
dehydrogenase polypeptide may be introduced into an organism. A
modified polypeptide can be expressed by a host organism that
includes a modified polynucleotide encoding the modified
polypeptide. Modified polypeptides often have an activity different
than the activity of an unmodified counterpart. A modified activity
sometimes is a different catalytic activity or a different
substrate specificity, or a different catalytic activity and a
different substrate specificity. A different activity sometimes is
an activity that is higher or lower than the activity of an
unmodified counterpart polypeptide. In some embodiments, the
catalytic activity of a modified polypeptide is higher or lower
than the catalytic activity of the unmodified counterpart for a
particular substrate. In certain embodiments, the substrate
specificity of a modified polypeptide is higher or lower than the
substrate specificity of the unmodified counterpart for a
particular substrate. A modified polypeptide often is active and an
activity of a modified polypeptide often can be detected (e.g.,
substrate turnover can be detected). A desired activity for a
particular polypeptide sometimes is referred to as a "target
activity."
[0296] In some embodiments a genetic modification in a genetically
modified organism alters a substrate specificity of an acyl-coA
oxidase polypeptide produced in the organism. Sometimes the
substrate specificity is reduced for a substrate having a
particular chain length. In some embodiments, a modified acyl-coA
oxidase substrate specificity is reduced for a C8, C10, C12, C14,
C16, C18, C20 substrate or combination thereof. In certain
embodiments, a modified acyl-coA oxidase substrate specificity is
reduced for a C10, C12, or C18 substrate.
[0297] In some embodiments a genetic modification in a genetically
modified organism alters a substrate specificity of an acyl-coA
dehydrogenase polypeptide produced in the organism. Sometimes a
co-factor specificity is modified, and in some embodiments the
modified polypeptide prefers to utilize oxygen as a co-factor.
[0298] One or more particular modifications can be selected to
generate a modified polypeptide having a target activity.
Modifications often are amino acid modifications (e.g., deletion,
insertion of one or more amino acids). Amino acid modifications
sometimes are amino acid substitutions. Amino acid substitutions
sometimes are conservative, non-limiting examples of which include
substitution of an amino acid containing an acidic moiety to
another amino acid containing an acidic moiety (e.g., D, E),
substitution of an amino acid containing a basic moiety to another
amino acid containing a basic moiety (e.g., H, K, R), substitution
of an amino acid containing an aliphatic chain moiety to another
amino acid containing an aliphatic chain moiety (V, L, I, A),
substitution of an amino acid containing a cyclic moiety to another
amino acid containing a cyclic moiety (e.g., W, F, Y), and
substitution of an amino acid containing a polar moiety to another
amino acid containing a polar moiety (e.g., S, T). Amino acid
substitutions sometimes are non-conservative, non-limiting examples
of which include substitution of an amino acid containing an acidic
moiety to an amino acid containing a basic moiety, substitution of
an amino acid containing a basic moiety to an amino acid containing
an acidic moiety, substitution of an amino acid containing
relatively small moiety (e.g., G, A) to another amino acid
containing a relatively large moiety (e.g., Y, W, F, I, L), and
substitution of an amino acid containing a relatively large moiety
to another amino acid containing an relatively small moiety.
[0299] Particular modifications can be selected using any suitable
method known in the art. In certain embodiments, a reference
structure is known for a related polypeptide with a known activity,
and modifications to a target polypeptide can be guided by
alignment of the target polypeptide structure to the reference
structure. A reference structure sometimes is a primary structure
(e.g., polynucleotide or polypeptide sequence) and the primary
structure of a target can be aligned to the reference structure
using an alignment method known in the art. Particular amino acids
in the target that align with (e.g., are identical to or homologous
to) or do not align with (e.g., are not identical to or not
homologous to) particular amino acids in the reference can be
selected for modification. Selections can be made by inspection of
an alignment or by software known in the art that identifies,
scores and/or ranks amino acids for modification based on an
alignment.
[0300] A reference structure sometimes is a secondary structure,
tertiary structure or quaternary structure, each of which are three
dimensional structures pertaining to a polypeptide. A primary
structure of a target polypeptide can be modeled to a secondary,
tertiary or quaternary reference structure using three-dimensional
modeling software known in the art. A secondary, tertiary or
quaternary structure of a target polypeptide can be compared to a
secondary, tertiary or quaternary reference structure using
three-dimensional comparative software known in the art. Particular
structures (e.g., a particular individual amino acid; a particular
group of contiguous or non-contiguous amino acids) in the target
that align with or map to, or do not align with or map to,
particular structures in the reference can be selected for
modification. Also, particular structures in the target that are in
proximity to a substrate or co-factor can be selected for
modification. Selections can be made by inspection of an alignment
or map or by software known in the art that identifies, scores
and/or ranks amino acids and/or structures for modification based
on an alignment and map.
[0301] After particular amino acids and/or structures are selected
for modification in a first polypeptide, amino acids and structures
in a second polypeptide that align with the selected amino acids
and structures in the first polypeptide may be identified. In a
non-limiting example, particular amino acid substitutions and
structural modifications (e.g., loop amino acid deletion/insertion)
for Candida spp. POX4 and POX5 polypeptides are disclosed herein. A
primary structure of another acyl-coA oxidase polypeptide can be
aligned with the amino acid sequence or modeled structure of a POX4
or POX5 polypeptide and some or all amino acids of the other
polypeptide that align with those selected for modification in the
POX4 or POX5 polypeptide also can be selected for modification.
Certain criteria for selecting acyl-coA dehydrogenase modifications
also are described herein.
[0302] One or more activities of a modified polypeptide can be
characterized using any suitable assay known in the art. A modified
polypeptide can be expressed in an organism other than a target
organism in which a target product will be produced, for assaying
activity. For example, a modified polypeptide can be expressed in a
bacterium (e.g., E. coli), assayed and then introduced into a yeast
(e.g., Candida spp. yeast) for production of a target diacid.
[0303] Feedstocks, Media, Supplements & Additives
[0304] Engineered microorganisms often are cultured under
conditions that optimize yield of a fatty dicarboxylic acid (e.g.,
an eight to eighteen-carbon fatty dicarboxylic acid). Non-limiting
examples of fatty dicarboxylic acids include suberic acid (i.e.,
octanedioic acid, 1,8-octanedioic acid, octanedioic acid,
octane-1,8-dioic acid, 1,6-hexanedicarboxylic acid, capryllic
diacids), sebacic acid (i.e., 1,10-decanedioic acid, decanedioic
acid, decane-1,10-dioic acid, 1,8-octanedicarboxylic acid, capric
diacid), dodecanedioic acid (i.e., DDDA, 1,12-dodecanedioic acid,
dodecanedioic acid, dodecane-1,12-dioic acid,
1,10-decanedicarboxylic acid, decamethylenedicarboxylic acid,
1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e.,
TDDA, 1,14-tetradecanedioic acid, tetradecanedioic acid,
tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid,
myristic diacid), thapsic acid (i.e., hexadecanedioic acid,
1,16-hexadecanedioic acid, hexadecanedioic acid,
hexadecane-1,16-dioic acid, 1,14-tetradecanedicarboxylic acid,
palmitic diacid), cis-9-hexadecenedioic acid (i.e., palmitoleic
diacids), octanedioic acid (i.e., 1,18-octadecanedioic acid,
octadecanedioic acid, octadecane-1,18-dioic acid,
1,16-hexadecanedicarboxylic acid, stearic diacid),
cis-9-octadecenedioic acid (i.e., oleic diacids),
cis-9,12-octadecenedioic acid (i.e., linoleic diacids),
cis-9,12,15-octadecenedioic acid (i.e., linolenic diacids),
arachidic diacid (i.e., eicosanoic diacid, icosanoic diacid),
11-eicosenoic diacid (i.e., cis-11-eicosenedioic acid),
13-eicosenoic diacids (i.e., cis-13-eicosenedioic acid),
arachidonic diacid (i.e., cis-5,8,11,14-eicosatetraenedioic acid).
Culture conditions often optimize activity of one or more of the
following activities: omega oxo fatty acid dehydrogenase activity,
omega hydroxyl fatty acid dehydrogenase activity, acetyl CoA
carboxylase activity, monooxygenase activity, monooxygenase
reductase activity, fatty alcohol oxidase, acyl-CoA ligase,
acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA
dehydrogenase, and/or acyltransferase (e.g., acetyl-CoA
C-acyltransferase) activities. In general, non-limiting examples of
conditions that may be optimized include the type and amount of
carbon source, the type and amount of nitrogen source, the
carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH,
length of the biomass production phase, length of target product
accumulation phase, and time of cell harvest.
[0305] Culture media generally contain a suitable carbon source.
Carbon sources useful for culturing microorganisms and/or
fermentation processes sometimes are referred to as feedstocks. The
term "feedstock" as used herein refers to a composition containing
a carbon source that is provided to an organism, which is used by
the organism to produce energy and metabolic products useful for
growth. A feedstock may be a natural substance, a "man-made
substance," a purified or isolated substance, a mixture of purified
substances, a mixture of unpurified substances or combinations
thereof. A feedstock often is prepared by and/or provided to an
organism by a person, and a feedstock often is formulated prior to
administration to the organism. A carbon source may comprise, but
is not limited to including, one or more of the following
substances: alkanes, alkenes, mono-carboxylic acids, di-carboxylic
acids, monosaccharides (e.g., also referred to as "saccharides,"
which include 6-carbon sugars (e.g., glucose, fructose), 5-carbon
sugars (e.g., xylose and other pentoses) and the like),
disaccharides (e.g., lactose, sucrose), oligosaccharides (e.g.,
glycans, homopolymers of a monosaccharide), polysaccharides (e.g.,
starch, cellulose, heteropolymers of monosaccharides or mixtures
thereof), sugar alcohols (e.g., glycerol), and renewable feedstocks
(e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses,
barley malt).
[0306] Carbon sources also can be selected from one or more of the
following non-limiting examples: paraffin (e.g., saturated
paraffin, unsaturated paraffin, substituted paraffin, linear
paraffin, branched paraffin, or combinations thereof); alkanes
(e.g., dodecane), alkenes or alkynes, each of which may be linear,
branched, saturated, unsaturated, substituted or combinations
thereof (described in greater detail below); linear or branched
alcohols (e.g., dodecanol); fatty acids (e.g., about 1 carbon to
about 60 carbons, including free fatty acids, soap stock, for
example); esters of fatty acids; monoglycerides; diglycerides;
triglycerides, phospholipids. Non-limiting commercial sources of
products for preparing feedstocks include plants, plant oils or
plant products (e.g., vegetable oils (e.g., almond oil, canola oil,
cocoa butter, coconut oil, corn oil, cottonseed oil, flaxseed oil,
grape seed oil, illipe, olive oil, palm oil, palm olein, palm
kernel oil, safflower oil, peanut oil, soybean oil, sesame oil,
shea nut oil, sunflower oil walnut oil, the like and combinations
thereof) and animal fats (e.g., beef tallow, butterfat, lard, cod
liver oil). A carbon source may include a petroleum product and/or
a petroleum distillate (e.g., diesel, fuel oils, gasoline,
kerosene, paraffin wax, paraffin oil, petrochemicals). In some
embodiments, a feedstock comprises petroleum distillate. A carbon
source can be a fatty acid distillate (e.g., a palm oil distillate
or corn oil distillate). Fatty acid distillates can be by-products
from the refining of crude plant oils. In some embodiments, a
feedstock comprises a fatty acid distillate.
[0307] In some embodiments, a feedstock comprises a soapstock (i.e.
soap stock). A widely practiced method for purifying crude
vegetable oils for edible use is the alkali or caustic refining
method. This process employs a dilute aqueous solution of caustic
soda to react with the free fatty acids present which results in
the formation of soaps. The soaps together with hydrated
phosphatides, gums and prooxidant metals are typically separated
from the refined oil as the heavy phase discharge from the refining
centrifuge and are typically known as soapstock.
[0308] A carbon source also may include a metabolic product that
can be used directly as a metabolic substrate in an engineered
pathway described herein, or indirectly via conversion to a
different molecule using engineered or native biosynthetic pathways
in an engineered microorganism. In certain embodiments, metabolic
pathways can be preferentially biased towards production of a
desired product by increasing the levels of one or more activities
in one or more metabolic pathways having and/or generating at least
one common metabolic and/or synthetic substrate. In some
embodiments, a metabolic byproduct (e.g., fatty acid) of an
engineered activity (e.g., omega oxidation activity) can be used in
one or more metabolic pathways selected from gluconeogenesis,
pentose phosphate pathway, glycolysis, fatty acid synthesis, beta
oxidation, and omega oxidation, to generate a carbon source that
can be converted to a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0309] The term "paraffin" as used herein refers to the common name
for alkane hydrocarbons, independent of the source (e.g., plant
derived, petroleum derived, chemically synthesized, fermented by a
microorganism), or carbon chain length. A carbon source sometimes
comprises a paraffin, and in some embodiments, a paraffin is
predominant in a carbon source (e.g., about 75%, 80%, 85%, 90% or
95% paraffin). A paraffin sometimes is saturated (e.g., fully
saturated), sometimes includes one or more unsaturations (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 unsaturations) and sometimes is
substituted with one or more non-hydrogen substituents.
Non-limiting examples of non-hydrogen substituents include halo,
acetyl, .dbd.O, .dbd.N--CN, .dbd.N--OR, .dbd.NR, OR, NR.sub.2, SR,
SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R, NRCONR.sub.2, NRCOOR,
NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and NO.sub.2, where each R
is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl,
C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8
alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl,
and each R is optionally substituted with halo, .dbd.O, .dbd.N--CN,
.dbd.N--OR', .dbd.NR', OR', NR'.sub.2, SR', SO.sub.2R',
SO.sub.2NR'.sub.2, NR'SO.sub.2R', NR'CONR'.sub.2, NR'COOR',
NR'COR', CN, COOR', CONR'.sub.2, OOCR', COR', and NO.sub.2, where
each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8
acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl.
[0310] In some embodiments a feedstock is selected according to the
genotype and/or phenotype of the engineered microorganism to be
cultured. For example, a feedstock rich in 12-carbon fatty acids,
12-carbon dicarboxylic acids or 12-carbon paraffins, or a mixture
of 10, 12 and 14-carbon compounds can be useful for culturing yeast
strains harboring an alteration that partially blocks beta
oxidation by disrupting POX4 activity, as described herein.
Non-limiting examples of carbon sources having 10 to 14 carbons
include fats (e.g., coconut oil, palm kernel oil), paraffins (e.g.,
alkanes, alkenes, or alkynes) having 10 to 14 carbons, (e.g.,
dodecane (also referred to as adakane12, bihexyl, dihexyl and
duodecane); tetradecane), alkene and alkyne derivatives), fatty
acids (dodecanoic acid, tetradecanoic acid), fatty alcohols
(dodecanol, tetradecanol), the like, non-toxic substituted
derivatives or combinations thereof.
[0311] A carbon source sometimes comprises an alkyl, alkenyl or
alkynyl compound or molecule (e.g., a compound that includes an
alkyl, alkenyl or alkynyl moiety (e.g., alkane, alkene, alkyne)).
In certain embodiments, an alkyl, alkenyl or alkynyl molecule, or
combination thereof, is predominant in a carbon source (e.g., about
75%, 80%, 85%, 90% or 95% of such molecules). As used herein, the
terms "alkyl," "alkenyl" and "alkynyl" include straight-chain
(referred to herein as "linear"), branched-chain (referred to
herein as "non-linear"), cyclic monovalent hydrocarbyl radicals,
and combinations of these, which contain only C and H atoms when
they are unsubstituted. Non-limiting examples of alkyl moieties
include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl,
2-propenyl, 3-butynyl, and the like. An alkyl that contains only C
and H atoms and is unsubstituted sometimes is referred to as
"saturated." An alkenyl or alkynyl generally is "unsaturated" as it
contains one or more double bonds or triple bonds, respectively. An
alkenyl can include any number of double bonds, such as 1, 2, 3, 4
or 5 double bonds, for example. An alkynyl can include any number
of triple bonds, such as 1, 2, 3, 4 or 5 triple bonds, for example.
Alkyl, alkenyl and alkynyl molecules sometimes contain between
about 2 to about 60 carbon atoms (C). For example, an alkyl,
alkenyl and alkynyl molecule can include about 1 carbon atom, about
2 carbon atoms, about 3 carbon atoms, about 4 carbon atoms, about 5
carbon atoms, about 6 carbon atoms, about 7 carbon atoms, about 8
carbon atoms, about 9 carbon atoms, about 10 carbon atoms, about 12
carbon atoms, about 14 carbon atoms, about 16 carbon atoms, about
18 carbon atoms, about 20 carbon atoms, about 22 carbon atoms,
about 24 carbon atoms, about 26 carbon atoms, about 28 carbon
atoms, about 30 carbon atoms, about 32 carbon atoms, about 34
carbon atoms, about 36 carbon atoms, about 38 carbon atoms, about
40 carbon atoms, about 42 carbon atoms, about 44 carbon atoms,
about 46 carbon atoms, about 48 carbon atoms, about 50 carbon
atoms, about 52 carbon atoms, about 54 carbon atoms, about 56
carbon atoms, about 58 carbon atoms or about 60 carbon atoms. In
some embodiments, paraffins can have a mean number of carbon atoms
of between about 8 to about 18 carbon atoms (e.g., about 8 carbon
atoms, about 9 carbon atoms, about 10 carbon atoms, about 11 carbon
atoms, about 12 carbon atoms, about 13 carbon atoms, about 14
carbon atoms, about 15 carbon atoms, about 16 carbon atoms, about
17 carbon atoms and about 18 carbon atoms). A single group can
include more than one type of multiple bond, or more than one
multiple bond. Such groups are included within the definition of
the term "alkenyl" when they contain at least one carbon-carbon
double bond, and are included within the term "alkynyl" when they
contain at least one carbon-carbon triple bond. Alkyl, alkenyl and
alkynyl molecules include molecules that comprise an alkyl, alkenyl
and/or alkynyl moiety, and include molecules that consist of an
alkyl, alkenyl or alkynyl moiety (i.e., alkane, alkene and alkyne
molecules).
[0312] Alkyl, alkenyl and alkynyl substituents sometimes contain
1-20C (alkyl) or 2-20C (alkenyl or alkynyl). They can contain about
8-20C or about 10-20C in some embodiments. A single group can
include more than one type of multiple bond, or more than one
multiple bond. Such groups are included within the definition of
the term "alkenyl" when they contain at least one carbon-carbon
double bond, and are included within the term "alkynyl" when they
contain at least one carbon-carbon triple bond.
[0313] Alkyl, alkenyl and alkynyl groups or compounds sometimes are
substituted to the extent that such substitution can be synthesized
and can exist. Typical substituents include, but are not limited
to, halo, acetyl, .dbd.O, .dbd.N--CN, .dbd.N--OR, .dbd.NR, OR,
NR.sub.2, SR, SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R,
NRCONR.sub.2, NRCOOR, NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and
NO.sub.2, where each R is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8
heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C11 aryl, or
C5-C11 heteroaryl, and each R is optionally substituted with halo,
.dbd.O, .dbd.N--CN, .dbd.N--OR', .dbd.NR', OR', NR'.sub.2, SR',
SO.sub.2R', SO.sub.2NR'.sub.2, NR'SO.sub.2R', NR'CONR'.sub.2,
NR'COOR', NR'COR', CN, COOR', CONR'.sub.2, OOCR', COR', and
NO.sub.2, where each R' is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10
heteroaryl. Alkyl, alkenyl and alkynyl groups can also be
substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10
heteroaryl, each of which can be substituted by the substituents
that are appropriate for the particular group.
[0314] "Acetylene" or "acetyl" substituents are 2-10C alkynyl
groups that are optionally substituted, and are of the formula
--C.ident.C--Ri, where Ri is H or C1-C8 alkyl, C2-C8 heteroalkyl,
C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8
heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C10
heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each
Ri group is optionally substituted with one or more substituents
selected from halo, .dbd.O, .dbd.N--CN, .dbd.N--OR', .dbd.NR', OR',
NR'2, SR', SO.sub.2R', SO.sub.2NR'.sub.2, NR'SO.sub.2R',
NR'CONR'.sub.2, NR'COOR', NR'COR', CN, COOR', CONR'.sub.2, OOCR',
COR', and NO.sub.2, where each R' is independently H, C1-C6 alkyl,
C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl,
C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each
of which is optionally substituted with one or more groups selected
from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6
heteroacyl, hydroxy, amino, and .dbd.O; and where two R' can be
linked to form a 3-7 membered ring optionally containing up to
three heteroatoms selected from N, O and S. In some embodiments, Ri
of --C.ident.C--Ri is H or Me.
[0315] A carbon source sometimes comprises a heteroalkyl,
heteroalkenyl and/or heteroalkynyl molecule or compound (e.g.,
comprises heteroalkyl, heteroalkenyl and/or heteroalkynyl moiety
(e.g., heteroalkane, heteroalkene or heteroalkyne)). "Heteroalkyl",
"heteroalkenyl", and "heteroalkynyl" and the like are defined
similarly to the corresponding hydrocarbyl (alkyl, alkenyl and
alkynyl) groups, but the `hetero` terms refer to groups that
contain one to three O, S or N heteroatoms or combinations thereof
within the backbone; thus at least one carbon atom of a
corresponding alkyl, alkenyl, or alkynyl group is replaced by one
of the specified heteroatoms to form a heteroalkyl, heteroalkenyl,
or heteroalkynyl group. The typical and sizes for heteroforms of
alkyl, alkenyl and alkynyl groups are generally the same as for the
corresponding hydrocarbyl groups, and the substituents that may be
present on the heteroforms are the same as those described above
for the hydrocarbyl groups. For reasons of chemical stability, it
is also understood that, unless otherwise specified, such groups do
not include more than two contiguous heteroatoms except where an
oxo group is present on N or S as in a nitro or sulfonyl group.
[0316] The term "alkyl" as used herein includes cycloalkyl and
cycloalkylalkyl groups and compounds, the term "cycloalkyl" may be
used herein to describe a carbocyclic non-aromatic compound or
group that is connected via a ring carbon atom, and
"cycloalkylalkyl" may be used to describe a carbocyclic
non-aromatic compound or group that is connected to a molecule
through an alkyl linker. Similarly, "heterocyclyl" may be used to
describe a non-aromatic cyclic group that contains at least one
heteroatom as a ring member and that is connected to the molecule
via a ring atom, which may be C or N; and "heterocyclylalkyl" may
be used to describe such a group that is connected to another
molecule through a linker. The sizes and substituents that are
suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl groups are the same as those described above for
alkyl groups. As used herein, these terms also include rings that
contain a double bond or two, as long as the ring is not
aromatic.
[0317] A carbon source sometimes comprises an acyl compound or
moiety (e.g., compound comprising an acyl moiety). As used herein,
"acyl" encompasses groups comprising an alkyl, alkenyl, alkynyl,
aryl or arylalkyl radical attached at one of the two available
valence positions of a carbonyl carbon atom, and heteroacyl refers
to the corresponding groups where at least one carbon other than
the carbonyl carbon has been replaced by a heteroatom chosen from
N, O and S. Thus heteroacyl includes, for example, --C(.dbd.O)OR
and --C(.dbd.O)NR.sub.2 as well as --C(.dbd.O)-heteroaryl.
[0318] Acyl and heteroacyl groups are bonded to any group or
molecule to which they are attached through the open valence of the
carbonyl carbon atom. Typically, they are C1-C8 acyl groups, which
include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl
groups, which include methoxyacetyl, ethoxycarbonyl, and
4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms
of such groups that comprise an acyl or heteroacyl group can be
substituted with the substituents described herein as generally
suitable substituents for each of the corresponding component of
the acyl or heteroacyl group.
[0319] A carbon source sometimes comprises one or more aromatic
moieties and/or heteroaromatic moieties. "Aromatic" moiety or
"aryl" moiety refers to a monocyclic or fused bicyclic moiety
having the well-known characteristics of aromaticity; examples
include phenyl and naphthyl. Similarly, "heteroaromatic" and
"heteroaryl" refer to such monocyclic or fused bicyclic ring
systems which contain as ring members one or more heteroatoms
selected from O, S and N. The inclusion of a heteroatom permits
aromaticity in 5 membered rings as well as 6 membered rings.
Typical heteroaromatic systems include monocyclic C5-C6 aromatic
groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl,
pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl and the
fused bicyclic moieties formed by fusing one of these monocyclic
groups with a phenyl ring or with any of the heteroaromatic
monocyclic groups to form a C8-C10 bicyclic group such as indolyl,
benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl,
quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused
ring bicyclic system which has the characteristics of aromaticity
in terms of electron distribution throughout the ring system is
included in this definition. It also includes bicyclic groups where
at least the ring which is directly attached to the remainder of
the molecule has the characteristics of aromaticity. Typically, the
ring systems contain 5-12 ring member atoms. The monocyclic
heteroaryls sometimes contain 5-6 ring members, and the bicyclic
heteroaryls sometimes contain 8-10 ring members.
[0320] Aryl and heteroaryl moieties may be substituted with a
variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8
alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of
which can itself be further substituted; other substituents for
aryl and heteroaryl moieties include halo, OR, NR.sub.2, SR,
SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R, NRCONR.sub.2, NRCOOR,
NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and NO.sub.2, where each R
is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl,
C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10
aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12
heteroarylalkyl, and each R is optionally substituted as described
above for alkyl groups. The substituent groups on an aryl or
heteroaryl group may be further substituted with the groups
described herein as suitable for each type of such substituents or
for each component of the substituent. Thus, for example, an
arylalkyl substituent may be substituted on the aryl portion with
substituents typical for aryl groups, and it may be further
substituted on the alkyl portion with substituents as typical or
suitable for alkyl groups.
[0321] Similarly, "arylalkyl" and "heteroarylalkyl" refer to
aromatic and heteroaromatic ring systems, which are stand-alone
molecules (e.g., benzene or substituted benzene, pyridine or
substituted pyridine), or which are bonded to an attachment point
through a linking group such as an alkylene, including substituted
or unsubstituted, saturated or unsaturated, cyclic or acyclic
linkers. A linker often is C1-C8 alkyl or a hetero form thereof.
These linkers also may include a carbonyl group, thus making them
able to provide substituents as an acyl or heteroacyl moiety. An
aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group
may be substituted with the same substituents described above for
aryl groups. An arylalkyl group sometimes includes a phenyl ring
optionally substituted with the groups defined above for aryl
groups and a C1-C4 alkylene that is unsubstituted or is substituted
with one or two C1-C4 alkyl groups or heteroalkyl groups, where the
alkyl or heteroalkyl groups can optionally cyclize to form a ring
such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, a
heteroarylalkyl group often includes a C5-C6 monocyclic heteroaryl
group optionally substituted with one or more of the groups
described above as substituents typical on aryl groups and a C1-C4
alkylene that is unsubstituted. A heteroarylalkyl group sometimes
is substituted with one or two C1-C4 alkyl groups or heteroalkyl
groups, or includes an optionally substituted phenyl ring or C5-C6
monocyclic heteroaryl and a C1-C4 heteroalkylene that is
unsubstituted or is substituted with one or two C1-C4 alkyl or
heteroalkyl groups, where the alkyl or heteroalkyl groups can
optionally cyclize to form a ring such as cyclopropane, dioxolane,
or oxacyclopentane.
[0322] Where an arylalkyl or heteroarylalkyl group is described as
optionally substituted, the substituents may be on the alkyl or
heteroalkyl portion or on the aryl or heteroaryl portion of the
group. The substituents optionally present on the alkyl or
heteroalkyl portion sometimes are the same as those described above
for alkyl groups, and the substituents optionally present on the
aryl or heteroaryl portion often are the same as those described
above for aryl groups generally.
[0323] "Arylalkyl" groups as used herein are hydrocarbyl groups if
they are unsubstituted, and are described by the total number of
carbon atoms in the ring and alkylene or similar linker. Thus a
benzyl group is a C7-arylalkyl group, and phenylethyl is a
C8-arylalkyl.
[0324] "Heteroarylalkyl" as described above refers to a moiety
comprising an aryl group that is attached through a linking group,
and differs from "arylalkyl" in that at least one ring atom of the
aryl moiety or one atom in the linking group is a heteroatom
selected from N, O and S. The heteroarylalkyl groups are described
herein according to the total number of atoms in the ring and
linker combined, and they include aryl groups linked through a
heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl
linker such as an alkylene; and heteroaryl groups linked through a
heteroalkyl linker. Thus, for example, C7-heteroarylalkyl includes
pyridylmethyl, phenoxy, and N-pyrrolyl methoxy.
[0325] "Alkylene" as used herein refers to a divalent hydrocarbyl
group. Because an alkylene is divalent, it can link two other
groups together. An alkylene often is referred to as
--(CH.sub.2).sub.n-- where n can be 1-20, 1-10, 1-8, or 1-4, though
where specified, an alkylene can also be substituted by other
groups, and can be of other lengths, and the open valences need not
be at opposite ends of a chain. Thus --CH(Me)-- and --C(Me).sub.2--
may also be referred to as alkylenes, as can a cyclic group such as
cyclopropan-1,1-diyl. Where an alkylene group is substituted, the
substituents include those typically present on alkyl groups as
described herein.
[0326] In some embodiments, a feedstock includes a mixture of
carbon sources, where each carbon source in the feedstock is
selected based on the genotype of the engineered microorganism. In
certain embodiments, a mixed carbon source feedstock includes one
or more carbon sources selected from sugars, cellulose, alkanes,
fatty acids, triacylglycerides, paraffins, the like and
combinations thereof.
[0327] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). In addition to appropriate carbon and nitrogen sources,
culture media also can contain suitable minerals, salts, cofactors,
buffers, vitamins, metal ions (e.g., Mn.sup.+2, Co.sup.+2,
Zn.sup.+2, Mg.sup.+2) and other components suitable for culture of
microorganisms.
[0328] Engineered microorganisms sometimes are cultured in complex
media (e.g., yeast extract-peptone-dextrose broth (YPD)). In some
embodiments, engineered microorganisms are cultured in a defined
minimal media that lacks a component necessary for growth and
thereby forces selection of a desired expression cassette (e.g.,
Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
[0329] Culture media in some embodiments are common commercially
prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,
Detroit, Mich.). Other defined or synthetic growth media may also
be used and the appropriate medium for growth of the particular
microorganism are known. A variety of host organisms can be
selected for the production of engineered microorganisms.
Non-limiting examples include yeast (e.g., Candida tropicalis
(e.g., ATCC20336, ATCC20913, ATCC20962), Yarrowia lipolytica (e.g.,
ATCC20228)) and filamentous fungi (e.g., Aspergillus nidulans
(e.g., ATCC38164) and Aspergillus parasiticus (e.g., ATCC 24690)).
In specific embodiments, yeast are cultured in YPD media (10 g/L
Bacto Yeast Extract, 20 g/L Bacto Peptone, and 20 g/L Dextrose).
Filamentous fungi, in particular embodiments, are grown in CM
(Complete Medium) containing 10 g/L Dextrose, 2 g/L Bacto Peptone,
1 g/L Bacto Yeast Extract, 1 g/L Casamino acids, 50 mL/L
20.times.Nitrate Salts (120 g/L NaNO.sub.3, 10.4 g/L KCl, 10.4 g/L
MgSO.sub.4.7H.sub.2O)O), 1 mL/L 1000.times. Trace Elements (22 g/L
ZnSO.sub.4.7H.sub.2O, 11 g/L H.sub.3BO.sub.3, 5 g/L
MnCl.sub.2.7H.sub.2O, 5 g/L FeSO.sub.4.7H.sub.2O, 1.7 g/L
CoCl.sub.2.6H.sub.2O, 1.6 g/L CuSO.sub.4.5H.sub.2O, 1.5 g/L
Na.sub.2MoO.sub.4.2H.sub.2O, and 50 g/L Na.sub.4EDTA), and 1 mL/L
Vitamin Solution (100 mg each of Biotin, pyridoxine, thiamine,
riboflavin, p-aminobenzoic acid, and nicotinic acid in 100 mL
water).
[0330] Growth Conditions & Fermentation
[0331] A suitable pH range for the fermentation often is between
about pH 4.0 to about pH 8.0, where a pH in the range of about pH
5.5 to about pH 7.0 sometimes is utilized for initial culture
conditions. Depending on the host organism, culturing may be
conducted under aerobic or anaerobic conditions, where microaerobic
conditions sometimes are maintained. A two-stage process may be
utilized, where one stage promotes microorganism proliferation and
another state promotes production of target molecule. In a
two-stage process, the first stage may be conducted under aerobic
conditions (e.g., introduction of air and/or oxygen) and the second
stage may be conducted under anaerobic conditions (e.g., air or
oxygen are not introduced to the culture conditions). In some
embodiments, the first stage may be conducted under anaerobic
conditions and the second stage may be conducted under aerobic
conditions. In certain embodiments, a two-stage process may include
two more organisms, where one organism generates an intermediate
product in one stage and another organism processes the
intermediate product into a target fatty dicarboxylic acid product
(e.g., sebacic or dodecanedioic acid) in another stage, for
example.
[0332] A variety of fermentation processes may be applied for
commercial biological production of a target fatty dicarboxylic
acid product. In some embodiments, commercial production of a
target fatty dicarboxylic acid product from a recombinant microbial
host is conducted using a batch, fed-batch or continuous
fermentation process, for example.
[0333] A batch fermentation process often is a closed system where
the media composition is fixed at the beginning of the process and
not subject to further additions beyond those required for
maintenance of pH and oxygen level during the process. At the
beginning of the culturing process the media is inoculated with the
desired organism and growth or metabolic activity is permitted to
occur without adding additional sources (i.e., carbon and nitrogen
sources) to the medium. In batch processes the metabolite and
biomass compositions of the system change constantly up to the time
the culture is terminated. In a typical batch process, cells
proceed through a static lag phase to a high-growth log phase and
finally to a stationary phase, wherein the growth rate is
diminished or halted. Left untreated, cells in the stationary phase
will eventually die.
[0334] A variation of the standard batch process is the fed-batch
process, where the carbon source is continually added to the
fermentor over the course of the fermentation process. Fed-batch
processes are useful when catabolite repression is apt to inhibit
the metabolism of the cells or where it is desirable to have
limited amounts of carbon source in the media at any one time.
Measurement of the carbon source concentration in fed-batch systems
may be estimated on the basis of the changes of measurable factors
such as pH, dissolved oxygen and the partial pressure of waste
gases (e.g., CO.sub.2).
[0335] Batch and fed-batch culturing methods are known in the art.
Examples of such methods may be found in Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd ed.,
(1989) Sinauer Associates Sunderland, Mass. and Deshpande, Mukund
V., Appl. Biochem. Biotechnol., 36:227 (1992).
[0336] In continuous fermentation process a defined media often is
continuously added to a bioreactor while an equal amount of culture
volume is removed simultaneously for product recovery. Continuous
cultures generally maintain cells in the log phase of growth at a
constant cell density. Continuous or semi-continuous culture
methods permit the modulation of one factor or any number of
factors that affect cell growth or end product concentration. For
example, an approach may limit the carbon source and allow all
other parameters to moderate metabolism. In some systems, a number
of factors affecting growth may be altered continuously while the
cell concentration, measured by media turbidity, is kept constant.
Continuous systems often maintain steady state growth and thus the
cell growth rate often is balanced against cell loss due to media
being drawn off the culture. Methods of modulating nutrients and
growth factors for continuous culture processes, as well as
techniques for maximizing the rate of product formation, are known
and a variety of methods are detailed by Brock, supra.
[0337] In some embodiments involving fermentation, the fermentation
can be carried out using two or more microorganisms (e.g., host
microorganism, engineered microorganism, isolated naturally
occurring microorganism, the like and combinations thereof), where
a feedstock is partially or completely utilized by one or more
organisms in the fermentation (e.g., mixed fermentation), and the
products of cellular respiration or metabolism of one or more
organisms can be further metabolized by one or more other organisms
to produce a desired target product (e.g., sebacic acid,
dodecanedioic acid, hexanoic acid). In certain embodiments, each
organism can be fermented independently and the products of
cellular respiration or metabolism purified and contacted with
another organism to produce a desired target product. In some
embodiments, one or more organisms are partially or completely
blocked in a metabolic pathway (e.g., beta oxidation, omega
oxidation, the like or combinations thereof), thereby producing a
desired product that can be used as a feedstock for one or more
other organisms. Any suitable combination of microorganisms can be
utilized to carry out mixed fermentation or sequential
fermentation.
Taget Product Production, Isolation and Yield
[0338] In various embodiments a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is isolated or purified from the culture media
or extracted from the engineered microorganisms. In some
embodiments, fermentation of feedstocks by methods described herein
can produce a target fatty dicarboxylic acid product (e.g., sebacic
or dodecanedioic acid) at a level of about 10% to about 100% of
theoretical yield (e.g., about 15%, about 20%, about 25% or more of
theoretical yield (e.g., 25% or more, 26% or more, 27% or more, 28%
or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or
more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or
more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or
more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or
more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or
more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or
more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or
more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or
more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or
more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or
more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or
more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or
more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or
more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or
more, or 99% or more of theoretical yield). The term "theoretical
yield" as used herein refers to the amount of product that could be
made from a starting material if the reaction is 100% complete.
Theoretical yield is based on the stoichiometry of a reaction and
ideal conditions in which starting material is completely consumed,
undesired side reactions do not occur, the reverse reaction does
not occur, and there are no losses in the work-up procedure.
Culture media may be tested for target product (e.g., sebacic or
dodecanedioic acid) concentration and drawn off when the
concentration reaches a predetermined level. Detection methods are
known in the art, including but not limited to chromatographic
methods (e.g., gas chromatography) or combined chromatographic/mass
spectrometry (e.g., GC-MS) methods. Target product (e.g., sebacic
or dodecanedioic acid) may be present at a range of levels as
described herein.
[0339] A target fatty dicarboxylic acid product sometimes is
retained within an engineered microorganism after a culture process
is completed, and in certain embodiments, the target product is
secreted out of the microorganism into the culture medium. For the
latter embodiments, (i) culture media may be drawn from the culture
system and fresh medium may be supplemented, and/or (ii) target
product may be extracted from the culture media during or after the
culture process is completed. Engineered microorganisms may be
cultured on or in solid, semi-solid or liquid media. In some
embodiments media is drained from cells adhering to a plate. In
certain embodiments, a liquid-cell mixture is centrifuged at a
speed sufficient to pellet the cells but not disrupt the cells and
allow extraction of the media, as known in the art. The cells may
then be resuspended in fresh media. Target product may be purified
from culture media according to known methods know in the art.
[0340] Provided herein are non-limiting examples of methods useful
for recovering target product from fermentation broth and/or
isolating/partially purifying a target fatty dicarboxylic acid
product from non-target products when utilizing mixed chain length
feedstocks. Recovery of a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) from fermentation broth can be accomplished
using a variety of methods. Optionally, one can first employ a
centrifugation step to separate cell mass and a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) from the aqueous phase. A fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) has limited solubility in water under
fermentation conditions, and has a density similar to that of
water. Upon centrifugation, the majority of fatty dicarboxylic acid
(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) will be pulled away from the water stream, and
be concentrated in the cell mass stream. The concentrated fatty
dicarboxylic acid stream will then be further concentrated via
filtration steps (e.g., solid dodecanedioic acid will be retained
on a filter, allowing water to pass through, concentrating the
product). Once the fatty dicarboxylic acid (e.g., octanedioic acid,
decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) is
concentrated to the desired level, the temperature will be
increased to above its melting point of 130 C. After the fatty
dicarboxylic acid is melted, the remaining impurities are removed
via filtration; the final product is recovered by decreasing the
temperature, allowing the fatty dicarboxylic acid to solidify, and
collecting the solid product.
[0341] Alternatively, a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) can
be recovered from fermentation broth by first extracting the broth
with an organic solvent in which a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is soluble (e.g., ethanol). The organic solvent
phase can then be filtered through various membranes to further
purify the fatty dicarboxylic acid. Subsequent extractions with the
same or a different organic solvent can then be performed and each
round of extraction can be followed by membrane filtration to
further concentrate the fatty dicarboxylic acid. The organic
solvent can be evaporated, leaving the fatty dicarboxylic acid
behind as a residue and the residue can be dried to provide the
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) in solid form.
[0342] In certain embodiments, target product is extracted from the
cultured engineered microorganisms.
[0343] The microorganism cells may be concentrated through
centrifugation at a speed sufficient to shear the cell membranes.
In some embodiments, the cells may be physically disrupted (e.g.,
shear force, sonication) or chemically disrupted (e.g., contacted
with detergent or other lysing agent). The phases may be separated
by centrifugation or other method known in the art and target
product may be isolated according to known methods.
[0344] Commercial grade target product sometimes is provided in
substantially pure form (e.g., 90% pure or greater, 95% pure or
greater, 99% pure or greater or 99.5% pure or greater). In some
embodiments, target product may be modified into any one of a
number of downstream products. For example, a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) may be polycondensed with hexamethylenediamine
to produce nylon. Nylon may be further processed into fibers for
applications in carpeting, automobile tire cord and clothing. A
fatty dicarboxylic acid
[0345] (e.g., octanedioic acid, decanedioic acid, dodecanedioic
acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic
acid, eicosanedioic acid) is also used for manufacturing
plasticizers, lubricant components and polyester polyols for
polyurethane systems. Various esters of food grade fatty
dicarboxylic acids (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) are used as components in
fragrance manufacture, gelling aids, flavorings, acidulant,
leavening and buffering agent. A fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) has two carboxylic acid (--COOH) groups, which
can yield two kinds of salts. Its derivatives, acyl halides,
anhydrides, esters, amides and nitriles, are used in making a
variety of downstream products through further reactions of
substitution, catalytic reduction, metal hydride reduction,
diborane reduction, keto formation with organometallic reagents,
electrophile bonding at oxygen, and condensation.
[0346] Target product may be provided within cultured microbes
containing target product, and cultured microbes may be supplied
fresh or frozen in a liquid media or dried. Fresh or frozen
microbes may be contained in appropriate moisture-proof containers
that may also be temperature controlled as necessary. Target
product sometimes is provided in culture medium that is
substantially cell-free. In some embodiments target product or
modified target product purified from microbes is provided, and
target product sometimes is provided in substantially pure form. In
certain embodiments crystallized or powdered target product is
provided. Dodecanedioic acid (1,12 dodecanedioic acid; DDDA) is a
white powder or crystal with a melting point of between 260.degree.
F. and 266.degree. F. Sebacic acid (1,8 ocatanedicarboxylic acid)
is also a white powder or crystal with a melting point of between
268.degree. F. and 274.degree. F. A crystallized or powdered fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) may be transported in a
variety of containers including one ton cartons, drums, 50 pound
bags and the like.
[0347] In certain embodiments, a fatty dicarboxylic acid target
product (e.g., dodecanedioic acid or sebacic acid) is produced with
a yield of about 0.50 grams of target product per gram of feedstock
added, or greater; 0.51 grams of target product per gram of
feedstock added, or greater; 0.52 grams of target product per gram
of feedstock added, or greater; 0.53 grams of target product per
gram of feedstock added, or greater; 0.54 grams of target product
per gram of feedstock added, or greater; 0.55 grams of target
product per gram of feedstock added, or greater; 0.56 grams of
target product per gram of feedstock added, or greater; 0.57 grams
of target product per gram of feedstock added, or greater; 0.58
grams of target product per gram of feedstock added, or greater;
0.59 grams of target product per gram of feedstock added, or
greater; 0.60 grams of target product per gram of feedstock added,
or greater; 0.61 grams of target product per gram of feedstock
added, or greater; 0.62 grams of target product per gram of
feedstock added, or greater; 0.63 grams of target product per gram
of feedstock added, or greater; 0.64 grams of target product per
gram of feedstock added, or greater; 0.65 grams of target product
per gram of feedstock added, or greater; 0.66 grams of target
product per gram of feedstock added, or greater; 0.67 grams of
target product per gram of feedstock added, or greater; 0.68 grams
of target product per gram of feedstock added, or greater; 0.69
grams of target product per gram of feedstock added, or greater;
0.70 grams of target product per gram of feedstock added or
greater; 0.71 grams of target product per gram of feedstock added,
or greater; 0.72 grams of target product per gram of feedstock
added, or greater; 0.73 grams of target product per gram of
feedstock added, or greater; 0.74 grams of target product per gram
of feedstock added, or greater; 0.75 grams of target product per
gram of feedstock added, or greater; 0.76 grams of target product
per gram of feedstock added, or greater; 0.77 grams of target
product per gram of feedstock added, or greater; 0.78 grams of
target product per gram of feedstock added, or greater; 0.79 grams
of target product per gram of feedstock added, or greater; 0.80
grams of target product per gram of feedstock added, or greater;
0.81 grams of target product per gram of feedstock added, or
greater; 0.82 grams of target product per gram of feedstock added,
or greater; 0.83 grams of target product per gram of feedstock
added, or greater; 0.84 grams of target product per gram of
feedstock added, or greater; 0.85 grams of target product per gram
of feedstock added, or greater; 0.86 grams of target product per
gram of feedstock added, or greater; 0.87 grams of target product
per gram of feedstock added, or greater; 0.88 grams of target
product per gram of feedstock added, or greater; 0.89 grams of
target product per gram of feedstock added, or greater; 0.90 grams
of target product per gram of feedstock added, or greater; 0.91
grams of target product per gram of feedstock added, or greater;
0.92 grams of target product per gram of feedstock added, or
greater; 0.93 grams of target product per gram of feedstock added,
or greater; 0.94 grams of target product per gram of feedstock
added, or greater; 0.95 grams of target product per gram of
feedstock added, or greater; 0.96 grams of target product per gram
of feedstock added, or greater; 0.97 grams of target product per
gram of feedstock added, or greater; 0.98 grams of target product
per gram of feedstock added, or greater; 0.99 grams of target
product per gram of feedstock added, or greater; 1.0 grams of
target product per gram of feedstock added, or greater; 1.1 grams
of target product per gram of feedstock added, or greater; 1.2
grams of target product per gram of feedstock added, or greater;
1.3 grams of target product per gram of feedstock added, or
greater; 1.4 grams of target product per gram of feedstock added,
or greater; or about 1.5 grams of target product per gram of
feedstock added, or greater.
[0348] In certain embodiments, a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is produced with a yield of greater than about
0.15 grams per gram of the feedstock (e.g., dodecane, mixed chain
length alkanes, lauric acid, mixed chain length fatty acids, oil,
the like or combinations of the foregoing). In some embodiments, a
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) is produced at between
about 10% and about 100% of maximum theoretical yield of any
introduced feedstock ((e.g., about 15%, about 20%, about 25% or
more of theoretical yield (e.g., 25% or more, 26% or more, 27% or
more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or
more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or
more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or
more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or
more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or
more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or
more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or
more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or
more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or
more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or
more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or
more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or
more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or
more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or
more, 98% or more, or 99% or more of theoretical maximum yield). In
certain embodiments, a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) is
produced in a concentration range of between about 50 g/L to about
1000 g/L of culture media (e.g., about 50 g/L, about 55 g/L, about
60 g/L, about 65 g/L, about 70 g/L, about 75 g/L, about 80 g/L,
about 85 g/L, about 90 g/L, about 95 g/L, about 100 g/L, about 110
g/L, about 120 g/L, about 130 g/L, about 140 g/L, about 150 g/L,
about 160 g/L, about 170 g/L, about 180 g/L, about 190 g/L, about
200 g/L, about 225 g/L, about 250 g/L, about 275 g/L, about 300
g/L, about 325 g/L, about 350 g/L, about 375 g/L, about 400 g/L,
about 425 g/L, about 450 g/L, about 475 g/L, about 500 g/L, about
550 g/L, about 600 g/L, about 650 g/L, about 700 g/L, about 750
g/L, about 800 g/L, about 850 g/L, about 900 g/L, about 950 g/L, or
about 1000 g/L).
[0349] In some embodiments, a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is produced at a rate of between about 0.5
g/L/hour to about 5 g/L/hour (e.g., about 0.5 g/L/hour, about 0.6
g/L/hour, about 0.7 g/L/hour, about 0.8 g/L/hour, about 0.9
g/L/hour, about 1.0 g/L/hour, about 1.1 g/L/hour, about 1.2
g/L/hour, about 1.3 g/L/hour, about 1.4 g/L/hour, about 1.5
g/L/hour, about 1.6 g/L/hour, about 1.7 g/L/hour, about 1.8
g/L/hour, about 1.9 g/L/hour, about 2.0 g/L/hour, about 2.25
g/L/hour, about 2.5 g/L/hour, about 2.75 g/L/hour, about 3.0
g/L/hour, about 3.25 g/L/hour, about 3.5 g/L/hour, about 3.75
g/L/hour, about 4.0 g/L/hour, about 4.25 g/L/hour, about 4.5
g/L/hour, about 4.75 g/L/hour, or about 5.0 g/L/hour.) In certain,
embodiments, the engineered organism comprises between about a
5-fold to about a 500-fold increase in a fatty dicarboxylic acid
(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) production when compared to wild-type or
partially engineered organisms of the same strain, under identical
fermentation conditions (e.g., about a 5-fold increase, about a
10-fold increase, about a 15-fold increase, about a 20-fold
increase, about a 25-fold increase, about a 30-fold increase, about
a 35-fold increase, about a 40-fold increase, about a 45-fold
increase, about a 50-fold increase, about a 55-fold increase, about
a 60-fold increase, about a 65-fold increase, about a 70-fold
increase, about a 75-fold increase, about a 80-fold increase, about
a 85-fold increase, about a 90-fold increase, about a 95-fold
increase, about a 100-fold increase, about a 125-fold increase,
about a 150-fold increase, about a 175-fold increase, about a
200-fold increase, about a 250-fold increase, about a 300-fold
increase, about a 350-fold increase, about a 400-fold increase,
about a 450-fold increase, or about a 500-fold increase).
[0350] In certain embodiments, the maximum theoretical yield
(Y.sub.max) of dodecanedioic acid in a fully beta-oxidation blocked
engineered microorganism is about 1.15 grams of dodecanedioic acid
produced per gram of lauric acid added. In some embodiments, the
maximum theoretical yield (Y.sub.max) of dodecanedioic acid in a
fully beta-oxidation blocked engineered microorganism is about 1.07
grams of dodecanedioic acid produced per gram of methyl laurate
added. In certain embodiments, the maximum theoretical yield
(Y.sub.max) of dodecanedioic acid in a partially beta-oxidation
blocked engineered microorganism is about 0.82 grams of
dodecanedioic acid produced per gram of oleic acid added. In some
embodiments, the maximum theoretical yield (Y.sub.max) of
dodecanedioic acid in a partially beta-oxidation blocked engineered
microorganism is about 0.95 grams of dodecanedioic acid produced
per gram of coconut oil added. The percentage of Y.sub.max for the
engineered microorganism under conditions in which dodecanedioic
acid is produced is calculated as (%
Y.sub.max)=Y.sub.p/s/Y.sub.max*100, where
(Y.sub.p/s)=[dodecanedioic acid (g/L)]*final volume of culture in
flask (L)]/[feedstock added to flask (g)]. In some embodiments, the
engineered microorganism produces dodecanedioic acid at about 10%
to about 100% of maximum theoretical yield.
[0351] In certain embodiments, the maximum theoretical yield
(Y.sub.max) of sebacic acid in a fully beta-oxidation blocked
engineered microorganism is about 1.42 grams of sebacic acid
produced per gram of decane added. In some embodiments, the maximum
theoretical yield (Y.sub.max) of sebacic acid in a fully
beta-oxidation blocked engineered microorganism is about 1.17 grams
of sebacic acid produced per gram of capric acid added. In certain
embodiments, the maximum theoretical yield (Y.sub.max) of sebacic
acid in a partially beta-oxidation blocked engineered microorganism
is about 0.83 grams of sebacic acid produced per gram of coconut
oil added. In some embodiments, the maximum theoretical yield
(Y.sub.max) of sebacic acid in a partially beta-oxidation blocked
engineered microorganism is about 0.72 grams of sebacic acid
produced per gram of oleic acid added. The percentage of Y.sub.max
for the engineered microorganism under conditions in which sebacic
acid is produced is calculated as MY Y/Y (%
Y.sub.max)=Y.sub.p/s/Y.sub.max*100, where (Y.sub.p/s)=[sebacic acid
(g/L)]*final volume of culture in flask (L)]/[feedstock added to
flask (g)]. In some embodiments, the engineered microorganism
produces sebacic acid at about 10% to about 100% of maximum
theoretical yield.
EXAMPLES
[0352] The examples set forth below illustrate certain embodiments
and do not limit the technology. Certain examples set forth below
utilize standard recombinant DNA and other biotechnology protocols
known in the art. Many such techniques are described in detail in
Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. DNA mutagenesis can be accomplished using the
Stratagene (San Diego, Calif.) "QuickChange" kit according to the
manufacturer's instructions.
[0353] Non-limiting examples of recombinant DNA techniques and
genetic manipulation of microorganisms are described herein. In
some embodiments, strains of engineered organisms described herein
are mated to combine genetic backgrounds to further enhance carbon
flux management through native and/or engineered pathways described
herein, for the production of a desired target product (e.g.,
sebacic or dodecanedioic acid).
Example 1
Conversion of Decane to Sebacic Acid in Shake Flask
Fermentation
[0354] 50 mL of SP92 medium (6.7 g/L yeast nitrogen base, 3.0 g/L
yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0 g/L
K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4, 75 g/L dextrose) was
inoculated with a single colony of a completely beta-oxidation
blocked strain of Candida tropicalis (ATCC20962) and the culture
was grown overnight at 30.degree. C., with shaking at about 300
rpm. Cells were pelleted by centrifugation for 10 minutes at
4.degree. C. and 1,050.times.g and the supernatant discarded. Cells
were resuspended in 20 mL TB-low nitrogen (low-N) media (1.7 g/L
yeast nitrogen base without ammonium sulfate, 3.0 g/L yeast
extract, 1.0 g/L K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4) and
transferred to a new sterile 250 mL glass baffled flask and
incubated at 30C, with shaking at about 250 rpm, utilizing the
following feeding schedule: dextrose fed to 0.1% at 0, 1, 2, 3, 4,
and 5 hours, dextrose fed to 5% at 30 hours, decane fed to 0.7% at
0, 5, 30, and 48 hours. Samples were removed for gas
chromatographic (GC) analysis at 0, 4, 30, and 72 hours. The GC
profile showed that the culture accumulated the C10 dicarboxylic
acid (sebacic acid) with very little accumulation of the C10
monocarboxylic acid (capric acid), as shown in FIG. 9. After 72
hours of incubation the concentration of sebacic acid was 0.94 g/L
and the capric acid concentration was 0.01 g/L. There was no
significant accumulation of any other monoacid or diacid.
Example 2
Conversion of Capric Acid to Sebacic Acid in Shake Flask
Fermentation
[0355] 5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) was inoculated with a single colony of
Candida tropicalis (ATCC20962) and the culture was grown overnight
at 30.degree. C., with shaking at about 250 rpm. This starter
culture was then used to inoculate 25 mL cultures in the same
medium to an initial OD.sub.600 nm of 0.4 and grown overnight at
30.degree. C., with shaking at about 300 rpm. Cells were pelleted
by centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g
and the supernatant discarded. Cells were resuspended in 12.5 mL
TB-lowN media+glycerol (1.7 g/L yeast nitrogen base without
ammonium sulfate, 3.0 g/L yeast extract, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) and transferred to a new sterile 250 mL
glass baffled flask. Cultures were fed 0.05% or 0.1% capric acid
and incubated at 30.degree. C., with shaking at about 300 rpm.
After 24 hours incubation cultures were fed glycerol to 75 g/L and
incubation continued before sampling for GC at 48 hours. GC
analysis showed that nearly all capric acid was converted to
sebacic acid under both starting concentrations of capric acid, as
shown in FIG. 10.
Example 3
Fermentation Procedure for Conversion of Decane to Sebacic Acid
[0356] Filter sterilized modified SP92-glycerol fermentation medium
(6.7 g/L yeast nitrogen base, 3.0 g/L yeast extract, 3.0 g/L
(NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 20 g/L glycerol) is
transferred to a sterile fermentation vessel. Growth of Candida
tropicalis (ATCC20962) is innoculated to an initial OD.sub.600 nm
of about 1.0 with a 5% inoculum and growth carried out under the
following conditions: 30.degree. C. with shaking at about 1000 rpm,
1 volume per volume per minute aeration (vvm), pH 5.8 and initial
volume of 0.3 L. Growth proceeds for approximately 8 hours and the
conversion phase is initiated by the addition of decane to 2 g/L.
Continuous feeds for decane (1 g/L-h) and glucose (1.5 g/L-h) are
initiated at the same time as the addition of the decane bolus.
Fermentation conditions are maintained at 30.degree. C., 1000 rpm,
1 vvm, and pH 5.8 for 44 hours.
[0357] Samples were collected for GC analysis at 44 hours after
initiating the conversion phase. The data, presented in FIG. 16,
show that the decane was converted exclusively to the C10
dicarboxylic acid, sebacic acid. Significant evaporative losses
from the decane feed bottles prevented an accurate determination of
product yield.
Example 4
Conversion of Mixed Fatty Acid Feedstock to Mixed Diacid Products
Containing Sebacic Acid in Shake Flask Fermentation
[0358] 5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) is inoculated with a single colony of
Candida tropicalis (ATCC20962) and grown as described in Example 2.
25 mL of the same media is inoculated using overnight cultures to
an initial OD.sub.600 nm of 0.4 and grown overnight at 30.degree.
C., with shaking at about 300 rpm. Cells are pelleted by
centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g and
the supernatant discarded. Cells are resuspended in 12.5 mL TB-lowN
media without carbon source (1.7 g/L yeast nitrogen base without
ammonium sulfate, 3.0 g/L yeast extract, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4) and transferred to a new sterile 250 mL glass baffled
flask. Cultures are fed 0.05% capric acid, 0.05% methyl laurate,
and 30 g/L glycerol and incubated at 30.degree. C., 300 rpm. After
24 hours of incubation cultures are sampled for GC analysis.
[0359] The results, presented in FIG. 17, show that the C12 and C10
fatty acids were converted to dicarboxylic acids of the same chain
length (e.g., C12 and C10 dicarboxylic acids), with no evidence of
chain shortening of the diacids (e.g., no significant levels of
monocarboxylic acids were detected).
Example 5
Conversion of Long Chain Fatty Acids to Mixed Diacids
[0360] SP92 fermentation medium was filter sterilized and
transferred to a sterile fermentation vessel. Growth of a partially
beta-oxidation blocked strain of Candida tropicalis (sAA106) was
initiated with a 10% inoculum (initial OD.sub.600 nm=3.0) and grown
under the following conditions: of 30.degree. C. with shaking at
about 1200 rpm, 1 vvm, pH 6.1 and initial volume of 0.3 L. Growth
continued until the glucose concentration dropped to less than 2
g/L at which time the conversion phase was initiated by increasing
the pH to 8.0 by the addition of 6N KOH and by the addition of
methyl myristate to 30 g/L. Immediately following the methyl
myristate bolus a continuous feed of glucose was initiated at a
rate of 1.5 g/L-h. Fermentation conditions were maintained at
30.degree. C., 1200 rpm, 1 vvm, and pH 8.0 for 90 hours with
boluses of 30 g/L methyl myristate at 24, 48, and 72 hours after
initiation of conversion. Samples for GC were collected at 24, 48,
72, and 90 hours. The diacid profile graphically illustrated in
FIG. 11 shows an accumulation of dicarboxylic acids ranging in
chain-length from 6 to 14 carbons long, including sebacic acid. The
methyl myristate substrate (methyl ester of myristic acid) is first
converted to the C14 dicarboxylic acid via the co-oxidation pathway
before being shortened by two carbon increments via the cyclic
.beta.-oxidation pathway. The glucose co-feed employed during the
fermentation represses the .beta.-oxidation pathway such that all
chain-lengths of diacid accumulate. Manipulation of diacid
chain-length distribution is being investigated by altering the
glucose co-feed rate in the fermentation medium, thereby allowing
growth under varying glucose concentrations.
Example 6
Fermentation Procedure for Conversion of Mixed Long-Chain Fatty
Acids to Mixed Diacids of Shorter Chain Length
[0361] SP92 fermentation medium without glycerol was filter
sterilized and transferred to a sterile fermentation vessel.
Autoclaved virgin coconut oil was added to the vessel to a final
concentration of 80 g/L. A partially beta-oxidation blocked Candida
tropicalis strain (sAA496) was inoculated to an initial OD.sub.600
nm of 1.0 with a 5% inoculum and grown under the following
conditions: 30.degree. C. with shaking at about 1200 rpm, 1 vvm,
initial pH 6.5 and initial volume of 1.0 L. The effect of pH on the
distribution of fatty acid chain lengths was determined by
manipulating the pH of the fermentation media. The pH of the
fermentation was either 1) increased to pH 7.5 and controlled at
that pH for the entire run, 2) allowed to drop naturally due to the
growth of the culture before controlling at pH 6.0 for the rest of
the run, or 3) allowed to drop naturally due to the growth of the
culture before controlling at pH 4.5 for the rest of the run.
Samples were collected for GC analysis after 140 hours of
fermentation time. The product diacid composition was shown to
shift to longer chain diacids with increasing pH, as shown in the
table below.
TABLE-US-00001 Diacid composition (fraction of total diacids) C12
Diacid Sebacic Acid Suberic Acid Adipic Acid pH 4.5 0.00 0.00 0.68
0.32 pH 6.0 0.03 0.10 0.75 0.12 pH 7.5 0.16 0.17 0.62 0.05
Example 7
Conversion of Capric Acid to Sebacic Acid in Shake Flask
Fermentations Using Fully Beta-Oxidation Blocked Strains Having
Additional Genetic Modifications in the Omega Oxidation Pathway
[0362] Various genetically modified strains of Candida tropicalis
were inoculated into 5 mL of SP92 medium (6.7 g/L yeast nitrogen
base, 3.0 g/L yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0
g/L K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4, 75 g/L glycerol).
The strains included a completely beta-oxidation blocked strain of
Candida tropicalis (sAA003), as well as derivatives of sAA003 with
amplified components of the omega-oxidation pathway (e.g., various
cytochrome P450s, cytochrome P450 reductase or combinations
thereof) and the cultures grown overnight at 30.degree. C., with
shaking at about 250 rpm.
[0363] These starter cultures were then used to inoculate 25 mL
cultures in the same medium and grown overnight at 30.degree. C.,
with shaking at about 250 rpm. Cells were pelleted by
centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g and
the supernatant discarded. Cells were resuspended in 12.5 mL
TB-lowN media+glycerol (1.7 g/L yeast nitrogen base without
ammonium sulfate, 3.0 g/L yeast extract, 1.0 g/L K.sub.2HPO.sub.4,
1.0 g/L KH.sub.2PO.sub.4, 75 g/L glycerol) and transferred to a new
sterile 250 mL glass baffled flask. Cultures were fed 0.05% from a
5% capric acid solution in ethanol and incubated at 30.degree. C.,
with shaking at about 300 rpm. After 24 hours incubation cultures
were fed glycerol to 30 g/L and an additional bolus of 0.05% capric
acid. Incubation continued before sampling for GC at 24, 48, and 72
hours. The results are shown in FIG. 12. GC analysis showed that a
greater proportion of capric acid was converted to sebacic acid
when particular elements of the omega-oxidation pathway are
amplified. The data are presented as % of theoretical maximum
yield. Strains which include genetic modifications to CYPA18 and
CYPA19 achieve approximately 80% of theoretical maximum yield in
conversion of capric acid to sebacic acid. The strain
designated+CPR+A18 has about 30 copies of CYPA18, whereas the
strain designated+CPR+A19 has about 7 copies of CYPA19.
Example 8
Conversion of Methyl-Laurate to Dodecanedioic Acid in Shake Flask
Fermentation
[0364] 5 mL of SP92 glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) was inoculated with a single colony of a
completely beta-oxidation blocked strain of Candida tropicalis
(ATCC20962), as well as, modified derivatives of this strain with
amplified components of the omega-oxidation pathway, and the
cultures grown overnight at 30.degree. C., with shaking at about
250 rpm. The starter cultures were then used to inoculate 25 mL
cultures of the same medium and grown overnight at 30.degree. C.,
with shaking at about 250 rpm. Cells were pelleted by
centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g and
the supernatant discarded. Cells were resuspended in 12.5 mL SP92
glycerol medium and transferred to a sterile 250 mL glass baffled
flask. Cultures were fed 2% (v/v) methyl laurate and incubated at
30.degree. C., with shaking at about 300 rpm. After 24 hours
incubation, cultures were fed glycerol to 60 g/L and incubation
continued before sampling for GC at 48 hours. GC analysis showed
that amplification of certain components of the omega oxidation
pathway allow for increased conversion to dodecanedioic acid (FIG.
13).
Example 9
Alteration of Acyl CoA Oxidase Substrate Specificity
[0365] The substrate specificity of the peroxisomal acyl-CoA
oxidase enzymes POX4 and POX5 have been shown to be involved in the
control of the diacid product chain-length in fermentations of
Candida tropicalis fed a mixed chain-length fatty acid feedstock.
Reduction or elimination of POX4 activity, POX5 activity or POX4
activity and POX5 activity, effects the carbon chain-length
distribution of dicarboxylic acids produced in Candida spp.
Acyl-CoA oxidase is the first enzyme in the cyclic beta-oxidation
pathway that shortens a substrate by two carbons each cycle. Thus
the acyl-CoA oxidase activity serves as the pathway entry point for
substrates entering into the beta-oxidation pathway. Altering the
substrate specificity an acyl-CoA oxidase activity such that it is
not active on substrate carbon chains shorter than a desired carbon
chain length (e.g., C8, C10, C12, C14 and the like), can inhibit
shortening of carbon chains below a chosen threshold, allowing
accumulation of a desired target chain length and product (e.g.,
C12, dodecanedioic acid).
[0366] The native acyl-CoA oxidase isozymes in Candida strain
ATCC20336, Pox4p and Pox5p have different substrate specificities.
The Pox4p isozyme has a broad substrate specificity while the Pox5p
isozyme has a narrow substrate specificity. In strains that are
Pox4.sup.-, Pox5.sup.- the chain length of the diacid product is
determined by the substrate specificity of the Pox5.beta. isozyme
and the main product is adipic acid.
[0367] To maximize production of desired diacid products of longer
chain lengths (e.g., C12) in fermentations, genetically modified
organisms containing an acyl-CoA oxidase activity with a substrate
chain-length specificity appropriate for the chain-length of the
desired diacid product can be engineered, in some embodiments. The
source of the acyl-CoA oxidase activity or the method of
engineering the acyl-CoA oxidase activity may vary. Non-limiting
examples of organisms which can be used to provide polynucleotide
sequences suitable for use in engineering altered substrate
specificity acyl-CoA oxidase activities include; plants (e.g.,
Arabidopsis, Cucurbita (e.g., pumpkin, squash), Oryza (e.g.,
rice)); animals (e.g., Bos (e.g., bovine), Cavia (e.g., guinea
pig), Mus (e.g., mouse), Rattus (e.g., rat), Phascolarctos (e.g.,
Koala), primates (e.g., orangutans)); molds (e.g., Dictyostelium
(e.g., slime molds)); insects (e.g., Drosophila); Yeast (e.g.,
Yarrowia lipolytica, Candida maltosa, Candida glabrata, Ashbya
gossypii, Debaryomyces hansenii, Kluyveromyces lactis, Pichia
pastoris, Saccharomyces cerevisiae); bacteria (e.g., Eschericia
coli); cyanobacteria; nematodes (e.g., Caenorhabditis); and
humans.
[0368] Acyl-CoA oxidase activities with different substrate
chain-length specificities can be identified by: [0369] 1)
Selecting acyl-CoA oxidase genes from heterologous organisms that
contain different substrate chain-length specificities. The
identified genes can be transferred into a Candida strain deleted
for all acyl-CoA oxidase activity. The only acyl-CoA oxidase
activity detectable in such a genetically modified organism may be
that imparted by the heterologous gene. [0370] 2) Engineering an
acyl-CoA oxidase gene library by domain swapping from multiple
acyl-CoA oxidase genes to produce a library of non-native chimeric
acyl-CoA oxidase genes. The library of chimeric genes can be
transferred into a strain of Candida deleted for all acyl-CoA
oxidase activity. The only detectable acyl-CoA oxidase activity may
be that imparted by an engineered gene from the library of
non-native chimeric acyl-CoA oxidase genes. [0371] 3) Engineering
an acyl-CoA oxidase gene library by random mutagenesis. A naturally
occurring or engineered acyl-CoA oxidase activity with a substrate
chain-length specificity close to that desired can be used as the
basis for random mutagenesis, followed by screening and/or
selection in an effort to generate and identify an altered activity
with the desired substrate chain-length specificity. The library of
genes can be transferred into a Candida strain deleted for all
acyl-CoA oxidase activity. The only detectable acyl-CoA oxidase
activity may be that imparted by the gene from the randomly
mutagenized library. [0372] 4) Engineering an acyl-CoA oxidase gene
by intelligent design and directed mutation using protein
structural information to guide the position and identity of the
amino acid(s) to be replaced. The engineered gene(s) can be
transferred into a Candida strain deleted for all acyl-CoA oxidase
activity. The only detectable acyl-CoA oxidase activity may be that
imparted by the engineered gene(s).
[0373] A non-limiting example of a post-engineering method for
selecting genes that impart the desired substrate chain-length
specificity is provided herein. Selection is performed by growth on
substrates of different chain lengths that are provided as the only
carbon source. Growth of the cells on certain substrates but not
others often reflects the substrate chain-length specificity of the
acyl-CoA oxidase enzyme present in the strain. Candida tropicalis
can utilize alkanes provided in the gas phase as its sole carbon
source for growth. Alkanes of different chain lengths are provided
by soaking a filter paper in the appropriate alkane, and inverting
a solid growth media without a carbon source over the filter paper,
with each specific carbon source (e.g., specific chain length
alkane) provided in a different petri dish. Serially diluted
Candida carrying the altered specificity acyl-CoA oxidase genes are
spotted on the solid growth media as a growth selection for the
chain-length specificity of the acyl-CoA oxidase enzyme in each
strain. Shown in FIGS. 14 and 15 are a schematic representation of
the selection process, which provides an alkane as a gas phase
carbon source, as described herein. The solid growth media is an
agar medium containing yeast nitrogen base without amino acids or
any other carbon source. The plated cells are inverted over a lid
containing a filter paper soaked with an alkane of appropriate
chain length that evaporates and provides the carbon source through
the gas phase, as shown in FIG. 14.
[0374] Candida strains containing altered acyl-CoA oxidase
activities generated as described herein are selected and/or
screened using the method described herein. Strains carrying
different altered acyl-CoA oxidase activities (e.g., strain 1 (S1),
strain 2 (S2), strain 3 (S3), strain 4 (S4)) are grown overnight in
a rich medium (e.g., YPD). Overnight cultures are centrifuged and
washed to remove any traces of residual rich medium and serial
dilutions of the cells are prepared in a phosphate buffered
solution. The serial dilutions of each strain are spotted onto
multiple YNB agar plates (growth medium having no amino acids or
other carbon sources), the individual plates inverted over filter
papers soaked in the appropriate chain length alkane, and the plate
incubated at 30.degree. C. The growth of the strains is dependent
upon the chain-length specificity of the acyl-CoA oxidase. In order
to utilize the particular alkane for growth the provided
chain-length must be able to enter the beta-oxidation pathway. The
shortest chain-length at which a certain strain is able to grow
indicates the shortest chain-length of the acyl-CoA oxidase
isozymes substrate specificity. An example is provided in FIG. 15.
FIG. 15 illustrates that strain S4 can grow on decane, but is
unable to grow on octane. Therefore the modified acyl-CoA oxidase
activity of strain S4 has a substrate chain-length specificity that
inhibits the utilization of 8 carbon molecules and the diacid
product from fermentations with this strain typically result in an
8 carbon diacid. Acyl-CoA oxidase activities with any desired
specificity can be selected and/or screened using the method
described herein.
[0375] It will be understood that the example presented herein is a
generalize method used to describe the selection/screening process.
The feedstocks used for the selection and screening process are
altered to suit the acyl-CoA oxidase activity being sought. For
example, for acyl-CoA oxidases having specificity for longer chain
substrates, feedstocks having longer carbon chain lengths could be
substituted to allow selection and or screening for acyl-CoA
oxidase activities with specificities for longer carbon chain
lengths.
Example 10
Transformation of Candida Spp. Procedure
[0376] 5 mL YPD start cultures were inoculated with a single colony
of Candida strain ATCC20336 and incubated overnight at 30.degree.
C., with shaking at about 200 rpm. The following day, fresh 25 mL
YPD cultures, containing 0.05% Antifoam B, were inoculated to an
initial OD.sub.600 nm of 0.4 and the culture incubated at
30.degree. C., with shaking at about 200 rpm until an OD.sub.600 nm
of 1.0-2.0 was reached. Cells were pelleted by centrifugation at
1,000.times.g, 4.degree. C. for 10 minutes. Cells were washed by
resuspending in 10 mL sterile water, pelleted, resuspended in 1 mL
sterile water and transferred to a 1.5 mL microcentrifuge tube. The
cells were then washed in 1 mL sterile TE/LiOAC solution, pH 7.5,
pelleted, resuspended in 0.25 mL TE/LiOAC solution and incubated
with shaking at 30.degree. C. for 30 minutes.
[0377] The cell solution was divided into 50 uL aliquots in 1.5 mL
tubes to which was added 5-8 ug of linearized DNA and 5 uL of
carrier DNA (boiled and cooled salmon sperm DNA, 10 mg/mL). 300 uL
of sterile PEG solution (40% PEG 3500, 1.times.TE, 1.times.LiOAC)
was added, mixed thoroughly and incubated at 30.degree. C. for 60
minutes with gentle mixing every 15 minutes. 40 uL of DMSO was
added, mixed thoroughly and the cell solution was incubated at
42.degree. C. for 15 minutes. Cells were then pelleted by
centrifugation at 1,000.times.g 30 seconds, resuspended in 500 uL
of YPD media and incubated at 30.degree. C. with shaking at about
200 rpm for 2 hours. Cells were then pelleted by centrifugation and
resuspended in 1 mL 1.times.TE, cells were pelleted again,
resuspended in 0.2 mL 1.times.TE and plated on selective media.
Plates were incubated at 30.degree. C. for growth of
transformants.
Example 11
Procedure for Recycling of the URA3 Marker
[0378] The URA3 gene was obtained from genomic DNA of Candida yeast
culture ATCC20336. Candida strain ATCC20336 has a limited number of
selectable marker, as compared to S. cerevisiae, therefore, the
URA3 marker is "recycled" to allow multiple rounds of selection
using URA3. To reutilize the URA3 marker for subsequent engineering
of Candida spp., a single colony having the Ura.sup.+ phenotype was
inoculated into 3 mL YPD and grown overnight at 30.degree. C. with
shaking. The overnight culture was then harvested by centrifugation
and resuspended in 1 mL YNB+YE (6.7 g/L Yeast Nitrogen Broth, 3 g/L
Yeast Extract). The resuspended cells were then serially diluted in
YNB+YE and 100 uL aliquots plated on YPD plates (incubation
overnight at 30.degree. C.) to determine titer of the original
suspension. Additionally, triplicate 100 uL aliquots of the
undiluted suspension were plated on SC Dextrose (Bacto Agar 20 g/L,
Uracil 0.3 g/L, Dextrose 20 g/L, Yeast Nitrogen Broth 6.7 g/L,
Amino Acid Dropout Mix 2.14 g/L) and 5-FOA. at 3 different
concentrations (0.5, 0.75, 1 mg/mL).
[0379] Plates were incubated for at least 5 days at 30.degree. C.
Colonies arising on the SC Dextrose+5-FOA plates were resuspended
in 50 uL sterile, distilled water and 5 uL utilized to streak on to
YPD and SC-URA (SC Dextrose medium without Uracil) plates. Colonies
growing only on YPD and not on SC-URA plates were then inoculated
into 3 mL YPD and grown overnight at 30.degree. C. with shaking.
Overnight cultures were harvested by centrifugation and resuspended
in 1.5 mL YNB (6.7 g/L Yeast Nitrogen Broth). The resuspended cells
were serially diluted in YNB and 100 uL aliquots plated on YPD
plates and incubation overnight at 30.degree. C. to determine
initial titer. 1 mL of each undiluted cell suspension also was
plated on SC-URA and incubated for up to 7 days at 30.degree. C.
Colonies on the SC-URA plates are revertants and the isolate with
the lowest reversion frequency (<10.sup.-7) was used for
subsequent strain engineering.
Example 12
Cloning and Analysis of Candida Fatty Alcohol Oxidase (FAO)
Alleles
[0380] Isolation of Fatty Alcohol Oxidase Genes from Candida
[0381] Candida strain (ATCC20336) fatty alcohol oxidase genes were
isolated by PCR amplification using primers generated to amplify
the sequence region covering promoter, fatty alcohol oxidase gene
(FAO) and terminator of the FAO1 sequence (Gen Bank accession
number of FAO1 AY538780). The primers used to amplify the fatty
alcohol oxidase nucleotide sequences from Candida strain ATCC20336
strain ATCC20336, are showing in the table below.
TABLE-US-00002 Oligonucleotides for cloning FAO alleles Oligo
Sequence oAA0144 AACGACAAGATTAGATTGGTTGAGA oAA0145
GTCGAGTTTGAAGTGTGTGTCTAAG oAA0268
AGATCTCATATGGCTCCATTTTTGCCCGACCAGGTCGACTAC AAACACGTC oAA0269
ATCTGGATCCTCATTACTACAACTTGGCTTTGGTCTTCAAGG AGTCTGCCAAACCTAAC
oAA0282 ACATCTGGATCCTCATTACTACAACTTGGCCTTGGTCT oAA0421
CACACAGCTCTTCTAGAATGGCTCCATTTTTGCCCGACCAG GTCGAC oAA0422
CACACAGCTCTTCCTTTCTACAACTTGGCTTTGGTCTTCAAG GAGTCTGC oAA0429
GTCTACTGATTCCCCTTTGTC oAA0281 TTCTCGTTGTACCCGTCGCA
[0382] PCR reactions contained 25 uL 2.times. master mix, 1.5 uL of
oAA0144 and oAA0145 (10 uM), 3.0 uL genomic DNA, and 19 uL sterile
H.sub.2O. Thermocycling parameters used were 98.degree. C. for 2
minutes, 35 cycles of 98.degree. C. 20 seconds, 52.degree. C. 20
seconds, 72.degree. C. 1 minute, followed by 72.degree. C. 5
minutes and a 4.degree. C. hold. PCR products of the correct size
were gel purified, ligated into pCR-Blunt II-TOPO (Invitrogen) and
transformed into competent TOP10 E. coli cells (Invitrogen). Clones
containing PCR inserts were sequenced to confirm correct DNA
sequence. Four FAO alleles were identified from sequence analysis
and designated as FAO-13, FAO-17, FAO-18 and FAO-20. The sequence
of the clone designated FAO-18 had a sequence that was
substantially identical to the sequence of FAO1 from Gen Bank. The
resulting plasmids of the four alleles were designated pAA083,
pAA084, pAA059 and pAA085, respectively. Sequence identity
comparisons of FAO genes isolated as described herein are shown in
the tables below.
TABLE-US-00003 DNA sequence identity FAO- FAO- FAO- FAO- FAO1 18 17
13 20 FAO2a FAO2b FAO1 100 100 98 96 95 83 82 FAO- 100 98 96 95 83
82 18 FAO- 100 98 98 83 82 17 FAO- 100 99 83 83 13 FAO- 100 83 83
20 FAO2a 100 96 FAO2b 100
TABLE-US-00004 Protein sequence identity FAO- FAO- FAO- FAO- FAO1
18 17 13 20 FAO2a FAO2b FAO1 100 100 99 98 98 81 80 FAO- 100 99 98
98 81 80 18 FAO- 100 99 99 82 81 17 FAO- 100 99 82 81 13 FAO- 100
82 81 20 FAO2a 100 97 FAO2b 100
TABLE-US-00005 Amino acid differences in FAO alleles 32 75 89 179
185 213 226 352 544 590 FAO1 E M G L Y T R H S P FAO-13 Q T A L Y A
K Q A A FAO-20 Q T A M D A K Q A A
[0383] Expression of FAO Alleles in E. Coli
[0384] To determine the levels of FAO enzyme activity with respect
to various carbon sources, the four isolated FAO alleles were
further cloned and over-expressed in E. coli. The FAOs were
amplified using the plasmids mentioned above as DNA template by PCR
with primers oAA0268 and oAA0269 for FAO-13 and FAO-20 and oAA0268
and oAA0282 for FAO-17 and FAO-18, using conditions as described
herein. PCR products of the correct size were gel purified and
ligated into pET11a vector between NdeI and BamHI sites and
transformed into BL21 (DE3) E. coli cells. The colonies containing
corresponding FAOs were confirmed by DNA sequencing. Unmodified
pET11a vector also was transformed into BL21 (DE3) cells, as a
control. The resulting strains and plasmids were designated sAA153
(pET11a), sAA154 (pAA079 containing FAO-13), sAA155 (pAA080
containing FAO-17), sAA156 (pAA081 containing FAO-18) and sAA157
(pAA082 containing FAO-20), respectively. The strains and plasmids
were used for FAO over-expression in E. coli. One colony of each
strain was transferred into 5 mL of LB medium containing 100
.mu.g/mL ampicillin and grown overnight at 37.degree. C., 200 rpm.
The overnight culture was used to inoculate a new culture to
OD.sub.600 nm 0.2 in 25 ml LB containing 100 pg/mlampicillin. Cells
were induced at OD.sub.600 nm 0.8 with 0.3 mM IPTG for 3 hours and
harvested by centrifugation at 4.degree. C. 1,050.times.g for 10
minutes. The cell pellet was stored at -20.degree. C.
[0385] Expression of FAOs in a Candida Strain
[0386] Two alleles, FAO-13 and FAO-20, were chosen for
amplification in Candida based on their substrate specificity
profile, as determined from enzyme assays of soluble cell extracts
of E. coli with over expressed FAOs. DNA fragments containing
FAO-13 and FAO-20 were amplified using plasmids pAA079 and pAA082
as DNA templates, respectively, by PCR with primers oAA0421 and
oAA0422. PCR products of the correct sizes were gel purified and
ligated into pCR-Blunt II-TOPO (Invitrogen), transformed into
competent TOP10 E. coli cells (Invitrogen) and clones containing
FAO inserts were sequenced to confirm correct DNA sequence.
Plasmids containing FAO-13 and FAO-20 were digested with SapI and
ligated into vector pAA105, which includes the Candida strain
ATCC20336 PGK promoter and terminator. The resulting plasmids were
confirmed by restriction digestion and DNA sequencing and
designated as pAA115 (FAO-13) and pAA116 (FAO-20), respectively.
Plasmids pAA115 and pAA116 were linearized with SpeI, transformed
into competent Candida spp. Ura.sup.- strains sAA002 (SU-2,
ATCC20913) and sAA103. The integration of FAO-13 and FAO-20 was
confirmed by colony PCR using primers oAA0429 and oAA0281. The
resulting strains were designated as sAA278 (pAA115 integrated in
strain sAA002), sAA280 (pAA116 integrated in sAA002), sAA282
(pAA115 integrated in sAA103), and sAA284 (pAA116 integrated in
sAA103), and were used for fatty alcohol oxidase over-expression in
Candida spp.
[0387] One colony of each strain was inoculated into 5 ml YPD and
grown overnight as described herein. The overnight culture was used
to inoculate a new 25 mL YPD culture to about OD.sub.600 nm 0.5.
FAO over-expression was regulated by the PGK promoter/terminator,
induced with glucose in the medium and expressed constitutively.
Strains sAA002 and sAA103 (e.g., untransformed starting strains)
were included as negative controls for FAO over-expression. Cells
were harvested at early log phase (OD.sub.600 nm=in the range of
between about 3 to about 5) by centrifugation at 4.degree. C. for
10 minutes at 1,050.times.g. Cell pellets were stored at
-20.degree. C.
[0388] Cell Extract Preparation from E. Coli
[0389] Cell pellets from 25 mL of FAO expressing E. coli cultures
were resuspended in 10 mL phosphate-glycerol buffer containing 50
mM potassium phosphate buffer (pH7.6), 20% glycerol, 1 mM
Phenylmethylsulfonyl fluoride (PMSF), 2 uL Benzonase 25 U/uL, 20 uL
10 mg/mL lysozyme. The cells were then lysed by incubation at room
temperature for 50 minutes on a rotating shaker, and the cell
suspension centrifuged for 30 minutes at 4.degree. C. using
15,000.times.g for. The supernatant was aliquoted in 1.5 ml
microcentrifuge tubes and stored at -20.degree. C. for FAO enzyme
activity assays.
[0390] Cell Extract Preparation from Candida
[0391] Frozen C. tropicalis cell pellets were resuspended in 1.2 ml
of phosphate-glycerol buffer containing 50 mM potassium phosphate
buffer (pH7.6), 20% glycerol, 1 mM Phenylmethylsulfonyl fluoride
(PMSF). Resuspended cells were transferred to 1.5 mL screw-cap
tubes containing about 500 uL of zirconia beads on ice. The cells
were lysed with a Bead Beater (Biospec) using 2 minute pulses and 1
minute rest intervals on ice. The process was repeated 3 times. The
whole cell extract was then transferred to a new 1.5 ml tube and
centrifuged at 16,000.times.g for 15 minutes at 4.degree. C. The
supernatant was transferred into a new tube and used for FAO enzyme
activity assays.
[0392] Protein Concentration Determination
[0393] Protein concentration of the cell extracts was determined
using the Bradford Reagent following manufacturers' recommendations
(Cat#23238, Thermo scientific).
[0394] FAO Enzyme Activity Assay
[0395] FAO enzyme activity assays were performed using a
modification of Eirich et al., 2004). The assay utilizes a
two-enzyme coupled reaction (e.g., FAO and horse radish peroxidase
(HRP)) and can be monitored by spectrophotometry. 1-Dodecanol was
used as a standard substrate for fatty alcohol oxidase enzymatic
activity assays. FAO oxidizes the dodecanol to dodecanal while
reducing molecular oxygen to hydrogen peroxide simultaneously. HRP
reduces (2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid;
ABTS) in the two-enzyme coupled reaction, where the electron
obtained from oxidizing hydrogen peroxide to ABTS, which can be
measured by spectrometry at 405 nm. The assay was modified using
aminotriazole (AT) to prevent the destruction of H.sub.2O.sub.2 by
endogenous catalase, thus eliminating the need for microsomal
fractionation. The final reaction mixture (1.0 mL) for FAO enzyme
assay consisted of 500 .mu.L of 200 mM HEPES buffer, pH 7.6; 50
.mu.L of a 10 mg/mL ABTS solution in deionized water; 10 .mu.L of 5
mM solution of dodecanol in acetone; 40 .mu.L of 1M AT and 5 .mu.L
of a 2 mg/mL horseradish peroxidase solution in 50 mM potassium
phosphate buffer, pH 7.6. Reaction activity was measured by
measuring light absorbance at 405 nm for 10 minutes at room
temperature after adding the extract. The amount of extract added
to the reaction mixture was varied so that the activity fell within
the range of 0.2 to 1.0 .DELTA.A.sub.405 nm/min. The actual amounts
of extract used were about 1.69 U/mg for E. coli expressed FAO-13,
0.018 U/mg for E. coli expressed FAO-17, 0.35 U/mg for E. coli
expressed FAO-18 (e.g., FAO1), 0.47 U/mg E. coli expressed FAO-20,
0.036 U/mg Candida (strain sAA278) expressed FAO-13, 0.016 U/mg
Candida (strain sAA282) expressed FAO-13, 0.032 U/mg Candida
(strain sAA280) expressed FAO-20 and 0.029 U/mg Candida (strain
sAA284) expressed FAO-20. FAO activity was reported as activity
units/mg of total protein (1 unit=1 .mu.mole substrate
oxidized/min). An extinction coefficient at 405 nm of 18.4 was used
for ABTS and was equivalent to 0.5 mM oxidized substrate. The
results of the activity assays are shown in the tables below.
TABLE-US-00006 FAO activity (units/mg total protein) on primary
alcohols 1- 1- 1- 1- 1- 1-Do- 1-Tetra- Hexa- Buta- Penta- Hexa-
Octa- Deca- deca- deca- deca- nol nol nol nol nol nol nol nol FAO-
0.01 0.09 1.17 82.67 70.94 100 79.35 58.88 13 FAO- 0.72 0.26 1.06
66.23 22.00 100 47.86 60.98 17 FAO- 0.07 0.11 0.26 60.56 54.56 100
114.47 50.65 18 FAO- 0.07 0.11 0.91 55.96 74.57 100 89.52 42.59
20
TABLE-US-00007 FAO activity (units/mg total protein) on omega
hydroxy fatty acids 1- 6- 10- 12- 16- Dodecanol OH-HA OH-DA OH-DDA
OH-HDA FAO-13 100 4.18 4.14 6.87 8.57 FAO-17 100 1.18 0.00 0.59
0.94 FAO-18 100 0.00 0.00 4.87 2.94 FAO-20 100 0.03 0.04 2.25
7.46
Example 13
Construction of Candida Shuttle Vector pAA061
[0396] Vector pAA061 was constructed from a pUC19 backbone to
harbor the selectable marker URA3 from Candida strain ATCC20336 as
well as modifications to allow insertion of Candida promoters and
terminators. A 1,507 bp DNA fragment containing the promoter, ORF,
and terminator of URA3 from Candida ATCC20336 was amplified using
primers oAA0124 and oAA0125, shown in the table below. The URA3 PCR
product was digested with NdeI/MluI and ligated into the 2,505 bp
fragment of pUC19 digested with NdeI/BsmBI (an MluI compatible
overhang was produced by BsmBI). In order to replace the lac
promoter with a short 21 bp linker sequence, the resulting plasmid
was digested with SphI/SapI and filled in with a linker produced by
annealing oligos oAA0173 and oAA0174. The resulting plasmid was
designated pAA061.
TABLE-US-00008 Oligonucleotides for construction of pAA061 PCR
product Oligos Sequence (bp) oAA0124
cacacacatatgCGACGGGTACAACGAGAATT 1507 oAA0125
cacacaacgcgtAGACGAAGCCGTTCTTCAAG oAA0173 ATGATCTGCCATGCCGAACTC 21
(linker) oAA0174 AGCGAGTTCGGCATGGCAGATCATCATG
Example 14
Cloning of Candida PGK Promoter and Terminator
[0397] Vector pAA105 was constructed from base vector pAA061 to
include the phosphoglycerate kinase (PGK) promoter and terminator
regions from Candida ATCC20336 with an intervening multiple cloning
site (MCS) for insertion of open reading frames (ORF's). The PGK
promoter region was amplified by PCR using primers oAA0347 and
oAA0348, shown in the table below. The 1,029 bp DNA fragment
containing the PGK promoter was digested with restriction enzymes
PstI/XmaI. The PGK terminator region was amplified by PCR using
primers oAA0351 and oAA0352, also shown in the table below. The 396
bp DNA fragment containing the PGK terminator was digested with
restriction enzymes XmaI/EcoRI. The 3,728 bp PstI/EcoRI DNA
fragment from pAA061 was used in a three piece ligation reaction
with the PGK promoter and terminator regions to produce pAA105. The
sequence between the PGK promoter and terminator contains
restriction sites for incorporating ORF's to be controlled by the
functionally linked constitutive PGK promoter.
TABLE-US-00009 Oligonucleotides for cloning Candida PGK promoter
and terminator PCR product Oligos Sequence (bp) oAA0347
CACACACTGCAGTTGTCCAATGTAATAATTTT 1028 oAA0348
CACACATCTAGACCCGGGCTCTTCTTCTGAATAG GCAATTGATAAACTTACTTATC oAA0351
GAGCCCGGGTCTAGATGTGTGCTCTTCCAAA 396 GTACGGTGTTGTTGACA oAA0352
CACACACATATGAATTCTGTACTGGTAGAGCT AAATT
Example 15
Cloning of the POX4 Locus
[0398] Primers oAA0138 and oAA0141 (shown in the table below) were
generated to amplify the entire sequence of NCBI accession number
M12160 for the YSAPOX4 locus from genomic DNA prepared from Candida
strain ATCC20336. The 2,845 bp PCR product was cloned into the
vector, pCR-BluntII-TOPO (Invitrogen), sequenced and designated
pAA052.
TABLE-US-00010 Oligonucleotides for cloning of POX4 Oligos Sequence
PCR product (bp) oAA0138 GAGCTCCAATTGTAATATTTCGGG 2845 oAA0141
GTCGACCTAAATTCGCAACTATCAA
Example 16
Cloning of the POX5 Locus
[0399] Primers oAA0179 and oAA0182 (shown in the table below) were
generated to amplify the entire sequence of NCBI accession number
M12161 for the YSAPOX5 locus from genomic DNA prepared from Candida
strain ATCC20336. The 2,624 bp PCR product was cloned into the
vector, pCR-BluntII-TOPO (Invitrogen), sequenced and designated
pAA049.
TABLE-US-00011 Oligonucleotides for cloning of POX5 Oligos Sequence
PCR product (bp) oAA0179 GAATTCACATGGCTAATTTGGCCTCG 2624
GTTCCACAACGCACTCAGCATTAAA AA oAA0182 GAGCTCCCCTGCAAACAGGGAAA
CACTTGTCATCTGATTT
Example 17
Construction of Strains with Amplified CPR and CYP52 Genes
[0400] Strains having an increased number of copies of cytochrome
P450 reductase (CPR) and/or for cytochrome P450 monooxygenase
(CYP52) genes were constructed to determine how over expression of
CPR and CYP52 affected diacid production.
[0401] Cloning and Integration of the CPR Gene.
[0402] A 3,019 bp DNA fragment encoding the CPR promoter, ORF, and
terminator from Candida ATCC750 was amplified by PCR using primers
oAA0171 and oAA0172 (see table below) incorporating unique SapI and
SphI sites. The amplified DNA fragment was cut with the indicated
restriction enzymes and ligated into plasmid pAA061, (described in
Example 13) to produce plasmid pAA067. Plasmid pAA067 was
linearized with ClaI and transformed into Candida Ura.sup.- strain
sAA103 (ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3).
Transformations were performed with plasmid pAA067 alone and in
combination with plasmids harboring the CYP52A15 or CYP52A16 genes,
described below.
[0403] Cloning and Integration of CYP52A15 Gene.
[0404] A 2,842 bp DNA fragment encoding the CYP52A15 promoter, ORF,
and terminator from Candida ATCC20336 was amplified by PCR using
primers oAA0175 and oAA0178 (see table below) and cloned into
pCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A15
DNA fragment was isolated by restriction digest with XbaI/BamHI
(2,742 bp) and ligated into plasmid pAA061, (described in Example
13), to produce plasmid pAA077. Plasmid pAA077 was linearized with
PmlI and transformed into Candida Ura.sup.- strain sAA103
(ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA077
was cotransformed with plasmid pAA067 harboring the CPR gene.
[0405] Cloning and Integration of CYP52A16 Gene.
[0406] A 2,728 bp DNA fragment encoding the CYP52A16 promoter, ORF,
and terminator from Candida ATCC20336 was amplified by PCR using
primers oAA0177 and oAA0178 (see table below) and cloned into
pCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A16
DNA fragment was amplified with primers oAA0260 and oAA0261(see
table below) which incorporated unique SacI/XbaI restriction sites.
The amplified DNA fragment was digested with SacI and XbaI
restriction enzymes and ligated into plasmid pAA061 to produce
plasmid pAA078. Plasmid pAA078 was linearized with ClaI and
transformed into Candida Ura.sup.- strain sAA103 (ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA078 was
cotransformed with plasmid pAA067 harboring the CPR gene.
TABLE-US-00012 Oligonucleotides for cloning of CPR, CYP52A15 and
CYP52A16 Oligos Sequence PCR product (bp) oAA0171
cacctcgctcttccAGCTGTCATGTCTATTCAATGCTTCGA 3019 oAA0172
cacacagcatgcTAATGTTTATATCGTTGACGGTGAAA oAA0175
cacaaagcggaagagcAAATTTTGTATTCTCAGTAGGATTT 2842 CATC oAA0178
cacacagcatgCAAACTTAAGGGTGTTGTAGATATCCC oAA0177
cacacacccgggATCGACAGTCGATTACGTAATCCATATT 2772 ATTT oAA0178
cacacagcatgCAAACTTAAGGGTGTTGTAGATATCCC oAA0260
cacacagagctcACAGTCGATTACGTAATCCAT 2772 oAA0261
cacatctagaGCATGCAAACTTAAGGGTGTTGTA
[0407] Preparation of Genomic DNA.
[0408] Genomic DNA was prepared from transformants for PCR
verification and for Southern blot analysis. Isolated colonies were
inoculated into 3 mL YPD and grown overnight at 30.degree. C. with
shaking. Cells were pelleted by centrifugation. To each pellet, 200
uL Breaking Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM
Tris pH 8 and, 1 mM EDTA) was added, and the pellet resuspended and
transferred to a fresh tube containing 200 uL 0.5 mm
Zirconia/Silica Beads. 200 uL Phenol:Chloroform:lsoamyl Alcohol
(25:24:1) was added to each tube, followed by vortexing for 1
minute. Sterile distilled water was added (200 uL) to each tube and
the tubes were centrifuged at 13000 rpm for 10 minutes. The aqueous
layer was ethanol precipitated and washed with 70% ethanol. The
pellet was resuspended in 100-200 .mu.l 10 mM Tris, after drying.
Genomic DNA preparation for southern blot analysis was performed
using the same procedure on 25 mL cultures for each colony
tested.
[0409] Characterization of Strains with Amplified CPR and CYP52
Genes.
[0410] Verification of integrants was performed by PCR using
primers oAA0252 and oAA0256 (CPR), oAA0231 and oAA0281 (CYP52A15),
and oAA242 and oAA0257 (CYP52A16). The primers used for
verification are shown in the table below.
TABLE-US-00013 Oligonucleotides for PCR verification of CPR,
CYP52A15 and CYP52A16 Oligos Sequence PCR product (bp) oAA0252
TTAATGCCTTCTCAAGACAA 743 oAA0256 GGTTTTCCCAGTCACGACGT oAA0231
CCTTGCTAATTTTCTTCTGTATAGC 584 oAA0281 TTCTCGTTGTACCCGTCGCA oAA0242
CACACAACTTCAGAGTTGCC 974 oAA0257 TCGCCACCTCTGACTTGAGC
[0411] Southern blot analysis was used to determine the copy number
of the CPR, CYP52A15 and CYP52A16 genes. Biotinylated DNA probes
were prepared with gene specific oligonucleotides using the NEBlot
Phototope Kit from New England BioLabs (Catalog #N7550S) on PCR
products generated from each gene target as specified in the table
below. Southern Hybridizations were performed using standard
methods (e.g., Sambrook, J. and Russell, D. W. (2001) Molecular
Cloning: A Laboratory Manual, (3.sup.rd ed.), pp. 6.33-6.64. Cold
Spring Harbor Laboratory Press). Detection of hybridized probe was
performed using the Phototope-Star Detection Kit from New England
BioLabs (Catalog #N7020S). Copy number was determined by
densitometry of the resulting bands.
TABLE-US-00014 Oligonucleotides for Probe Template PCR of CPR,
CYP52A15 and CYP52A16 Oligos Sequence Gene Template PCR product
(bp) oAA0250 AATTGAACATCAGAAGAGGA CPR pAA067 1313 oAA0254
CCTGAAATTTCCAAATGGTGTCTAA oAA0227 TTTTTTGTGCGCAAGTACAC CYP52A15
pAA077 905 oAA0235 CAACTTGACGTGAGAAACCT oAA0239
AGATGCTCGTTTTACACCCT CYP52A16 pAA078 672 oAA0247
ACACAGCTTTGATGTTCTCT
Example 18
Addition and/or Amplification of Monooxygenase and Monooxygenase
Reductase Activities
[0412] Cytochrome P450's often catalyze a monooxygenase reaction,
e.g., insertion of one atom of oxygen into an organic substrate
(RH) while the other oxygen atom is reduced to water:
RH+O2+2H++2e-.fwdarw.ROH+H2O
[0413] The substrates sometimes are of a homogeneous carbon chain
length. Enzymes with monooxygenase activity sometimes recognize
substrates of specific carbon chain lengths, or a subgroup of
carbon chain lengths with respect to organic substrates of
homogenous carbon chain length. Addition of novel cytochrome
activities (e.g., B. megaterium BM3) and/or amplification of
certain or all endogenous or heterologous monooxygenase activities
(e.g., CYP52A12 polynucleotide, CYP52A13 polynucleotide, CYP52A14
polynucleotide, CYP52A15 polynucleotide, CYP52A16 polynucleotide,
CYP52A17 polynucleotide, CYP52A18 polynucleotide, CYP52A19
polynucleotide, CYP52A20 polynucleotide, CYP52D2 polynucleotide,
BM3 polynucleotide) can contribute to an overall increase in carbon
flux through native and/or engineered metabolic pathways, in some
embodiments. In certain embodiments, adding a novel monooxygenase
or increasing certain or all endogenous or heterologous
monooxygenase activities can increase the flux of substrates of
specific carbon chain length or subgroups of substrates with
mixtures of specific carbon chain lengths. In some embodiments, the
selection of a monooxygenase activity for amplification in an
engineered strain is related to the feedstock utilized for growth
of the engineered strain, pathways for metabolism of the chosen
feedstock and the desire end product (e.g., dodecanedioic
acid).
[0414] Strains engineered to utilize plant-based oils for
conversion to dodecanedioic acid can benefit by having one or more
monooxygenase activities with substrate specificity that matches
the fatty acid chain-length distribution of the oil. For example,
the most prevalent fatty acid in coconut oil is lauric acid (12
carbons long), therefore, the monooxygenase activity chosen for a
coconut oil-utilizing strain can have a substrate preference for
C12 fatty acids. For strains engineered to utilize other plant
based oils with different fatty acid chain-length distributions it
may be desirable to amplify a monooxygenase activity that has a
matching substrate preference. In some embodiments, a genetic
modification that alters monooxygenase activity increases the
activity of one or more monooxygenase activities with a substrate
preference for feedstocks having carbon chain lengths of between
about 12 and about 24 carbons (e.g., mixed chain length alkanes,
mixed chain length fatty acids, soapstocks, the like and
combinations thereof). In certain embodiments, the genetic
modification increases the activity of a monooxygenase activity
with a preference for fatty acids having a carbon chain-length
distribution of between about 10 carbons and about 16 carbons.
[0415] As mentioned previously, the enzymes that carry out the
monooxygenase activity are reduced by the activity of monooxygenase
reductase, thereby regenerating the enzyme. Selection of a CPR for
amplification in an engineered strain depends upon which P450 is
amplified, in some embodiments. A particular CPR may interact
preferentially with one or more monooxygenase activities, in some
embodiments, but not well with other monooxygenases. A
monooxygenase reductase from Candida strain ATCC750, two
monooxygenase reductase activities from Candida strain ATCC20336
and a monooxygenase reductase activity from Bacillus megaterium are
being evaluated for activity with the added and/or amplified
monooxygenases described herein. Provided in the tables below are
nucleotide sequences used to add or amplify monooxygenase and
monooxygenase reductase activities.
Example 19
Amplification of Selected Beta Oxidation Activities
[0416] Described herein are methods of amplifying a POX5 beta
oxidation activity. Substantially similar methods can be utilized
to amplify different beta oxidation activities including non-PDX
(e.g., acyl-CoA oxidase) activities and/or acyl-CoA oxidase
activities with altered substrate specificities, as described
herein.
Construction of POX5 Amplified Strains
[0417] Plasmid pAA166 (P.sub.POX4POX5T.sub.POX4)
[0418] A PCR product containing the nucleotide sequence of POX5 was
amplified from Candida 20336 genomic DNA using primers oAA540 and
oAA541. The PCR product was gel purified and ligated into pCR-Blunt
II-TOPO (Invitrogen), transformed into competent TOP10 E. coli
cells (Invitrogen) and clones containing PCR inserts were sequenced
to confirm correct DNA sequence. One such plasmid was designated,
pAA165. Plasmid pAA165 was digested with BspQI and a 2-kb fragment
was isolated. Plasmid pAA073 which contained a POX4 promoter and
POX4 terminator was also digested with BspQI and gel purified. The
isolated fragments were ligated together to generate plasmid
pAA166. Plasmid pAA166 contains a P.sub.POX4POX5T.sub.POX4
fragment.
[0419] Plasmid pAA204 (Thiolase Deletion Construct)
[0420] A PCR product containing the nucleotide sequence of a
short-chain thiolase (e.g., acetyl-coA acetyltransferase) was
amplified from Candida 20336 genomic DNA using primers oAA640 and
oAA641. The PCR product was gel purified and ligated into pCR-Blunt
II-TOPO (Invitrogen), transformed into competent TOP10 E. coli
cells (Invitrogen) and clones containing PCR inserts were sequenced
to confirm correct DNA sequence. One such plasmid was designated,
pAA184. A URA3 PCR product was amplified from pAA061 using primers
oAA660 and oAA661. The PCR product was gel purified and ligated
into pCR-Blunt II-TOPO (Invitrogen), transformed as described and
clones containing PCR inserts were sequenced to confirm the correct
DNA sequence. One such plasmid was designated pAA192. Plasmid
pAA184 was digested with BglII/SalI and gel purified. Plasmid
pAA192 was digested with BglII/SalI and a 1.5 kb fragment was gel
purified. The isolate fragments were ligated together to generate
pAA199. An alternative P.sub.URA3 PCR product was amplified from
plasmid pAA061 using primers oAA684 and oAA685. The PCR product was
gel purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed as described and clones containing PCR inserts were
sequenced. One such plasmid was designated, pAA201. Plasmid pAA199
was digested with SalI and gel purified. Plasmid pAA201 was
digested with SalI and a 0.43 kb P.sub.URA3 was gel purified. The
isolated fragments were ligated to generate plasmid pAA204 that
contains a direct repeat of P.sub.URA3.
[0421] Plasmid pAA221 (P.sub.POX4POX5T.sub.POX4 in Thiolase
Deletion Construct)
[0422] A PCR product containing the nucleotide sequence of
P.sub.POX4POX5T.sub.POX4 was amplified from plasmid pAA166 DNA
using primers oAA728 and oAA729. The PCR product was gel purified
and ligated into pCR-Blunt II-TOPO, transformed as described and
clones containing PCR inserts were sequenced to confirm the
sequence of the insert. One such plasmid was designated, pAA220.
Plasmid pAA204 was digested with BglII, treated with shrimp
alkaline phosphatase (SAP), and a 6.5 kb fragment was gel purified.
Plasmid pAA220 was digested with BglII and a 2.7 kb fragment
containing P.sub.POX4POX5T.sub.POX4 was gel purified. The isolated
fragments were ligated to generate plasmid pAA221.
[0423] Strain sAA617 (P.sub.POX4POX5T.sub.POX4 in sAA451)
[0424] Strain sAA451 is a ura-, partially .beta.-oxidation blocked
strain (ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5). Plasmid
pAA221 was digested with EcoRI to release a DNA fragment containing
P.sub.POX4POX5T.sub.POX4 in a thiolase deletion construct. The DNA
was column purified and transformed to strain sAA451 to plate on
SCD-ura plate. After two days, colonies were streaked out on YPD
plates, single colonies selected and again streaked out on YPD
plates. Single colonies were selected from the second YPD plates
and characterized by colony PCR. The insertion of
P.sub.POX4POX5T.sub.POX4 in strain sAA451, disrupting the
short-chain thiolase gene, was confirmed by PCR and one such strain
was designated sAA617.
[0425] Strain sAA620
[0426] Strain sAA617 was grown overnight on YPD medium and plated
on SCD+URA+5-FOA, to select for loop-out of URA3. Colonies were
streaked out onto YPD plates twice as described for strain sAA617,
and single colonies characterized by colony PCR. The loop-out of
URA3 by direct repeats of PURA3 was confirmed by PCR. One such
strain was designated sAA620. Strain sAA620 has one additional copy
of POX5 under control of the POX4 promoter.
[0427] Plasmid pAA156
[0428] A PCR product containing the nucleotide sequence of CYP52A19
was amplified from Candida strain 20336 genomic DNA, using primers
oAA525 and oAA526. The PCR product was gel purified and ligated
into pCR-Blunt II-TOPO, transformed as described, and clones
containing PCR inserts were sequenced to confirm correct DNA
sequence. One such plasmid was designated, pAA144. Plasmid pAA144
was digested with BspQI and a 1.7-kb fragment was isolated. Plasmid
pAA073, which includes a POX4 promoter and POX4 terminator, also
was digested with BspQI and gel purified. The isolated fragments
were ligated together to generate plasmid, pAA156. Plasmid pAA156
included P.sub.POX4CYP52A19T.sub.POX4 fragment and URA3.
[0429] Strain sAA496
[0430] Plasmid pAA156 was digested with ClaI and column purified.
Strain sAA451 was transformed with this linearized DNA and plated
on SCD-ura plate. Colonies were checked for CYP52A19 integration.
Colonies positive for plasmid integration were further analyzed by
qPCR to determine the number of copies of CYP52A19 integrated. One
such strain, designated sAA496 contained about 13 copies of the
monooxygenase activity encoded by CYP52A19.
[0431] Strains sAA632 and sAA635
[0432] Strain sAA620 was transformed with linearized pAA156 DNA and
plated on SCD-ura plates. Several colonies were checked for
CYP52A19 integration. Colonies positive for plasmid integration
were further analyzed by qPCR to determine the number of copies of
CYP52A19 integrated. One such strain, designated sAA632 contained
about 27 copies of the monooxygenase activity encoded by CYP52A19.
Another strain, designated sAA635, contained about 12 copies of the
monooxygenase activity encoded by CYP52A19.
Example 20
Cloning of Candida ACH Genes
[0433] ACH PCR product was amplified from Candida strain ATCC20336
genomic DNA using primers oAA1095 and oAA1096, shown in the table
below. The PCR product was gel purified and ligated into pCR-Blunt
II-TOPO (Invitrogen), transformed into competent TOP10 E. coli
cells (Invitrogen) and clones containing PCR inserts were sequenced
to confirm correct DNA sequence.
[0434] Sequence analysis of multiple transformants revealed the
presence of allelic sequences for the ACH gene, which were
designated ACHA and ACHB. A vector containing the DNA sequence for
the ACHA allele was generated and designated pAA310. A vector
containing the DNA sequence for the ACHB allele was generated and
designated pAA311.
TABLE-US-00015 Primer sequence oAA1095
CACACACCCGGGATGATCAGAACCGTCCGTTATCAAT oAA1096
CACACATCTAGACTCTCTTCTATTCTTAATTGCCGCTTCCAC TAAACGGCAAAGTCTCCACG
Example 21
Cloning of Candida FAT1 Gene
[0435] FAT1 PCR product was amplified from Candida 20336 genomic
DNA using primers oAA1023 and oAA1024, shown in the table below.
The PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
for the FAT1 gene was designated pAA296.
TABLE-US-00016 Primer sequence oAA1023 GATATTATTCCACCTTCCCTTCATT
oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA
Example 22
Cloning of Candida ARE1 and ARE2 Genes
[0436] ARE1 and ARE2 PCR products were amplified from Candida 20336
genomic DNA using primers oAA2006/oAA2007 and oAA1012/oAA1018,
respectively, shown in the table below. The PCR products were gel
purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed into competent TOP10 E. coli cells (Invitrogen) and
clones containing PCR inserts were sequenced to confirm correct DNA
sequence. A vector containing the DNA sequence for the ARE1 gene
was designated pAA318. A vector containing the DNA sequence for the
ARE2 gene was designated pAA301.
TABLE-US-00017 Primer sequence oAA1012
ATGTCCGACGACGAGATAGCAGGAATAGTCAT oAA1018
TCAGAAGAGTAAATACAACGCACTAACCAAGCT oAA2006
ATGCTGAAGAGAAAGAGACAACTCGACAAG oAA2007
GTGGTTATCGGACTCTACATAATGTCAACG
Example 23
Construction of an Optimized TESA Gene for Expression in
Candida
[0437] The gene sequence for the E. coli TESA gene was optimized
for expression in Candida by codon replacement. A new TESA gene
sequence was constructed using codons from Candida with similar
usage frequency for each of the codons in the native E. coli TESA
gene (avoiding the use of the CTG codon due to the alternative
yeast nuclear genetic code utilized by Candida). The optimized TESA
gene was synthesized with flanking BspQI restriction sites and
provided in vector pIDTSMART-Kan (Integrated DNA Technologies). The
vector was designated as pAA287. Plasmid pAA287 was cut with BspQI
and the 555 bp DNA fragment was gel purified. Plasmid pAA073 also
was cut with BspQI and the linear DNA fragment was gel purified.
The two DNA fragments were ligated together to place the optimized
TESA gene under the control of the Candida POX4 promoter. The
resulting plasmid was designated pAA294.
Example 24
Cloning of Candida DGA1 Gene
[0438] DGA1 PCR product was amplified from Candida 20336 genomic
DNA using primers oAA996 and oAA997, shown in the table below. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
of the DGA1 gene was designated pAA299.
TABLE-US-00018 Primer Sequence oAA996
ATGACTCAGGACTATAAAGACGATAGTCCTACGTCCACTGAGT TG oAA997
CTATTCTACAATGTTTAATTCAACATCACCGTAGCCAAACCT
Example 25
Cloning of Candida LRO1 Gene
[0439] LRO1 PCR product was amplified from Candida 20336 genomic
DNA using primers oAA998 and oAA999, shown in the table below. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
of the LRO1 gene was designated pAA300.
TABLE-US-00019 Primer sequence oAA998 ATGTCGTCTTTAAAGAACAGAAAATC
oAA999 TTATAAATTTATGGCCTCTACTATTTCT
Example 26
Cloning of Candida ACS1 Gene and Construction of Deletion
Cassette
[0440] ACS1 PCR product was amplified from Candida 20336 genomic
DNA using primers oAA951 and oAA952, shown in the table below. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm the DNA sequence. One such plasmid was designated pAA275.
Plasmid pAA280 was digested with BamHI to release a 2.0 kb
P.sub.URA3URA3T.sub.URA3P.sub.URA3 cassette. Plasmid pAA275 was
digested with BglII and gel purified. The two pieces were ligated
together to generate plasmid pAA276 and pAA282. Plasmid pAA276 and
pAA282 have the P.sub.URA3URA3T.sub.URA3P.sub.URA3 cassette
inserted into the ACS gene in opposite orientations.
TABLE-US-00020 Primer sequence oAA951
CCTACTTCCACAGCTTTAATCTACTATCAT oAA952
TTTAAGAAAACAACTAAGAGAAGCCAC
Example 27
Construction of Strain sAA722 (pox4a::ura3/pox4b::ura3 POX5/POX5
ACS1/acs1:: P.sub.URA3URA3T.sub.URA3P.sub.URA3)
[0441] Plasmid pAA276 was digested with BamHI/XhoI and column
purified. Strain sAA329 (ura3/ura3 pox4a::ura3/pox4b::ura3
POX5/POX5) was transformed with the linearized DNA and plated on
SCD-ura plate. Several colonies were checked for ACS1 disruption.
One such strain was designated sAA722.
Example 28
Construction of Strain sAA741 (pox4a::ura3/pox4b::ura3 POX5/POX5
ACS1/acs/::P.sub.URA3)
[0442] Strain sAA722 was grown in YPD media overnight and plated on
5-FOA plate. Colonies that grew in the presence of 5-FOA were PCR
screened for the looping out of the URA3 gene leaving behind only
the URA3 promoter (P.sub.URA3) in the ACS1 site. Out of 30 colonies
analyzed, only one strain showed the correct genetic modification.
The strain was designated sAA741.
Example 29
Construction of Strain sAA776 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3URA3T.sub.URA3P.sub.URA3/acs1::P.sub.URA3)
[0443] Plasmid pAA282 was digested with BamHI/XhoI and column
purified. Strain sAA741 (see Example 28) was transformed with the
linearized DNA and plated on SCD-ura plate. Several colonies were
checked for double ACS1 knockout by insertional inactivation. One
such strain was designated sAA776.
Example 30
Construction of Strain sAA779 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1:: P.sub.URA3)
[0444] Strain sAA776 (see Example 29) was grown in YPD media
overnight and plated on 5-FOA plates. Colonies that grew in the
presence of 5-FOA were PCR screened for the looping out of the URA3
gene leaving behind only the URA3 promoter (P.sub.URA3) in both
ACS1 copies. One such strain was designated sAA779.
Example 31
Construction of Strain sAA811 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1:: P.sub.URA3
ura3::3.times.P.sub.POX4P450A19)
[0445] Plasmid pAA156 containing a P450A19 integration cassette was
digested with ClaI and column purified. Strain sAA779 (see Example
30) was transformed with the linearized DNA and plated on SCD-ura
plate. Several colonies were checked for P450A19 integration. From
those colonies, qPCR was performed to check the copy number of
P450A19 integration. One strain, designated sAA811, contained 3
copies of P450A19.
Example 32
Construction of Strain sAA810 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1:: P.sub.URA3 ura3::5.times.P.sub.POX4P450A19
ura3::8.times.P.sub.POX4TESA)
[0446] Plasmid pAA156 containing a P450-A19 integration cassette
was digested with ClaI and column purified. Plasmid pAA294
containing a TESA integration cassette also was digested with ClaI
and column purified. Strain sAA779 was cotransformed with both
linearized DNAs and plated on SCD-ura plate. Several colonies were
checked for both P450A19 integration and TESA integration. Colonies
that were positive for both TESA and P450A19 were further analyzed
by qPCR. qPCR was performed to check the copy number of the P450A19
and TESA integration events. One strain, designated sAA810,
contained 5 copies of P450A19 and 8 copies of TESA.
Example 33
General Techniques & Methods
Growth Media, Reagents and Conditions
[0447] YPD, ScD-ura media and plates, and 5-FOA containing plates
were made as described in Methods in Yeast Genetics: a Cold Spring
Harbor Laboratory Manual/David C. Amberg, Daniel J. Burke, Jeffrey
Strathern, -2005 ed.).
[0448] SP92+ glycerol was made by adding 6.7 g of Bacto yeast
nitrogen base without amino acids (BD, Franklin Lakes, N.J., USA),
3.0 g of Bacto yeast extract (BD, Franklin Lakes, N.J., USA), 3.0 g
of ammonium sulfate, 1.0 g of potassium phosphate monobasic, 1.0 g
of potassium phosphate dibasic, and 75 g of glycerol to water to a
final volume of one liter. The media was then filtered
sterilized.
[0449] TB-low N Media was made by adding 1.7 g Bacto yeast nitrogen
base without ammonium sulfate, 3 g of Bacto yeast extract, 1 g of
potassium phosphate monobasic and 1 g potassium phosphate dibasic
per liter of water. The media was filtered sterilized.
[0450] Overnight cultures were typically grown in 2 to 5 ml of
either ScD-ura media or YPD media in standard culture tubes at 30 C
on a shaker at about 250 rpm.
Molecular Methods
[0451] Gel purifications of DNA fragments were done as recommended
by the manufacturer using either the GeneJET Gel Extraction Kit
(Fermentas Inc, Glen Burnie, Md. USA) or the Zymoclean Gel DNA
Recovery Kit (ZymoResearch, Irvine, Calif.).
[0452] PCR was performed using either PFU Ultra II DNA Polymerase
(Agilent Technologies, Santa Clara, Calif.), Taq DNA polymerase
(New England Biolabs, Ipswich, Mass., USA), DreamTaq PCR Master Mix
(Fermentas Inc, Glen Burnie, Md. USA) or Quick Load Midas Mix
(Monserate, San Diego, Calif. USA). Each enzyme was used according
to the manufacturer's instructions.
[0453] Restriction enzyme digestions were conducted as recommended
by each manufacturer (New England Biolabs, Ipswich, Mass., USA or
Fermentas Inc, Glen Burnie, Md. USA). DNA ligations were conducted
using either the Rapid Ligation Kit (Fermentas Inc, Glen Burnie,
Md. USA) or using T4 DNA Ligase (New England Biolabs, Ipswich,
Mass., USA) according to the manufacturer's instructions.
[0454] Yeast transformations were performed as described in Example
10.
[0455] Genomic DNA Preparation
[0456] The URA3 gene was obtained from genomic DNA of Candida yeast
culture ATCC20336. Genomic DNA from Candida strain ATCC20336 was
prepared as follows: About 1.5 ml of an overnight culture of cells
was and the pellet was resuspended in about 200 .mu.l of a solution
containing 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0,
and 1 mM EDTA. About 200 .mu.l of acid washed glass beads were
added with about 200 .mu.l of phenol:chloroform:isoamyl alcohol
(25:24:1) at a pH of about 8.0. The sample was vortexed for about 2
minutes after which about 200 .mu.l of water was added. The sample
was then centrifuged at 13000 rpm for about 10 minutes. The aqueous
layer was transferred to a new microcentrifuge tube and an equal
volume of chloroform:isoamyl alcohol (24:1) solution was added.
This sample was vortexed for 10 seconds and then centrifuged at
13000 rpm for about 2 minutes. The aqueous layer was transferred to
a new microfuge tube and 1 ml of ethanol was added. The tube was
then placed at -80.degree. C. for about 15 minutes and then spun at
13000 rpm for 15 minutes to pellet the DNA The DNA was washed with
70% ethanol and air-dried. The DNA was then resuspended in about
500 .mu.l of water.
[0457] Genomic DNA for Klyveromyces lactis (ATCC8585) was purchased
from the American Type Culture Collection (Manassas, Va., USA).
[0458] To calculate gene copy number, a qPCR method was used as
described by Jin et al (Appl. Environ. Microbiol. January 2003 vol.
69, no. 1, 495-503). qPCR was performed according to the
manufacturer's instructions using either the Brilliant III
Ultra-Fast SYBR.RTM. Green QPCR Master Mix (Agilent Technologies,
Englewood, Colo. USA) or the QuantiTect Multiplex PCR NoROX Kit
(Qiagen). Genomic DNA from Candida strain ATCC20336 or plasmid DNA
containing the actin gene from ATCC20336 and the gene of interest
were used as standards.
[0459] Primers and probes used throughout these Examples were made
via standard DNA synthesis techniques by Integrated DNA
Technologies (Coralville, Iowa, USA).
Example 34
Construction of Cloning Plasmid AA073
[0460] The plasmid pAA073 was designed to contain the POX4 promoter
and terminator from Candida strain ATCC20336 (this strain is also
referred to herein as strain sAA001). This plasmid was derived from
the publicly available plasmid pUC19 which contains an ampicillin
resistance marker. pAA073 was designed to have two SapI restriction
enzyme sites located between the POX4 promoter and terminator which
allows unidirectional cloning of any gene of interest in tandem
with the POX4 promoter. The Candida strain ATCC20336 URA3 gene
including the open reading frame and the endogenous regulatory
regions was also placed into pAA073 as a selection marker for
transformants. Plasmid pAA073 allows the direct integration of
multiple copies of any gene of interest by digesting the plasmid
with a unique restriction enzyme such as SpeI, ClaI or BstZ171.
These multiple cloning sites for are contained in the URA3
auxotrophic marker region and can be selectively be used to avoid
cutting the gene of interest (i.e., the DNA sequence for the gene
of interest can be searched for particular restriction enzyme cut
sites and those enzymes can be avoided). In addition, this plasmid
can serve as a template to generate an antibiotic free-DNA cassette
containing the gene of interest and the PDX 4 regulatory regions
inserted between the 3' and 5' regions of the URA3 gene; this
cassette can be PCR amplified using the plasmid as a template, and
the isolated PCR product can be inserted into any microorganism
strain.
[0461] A diagram of pAA073 is set forth in FIG. 18 and the sequence
of pAA073 is set forth as SEQ ID NO. 3704.
Example 35
Cloning Enoyl-CoA Isomerase (ECI) Genes from ATCC 20336
[0462] The amino acid sequence for Eci1 (i.e. Eci) from S.
cerevisiae S288c (SEQ ID NO. 3705) was used to identify homologs
from Candida species ATCC MYA-3404 and ATCC20336. The BLAST search
revealed two Eci1p homologs in each strain of Candida, which have
been named Eci1p and Eci2p (see TABLE 1). The percent amino acid
identities for the homologs are shown below:
TABLE-US-00021 TABLE 1 Amino acid percent identity Eci2p_MYA-
Eci1p_MYA- Eci1p_S.c. Eci2p_20336 Eci1p_20336 3404 SEQ ID 3404 SEQ
ID SEQ ID SEQ ID SEQ ID NO. 3707 NO. 3706 NO. 3705 NO. 3709 NO.
3708 Eci2p_MYA- 58 36 84 57 3404 SEQ ID NO. 3707 Eci1p_MYA- 39 57
92 3404 SEQ ID NO. 3706 Eci1p_S.c. 37 40 SEQ ID NO. 3705
Eci2p_20336 57 SEQ ID NO. 3709 Eci1p_20336 SEQ ID NO. 3708
[0463] The ECI1 gene encoding the N-terminal 241 residues of SEQ ID
NO. 3708 was amplified from genomic DNA (ATCC 20336) using
oligonucleotides oAA2835 (SEQ ID NO. 3712) and oAA2836 (SEQ ID NO.
3713) that also incorporated unique SapI restriction sites. The 770
bp PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Life Technologies), transformed into competent TOP10 E. coli cells
(Life Technologies) and clones containing PCR inserts were
sequenced to confirm the correct DNA sequence. One such plasmid was
named pAA574 (SEQ ID NO. 3710).
[0464] The full length ECI2 gene encoding Eci2p (SEQ ID NO. 3709)
was amplified from genomic DNA (ATCC 20336) using oligonucleotides
oAA2837 (SEQ ID NO. 3714) and oAA2838 (SEQ ID NO. 3715) that also
incorporated unique SapI restriction sites. The 851 bp PCR product
was gel purified and ligated into pCR-Blunt II-TOPO (Life
Technologies), transformed into competent TOP10 E. coli cells (Life
Technologies) and clones containing PCR inserts were sequenced to
confirm the correct DNA sequence. One such plasmid was named pAA575
(SEQ ID NO. 3711).
Example 36
Generation of Strain sAA1764 (ura3/ura3 pox4a::ura3/pox4b::ura3
POX5/POX5 acs1:: PURA3/acs1:: PURA3
fat1-.DELTA.1::PURA3/fat1-.DELTA.62::PURA3
eci1-.DELTA.1::URA3/ECI1)
[0465] Deletion of the first allele of ECI1 was achieved by
transforming cells (strain sAA886 (pox4a::ura3/pox4b::ura3
POX5/POX5 acs1:: PURA3/acs1:: PURA3
fat1-1::PURA3/fat1-.DELTA.2::PURA3 ura3/ura3)) with linear DNA
cassettes constructed by overlap extension PCR (OE-PCR). A deletion
cassette for the first ECI1 allele in strain sAA886 was generated
from three DNA fragments. A first DNA fragment (ECI1 5' homology)
was amplified from ATCC20336 gDNA using primers oAA3085 (SEQ ID NO.
3716) and oAA3086 (SEQ ID NO. 3717). A second DNA fragment
(PURA3URA3TURA3PURA3) was amplified from plasmid pAA298 (FIG. 29,
and SEQ ID NO: 3784) using primers oAA3087 (SEQ ID NO. 3718) and
oAA3088 (SEQ ID NO. 3719). The third DNA fragment (ECI1 3'
homology) was amplified from ATCC20336 gDNA using primers oAA3089
(SEQ ID NO. 3720) and oAA3090 (SEQ ID NO. 3721). All three DNA
fragments were combined in the same reaction to generate the
full-length deletion cassette (FIG. 19) by OE-PCR using primers
oAA3085 (SEQ ID NO. 3716) and oAA3090 (SEQ ID NO. 3721).
[0466] Strain sAA886 was transformed with the full-length deletion
cassette and plated on SCD-Ura plate. Several colonies were
screened by PCR for integration of the deletion cassette at the
first ECI1 allele. One such strain was named sAA1764.
Example 37
Generation of Strain sAA1860 (ura3/ura3 pox4a::ura3/pox4b::ura3
POX5/POX5 acs1:: PURA3/acs1:: PURA3
fat1-.DELTA.1::PURA3/fat1-.DELTA.2::PURA3
eci1-.DELTA.1::PURA3/ECI1)
[0467] Strain sAA1764 was grown in YPD media overnight and plated
on 5-FOA plate. Colonies that grew in the presence of 5-FOA were
PCR screened for the looping out of the URA3 gene leaving behind
only the URA3 promoter (PURA3) in the first ECI1 allele. One such
strain was named sAA1860.
Example 38
Construction of a Double ECI1 Knockout Strain (Ura3/Ura3
Pox4a::Ura3/Pox4b::Ura3 POX5/POX5 acs1:: PURA3/acs1:: PURA3
fat1-.DELTA.1::PURA3/fat1-.DELTA.2::PURA3
eci1-.DELTA.1::PURA3/eci1-.DELTA.2::URA3)
[0468] Deletion of the second allele of ECI1 is achieved by
transforming cells with linear DNA cassettes constructed by overlap
extension PCR (OE-PCR). A deletion cassette for the second ECI1
allele in sAA1860 (ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5
acs1:: PURA3/acs1:: PURA3 fat1-.DELTA.1::PURA3/fat1-.DELTA.2::PURA3
eci1-.DELTA.1::PURA3/ECI1) was generated from three DNA fragments.
A first DNA fragment (ECI1 5' homology) was amplified from
ATCC20336 gDNA using primers oAA3212 (SEQ ID NO. 3722) and oAA3213
(SEQ ID NO. 3723). A second DNA fragment (PURA3URA3TURA3PURA3) was
amplified from plasmid pAA298 (FIG. 29, and SEQ ID NO: 3784) using
primers oAA3214 (SEQ ID NO. 3724) and oAA3215 (SEQ ID NO. 3725). A
third DNA fragment (ECI1 3' homology) was amplified from ATCC20336
gDNA using primers oAA3216 (SEQ ID NO. 3726) and oAA3217 (SEQ ID
NO. 3727). All three DNA fragments were combined in the same
reaction to generate the full-length deletion cassette (FIG. 20) by
OE-PCR using primers oAA3212 (SEQ ID NO. 3722) and oAA3217 (SEQ ID
NO. 3727).
[0469] To generate a double ECI1 knockout strain, sAA1860 is
transformed with the full-length deletion cassette and plated on
SCD-Ura plate. Several colonies are screened by PCR for integration
of the deletion cassette at the second ECI1 allele.
Example 39
Cloning of Acyl CoA Oxidase Proteins
[0470] Acyl-CoA oxidases from a range of organisms were cloned into
the E. coli expression vector pET26b (EMD4Biosciences, Darmstadt,
Germany), which contains a kanamycin resistance cassette. The
source of the acyl-CoA oxidase, the name of the gene, the primers
and restriction enzymes used to clone the acyl CoA oxidase coding
sequence into pET26b and the coding sequence are described herein.
The acyl CoA oxidase coding sequences were amplified by PCR using
the appropriate primers designed from cDNA libraries, published
cDNA or genomic DNA sequences corresponding to the organism. In the
event that a template source was not available, the coding
sequences were synthesized as gBlocks (IDT) and stitched together
by standard overlap extension PCR. The PCR products were then
cloned into pCRII-Blunt TOPO vector (Life Sciences) and the
products were sequenced to verify that they did not contain
undesired mutations. The coding sequences were released from the
TOPO vector using the appropriate restriction enzymes and ligated
into pET26b that had been digested with the same restriction
enzymes. The resulting expression plasmids were then transformed
into Rosetta II BL21 cells (Novagen).
Example 40
Expression of Acyl-CoA Oxidases in E. Coli
[0471] To express an enzyme, a colony from each transformation of
Rosetta cells was used to start a 5 ml overnight culture of LB
containing the antibiotics kanamycin (to select for pET26b) and
chloramphenicol (to select for a second plasmid found in Rosetta II
cells that mediates improved translation of eukaryotic proteins
expressed in E. coli) grown at 37.degree. C. The next morning, the
overnight culture was used to seed 30 ml of LB containing kanamycin
and chloramphenicol to an OD.sub.600 reading of 0.1. The 30 ml
cultures were grown at 37.degree. C. for 2 hours and then placed on
ice for 10 minutes. To induce expression,
isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the
culture to a final concentration of 0.1 mM. In some cases induction
was performed using Novagen Overnight Express Autoinduction System
1 (CAT#71300-3). The cells were then shaken at 15.degree. C.
overnight to express the acyl CoA oxidase.
Example 41
Acyl-CoA Oxidase Activity Assay
[0472] To test the activity of the acyl CoA oxidase, cells from the
overnight induction were pelleted at 1046.times.g at 4.degree. C.
in 50 ml conical tubes. The cell pellets were resuspended in 1 ml
of 50 mM KPO.sub.4, pH 7.6, 50 .mu.M FAD buffer and then
transferred to a 2 ml centrifuge tube. A Misonix Sonicator 3000
(QSonica, Newtown, Conn.) was used to lyse the cells, which were
sonicated at a power setting of 2 for 2 pulses of 20 seconds each.
The lysates were placed on ice for 30 seconds in between each
pulse. To obtain a supernatant, cell debris was pelleted at
16,100.times.g for 10 mins in a 4.degree. C. microcentrifuge. The
supernatant was transferred to a 1.5 ml centrifuge tube and
Bradford assays (Thermoscientific) were performed on cell lysates
according to manufacturer's specifications to determine protein
concentration in preparation for the acyl-CoA oxidase assays. A
Beckman Coulter DTX-800 Multimode Detector spectrophotometer was
used for the assays. The spectrophotometer was set to read for 5
minutes at 500 nm, 30.degree. C. Each reaction was 200 .mu.l in
volume and contained 10 .mu.g of cell lysate in 50 mM KPO.sub.4, pH
7.6, 200 .mu.g/ml BSA, 0.05% Triton X-100, 250 .mu.M fatty acyl-coA
substrate, 50 .mu.M FAD, 10 U horseradish peroxidase, 25 mM
p-hydroxybenzoic acid and 1 mM 4-aminoantipyrine. Fatty acyl-CoA
substrates covered a range from hexanoyl CoA (six carbon chain
length) to oleoyl CoA (eighteen carbon chain length).
[0473] In TABLE 2 unshaded blocks indicated the sample was not
tested. Dark shading indicates that no activity was detected. Light
shading indicates that minimal activity (i.e. poor activity) was
detected at less than or equal to 0.1 umol/min/ug (umol
substrate/minute/ug total protein). Medium shading indicates that
good activity was detected at >0.1 umol/min/ug.
[0474] The results in TABLE 2 indicated that several enzymes are
not functional when expressed in E. coli. Furthermore, the
remaining enzymes that are functional when expressed in E. coli
showed broad substrate specificity or were similar in their
substrate specificity to Pox5 from Candida strain ATCC20336 (i.e.
not very active on a C6 substrate, show peak activity on a C12
substrate and are active from C8 all the way to C18:1).
TABLE-US-00022 TABLE 2 Activity of Heterologous Acyl CoA Oxidases
##STR00001## ##STR00002##
Example 42
Genetic Modification of Candida Pox4, Pox5 and R. norvegicus
VLCAD
[0475] The objective was to design mutations in 1) the Pox4 and
Pox5 acyl CoA oxidases of Candida strain ATCC20336 (Pox4 and Pox5,
respectively) to alter their respective substrate specificities and
2) the R. norvegicus very long chain acyl-CoA dehydrogenase (VLCAD)
to convert it into an acyl CoA oxidase. When introduced into
Candida, these mutant enzymes may mediate selective conversion of
fatty acid substrates to sebacic, dodecanedioic acid or longer
chain diacids by beta oxidation.
[0476] Site-Directed Mutagenesis of Pox4 and Pox5-Methodology
[0477] Several approaches were used to identify regions and/or
residues of Pox4 and Pox5 of Candida strain ATCC20336 that
determine the substrate specificities of these enzymes. In rat
liver, a single gene with an alternatively spliced third exon
produces two spliceoforms, AcoI (acyl CoA oxidase-I, R. norvegicus,
RnAcoI) and AcoII (acyl CoA oxidase-II, R. norvegicus, RnAcoII),
which are identical in amino acid length and differ in amino acid
sequence only at the region encoded by the differentially spliced
exon (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta,
S., Kagamiyama, H., Osumi, T., Hashimoto, T. (1987) Complete
nucleotide sequence of cDNA and predicted amino acid sequence of
rat acyl-CoA oxidase. J. Biol. Chem. 262(17):8138-43.; Osumi, T.,
Ishii, N., Miyazawa, S., Hashimoto, T. (1987) Isolation and
structural characterization of the rat acyl-CoA oxidase gene. J.
Biol. Chem. 262 (17):8138-43; Setoyama, C., Tamaoki, H., Nishina,
Y., Shiga, K., Miura, R. (1995) Functional expression of two forms
of rat acyl-CoA oxidase and their substrate specificities. Biochem.
Biophys. Res. Commun. 217(2):482-7). A comparison of the primary
amino acid sequences of AcoI and AcoII revealed differences in
residues 90 to 133 as a result of the alternatively spliced exon
(underlined residues, FIG. 23). The splicing event resulted in two
enzymes, AcoI and AcoII, that display different substrate activity
profiles. RnAcoI prefers substrates with few carbons (e.g., fatty
acids with 8 or 10 carbons). RnAcoII prefers substrates with longer
carbon chains (e.g., 14 carbons). The crystal structure of RnAcoII
has been solved (PDB: 11S2 (without substrate); PDB: 2DDH (with
dodecanoate substrate)) and the region encoded by the alternatively
spliced exon ends at the boundary between the N-terminal alpha
helical domain and the subsequent beta sheet domain, both of which
are characteristic structural features of acyl CoA oxidases
(Acyl-CoA dehydrogenase (ACAD) superfamily, NCBI Conserved Domains
Accession cI0993) and have been identified as a region that may
determine substrate specificity.
[0478] To verify that this region of an acyl CoA oxidase plays a
role in determining substrate specificity, the HotSpot Wizard
algorithm was utilized (Pavelka, A., Chovancova, E., Damborsky, J.
HotSpot Wizard: a Web Server for Identification of Hot Spots in
Protein Engineering, Nucleic Acids Research 37: W376-W383, 2009.
http://loschmidt.chemi.muni.cz/hotspotwizard/). HotSpot Wizard is a
program that identifies regions of a protein for engineering of
substrate specificity or activity. The program utilizes structural,
functional and sequence homology data from numerous databases, such
as PDB, UniProt and NCBI, to identify regions and/or residues that
are "hot spots" for mutagenesis. The search relies on a PDB file
corresponding to a crystal structure of the enzyme of interest. In
the case of Pox4 or Pox5, no such structure was available.
Therefore, the structures of both proteins were determined by
modeling with the crystal structure of R. norvegicus AcoII as the
template (PDB:11S2). The SWISS-MODEL program was used to generate
these models (Arnold K., Bordoli L., Kopp J., and Schwede T.
(2006). The SWISS-MODEL Workspace: A web-based environment for
protein structure homology modelling. Bioinformatics, 22,195-201;
Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The
SWISS-MODEL Repository and associated resources. Nucleic Acids
Research. 37, D387-D392. Peitsch, M. C. (1995) Protein modeling by
E-mail Bio/Technology 13: 658-660; http://swissmodel.expasy.org/).
The resulting models, which were PDB files, were entered in HotSpot
Wizard as the "Query structure". The results of a HotSpot Wizard
analysis are summarized in FIG. 24A, 24B and 25). Residues
highlighted in grey are proposed mutagenic "hot spots". Dark grey
shading indicates residues with greater variability than those with
light grey shading. Residues shown in bold are found within or
close to the substrate binding pocket (discussed below).
[0479] All three enzymes were aligned to show areas of homology
(FIG. 26). In FIG. 26 light grey shading indicates identity between
all three enzymes. Darker shades of grey indicate partial identity
or homology between the three enzymes (e.g., a dark grey shading
may indicate identity for two of the three proteins in the
alignment). In some cases, dark grey shading indicates sequence
similarity (i.e., the residues are similar because they are acidic,
basic, polar or non-polar). The underlined region (FIG. 23 and FIG.
26) indicates the alternatively spliced exon of AcoII.
[0480] Molecular modeling alignments were used to identify residues
in Pox4 and Pox5 that are found within or close to the substrate
binding pocket (residues shown in bold, FIGS. 24A and 24B). The
molecular structure of AcoII complexed with its substrate
dodecanoate (PDB:2DDH) as determined from its crystal structure,
was aligned with the predicted molecular models of Pox4 and Pox5.
Residues located in the N-terminal loop and first part of alpha
helix D (TABLE 3) appear at the surface and lining of the substrate
entry/exit channel. In Pox4 these residues correspond to the
sequence IDTFNK (a.a. 95-100 of Pox4 from Candida strain ATCC20336)
and PDQQAQ (a.a. 80-85 of Pox5 from Candida strain ATCC20336).
According to HotSpot Wizard this entire sequence is category 9,
which means highly variable.
TABLE-US-00023 TABLE 3 N-terminal loop and first part of alpha
helix D Protein Sequence Residue(s) 1IS2 (ACOII) ISDPEE 79-84 ACOI
ISDPEE 79-84 Pox4 IDTFNK 95-100 Pox5 PDQQAQ 80-85
[0481] Residues located in the loop between alpha helices D and E'
form part of the substrate binding pocket. None of these residues
were identified as contact residues for the 12 carbon substrate but
may be contact residues for longer substrates. This stretch of four
amino acids is located within the divergent exon splice site of
ACO-I and ACO-II. In Pox4 and Pox5, three of these amino acids are
highly conserved (TABLE 4). The fourth amino acid is different
(113G in Pox4 & F98 in Pox5). Of the four amino acids in this
region, the divergent residue is closest to the substrate and the
amino acid character of this residue is drastically different
between Pox4 and Pox5. For this reason, this residue is of
particular interest. Additionally, according to HotSpot this
residue is highly variable.
TABLE-US-00024 TABLE 4 Loop between alpha helices D and E' Protein
Sequence Residue(s) 1IS2 (ACOII) RGHP 94-97 ACOI ANFV 94-97 Pox4
PQVG 110-113 Pox5 PQVF 95-98
[0482] The residue D101 is a contact residue for the substrate
carbons 6 through 9 in the 2DDH crystal structure of RnACOII. This
residue is located at the beginning of alpha helix E' which is part
of the substrate binding pocket. Since this is a contact residue
and is located in the small region of sequence that differs between
ACO-I and ACO-II, the corresponding amino acid in Pox4 and Pox5
(TABLE 5) is of interest. This residue is of interest since it
contacts the substrate at carbons 6-9 and ACO-II has lower activity
on substrates of chain-length 6-12 compared to ACO-I. If either
Pox4 or Pox5 is modified at this position to aspartate, it is
expected that there would be a decrease in activity on adipic acid
and lead to increases in yield of larger diacids. This residue has
a score of 6 from HotSpot.
TABLE-US-00025 TABLE 5 Residue making contact with substrate
carbons 6-9 Protein Sequence Residue(s) 1IS2 (ACOII) D 101 ACOI G
101 Pox4 G 117 Pox5 G 102
[0483] Residue F284 is a contact residue for the substrate carbons
10 through 12 in the 2DDH crystal structure of RnACOII. This
residue is conserved between RnAcoI and RnAcoII. The corresponding
amino acid in Pox4 and Pox5 (TABLE 6) is one of the very few
substrate contact residues that differ between Pox4 and Pox5. The
ACOI, ACOII, and Pox4 enzymes all have a large hydrophobic residue
at this location whereas the Pox5 enzyme has a small polar residue.
The HotSpot score for this residue is 9.
TABLE-US-00026 TABLE 6 Residue making contact with substrate
carbons 10-12 Protein Sequence Residue(s) 1IS2 (ACOII) F 284 ACOI F
284 Pox4 M 302 Pox5 T 287
[0484] The loop C-terminal to alpha helix L is much smaller in Pox5
than it is in Pox4 or ACO-1/ACO-II (TABLE 7). This loop appears to
display structural flexibility and may have implications for the
structure of the substrate pocket and how much the
substrate-binding pocket "breathes". The residues in this region
vary in HotSpot analysis, however the residues just downstream of
this region in Pox5 are all highly variable (shown next).
TABLE-US-00027 TABLE 7 Alpha helix L and loop C-terminal to alpha
helix L Protein Sequence Residue(s) 1IS2 (ACOII)
IYDQVRSGKLVGGMVSYLNDLPSQRI 438-469 QPQQVA ACOI
IYDQVRSGKLVGGMVSYLNDLPSQRI 438-469 QPQQVA Pox4
QVISIEDAGKTVRGSTAFLNQLKDYT 473-505 GSNSSKV Pox5 DLLKEPEQKGL
453-463
[0485] The loop between alpha helix L and M does not appear to be
as variable between Pox4 and Pox5 (TABLE 8), although HotSpot
analysis assigns this stretch of residues with scores of 9 with
high variability. It is expected that this loop, including the
previous section mentioned above, is a target for mutagenesis.
TABLE-US-00028 TABLE 8 Loop between a-helix L and M Protein
Sequence Residue(s) 1IS2 (ACOII) VWPTMV 470-475 ACOI VWPTMV 470-475
Pox4 VLNTVA 506-511 Pox5 VLSSVA 464-469
[0486] For both Pox4 and Pox5, the HotSpot Wizard analyses,
combined with molecular modeling alignments, determined that
residues within the same approximate regions are good targets for
mutagenesis. The multiple sequence alignment shows that the
alternatively spliced exon of AcoII overlaps with hot spot residues
in all three acyl CoA oxidases (FIG. 26).
Site-Directed Mutagenesis of Candida Pox4 and Pox5 to Alter
Substrate Specificity--Method
[0487] Using the HotSpot Wizard and molecular modeling results as a
guide, specific amino acids in Pox4 and Pox5 were mutated (i.e.
added, deleted or substituted) by converting primarily polar or
charged residues in the hot spot regions to alanine. TABLES 9A and
9B below show a summary of Candida strain ATCC20336 Pox5 and Pox4
mutations that were made and tested. The summary of the acyl CoA
activity profile associated with some of the mutants in TABLES 9A
and 9B are shown in FIG. 27 (Pox5) and FIG. 28 (Pox4). The number
of carbons in each substrate tested is shown below each bar in FIG.
27 and FIG. 28. Pox5 Mutant I (grey highlight in TABLE 9A) results
from "ACAD-based mutagenesis" (see discussion below).
TABLE-US-00029 TABLE 9A ##STR00003##
TABLE-US-00030 TABLE 9B Pox4 MUTANT POSITION AMINO ACID(S) MUTATION
A 98, 99, 100 FNK AAA B 102, 103 LS AA C 96 D A D 90 R A E 88 R A F
302 M A G 309, 310 RM A H 98 F A I 99 N A J 100 K A K 102 L A L 103
S A CT3 473-505 QVISIEDAGKTVRGSTAF DLLKEPEQKGL LNQLKDYTGSNSSKV
[0488] Pox4 and Pox5 from Candida strain ATCC20336 were cloned into
pET26b for expression in E. coli and assayed for acyl CoA oxidase
activity in vitro. The activity profiles of the genetically
modified Pox4 and Pox5 were compared to the activity profile of the
wild type enzymes. To alter their substrate activity profile, site
directed mutagenesis was performed on several locations in Pox4 and
Pox5. Complementary primers encoding the point mutation(s) were
used to amplify the coding sequences of Pox4 or Pox5 generating two
to four PCR products that were then "stitched" together to
regenerate the entire coding region using overlap extension PCR
(FIG. 22). As shown in FIG. 22, overlap extension PCR was performed
using primers A, B, C and D. Primers B and C are complementary and
contain the introduced genetic modifications (e.g. mutations). PCR
was performed using oligonucleotides A and B to produce a product
with overlap to a PCR product generated using oligonucleotides C
and D. The A-B product was used as a primer for the C-D product,
and vice versa, for overlap extension. Several mutagenic primer
pairs, for example, like the B-C primer pair, were used to produce
mutations at different locations that were "stitched" together,
i.e. A-B, C-D, E-F, etc. to generate an intact, full length coding
region. To produce more of the final product containing the
mutation(s), a PCR using the A primer and the most 3' reverse
primer was performed. Primers A and D were used to amplify the
entire coding sequence of Pox4 and Pox5 and to incorporate the
restriction enzyme sites (RE1 and RE2) for cloning into an E. coli
expression vector. The primers used for the site-directed
mutagenesis for Pox5 (Candida strain ATCC20336) are listed in TABLE
10 to TABLE 19. The primers used for the site-directed mutagenesis
for Pox4 (Candida strain ATCC20336) are listed in TABLE 20 to TABLE
26.
TABLE-US-00031 TABLE 10 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid(s) Mutation
Primer Primer Sequence (5'-3') Sites A PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC A 81, 82 DQ AA
PRIMER B GATCGACAATCTCTGGGCCTGAGCAGCTGGGTACTCGT GCTCAAAG A 81, 82
DQ AA PRIMER C CTTTGAGCACGAGTACCCAGCTGCTCAGGCCCAGAGAT TGTCGATC A
PRIMER D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG B
PRIMER A GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC B 86,
88 RLS ALA PRIMER B GTGGGTCAAAGACACCGAGGATAGCCAAAGCCTGGGC
CTGTTGGTCTGGGTAC B 86, 88 RLS ALA PRIMER C
GTACCCAGACCAACAGGCCCAGGCTTTGGCTATCCTCG GTGTCTTTGACCCAC B PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00032 TABLE 11 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid(s) Mutation
Primer Primer Sequence (5'-3') Sites C PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC C 93, 94 FD AA
PRIMER B GATTCTGGTGAAGACTTGTGGAGCAGCGACACCGAGG ATCGACAATC C 93, 94
FD AA PRIMER C GATTGTCGATCCTCGGTGTCGCTGCTCCACAAGTCTTC ACCAGAATC C
PRIMER D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG D
PRIMER A GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC D 291,
292 DS AA PRIMER B GAATCTACTGGTCATTCTGTAAGCAGCCATCATCATGG
TGACTCTACC D 291, 292 DS AA PRIMER C
GGTAGAGTCACCATGATGATGGCTGCTTACAGAATGA CCAGTAGATTC D PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00033 TABLE 12 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid(s) Mutation
Primer Primer Sequence (5'-3') Sites E PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC E 95, 96 PQ AA
PRIMER B CACCGATTCTGGTGAAGACAGCAGCGTCAAAGACACC GAGGATCG E 95, 96 PQ
AA PRIMER C CGATCCTCGGTGTCTTTGACGCTGCTGTCTTCACCAGA ATCGGTG E PRIMER
D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG F PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC F 294, 295 RM
AA PRIMER B GGTGATGAATCTACTGGTCGCGGCGTAGGAGTCCATC ATCATG F 294, 295
RM AA PRIMER C CATGATGATGGACTCCTACGCCGCGACCAGTAGATTCA TCACC F
PRIMER D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00034 TABLE 13 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites G PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC G 287 T A
PRIMER B GAGTCCATCATCATGGCGACTCTACCACCAATC G 287 T A PRIMER C
GATTGGTGGTAGAGTCGCCATGATGATGGACTC G PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG H PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC H 290, 291 MD
AA PRIMER B CTGGTCATTCTGTAGGAGGCTGCCATCATGGTGACTCT ACC H 290, 291
MD AA PRIMER C GGTAGAGTCACCATGATGGCAGCCTCCTACAGAATGAC CAG H PRIMER
D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00035 TABLE 14 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites I PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC I 284/436 GE EG
PRIMER B CATCATCATGGTGACTCTTTCACCAATCAAAGCCGAG I 284/436 GE EG
PRIMER C CTCGGCTTTGATTGGTGAAAGAGTCACCATGATGATG I 284/436 GE EG
PRIMER D GTTGTTGTCACCTCCCCAGGTACATTGG I 284/436 GE EG PRIMER E
CCAATGTACCTGGGGAGGTGACAACAAC I PRIMER F
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAG Not1 CAGCTTCTTCG J PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC J 291 D G
PRIMER B GGTCATTCTGTAGGAGCCCATCATCATGGTGAC J 291 D G PRIMER C
GTCACCATGATGATGGGCTCCTACAGAATGACC J PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAG Not1 CAGCTTCTTCG
TABLE-US-00036 TABLE 15 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites K PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC K 292 S A
PRIMER B CTGGTCATTCTGTAGGCGTCCATCATCATGGTG K 292 S A PRIMER C
CACCATGATGATGGACGCCTACAGAATGACCAG K PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG L PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC L 93 F A PRIMER
B GTGAAGACTTGTGGGTCTGCGACACCGAGGATCGAC L 93 F A PRIMER C
GTCGATCCTCGGTGTCGCAGACCCACAAGTCTTCAC L PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00037 TABLE 16 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites M PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC M 94 D G PRIMER
B GGTGAAGACTTGTGGGCCAAAGACACCGAGGATC M 94 D G PRIMER C
GATCCTCGGTGTCTTTGGCCCACAAGTCTTCACC M PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG CT1* PRIMER
A CACACAAGGGGAATTGTGAGCGGATAAC XbaI CT1* 102 G D PRIMER B
AACCCAAGTTGACGTCGATTCTGG CT1* 102 G D PRIMER C
CCAGAATCGACGTCAACTTGGGTT CT1* PRIMER D CACACAAACTGGATCCAACCGTTATCG
BamHI *Mutant CT1 produces a smaller fragment that is used to
replace the sequence in between the XbaI nd BamHI sites of wildtype
Pox5 cloned into pET26b.
TABLE-US-00038 TABLE 17 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites N PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC N 86 R A PRIMER
B CAAAGACACCGAGGATCGACAAAGCCTGGGCCTGTTG GTCTGGGTAC N 86 R A PRIMER
C GTACCCAGACCAACAGGCCCAGGCTTTGTCGATCCTCG GTGTCTTTG N PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG O PRIMER A
GITCACTGCCATATGCCTACCGAACTICAAAAAGAAAG Nde1 AGAACTC O 88 S A PRIMER
B CAAAGACACCGAGGATCGCCAATCTCTGGGCCTGTTG O 88 S A PRIMER C
CAACAGGCCCAGAGATTGGCGATCCTCGGTGTCTTTG O PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00039 TABLE 18 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites P PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC P 98 F G PRIMER
B GTTGACACCGATTCTGGTTCCGACTTGTGGGTCAAAGAC P 98 F G PRIMER C
GTCTTTGACCCACAAGTCGGAACCAGAATCGGTGTCAAC P PRIMER D
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG Q PRIMER A
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAG Nde1 AGAACTC Q 83, 85 QAQ
AAA PRIMER B CAAAGACACCGAGGATCGACAATCTAGCCGCAGCTTG
GTCTGGGTACTCGTGCTCAAAG Q 83, 85 QAQ AAA PRIMER C
CTTTGAGCACGAGTACCCAGACCAAGCTGCCGCTAGAT TGTCGATCCTCGGTGTCTTTG Q
PRIMER D CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGC Not1 AGCTTCTTCG
TABLE-US-00040 TABLE 19 Pox5 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites CT2 PRIMERA
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAA Nde1 GAGAACTC CT2 453-463
DLLKE QVISIEDAG PRIMER B CGGCATCTTCAATGCTGATAACTTGCTCTAACCATTG PEQK
KTVRGSTAF GCTTGGCA GL LNQLKDYT GSNSSKV CT2 453-463 DLLKE QVISIEDAG
PRIMER C TGCCAAGCCAATGGTTAGAGCAAGTTATCAGCATTGA PEQK KTVRGSTAF
AGATGCC GL LNQLKDYT GSNSSKV CT2 453-463 DLLKE QVISIEDAG PRIMER D
TCGGCAACGCTGGAGAGAACAACCTTGGAGCTGTTG PEQK KTVRGSTAF GAACCAGTGT GL
LNQLKDYT GSNSSKV CT2 453-463 DLLKE QVISIEDAG PRIMER E
ACACTGGTTCCAACAGCTCCAAGGTTGTTCTCTCCAG PEQK KTVRGSTAF CGTTGCCGA GL
LNQLKDYT GSNSSKV CT2 PRIMER F CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAG
Not1 CAGCTTCTTCG
TABLE-US-00041 TABLE 20 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites A PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA A 98, 99, FNK AAA
PRIMER B CAAAGATACCAATCAAGGACAATCTAGCAGCAGCA 100
GTGTCGATGGATTCTTGTTCTCTG A 98, 99, FNK AAA PRIMER C
CAGAGAACAAGAATCCATCGACACTGCTGCTGCTA 100 GATTGTCCTTGATTGGTATCTTTG A
PRIMER D CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1
AGCAGCGGTTTCATCAGA B PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT
Nde1 AGTGTA B 102, 103 LS AA PRIMER B
GTGGGTCAAAGATACCAATCAAAGCAGCTCTCTTG TTGAAAGTGTCGATG B 102, 103 LS
AA PRIMER C CATCGACACTTTCAACAAGAGAGCTGCTTTGATTG GTATCTTTGACCCAC B
PRIMER D CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1
AGCAGCGGTTTCATCAGA
TABLE-US-00042 TABLE 21 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites C PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA C 96 D A PRIMER B
GGACAATCTCTTGTTGAAAGTAGCGATGGATTCTT GTTCTCTG C 96 D A PRIMER C
CAGAGAACAAGAATCCATCGCTACTTTCAACAAGA GATTGTCC C PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA D
PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA D 90 R A
PRIMER B GTGTCGATGGATTCTTGTTCAGCGTATCTGGCGAT TCTGTTG D 90 R A
PRIMER C CAACAGAATCGCCAGATACGCTGAACAAGAATCCA TCGACAC D PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
TABLE-US-00043 TABLE 22 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites E PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA E 88 R A PRIMER B
GATGGATTCTTGTTCTCTGTAAGCGGCGATTCTGTT GATCTTGAC E 88 R A PRIMER C
GTCAAGATCAACAGAATCGCCGCTTACAGAGAACA AGAATCCATC E PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA F
PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA F 302 M A
PRIMER B GAGTCCAAAACCATCGCGACTCTACCACCCAAC F 302 M A PRIMER C
GTTGGGTGGTAGAGTCGCGATGGTTTTGGACTC F PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
TABLE-US-00044 TABLE 23 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites G PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA G 309, 310 RM A
PRIMER B GTGGACATTCTAGCCAACGCGGCGTAGGAGTCCA AAACCATC G 309, 310 RM
A PRIMER C GATGGTTTTGGACTCCTACGCCGCGTTGGCTAGAA TGTCCAC G PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA H
PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA H 98 F A
PRIMER B CAAGGACAATCTCTTGTTGGCAGTGTCGATGGATT CTTG H 98 F A PRIMER C
CAAGAATCCATCGACACTGCCAACAAGAGATTGTC CTTG H PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
TABLE-US-00045 TABLE 24 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites I PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA I 99 N A PRIMER B
CAATCAAGGACAATCTCTTCGCGAAAGTGTCGATG GATTC I 99 N A PRIMER C
GAATCCATCGACACTTTCGCGAAGAGATTGTCCTT GATTG I PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA J
PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA J 100 K A
PRIMER B CAATCAAGGACAATCTCGCGTTGAAAGTGTCGATG J 100 K A PRIMER C
CATCGACACTTTCAACGCGAGATTGTCCTTGATTG J PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
TABLE-US-00046 TABLE 25 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites K PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA K 102 L A PRIMER B
CAAAGATACCAATCAAGGAGGCTCTCTTGTTGAAA GTGTCG K 102 L A PRIMER C
CGACACTTTCAACAAGAGAGCCTCCTTGATTGGTA TCTTTG K PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA L
PRIMER A GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA L 103 S A
PRIMER B GTCAAAGATACCAATCAAGGCCAATCTCTTGTTGA AAGTG L 103 S A PRIMER
C CACTTTCAACAAGAGATTGGCCTTGATTGGTATCTT TGAC L PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
TABLE-US-00047 TABLE 26 Pox4 (Candida strain ATCC20336) Native
Mutant Amino Introduced Restriction Name Position Acid (s) Mutation
Primer Primer Sequence (5'-3') Sites CT3 PRIMER A
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTT Nde1 AGTGTA CT3 473-505 QVISIE
DLLKEPEQK PRIMER B CAATCCCTTTTGTTCTGGCTCCTTCAACAAGTCCTT DAGKT GL
GACAATTGGCTTACCAA VRGST AFLNQL KDYTGS NSSKV CT3 473-505 QVISIE
DLLKEPEQK PRIMER C GACTTGTTGAAGGAGCCAGAACAAAAGGGATTGG DAGKT GL
TTTTGAACACTGTTGCTGA VRGST AFLNQL KDYTGS NSSKV CT3 PRIMER D
CTTCGAGATGCGGCCGCTTATTACTTGGACAAGAT Not1 AGCAGCGGTTTCATCAGA
[0489] In Vitro Acyl CoA Oxidase Assay
[0490] E. coli lysates were tested for acyl CoA oxidase activity as
described in Example 41.
[0491] In Vitro Activity Assay for Pox4 Mutants
[0492] TABLE 27 shows the acyl CoA oxidase activity profile
associated with Pox4 mutants and TABLE 28 shows the acyl CoA
oxidase activity profile associated with Pox5 mutants. The carbon
length of the substrates tested is indicated above the data as C6
(6 carbons), C8 (8 carbons), 010 (10 carbons), C12 (twelve
carbons), C14 (fourteen carbons), C16 (sixteen carbons) and C18.1
(eighteen carbons). In TABLE 27 and 28, unshaded blocks indicated
the sample was not tested. Dark shading indicates that no activity
was detected. Light shading indicates that minimal activity (i.e.
poor activity) was detected at less than or equal to 0.1
umol/min/ug (umol substrate/minute/ug total protein). Medium
shading indicates that good activity was detected at >0.1
umol/min/ug.
[0493] The Pox4 Mutant C, although displaying good activity across
all substrates tested, demonstrated reduced overall activity for
all substrates (TABLE 27). Pox4 Mutant D showed a similar result.
Activity on C12 and C18:1 substrates was abolished in Pox4 Mutants
B, A, E and G (TABLE 27) and CT3 (not shown).
TABLE-US-00048 TABLE 27 In vitro Activity Assay for Pox4 Mutants
##STR00004## *Indicates a secondary mutation (I692S) that occurred
during PCR for cloning Candida strain ATCC20446 POX5
[0494] In Vitro Activity Assay for Pox5 Mutants--Results
[0495] Acyl CoA oxidase activity was abolished in Pox5 Mutants B,
C, F and M at least on substrates C6, C12 and C18:1 (TABLE 28).
Mutants CT1 and CT2 were also inactive (not shown). Mutants A, E
and I showed no change when compared to the activity of the wild
type protein. However, Pox5 Mutants D, H, G, and J displayed
altered substrate specificity when compared to wild type Pox5. Pox5
mutants D, H and J demonstrated reduced activity on C6 and/or C8
substrates. Pox5 mutant G displayed increased activity on C18:1
substrates.
TABLE-US-00049 TABLE 28 In vitro Activity Assay for Pox5 Mutants
##STR00005##
[0496] Acyl-CoA Dehydrogenase-Based Mutagenesis
[0497] Acyl-CoA oxidases and acyl-CoA dehydrogenases (ACAD) both
utilize similar but distinct mechanisms to catalyze dehydrogenation
of an acyl-CoA substrate to produce a 2-trans-enoyl-CoA, the first
step in 13-oxidation (Arent, S., Pye, V. E., Henriksen, A. (2008).
Structure and function of plant acyl CoA oxidases. Plant Phys.
Biochem. 46:292-301). There are acyl-CoA dehydrogenases of
different classes and they are grouped according to their substrate
specificities: very long, long, medium and short chain (VLCAD,
LOAD, MCAD, SCAD, respectively) (Kim, J. J., Miura, R. (2004).
Acyl-CoA dehydrogenases and acyl CoA oxidases. Structural basis for
mechanistic similarities and differences. Eur. J. Biochem.,
271(3):483-93.). The crystal structures of several of these enzymes
have been solved and these data show structural differences that
very likely contribute to their respective differences in substrate
specificity. The crystal structure of VLCAD (PDB: 3B96) has
revealed regions and amino acid residues of the protein that make
it structurally, and more than likely functionally, different from
MCAD (PDB: 3MDE) (McAndrew, R. P., Wang, Y., Mohsen, A. W., He, M.,
Vockley, J., Kim, J. J. (2008). Structural basis for substrate
fatty acyl chain specificity: crystal structure of human
very-long-chain acyl-CoA dehydrogenase. J. Biol. Chem.
283(14):9435-43). In some cases, a more significant difference is
the location of the catalytic residue. In MCAD, the catalytic
glutamate is located at position 376 on the loop connecting helix J
and K while in LOAD, the catalytic glutamate is at position 255 on
the adjacent helix G (Nandy, A., Kieweg, V., Krautle, F. G., Vock,
P., Kuchler, B., Bross, P., Kim, J. J., Rasched, I., Ghisla, S.
(1996). Medium-long-chain chimeric human Acyl-CoA dehydrogenase:
medium-chain enzyme with the active center base arrangement of
long-chain Acyl-CoA dehydrogenase. Biochemistry, 35(38):12402-11;
Lee, H. J., Wang, M., Paschke, R., Nandy, A., Ghisla, S., Kim, J.
J. (1996). Crystal structures of the wild type and the
Glu376Gly/Thr255Glu mutant of human medium-chain acyl-CoA
dehydrogenase: influence of the location of the catalytic base on
substrate specificity. Biochemistry, 35(38):12412-20).
[0498] The crystal structure of VLCAD (PDB: 3B96) has also revealed
regions and amino acid residues of the protein that make it
structurally, and more than likely functionally, different from
MCAD (PDB: 3MDE) (McAndrew et al., 2008). VLCAD is larger than
other acyl-CoA dehydrogenase proteins and forms a dimer, like a
typical acyl CoA oxidase. Its substrate binding cavity is larger
compared to other acyl-CoA dehydrogenase proteins and resembles an
acyl CoA oxidase substrate binding pocket. The larger and more
spacious pocket is necessary for accommodating the longer fatty
acyl-CoA substrates that it acts upon. However, the crystal
structures of rat AcoII and Arabidopsis thaliana ACX1 (PDB ID:
1WO7) also reveal large substrate binding pockets and this feature
does not necessarily explain the substrate specificities of each
enzyme (Arent et al., 2008). Structural differences between MCAD
and VLCAD offer some insight. At the base of the MCAD substrate
binding pocket, there are two polar/charged residues (Q95 and E99)
that are different from the analogous residues in VLCAD (G175 and
G178). The increased hydrophobicity of the base of the VLCAD
substrate binding pocket may be a factor, in addition to pocket
size and depth, which contributes to substrate specificity. The
corresponding residues in Candida strain ATCC20336 Pox5 are F98 and
G102. Mutant P (F98G) is the mutation that should more closely
reproduce the base of the VLCAD substrate binding pocket.
[0499] A double mutation in MCAD (e.g., E376G, T255E) can change
its substrate specificity profile. This double mutation produced
somewhat of a chimeric enzyme (MLCAD) (Nandy, et. al. 1996). MCAD
has a broad substrate profile (C4-C18) with peak activity at C6 and
C8. LCAD has a similarly broad profile with peak activity at C10
and C12. MLCAD has a more defined substrate profile (C10-C18)
compared to MCAD or LCAD with peak activity at C12. However, the
overall enzymatic activity of the MLCAD was also reduced (V.sub.max
of MLCAD for C12 substrate is approximately 25% of V.sub.max of
LCAD for C12 substrate).
[0500] Based in part on the results of the above studies, Pox5 was
mutated as described in TABLE 14 (Mutant I) to shift its substrate
profile to preferentially act on longer chain substrates.
[0501] VLCAD Mutagenesis
[0502] VLCAD has a substrate profile that is appropriate for
production of longer chain diacids, such as sebacic or
dodecanedioic acid. Activity of the enzyme ranges from acyl
substrates that are 10 carbons to 22 carbons long and peak activity
is on a C16 substrate. However, the enzymatic mechanism of VLCAD
differs from that of a typical acyl CoA oxidase with respect to the
final electron acceptor; in VLCAD, the enzyme is reoxidized by
electron transfer ferroprotein (ETF) and AOXs are reoxidized by
oxygen to produce hydrogen peroxide (Arent et al., 2008; Kim and
Miura, 2004). To accommodate the difference in mechanisms, the
substrate binding pocket of an acyl CoA oxidase, such as A.
thaliana ACX1, is more spacious than that of VLCAD to allow oxygen
into the pocket to act as the final electron acceptor and reoxidize
the flavine adenine dinucleotide, or FAD, cofactor required for
dehydrogenation of the acyl-CoA substrate. ETF performs this
function in a typical acad and reoxidation of FAD by oxygen is
inhibited while substrate is bound (Kumar, N. R., Srivastava, D. K.
(1995). Facile and restricted pathways for the dissociation of
octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase
(MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and
mechanism of suppression of the enzyme's oxidase activity.
Biochemistry, 34(29): 9434-43). This is reflected in the shape of
the substrate binding pocket with respect to FAD. In an acyl CoA
oxidase, FAD is more solvent exposed, but in MCAD, the entire
flavin ring is embedded in the protein and is only accessible to
solvent when substrate is not present (Kim and Miura, 2004). In
order for an acid to have oxidase activity, the substrate binding
pocket must become more solvent accessible and permit oxidation of
the reduced FAD cofactor by oxygen. Mutagenesis studies of MCAD
have identified a residue that can achieve this result. Tyrosine
375 in MCAD, when changed to a lysine, confers significantly
increased (.about.200-fold increase relative to wild type MCAD)
acyl CoA oxidase activity (Zeng, J., Liu, Y., Wu, L., Li, D.
(2007). Mutation of Tyr375 to Lys375 allows medium-chain acyl-CoA
dehydrogenase to acquire acyl CoA oxidase activity. Biochim.
Biophys. Acta, 1774(12): 1628-34).
[0503] Molecular modeling suggests that the mutation increases
solvent accessibility near the FAD moiety in the active site. In
order for VLCAD to function as an acyl CoA oxidase with the
appropriate substrate specificity profile, an analogous mutation in
VLCAD is made. Tyrosine 375 in MCAD corresponds to phenylalanine
461 in human and rat VLCAD. A F461K mutation in VLCAD is tested to
see if it will now have acyl CoA oxidase activity.
Example 43
Nucleotide and Amino Acid Sequences Used for Manipulations
Described Herein
TABLE-US-00050 [0504] SEQ ID NO: Description Sequence SEQ ID NO: 1
Thioesterase MVAAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNAGFQVKA
activity NASAHPKANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAIT Cuphea
TVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIVRDGLVSRQSFLIRSYEI lanceolata Amino
GADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGMCKNDLIWVLTK acid (A.A. Seq)
MQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSV
WAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLHKFDVKTGD
SIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRR
ECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNA
GTNGAISTSTAKTSNGNSAS SEQ ID NO: 2 FAO-13 (fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgt
alcohol oxidase
gacgggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccct
activity)
gctgacaagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagg C.
Tropicalis
gaaaccgtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttg
Nucleotide (Nuc.
accaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaag
Seq)
gacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgcc
aaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaat
gagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacc
caggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttg
tacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccac
actttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactcc
gagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgact
acagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaat
tggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgtt
gactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagct
tctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaatta
ggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttc
tgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgca
gctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaagggg
atcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggcccc
aaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttc
aagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgatttt
ggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagcc
gctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatc
caggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaac
aacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatcca
accaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttg
caggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtcca
caggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgag
gactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttat
ggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgtt
gacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgcca
actgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggt
ttggcagactccttgaagaccaaagccaagttgtag SEQ ID NO: 3 FAO-13 (fatty
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVR alcohol oxidase
TFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPI activity)
KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY C. Tropicalis
PGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVV A.A. Seq
AHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQ
LFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQ
DYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRC
GFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGIL
CEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVS
VVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQAS
FLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEY
DVNKFDRNSILQALLVTADLLYIQGAKRILSPQAVWPIFESDKPKDKRSIKD
EDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGR
LFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ ID NO: 4
FAO-17 (fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgt
alcohol oxidase
gacgggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttcccc
activity)
gccgacaaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagg C.
Tropicalis
gaaaccgtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttg
Nuc. Seq
accaatgttttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaag
gacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgct
aaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaat
gagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacc
caggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttg
tacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccac
actttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactcc
gagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgact
acagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaat
tggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgtt
gactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagct
tctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaatta
ggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttc
tgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgca
gctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggc
atcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggcccc
aaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattc
aagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgatttt
ggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagcc
gctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatc
caggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaac
aacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatcca
accaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttg
caggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtcca
caggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgag
gactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttat
ggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgtt
gacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgcca
actgcaagcggtgccaaccctatggtcaccaccatgactcttgcaagacatgttgcgttaggt
ttggcagactccttgaagaccaaggccaagttgtag SEQ ID NO: 5 FAO-17 (fatty
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRT alcohol
oxidase FTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPI
activity) KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY C.
Tropicalis PGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVV
A.A. Seq AHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQ
LFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQ
DYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRC
GFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGIL
CEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVS
VVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQAS
FLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEY
DVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKD
EDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGR
LFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKI SEQ ID NO: 6
FAO-20 (fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgt
alcohol oxidase
gacgggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccct
activity)
gctgacaagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagg C.
Tropicalis
gaaaccgtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttg
Nuc. Seq
accaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaag
gacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgcc
aaaaggaaattgttcaggttggtttccacgcttaccttggttactttcacgagattggccaat
gagttgcatttgaaagccattcactatccaggaagagaagaccgtgaaaaggcttatgaaacc
caggagattgaccctttcaagtaccagtttatggaaaagccaaagtttgacggcgctgagttg
tacttgccagatattgatgttatcattattggatctggtgccggtgctggtgttgtggcccac
actttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactcc
gagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgact
acagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaat
tggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgtt
gactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagct
tctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaatta
ggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttc
tgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgca
gctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaagggg
atcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggcccc
aaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttc
aagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgatttt
ggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagcc
gctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatc
caggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaac
aacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatcca
accaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttg
caggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtcca
caggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgag
gactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttat
ggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgtt
gacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgcca
actgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggt
ttggcagactccttgaagaccaaagccaagttgtag SEQ ID NO: 7 FAO-20 (fatty
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVR alcohol oxidase
TFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPI activity)
KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY C. Tropicalis
PGREDREKAYETQEIDPFKYQFMEKPKFDGAELYLPDIDVIIIGSGAGAGV A.A. Seq
VAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQ
QLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKA
QDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHR
CGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYG
ILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPV
SVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQA
SFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVE
YDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIK
DEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDG
RLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ ID NO: 8
FAO-2a (fatty
atgaataccttcttgccagacgtgctcgaatacaaacacgtcgacacccttttgttattgtgt
alcohol oxidase
gacgggatcatccacgaaaccacagtcgatcagatcaaggacgccattgctcccgacttccct
activity)
gaggaccagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcaga C.
Tropicalis
gaagccgtctacgacacgatcaacgccaccccaaccgatgccgtgcacatgtgtattgtcttg
Nuc. Seq
accaccgcattggactccagaatcttggcccccacgttgaccaactcgttgacgcctatcaag
gatatgaccttgaaggagcgtgaacaattgttggcctcttggcgtgattccccgattgcggca
aagagaagattgttcagattgatttcctcgcttaccttgacgacgtttacgagattggccagc
gaattgcacttgaaagccatccactaccctggcagagacttgcgtgaaaaggcgtatgaaacc
caggtggttgaccctttcaggtacctgtttatggagaaaccaaagtttgacggcgccgaattg
tacttgccagatatcgacgtcatcatcattggatcaggcgccggtgctggtgtcatggcccac
actctcgccaacgacgggttcaagaccttggttttggaaaagggaaagtatttcagcaactcc
gagttgaactttaatgacgctgatggcgtgaaagagttgtaccaaggtaaaggtgctttggcc
accaccaatcagcagatgtttattcttgccggttccactttgggcggtggtaccactgtcaac
tggtctgcttgccttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtctt
gaatttgctgccgatgaagcctacgacaaagcgcaggattatgtttggaaacaaatgggtgct
tcaacagatggaatcactcactccttggccaacgaagttgtggttgaaggaggtaagaagttg
ggctacaagagcaaggaaattgagcagaacaacggtggccaccctgaccacccatgtggtttc
tgttacttgggctgtaagtacggtattaaacagggttctgtgaataactggtttagagacgca
gctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctcaacaagaatggc
gtcgcttatggtatcttgtgtgaggatgtcgaaaccggagtcaggttcactattagtggcccc
aaaaagtttgttgtttctgctggttctttgaacacgccaactgtgttgaccaactccggattc
aagaacaagcacattggtaagaacttgacgttgcacccagtttccaccgtgtttggtgacttt
ggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctttgttacgaggtt
gctgacttggacggcaagggccacggatgcagaatcgaaaccatcttgaacgctccattcatc
caagcttctttgttgccatggagaggaagtgacgaggtcagaagagacttgttgcgttacaac
aacatggtggccatgttgcttatcacgcgtgataccaccagtggttcagtttctgctgaccca
aagaagcccgacgctttgattgtcgactatgagattaacaagtttgacaagaatgccatcttg
caagctttcttgatcacttccgacatgttgtacattgaaggtgccaagagaatcctcagtcca
cagccatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacgatcaaggacaag
gactatgttgagtggagagccaaggctgctaagatacctttcgacacctacggttctgcatat
gggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggtgctgtt
gatactgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcct
actgccagcggtgccaacccaatgatatccaccatgacctttgctagacagattgcgttaggt
ttggctgactccttgaagaccaaacccaagttgtag SEQ ID NO: 9 FAO-2a (fatty
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPEDQYEEYLKT alcohol
oxidase FTKPSETPGFREAVYDTINATPTDAVHMCIVLITALDSRILAPTLTNSLTPIK
activity) DMTLKEREQLLASWRDSPIAAKRRLFRLISSLTLTTFTRLASELHLKAIHYP C.
Tropicalis GRDLREKAYETQVVDPFRYSFMEKPKFDGAELYLPDIDVIIIGSGAGAGVM A.A.
Seq AHTLANDGFKTLVLEKGKYFSNSELNFNDADGVKELYQGKGALATTNQQ
MFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQ
DYVWKQMGASTDGITHSLANEVVVEGGKKLGYKSKEIEQNNGGHPDHPC
GFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILNKNGVAYGI
LCEDVETGVRFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPV
STVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQA
SLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDY
EINKFDKNAILQAFLITSDMLYIEGAKRILSPQPWVPIFESNKPKEQRTIKDK
DYVEWRAKAAKIPFDTYGSAYGSAHQMSTCRMSGKGPKYGAVDTDGRL
FECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL SEQ ID NO: 10
FAO-2b (fatty
atgaataccttcttgccagacgtgctcgaatacaaacacgtcgatacccttttgttattatgt
alcohol oxidase
gacgggatcatccacgaaaccacagtcgaccagatcagggacgccattgctcccgacttccct
activity)
gaagaccagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcaga C.
Tropicalis
gaagccgtctacgacacgatcaacagcaccccaaccgaggctgtgcacatgtgtattgtattg
Nuc. Seq
accaccgcattggactcgagaatcttggcccccacgttgaccaactcgttgacgcctatcaag
gatatgaccttgaaagagcgtgaacaattgttggctgcctggcgtgattccccgatcgcggcc
aagagaagattgttcagattgatttcctcacttaccttgacgacctttacgagattggccagc
gacttgcacttgagagccatccactaccctggcagagacttgcgtgaaaaggcatatgaaacc
caggtggttgaccctttcaggtacctgtttatggaaaaaccaaagtttgacggcaccgagttg
tacttgccagatatcgacgtcatcatcattggatccggtgccggtgctggtgtcatggcccac
actttagccaacgacgggtacaagaccttggttttggaaaagggaaagtatttcagcaactcc
gagttgaactttaatgatgccgatggtatgaaagagttgtaccaaggtaaatgtgcgttgacc
accacgaaccagcagatgtttattcttgccggttccactttgggcggtggtaccactgttaac
tggtctgcttgtcttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtctt
gaatttgctgccgacgaagcctacgacaaagcacaagactatgtttggaaacaaatgggcgct
tctaccgaaggaatcactcactctttggcgaacgcggttgtggttgaaggaggtaagaagttg
ggttacaagagcaaggaaatcgagcagaacaatggtggccatcctgaccacccctgtggtttc
tgttacttgggctgtaagtacggtattaagcagggttctgtgaataactggtttagagacgca
gctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctccacaataaaggc
gtcgcttatggcatcttgtgtgaggatgtcgagaccggagtcaaattcactatcagtggcccc
aaaaagtttgttgtttctgcaggttctttgaacacgccaacggtgttgaccaactccggattc
aagaacaaacacatcggtaagaacttgacgttgcacccagtttcgaccgtgtttggtgacttt
ggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctctgttacgaagtc
gctgacttggacggcaagggccacggatgcagaatcgagaccatcttgaacgctccattcatc
caagcttctttgttgccatggagaggaagcgacgaggtcagaagagacttgttgcgttacaac
aacatggtggccatgttgcttatcacccgtgacaccaccagtggttcagtttctgctgaccca
aagaagcccgacgctttgattgtcgactatgacatcaacaagtttgacaagaatgccatcttg
caagctttcttgatcacctccgacatgttgtacatcgaaggtgccaagagaatcctcagtcca
caggcatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacaatcaaggacaag
gactatgtcgaatggagagccaaggctgccaagatacctttcgacacctacggttctgcctat
gggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggcgccgtt
gataccgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcct
actgccagcggtgccaacccaatgatctccaccatgacgtttgctagacagattgcgttaggt
ttggctgactctttgaagaccaaacccaagttgtag SEQ ID NO: 11 FAO-2b (fatty
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIRDAIAPDFPEDQYEEYLKT alcohol
oxidase FTKPSETPGFREAVYDTINSTPTEAVHMCIVLTTALDSRILAPTLTNSLTPIK
activity) DMTLKEREQLLAAWRDSPIAAKRRLFRLISSLTLTTFTRLASDLHLRAIHYP C.
Tropicalis A.A. GRDLREKAYETQVVDPFRYSFMEKPKFDGTELYLPDIDVIIIGSGAGAGVM
Seq AHTLANDGYKTLVLEKGKYFSNSELNFNDADGMKELYQGKCALTTTNQQ
MFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQ
DYVWKQMGASTEGITHSLANAVVVEGGKKLGYKSKEIEQNNGGHPDHPC
GFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILHNKGVAYGI
LCEDVETGVKFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPV
STVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQA
SLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDY
DINKFDKNAILQAFLITSDMLYIEGAKRILSPQAWVPIFESNKPKEQRTIKDK
DYVEWRAKAAKIPFDTYGSAYGSAHQMST SEQ ID NO: 12 FAO-18 (fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgt
alcohol oxidase
gacgggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttcccc
activity)
gccgacaaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagg C.
Tropicalis
gaaaccgtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttg
Nuc. Seq
accaatgttttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaag
gacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgct
aaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaat
gagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacc
caggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttg
tacttgccagatattgatgtgatcattattggatctggggccggtgctggtgtcgtggcccac
actttgaccaacgacggcttcaagagtttggttttggaaaagggcagatactttagcaactcc
gagttgaactttgatgacaaggacggggttcaagaattataccaaagtggaggtactttgacc
accgtcaaccagcagttgtttgttcttgctggttccacttttggtggtggtaccactgtcaat
tggtcggcctgtcttaaaacgccattcaaggtgcgtaaggaatggtatgatgagtttggcgtt
gactttgctgccgatgaagcctacgacaaagcacaggattatgtttggcagcaaatgggagct
tctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggcaagaaatta
ggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcatcacagatgcggtttc
tgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgca
gctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggc
atcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggcccc
aaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattc
aagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgatttt
ggcaaagacgttcaagcagatcacttccacaactccatcatgactgctctttgttcagaagcc
gctgatttagacggcaagggtcatggatgcagaattgaaaccatcttgaacgctccattcatc
caggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaac
aacatggtggccatgttacttcttagtcgtgataccaccagtggttccgtttcgtcccatcca
actaaacctgaagcattagttgtcgagtacgacgtgaacaagtttgacagaaactccatcttg
caggcattgttggtcactgctgacttgttgtacattcaaggtgccaagagaatccttagtccc
caaccatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgag
gactatgtcgaatggagagccaaggttgccaagattccttttgacacctacggctcgccttat
ggttcggcgcatcaaatgtcttcttgtcgtatgtcaggtaagggtcctaaatacggtgctgtt
gataccgatggtagattgtttgaatgttcgaatgtttatgttgctgacgctagtcttttgcca
actgctagcggtgctaatcctatggtcaccaccatgactcttgcaagacatgttgcgttaggt
ttggcagactccttgaagaccaaggccaagttgtag SEQ ID NO: 13 FAO-1 (fatty
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRT alcohol
oxidase FTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPI
activity) KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY C.
Tropicalis A.A.
PGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVV Seq
AHTLTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLTTVNQQ
LFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQ
DYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCG
FCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILC
EDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSV
VFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASF
LPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYD
VNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDE
DYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRL
FECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ ID NO: 14
cytochrome P450
atggccacacaagaaatcatcgattctgtacttccgtacttgaccaaatggtacactgtgatt A12
(CYP52A12)
actgcagcagtattagtcttccttatctccacaaacatcaagaactacgtcaaggcaaagaaa
Nuc. Seq
ttgaaatgtgtcgatccaccatacttgaaggatgccggtctcactggtattctgtctttgatc
gccgccatcaaggccaagaacgacggtagattggctaactttgccgatgaagttttcgacgag
tacccaaaccacaccttctacttgtctgttgccggtgctttgaagattgtcatgactgttgac
ccagaaaacatcaaggctgtcttggccacccaattcactgacttctccttgggtaccagacac
gcccactttgctcctttgttgggtgacggtatcttcaccttggacggagaaggttggaagcac
tccagagctatgttgagaccacagtttgctagagaccagattggacacgttaaagccttggaa
ccacacatccaaatcatggctaagcagatcaagttgaaccagggaaagactttcgatatccaa
gaattgttctttagatttaccgtcgacaccgctactgagttcttgtttggtgaatccgttcac
tccttgtacgatgaaaaattgggcatcccaactccaaacgaaatcccaggaagagaaaacttt
gccgctgctttcaacgtttcccaacactacttggccaccagaagttactcccagactttttac
tttttgaccaaccctaaggaattcagagactgtaacgccaaggtccaccacttggccaagtac
tttgtcaacaaggccttgaactttactcctgaagaactcgaagagaaatccaagtccggttac
gttttcttgtacgaattggttaagcaaaccagagatccaaaggtcttgcaagatcaattgttg
aacattatggttgccggaagagacaccactgccggtttgttgtcctttgctttgtttgaattg
gctagacacccagagatgtggtccaagttgagagaagaaatcgaagttaactttggtgttggt
gaagactcccgcgttgaagaaattaccttcgaagccttgaagagatgtgaatacttgaaggct
atccttaacgaaaccttgcgtatgtacccatctgttcctgtcaactttagaaccgccaccaga
gacaccactttgccaagaggtggtggtgctaacggtaccgacccaatctacattcctaaaggc
tccactgttgcttacgttgtctacaagacccaccgtttggaagaatactacggtaaggacgct
aacgacttcagaccagaaagatggtttgaaccatctactaagaagttgggctgggcttatgtt
ccattcaacggtggtccaagagtctgcttgggtcaacaattcgccttgactgaagcttcttat
gtgatcactagattggcccagatgtttgaaactgtctcatctgatccaggtctcgaataccct
ccaccaaagtgtattcacttgaccatgagtcacaacgatggtgtctttgtcaagatgtaa SEQ ID
NO: 15 cytochrome P450
atgactgtacacgatattatcgccacatacttcaccaaatggtacgtgatagtaccactcgct A13
(CYP52A13)
ttgattgcttatagagtcctcgactacttctatggcagatacttgatgtacaagcttggtgct
Nuc. Seq
aaaccatttttccagaaacagacagacggctgtttcggattcaaagctccgcttgaattgttg
aagaagaagagcgacggtaccctcatagacttcacactccagcgtatccacgatctcgatcgt
cccgatatcccaactttcacattcccggtcttttccatcaaccttgtcaatacccttgagccg
gagaacatcaaggccatcttggccactcagttcaacgatttctccttgggtaccagacactcg
cactttgctcctttgttgggtgatggtatctttacgttggatggcgccggctggaagcacagc
agatctatgttgagaccacagtttgccagagaacagatttcccacgtcaagttgttggagcca
cacgttcaggtgttcttcaaacacgtcagaaaggcacagggcaagacttttgacatccaggaa
ttgtttttcagattgaccgtcgactccgccaccgagtttttgtttggtgaatccgttgagtcc
ttgagagatgaatctatcggcatgtccatcaatgcgcttgactttgacggcaaggctggcttt
gctgatgcttttaactattcgcagaattatttggcttcgagagcggttatgcaacaattgtac
tgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtgcacaagtttgctgactac
tacgtcaacaaggctttggacttgacgcctgaacaattggaaaagcaggatggttatgtgttt
ttgtacgaattggtcaagcaaaccagagacaagcaagtgttgagagaccaattgttgaacatc
atggttgctggtagagacaccaccgccggtttgttgtcgtttgttttctttgaattggccaga
aacccagaagttaccaacaagttgagagaagaaattgaggacaagtttggactcggtgagaat
gctagtgttgaagacatttcctttgagtcgttgaagtcctgtgaatacttgaaggctgttctc
aacgaaaccttgagattgtacccatccgtgccacagaatttcagagttgccaccaagaacact
accctcccaagaggtggtggtaaggacgggttgtctcctgttttggtgagaaagggtcagacc
gttatttacggtgtctacgcagcccacagaaacccagctgtttacggtaaggacgctcttgag
tttagaccagagagatggtttgagccagagacaaagaagcttggctgggccttcctcccattc
aacggtggtccaagaatctgtttgggacagcagtttgccttgacagaagcttcgtatgtcact
gtcaggttgctccaggagtttgcacacttgtctatggacccagacaccgaatatccacctaag
aaaatgtcgcatttgaccatgtcgcttttcgacggtgccaatattgagatgtattag SEQ ID
NO: 16 cytochrome P450
atgactgcacaggatattatcgccacatacatcaccaaatggtacgtgatagtaccactcgct A14
(CYP52A14)
ttgattgcttatagggtcctcgactacttttacggcagatacttgatgtacaagcttggtgct
Nuc. Seq
aaaccgtttttccagaaacaaacagacggttatttcggattcaaagctccacttgaattgtta
aaaaagaagagtgacggtaccctcatagacttcactctcgagcgtatccaagcgctcaatcgt
ccagatatcccaacttttacattcccaatcttttccatcaaccttatcagcacccttgagccg
gagaacatcaaggctatcttggccacccagttcaacgatttctccttgggcaccagacactcg
cactttgctcctttgttgggcgatggtatctttaccttggacggtgccggctggaagcacagc
agatctatgttgagaccacagtttgccagagaacagatttcccacgtcaagttgttggagcca
cacatgcaggtgttcttcaagcacgtcagaaaggcacagggcaagacttttgacatccaagaa
ttgtttttcagattgaccgtcgactccgccactgagtttttgtttggtgaatccgttgagtcc
ttgagagatgaatctattgggatgtccatcaatgcacttgactttgacggcaaggctggcttt
gctgatgcttttaactactcgcagaactatttggcttcgagagcggttatgcaacaattgtac
tgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtgcacaagtttgctgactat
tacgtcagcaaggctttggacttgacacctgaacaattggaaaagcaggatggttatgtgttc
ttgtacgagttggtcaagcaaaccagagacaggcaagtgttgagagaccagttgttgaacatc
atggttgccggtagagacaccaccgccggtttgttgtcgtttgttttctttgaattggccaga
aacccagaggtgaccaacaagttgagagaagaaatcgaggacaagtttggtcttggtgagaat
gctcgtgttgaagacatttcctttgagtcgttgaagtcatgtgaatacttgaaggctgttctc
aacgaaactttgagattgtacccatccgtgccacagaatttcagagttgccaccaaaaacact
acccttccaaggggaggtggtaaggacgggttatctcctgttttggtcagaaagggtcaaacc
gttatgtacggtgtctacgctgcccacagaaacccagctgtctacggtaaggacgcccttgag
tttagaccagagaggtggtttgagccagagacaaagaagcttggctgggccttccttccattc
aacggtggtccaagaatttgcttgggacagcagtttgccttgacagaagcttcgtatgtcact
gtcagattgctccaagagtttggacacttgtctatggaccccaacaccgaatatccacctagg
aaaatgtcgcatttgaccatgtcccttttcgacggtgccaacattgagatgtattag SEQ ID
NO: 17 cytochrome P450
atgtcgtcttctccatcgtttgcccaagaggttctcgctaccactagtccttacatcgagtac A15
(CYP52A15)
tttcttgacaactacaccagatggtactacttcatacctttggtgcttctttcgttgaacttt
Nuc. Seq
ataagtttgctccacacaaggtacttggaacgcaggttccacgccaagccactcggtaacttt
gtcagggaccctacgtttggtatcgctactccgttgcttttgatctacttgaagtcgaaaggt
acggtcatgaagtttgcttggggcctctggaacaacaagtacatcgtcagagacccaaagtac
aagacaactgggctcaggattgttggcctcccattgattgaaaccatggacccagagaacatc
aaggctgttttggctactcagttcaatgatttctctttgggaaccagacacgatttcttgtac
tccttgttgggtgacggtattttcaccttggacggtgctggctggaaacatagtagaactatg
ttgagaccacagtttgctagagaacaggtttctcacgtcaagttgttggagccacacgttcag
gtgttcttcaagcacgttagaaagcaccgcggtcaaacgttcgacatccaagaattgttcttc
aggttgaccgtcgactccgccaccgagttcttgtttggtgagtctgctgaatccttgagggac
gaatctattggattgaccccaaccaccaaggatttcgatggcagaagagatttcgctgacgct
ttcaactattcgcagacttaccaggcctacagatttttgttgcaacaaatgtactggatcttg
aatggctcggaattcagaaagtcgattgctgtcgtgcacaagtttgctgaccactatgtgcaa
aaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgttcttgtacgag
ttggctaagcaaaccagagacccaaaggtcttgagagaccagttattgaacattttggttgcc
ggtagagacacgaccgccggtttgttgtcatttgttttctacgagttgtcaagaaaccctgag
gtgtttgctaagttgagagaggaggtggaaaacagatttggactcggtgaagaagctcgtgtt
gaagagatctcgtttgagtccttgaagtcttgtgagtacttgaaggctgtcatcaatgaaacc
ttgagattgtacccatcggttccacacaactttagagttgctaccagaaacactaccctccca
agaggtggtggtgaagatggatactcgccaattgtcgtcaagaagggtcaagttgtcatgtac
actgttattgctacccacagagacccaagtatctacggtgccgacgctgacgtcttcagacca
gaaagatggtttgaaccagaaactagaaagttgggctgggcatacgttccattcaatggtggt
ccaagaatctgtttgggtcaacagtttgccttgaccgaagcttcatacgtcactgtcagattg
ctccaggagtttgcacacttgtctatggacccagacaccgaatatccaccaaaattgcagaac
accttgaccttgtcgctctttgatggtgctgatgttagaatgtactaa SEQ ID NO: 18
cytochrome P450
atgtcgtcttctccatcgtttgctcaggaggttctcgctaccactagtccttacatcgagtac A16
tttcttgacaactacaccagatggtactacttcatccctttggtgcttctttcgttgaacttc
(CYP52A16)
atcagcttgctccacacaaagtacttggaacgcaggttccacgccaagccgctcggtaacgtc
Nuc. Seq
gtgttggatcctacgtttggtatcgctactccgttgatcttgatctacttaaagtcgaaaggt
acagtcatgaagtttgcctggagcttctggaacaacaagtacattgtcaaagacccaaagtac
aagaccactggccttagaattgtcggcctcccattgattgaaaccatagacccagagaacatc
aaagctgtgttggctactcagttcaacgatttctccttgggaactagacacgatttcttgtac
tccttgttgggcgatggtatttttaccttggacggtgctggctggaaacacagtagaactatg
ttgagaccacagtttgctagagaacaggtttcccacgtcaagttgttggaaccacacgttcag
gtgttcttcaagcacgttagaaaacaccgcggtcagacttttgacatccaagaattgttcttc
agattgaccgtcgactccgccaccgagttcttgtttggtgagtctgctgaatccttgagagac
gactctgttggtttgaccccaaccaccaaggatttcgaaggcagaggagatttcgctgacgct
ttcaactactcgcagacttaccaggcctacagatttttgttgcaacaaatgtactggattttg
aatggcgcggaattcagaaagtcgattgccatcgtgcacaagtttgctgaccactatgtgcaa
aaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgttcttgtacgag
ttggctaagcaaactagagacccaaaggtcttgagagaccagttgttgaacattttggttgcc
ggtagagacacgaccgccggtttgttgtcgtttgtgttctacgagttgtcgagaaaccctgaa
gtgtttgccaagttgagagaggaggtggaaaacagatttggactcggcgaagaggctcgtgtt
gaagagatctcttttgagtccttgaagtcctgtgagtacttgaaggctgtcatcaatgaagcc
ttgagattgtacccatctgttccacacaacttcagagttgccaccagaaacactacccttcca
agaggcggtggtaaagacggatgctcgccaattgttgtcaagaagggtcaagttgtcatgtac
actgtcattggtacccacagagacccaagtatctacggtgccgacgccgacgtcttcagacca
gaaagatggttcgagccagaaactagaaagttgggctgggcatatgttccattcaatggtggt
ccaagaatctgtttgggtcagcagtttgccttgactgaagcttcatacgtcactgtcagattg
ctccaagagtttggaaacttgtccctggatccaaacgctgagtacccaccaaaattgcagaac
accttgaccttgtcactctttgatggtgctgacgttagaatgttctaa SEQ ID NO: 19
cytochrome P450
atgattgaacaactcctagaatattggtatgtcgttgtgccagtgttgtacatcatcaaacaactccttgcat
A17 (CYP52A17)
acacaaagactcgcgtcttgatgaaaaagttgggtgctgctccagtcacaaacaagttgtacgacaacg
Nuc. Seq
ctttcggtatcgtcaatggatggaaggctctccagttcaagaaagagggcagggctcaagagta-
caac
gattacaagtttgaccactccaagaacccaagcgtgggcacctacgtcagtattcttttcggcaccagga
tcgtcgtgaccaaagatccagagaatatcaaagctattttggcaacccagtttggtgatttttctttgggca-
a
gaggcacactctttttaagcctttgttaggtgatgggatcttcacattggacggcgaaggctggaagcaca
gcagagccatgttgagaccacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacactt
ccagttgttgaagaagcatattcttaagcacaagggtgaatactttgatatccaggaattgttctttagatt-
ta
ccgttgattcggccacggagttcttatttggtgagtccgtgcactccttaaaggacgaatctattggtatca-
a
ccaagacgatatagattttgctggtagaaaggactttgctgagtcgttcaacaaagcccaggaatacttg
gctattagaaccttggtgcagacgttctactggttggtcaacaacaaggagtttagagactgtaccaagct
ggtgcacaagttcaccaactactatgttcagaaagctttggatgctagcccagaagagcttgaaaagca
aagtgggtatgtgttcttgtacgagcttgtcaagcagacaagagaccccaatgtgttgcgtgaccagtcttt
gaacatcttgttggccggaagagacaccactgctgggttgttgtcgtttgctgtctttgagttggccagaca-
c
ccagagatctgggccaagttgagagaggaaattgaacaacagtttggtcttggagaagactctcgtgttg
agagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaatgaaaccttgcgtattt
acccaagtgtcccaagaaacttcagaatcgccaccaagaacacgacattgccaaggggcggtggttc
agacggtacctcgccaatcttgatccaaaagggagaagctgtgtcgtatggtatcaactctactcatttgg
accctgtctattacggccctgatgctgctgagttcagaccagagagatggtttgagccatcaaccaaaaa
gctcggctgggcttacttgccattcaacggtggtccaagaatctgtttgggtcagcagtttgccttgacgga
agctggctatgtgttggttagattggtgcaagagttctcccacgttaggctggacccagacgaggtgtacc
cgccaaagaggttgaccaacttgaccatgtgtttgcaggatggtgctattgtcaagtttgactag
SEQ ID NO: 20 cytochrome P450
atgattgaacaaatcctagaatattggtatattgttgtgcctgtgttgtacatcatcaaacaactcattgcct-
a A18 (CYP52A18)
cagcaagactcgcgtcttgatgaaacagttgggtgctgctccaatcacaaaccagttgtacgacaacgtt
Nuc. Seq
ttcggtatcgtcaacggatggaaggctctccagttcaagaaagagggcagagctcaagagtaca-
acg
atcacaagtttgacagctccaagaacccaagcgtcggcacctatgtcagtattctttttggcaccaagatt
gtcgtgaccaaggatccagagaatatcaaagctattttggcaacccagtttggcgatttttctttgggcaag
agacacgctctttttaaacctttgttaggtgatgggatcttcaccttggacggcgaaggctggaagcatag
cagatccatgttaagaccacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacacttc
cagttgttgaagaagcatatccttaaacacaagggtgagtactttgatatccaggaattgttctttagattt-
ac
tgtcgactcggccacggagttcttatttggtgagtccgtgcactccttaaaggacgaaactatcggtatcaa
ccaagacgatatagattttgctggtagaaaggactttgctgagtcgttcaacaaagcccaggagtatttgt
ctattagaattttggtgcagaccttctactggttgatcaacaacaaggagtttagagactgtaccaagctgg
tgcacaagtttaccaactactatgttcagaaagctttggatgctaccccagaggaacttgaaaagcaag
gcgggtatgtgttcttgtatgagcttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttg
aacatcttgttggcaggaagagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaac
ccacacatctgggccaagttgagagaggaaattgaacagcagtttggtcttggagaagactctcgtgttg
aagagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaacgaaaccttgcgtgttt
acccaagtgtcccaagaaacttcagaatcgccaccaagaatacaacattgccaaggggtggtggtcc
agacggtacccagccaatcttgatccaaaagggagaaggtgtgtcgtatggtatcaactctacccactta
gatcctgtctattatggccctgatgctgctgagttcagaccagagagatggtttgagccatcaaccagaaa
gctcggctgggcttacttgccattcaacggtgggccacgaatctgtttgggtcagcagtttgccttgaccga
agctggttacgttttggtcagattggtgcaagagttctcccacattaggctggacccagatgaagtgtatcc
accaaagaggttgaccaacttgaccatgtgtttgcaggatggtgctattgtcaagtttgactag
SEQ ID NO: 21 cytochrome P450
atgctcgatcagatcttacattactggtacattgtcttgccattgttggccattatcaaccagatcgtggctc-
a A19 (CYP52A19)
gtcaggaccaattatttgatgaagaaattgggtgctaagccattcacacacgtccaacgtgacgggtggt
Nuc. Seq
tgggcttcaaattcggccgtgaattcctcaaagcaaaaagtgctgggagactggttgatttaat-
catctccc
gtttccacgataatgaggacactttctccagctatgcttttggcaaccatgtggtgttcaccagggaccccg
agaatatcaaggcgcttttggcaacccagtttggtgatttttcattgggcagcagggtcaagttcttcaaac
cattattggggtacggtatcttcacattggacgccgaaggctggaagcacagcagagccatgttgagac
cacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcat
atccttaaacacaagggtgagtactttgatatccaggaattgttctttagatttactgtcgactcggccacg-
g
agttcttatttggtgagtccgtgcactccttaaaggacgaggaaattggctacgacacgaaagacatgtct
gaagaaagacgcagatttgccgacgcgttcaacaagtcgcaagtctacgtggccaccagagttgcttta
cagaacttgtactggttggtcaacaacaaagagttcaaggagtgcaatgacattgtccacaagtttacca
actactatgttcagaaagccttggatgctaccccagaggaacttgaaaagcaaggcgggtatgtgttctt
gtatgagcttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttgaacatcttgttggca
ggaagagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaacccacacatctgggc
caagttgagagaggaaattgaacagcagtttggtcttggagaagactctcgtgttgaagagattacctttg
agagcttgaagagatgtgagtacttgaaggccgtgttgaacgaaactttgagattacacccaagtgtccc
aagaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggccccaacggcaagga
tcctatcttgatcaggaaggatgaggtggtgcagtactccatctcggcaactcagacaaatcctgcttatta
tggcgccgatgctgctgattttagaccggaaagatggtttgaaccatcaactagaaacttgggatgggctt
tcttgccattcaacggtggtccaagaatctgtttgggacaacagtttgctttgactgaagccggttacgttt-
tg
gttagacttgttcaggagtttccaaacttgtcacaagaccccgaaaccaagtacccaccacctagattgg
cacacttgacgatgtgcttgtttgacggtgcacacgtcaagatgtcatag SEQ ID NO: 22
cytochrome P450
atgctcgaccagatcttccattactggtacattgtcttgccattgttggtcattatcaagcagatcgtggctc-
a A20 (CYP52A20)
gccaggaccaattatttgatgaagaagttgggcgctaagccattcacacatgtccaactagacgggtgg
Nuc. Seq
tttggcttcaaatttggccgtgaattcctcaaagctaaaagtgctgggaggcaggttgatttaa-
tcatctccc
gtttccacgataatgaggacactttctccagctatgcttttggcaaccatgtggtgttcaccagggaccccg
agaatatcaaggcgcttttggcaacccagtttggtgatttttcattgggaagcagggtcaaattcttcaaac
cattgttggggtacggtatcttcaccttggacggcgaaggctggaagcacagcagagccatgttgagac
cacagtttgccagagagcaagttgctcatgtgacgtcgttggaaccacatttccagttgttgaagaagcat
attcttaagcacaagggtgaatactttgatatccaggaattgttctttagatttaccgttgattcagcgacg-
ga
gttcttatttggtgagtccgtgcactccttaagggacgaggaaattggctacgatacgaaggacatggctg
aagaaagacgcaaatttgccgacgcgttcaacaagtcgcaagtctatttgtccaccagagttgctttaca
gacattgtactggttggtcaacaacaaagagttcaaggagtgcaacgacattgtccacaagttcaccaa
ctactatgttcagaaagccttggatgctaccccagaggaacttgaaaaacaaggcgggtatgtgttcttgt
acgagcttgccaagcagacgaaagaccccaatgtgttgcgtgaccagtctttgaacatcttgttggctgg
aagggacaccactgctgggttgttgtcctttgctgtgtttgagttggccaggaacccacacatctgggcca
agttgagagaggaaattgaatcacactttgggctgggtgaggactctcgtgttgaagagattacctttgag
agcttgaagagatgtgagtacttgaaagccgtgttgaacgaaacgttgagattacacccaagtgtccca
agaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggccccaacggcaaggatc
ctatcttgatcagaaagaatgaggtggtgcaatactccatctcggcaactcagacaaatcctgcttattatg
gcgccgatgctgctgattttagaccggaaagatggtttgagccatcaactagaaacttgggatgggctta
cttgccattcaacggtggtccaagaatctgcttgggacaacagtttgctttgaccgaagccggttacgtttt-
g
gttagacttgttcaggaattccctagcttgtcacaggaccccgaaactgagtacccaccacctagattggc
acacttgacgatgtgcttgtttgacggggcatacgtcaagatgcaatag SEQ ID NO: 23
cytochrome P450
atggctatatctagtttgctatcgtgggatgtgatctgtgtcgtcttcatttgcgtttgtgtttatttcgggt-
a D2 (CYP52D2)
attgttatactaaatacttgatgcacaaacatggcgctcgagaaatcgagaatgtgatcaacgatgggttc
Nuc. Seq
tttgggttccgcttacctttgctactcatgcgagccagcaatgagggccgacttatcgagttca-
gtgtcaag
agattcgagtcggcgccacatccacagaacaagacattggtcaaccgggcattgagcgttcctgtgata
ctcaccaaggacccagtgaatatcaaagcgatgctatcgacccagtttgatgacttttcccttgggttgag
actacaccagtttgcgccgttgttggggaaaggcatctttactttggacggcccagagtggaagcagagc
cgatctatgttgcgtccgcaatttgccaaagatcgggtttctcatatcctggatctagaaccgcattttgtg-
tt
cttcggaagcacattgatggccacaatggagactacttcgacatccaggagctctacttccggttctcgat
ggatgtggcgacggggtttttgtttggcgagtctgtggggtcgttgaaagacgaagatgcgaggttcctgg
aagcattcaatgagtcgcagaagtatttggcaactagggcaacgttgcacgagttgtactttctttgtgacg
ggtttaggtttcgccagtacaacaaggttgtgcgaaagttctgcagccagtgtgtccacaaggcgttagat
gttgcaccggaagacaccagcgagtacgtgtttctccgcgagttggtcaaacacactcgagatcccgtt
gttttacaagaccaagcgttgaacgtcttgcttgctggacgcgacaccaccgcgtcgttattatcgtttgca
acatttgagctagcccggaatgaccacatgtggaggaagctacgagaggaggttatcctgacgatggg
accgtccagtgatgaaataaccgtggccgggttgaagagttgccgttacctcaaagcaatcctaaacga
aactcttcgactatacccaagtgtgcctaggaacgcgagatttgctacgaggaatacgacgcttcctcgt
ggcggaggtccagatggatcgtttccgattttgataagaaagggccagccagtggggtatttcatttgtgct
acacacttgaatgagaaggtatatgggaatgatagccatgtgtttcgaccggagagatgggctgcgtta
gagggcaagagtttgggctggtcgtatcttccattcaacggcggcccgagaagctgccttggtcagcagt
ttgcaatccttgaagcttcgtatgttttggctcgattgacacagtgctacacgacgatacagcttagaacta-
c
cgagtacccaccaaagaaactcgttcatctcacgatgagtcttctcaacggggtgtacatccgaactag
aacttga SEQ ID NO: 24 cytochrome
atgacaattaaagaaatgcctcagccaaaaacgtttggagagcttaaaaatttaccgttattaaacacag
P450:NADPH
ataaaccggttcaagctttgatgaaaattgcggatgaattaggagaaatctttaaattcgaggcgcctggt
P450 reductase
cgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatcacgctttgataaaa
(Bacillus
acttaagtcaagcgcttaaatttgtacgtgattttgcaggagacgggttatttacaagctgga-
cgcatgaaa megaterium)
aaaattggaaaaaagcgcataatatcttacttccaagcttcagtcagcaggcaatgaaaggctatcatg
nucleotide
cgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagcgtctaaatgcagatgagcatattga
Nuc. Seq
agtaccggaagacatgacacgtttaacgcttgatacaattggtctttgcggctttaactatcgc-
tttaacagc
ttttaccgagatcagcctcatccatttattacaagtatggtccgtgcactggatgaagcaatgaacaagctg
cagcgagcaaatccagacgacccagcttatgatgaaaacaagcgccagtttcaagaagatatcaagg
tgatgaacgacctagtagataaaattattgcagatcgcaaagcaagcggtgaacaaagcgatgatttat
taacgcatatgctaaacggaaaagatccagaaacgggtgagccgcttgatgacgagaacattcgctat
caaattattacattcttaattgcgggacacgaaacaacaagtggtcttttatcatttgcgctgtatttctta-
gt
aaaaatccacatgtattacaaaaagcagcagaagaagcagcacgagttctagtagatcctgttccaag
ctacaaacaagtcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatggccaact
gctcctgcgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaaaaaggcg
acgaactaatggttctgattcctcagcttcaccgtgataaaacaatttggggagacgatgtggaagagttc
cgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaaacggtcagc
gtgcgtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatgctaaaacactttg
actttgaagatcatacaaactacgagctggatattaaagaaactttaacgttaaaacctgaaggctttgtg
gtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctagcactgaacagtctgctaaaa
aagtacgcaaaaaggcagaaaacgctcataatacgccgctgcttgtgctatacggttcaaatatggga
acagctgaaggaacggcgcgtgatttagcagatattgcaatgagcaaaggatttgcaccgcaggtcgc
aacgcttgattcacacgccggaaatcttccgcgcgaaggagctgtattaattgtaacggcgtcttataacg
gtcatccgcctgataacgcaaagcaatttgtcgactggttagaccaagcgtctgctgatgaagtaaaag
gcgttcgctactccgtatttggatgcggcgataaaaactgggctactacgtatcaaaaagtgcctgctttta-
t
cgatgaaacgcttgccgctaaaggggcagaaaacatcgctgaccgcggtgaagcagatgcaagcga
cgactttgaaggcacatatgaagaatggcgtgaacatatgtggagtgacgtagcagcctactttaacctc
gacattgaaaacagtgaagataataaatctactctttcacttcaatttgtcgacagcgccgcggatatgcc
gcttgcgaaaatgcacggtgcgttttcaacgaacgtcgtagcaagcaaagaacttcaacagccaggca
gtgcacgaagcacgcgacatcttgaaattgaacttccaaaagaagcttcttatcaagaaggagatcattt
aggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaaggttcggcctagatgcatca
cagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccactcgctaaaacagtatccgta
gaagagcttctgcaatacgtggagcttcaagatcctgttacgcgcacgcagcttcgcgcaatggctgcta
aaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaaagcaagcctacaaagaaca
agtgctggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccggcgtgtgaaatgaaattcagc
gaatttatcgcccttctgccaagcatacgcccgcgctattactcgatttcttcatcacctcgtgtcgatgaa-
a
aacaagcaagcatcacggtcagcgttgtctcaggagaagcgtggagcggatatggagaatataaagg
aattgcgtcgaactatcttgccgagctgcaagaaggagatacgattacgtgctttatttccacaccgcagt
cagaatttacgctgccaaaagaccctgaaacgccgcttatcatggtcggaccgggaacaggcgtcgc
gccgtttagaggctttgtgcaggcgcgcaaacagctaaaagaacaaggacagtcacttggagaagca
catttatacttcggctgccgttcacctcatgaagactatctgtatcaagaagagcttgaaaacgcccaaag
cgaaggcatcattacgcttcataccgctttttctcgcatgccaaatcagccgaaaacatacgttcagcacg
taatggaacaagacggcaagaaattgattgaacttcttgatcaaggagcgcacttctatatttgcggaga
cggaagccaaatggcacctgccgttgaagcaacgcttatgaaaagctatgctgacgttcaccaagtga
gtgaagcagacgctcgcttatggctgcagcagctagaagaaaaaggccgatacgcaaaagacgtgt
gggctgggtaa SEQ ID NO: 25 NADPH
atggcattagataagttagatttatatgttattataacattggtggttgcaattgcagcttattttgcaaaga-
a cytochrome P450
agtttcttgaccaacaacaagataccgggttccttaatactgatagtggagatggtaattcaagagatatct
reductase, CPR
tacaagctttgaagaagaacaataaaaatacgttattattatttggatcccaaacaggtacagcagaag
(C. tropicalis
attatgccaacaaattgtcaagagaattgcattcaagatttggtttgaaaaccatggttgctgatttcgctga-
t strain ATCC750)
tatgatttcgaaaacttcggagatattactgaagatatcttggttttctttattgttgctacttatggtgaag-
g Nuc. Seq
accaaccgataatgctgacgaatttcacacttggttgactgaagaagctgacaccttgagtact-
ttgaaat
atactgtttttggtttgggtaattcaacttatgaattcttcaatgctattggtagaaaatttgacagattgt-
tg
gaaaaaggtggtgacagatttgctgaatacggtgaaggtgacgatggtactggtactttagatgaagattt
cttggcctggaaggataacgtgtttgattccttaaagaatgatttgaattttgaagaaaaagagttgaaata
cgaaccaaatgttaaattgactgaaagagatgatttatctggcaatgatccagatgtctccttgggtgaac
caaatgtcaaatacattaaatctgaaggtgttgacttaactaaaggtccatttgatcatactcatccatttt-
tg
gctagaattgttaaaactaaagaattgtttacttctgaagacagacattgtgttcatgttgaatttgatatt-
tc
aatcaaacttgaaatataccaccggtgatcatcttgcaatctggccatctaactctgatgaaaacattaag
caatttgccaaatgttttggtttagaagacaaacttgatactgttattgaattgaaagctttggattccact-
ta
ccatcccattccctaatccaatcacttatggagctgttattagacaccatttggaaatttcaggtcctgttt-
ct
gacaatttttcttatctattgctggatttgcccctgatgaagaaactaaaaagtcatttactagaattggtg-
gt
ataagcaagaatttgctagtaaagtcacccgtagaaaattcaacattgccgatgctttattatttgcttcca-
a
caacagaccatggtccgatgttccattcgaattccttattgaaaatgtccaacacttaactcctcgttatta-
ct
ccatttcttcttcctcattaagtgaaaagcaaaccattaatgttactgctgttgttgaagccgaagaagaag
ctgatggaagaccagttactggtgttgtcaccaacttgttgaagaatattgaaattgaacaaaacaaaact
ggtgaaaccccaatggttcattatgatttgaatggtccaagaggcaaatttagcaagttcagattgccagtt
cacgttagaagatctaatttcaaattaccaaagaatagcactaccccagttattttgattggtccaggtacc
ggtgttgcaccattgagaggttttgttagagaaagagttcaacaagttaaaaatggtgttaatgttggtaag
actgtattgttttatggatgtagaaattccgaacaagatttcttgtacaaacaagaatggagtgaatatgcc-
t
cagtattgggagaaaatttcgaaatgtttaatgccttctcaagacaagatccaactaagaaagtttatgttc
aagataagattttagaaaatagtgctcttgttgatgagttattatctagtggagcaattatttatgtttgtg-
gt
tgccagtagaatggctagagatgttcaagctgcaattgccaagattgttgccaaaagtagagatatccac
gaagataaagctgctgaattggttaaatcttggaaagttcaaaatagataccaagaagatgtctggtaa
SEQ ID NO: 26 NADPH
atggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtagccgcctattttgctaaga-
a cytochrome P450
ccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaactccagagac
reductase A,
gtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccagacgggtacggcaga
CPRA (Candida
agattacgccaacaaattgtccagagaattgcactccagatttggcttgaaaacgatggttgcagatttcg
strain
ctgattacgattgggataacttcggagatatcaccgaagacatcttggtgtttttcattgttgcca-
cctatggt ATCC20336)
gagggtgaacctaccgataatgccgacgagttccacacctggttgactgaagaagctgacactttgagt
Nuc. Seq
accttgaaatacaccgtgttcgggttgggtaactccacgtacgagttcttcaatgccattggta-
gaaagttt
gacagattgttgagcgagaaaggtggtgacaggtttgctgaatacgctgaaggtgatgacggtactggc
accttggacgaagatttcatggcctggaaggacaatgtctttgacgccttgaagaatgatttgaactttgaa
gaaaaggaattgaagtacgaaccaaacgtgaaattgactgagagagacgacttgtctgctgctgactc
ccaagtttccttgggtgagccaaacaagaagtacatcaactccgagggcatcgacttgaccaagggtc
cattcgaccacacccacccatacttggccagaatcaccgagacgagagagttgttcagctccaaggac
agacactgtatccacgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagct
atctggccatccaactccgacgaaaacattaagcaatttgccaagtgtttcggattggaagataaactcg
acactgttattgaattgaaggcgttggactccacttacaccatcccattcccaaccccaattacctacggtg
ctgtcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcaattgctgggtttg-
ct
ctgatgaagaaacaaagaaggcttttaccagacttggtggtgacaagcaagaattcgccgccaaggtc
acccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacgctccatggtccgatgttcct-
t
ttgaattccttattgaaaacgttccacacttgactccacgttactactccatttcgtcttcgtcattgagtg-
aa
agcaactcatcaacgttactgcagttgttgaagccgaagaagaagctgatggcagaccagtcactggt
gttgtcaccaacttgttgaagaacgttgaaattgtgcaaaacaagactggcgaaaagccacttgtccact
acgatttgagcggcccaagaggcaagttcaacaagttcaagttgccagtgcatgtgagaagatccaac
tttaagttgccaaagaactccaccaccccagttatcttgattggtccaggtactggtgttgccccattgaga
ggttttgtcagagaaagagttcaacaagtcaagaatggtgtcaatgttggcaagactttgttgttttatggt-
tg
cagaaactccaacgaggactttttgtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaac
tttgagatgttcaatgccttctccagacaagacccatccaagaaggtttacgtccaggataagattttaga
aaacagccaacttgtgcacgagttgttgactgaaggtgccattatctacgtctgtggtgatgccagtagaa
tggctagagacgtgcagaccacaatttccaagattgttgctaaaagcagagaaattagtgaagacaag
gctgctgaattggtcaagtcctggaaggtccaaaatagataccaagaagatgtttggtag SEQ ID
NO: 27 NADPH
atggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtggccgcctattttgctaaga-
a cytochrome P450
ccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaactccagagac
reductase B,
gtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccagaccggtacggcaga
CPRB (Candida
agattacgccaacaaattgtcaagagaattgcactccagatttggcttgaaaaccatggttgcagatttcg
strain
ctgattacgattgggataacttcggagatatcaccgaagatatcttggtgtttttcatcgttgcca-
cctacggt ATCC20336)
gagggtgaacctaccgacaatgccgacgagttccacacctggttgactgaagaagctgacactttgagt
Nuc. Seq
actttgagatataccgtgttcgggttgggtaactccacctacgagttcttcaatgctattggta-
gaaagtttga
cagattgttgagtgagaaaggtggtgacagatttgctgaatatgctgaaggtgacgacggcactggcac
cttggacgaagatttcatggcctggaaggataatgtctttgacgccttgaagaatgacttgaactttgaaga
aaaggaattgaagtacgaaccaaacgtgaaattgactgagagagatgacttgtctgctgccgactccc
aagtttccttgggtgagccaaacaagaagtacatcaactccgagggcatcgacttgaccaagggtccat
tcgaccacacccacccatacttggccaggatcaccgagaccagagagttgttcagctccaaggaaag
acactgtattcacgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagccat
ctggccatccaactccgacgaaaacatcaagcaatttgccaagtgtttcggattggaagataaactcga
cactgttattgaattgaaggcattggactccacttacaccattccattcccaactccaattacttacggtgc-
tg
tcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcgattgctgggtttgctc-
ct
atgaagaaacaaagaagactttcaccagacttggtggtgacaaacaagaattcgccaccaaggttac
ccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacactccatggtccgatgttccttt-
tg
agttccttattgaaaacatccaacacttgactccacgttactactccatttcttcttcgtcgttgagtgaaa-
aa
caactcatcaatgttactgcagtcgttgaggccgaagaagaagccgatggcagaccagtcactggtgtt
gttaccaacttgttgaagaacattgaaattgcgcaaaacaagactggcgaaaagccacttgttcactac
gatttgagcggcccaagaggcaagttcaacaagttcaagttgccagtgcacgtgagaagatccaacttt
aagttgccaaagaactccaccaccccagttatcttgattggtccaggtactggtgttgccccattgagagg
tttcgttagagaaagagttcaacaagtcaagaatggtgtcaatgttggcaagactttgttgttttatggttg-
ca
gaaactccaacgaggactttttgtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaacttt
gagatgttcaatgccttctctagacaagacccatccaagaaggtttacgtccaggataagattttagaaa
acagccaacttgtgcacgaattgttgaccgaaggtgccattatctacgtctgtggtgacgccagtagaatg
gccagagacgtccagaccacgatctccaagattgttgccaaaagcagagaaatcagtgaagacaag
gccgctgaattggtcaagtcctggaaagtccaaaatagataccaagaagatgtttggtag SEQ ID
NO: 28 cytochrome
mtikempqpktfgelknlpllntdkpvqalmkiadelgeifkfeapgrvtrylssqrlikeacdesrfdknls
P450:NADPH
qalkfvrdfagdglftswtheknwkkahnillpsfsqqamkgyhammvdiavqlvqkwerlnadehie
P450 reductase
vpedmtrltldtiglcgfnyrfnsfyrdqphpfitsmvrasdeamnksqranpddpaydenkrqfqedik
(Bacillus
vmndlvdkiiadrkasgeqsddllthmlngkdpetgeplddeniryqiitfliaghettsgll-
sfasyflvknp megaterium)
hvlqkaaeeaarvlvdpvpsykqvkqlkyvgmvlneasrlwptapafslyakedtvlggeyplekgdel
amino acid [P450
mvsipqlhrdktiwgddveefrperfenpsaipqhafkpfgngqracigqqfalheatsvlgmmlkhfdf
activity shown in
edhtnyesdiketltlkpegfvvkakskkiplggipspsteqsakkvrkkaenahntpslvlygsnmgta
italics, P450
egtardladiamskgfapqvatldshagnlpregavlivtasynghppdnakqfvdwldqasadevkg
reductase activity
vrysvfgcgdknwattyqkvpafidetlaakgaeniadrgeadasddfegtyeewrehmwsdvaayf
shown in normal
nldiensednkstlslqfvdsaadmplakmhgafstnvvaskelqqpgsarstrhleielpkeasyqeg
font]
dhlgviprnyegivnrvtarfgldasqqirseaeeeklahlplaktvsveelsqyvelqdpvtrtql-
ramaak A.A. Seq
tvcpphkveleallekqaykeqvsakrltmleslekypacemkfsefialspsirpryysisss-
prvdekq
asitvsvvsgeawsgygeykgiasnylaesqegdtitcfistpqseftspkdpetplimvgpgtgvapfrg
fvqarkqlkeqgqslgeahlyfgcrsphedysyqeelenaqsegiitlhtafsrmpnqpktyvqhvmeq
dgkklielldqgahfyicgdgsqmapaveatlmksyadvhqvseadarlwsqqleekgryakdvwag*
SEQ ID NO: 29 acyl CoA
ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGGTCCTGACCCTAGATCATCCATCCAAAAGGAAAGA
oxidase, POX4
GACAGCTCCAAATGGAACCCTCAACAAATGAACTACTTCTTGGAAGGCTCCGTCGAAAGAAGTGAGTTGATGA
(Candida strain
AGGCTTTGGCCCAACAAATGGAAAGAGACCCAATCTTGTTCACAGACGGCTCCTACTACGACTTGACCAAG
ATCC20336)
GACCAACAAAGAGAATTGACCGCCGTCAAGATCAACAGAATCGCCAGATACAGAGAACAAGAATCCATCGACA
nucleotide
CTTTCAACAAGAGATTGTCCTTGATTGGTATCTTTGACCCACAGGTCGGTACCAGAATTGGTGTCAACCTCGG
TTTGTTCCTTTCTTGTATCAGAGGTAACGGTACCACTTCCCAATTGAACTACTGGGCTAACGAAAAGGAAAC
CGCTGACGTTAAAGGTATCTACGGTTGTTTCGGTATGACCGAATTGGCCCACGGTTCCAACGTTGCTGGTTT
GGAAACCACCGCCACATTTGACAAGGAATCTGACGAGTTTGTCATCAACACCCCACACATTGGTGCCACCAA
GTGGTGGATTGGTGGTGCTGCTCACTCCGCCACCCACTGTTCTGTCTACGCCAGATTGATTGTTGACGGTCA-
AG
ATTACGGTGTCAAGACTTTTGTTGTCCCATTGAGAGACTCCAACCACGACCTCATGCCAGGTGTCACTGT
TGGTGACATTGGTGCCAAGATGGGTAGAGATGGTATCGATAACGGTTGGATCCAATTCTCCAACGTCAGAAT-
CC
CAAGATTCTTTATGTTGCAAAAGTTCTGTAAGGTTTCTGCTGAAGGTGAAGTCACCTTGCCACCTTTGGAA
CAATTGTCTTACTCCGCCTTGTTGGGTGGTAGAGTCATGATGGTTTTGGACTCCTACAGAATGTTGGCTA
GAATGTCCACCATTGCCTTGAGATACGCCATTGGTAGAAGACAATTCAAGGGTGACAATGTCGATCCAAAAG-
AT
CCAAACGCTTTGGAAACCCAATTGATAGATTACCCATTGCACCAAAAGAGATTGTTCCCATACTTGGCTGCT
GCCTACGTCATCTCCGCTGGTGCCCTCAAGGTTGAAGACACCATCCATAACACCTTGGCTGAATTGGACGCT
GCCGTTGAAAAGAACGACACCAAGGCTATCTTTAAGTCTATTGACGACATGAAGTCATTGTTTGTTGACTCT-
G
GTTCCTTGAAGTCCACTGCCACTTGGTTGGGTGCTGAAGCCATTGACCAATGTAGACAAGCCTGTGGTGGT
CACGGTTACTCGTCCTACAACGGCTTCGGTAAAGCCTACAACGATTGGGTTGTCCAATGTACTTGGGAAG
GTGACAACAATGTCTTGGCCATGAGTGTTGGTAAGCCAATTGTCAAGCAAGTTATCAGCATTGAAGATGCCG-
GC
AAGACCGTCAGAGGTTCCACCGCTTTCTTGAACCAATTGAAGGACTACACTGGTTCCAACAGCTCCAAGGTT
GTTTTGAACACTGTTGCTGACTTGGACGACATCAAGACTGTCATCAAGGCTATTGAAGTTGCCATCATCAGA
TTGTCCCAAGAAGCTGCTTCTATTGTCAAGAAGGAATCTTTCGACTATGTCGGCGCTGAATTGGTTCAACTC-
T
CCAAGTTGAAGGCTCACCACTACTTGTTGACTGAATACATCAGAAGAATTGACACCTTTGACCAAAAGGACT-
T
GGTTCCATACTTGATCACCCTCGGTAAGTTGTACGCTGCCACTATTGTCTTGGACAGATTTGCCGGTGTCTT
CTTGACTTTCAACGTTGCCTCCACCGAAGCCATCACTGCTTTGGCCTCTGTGCAAATTCCAAAGTTGTGTG
CTGAAGTCAGACCAAACGTTGTTGCTTACACCGACTCCTTCCAACAATCCGACATGATTGTCAATTCTGCT
ATTGGTAGATACGATGGTGACATCTATGAGAACTACTTTGACTTGGTCAAGTTGCAGAACCCACCATCCAAG
ACCAAGGCTCCTTACTCTGATGCTTTGGAAGCCATGTTGAACAGACCAACCTTGGACGAAAGAGAAAGATT
TGAAAAGTCTGATGAAACCGCTGCTATCTTGTCCAAGTAA SEQ ID NO: 30 acyl CoA
MTFTKKNVSVSQGPDPRSSIQKERDSSKWNPQQMNYFLEGSVERSELMKALAQQMERDPILFTDGSYYDLT
oxidase, POX4
KDQQRELTAVKINRIARYREQESIDTFNKRLSLIGIFDPQVGTRIGVNLGLFLSCIRGNGTTSQLNYWANEK
(Candida strain
ETADVKGIYGCFGMTELAHGSNVAGLETTATFDKESDEFVINTPHIGATKWWIGGAAHSATHCSVYARLI
ATCC20336)
VDGQDYGVKTFVVPLRDSNHDLMPGVTVGDIGAKMGRDGIDNGWIQFSNVRIPRFFMLQKFCKVSAEGEVT
amino acid
LPPLEQLSYSALLGGRVMMVLDSYRMLARMSTIALRYAIGRRQFKGDNVDPKDPNALETQLIDYPLHQKRLFP
YLAAAYVISAGALKVEDTIHNTLAELDAAVEKNDTKAIFKSIDDMKSLFVDSGSLKSTATWLGAEAIDQCR
QACGGHGYSSYNGFGKAYNDWVVQCTWEGDNNVLAMSVGKPIVKQVISIEDAGKTVRGSTAFLNQLKDYTGS
NSSKVVLNTVADLDDIKTVIKAIEVAIIRLSQEAASIVKKESFDYVGAELVQLSKLKAHHYLLTEYIRRIDT
FDQKDLVPYLITLGKLYAATIVLDRFAGVFLTFNVASTEAITALASVQIPKLCAEVRPNVVAYTDSFQQSDM
IVNSAIGRYDGDIYENYFDLVKLQNPPSKTKAPYSDALEAMLNRPTLDERERFEKSDETAAILSK*
SEQ ID NO: 31 acyl CoA
ATGCCTACCGAACTTCAAAAAGAAAGAGAACTCACCAAGTTCAACCCAAAGGAGTTGAACTACTTCTTGGAA
oxidase, POX5
GGTTCCCAAGAAAGATCCGAGATCATCAGCAACATGGTCGAACAAATGCAAAAAGACCCTATCTTGAAGGTC
(Candida strain
GACGCTTCATACTACAACTTGACCAAAGACCAACAAAGAGAAGTCACCGCCAAGAAGATTGCCAGACTCTC
ATCC20336)
CAGATACTTTGAGCACGAGTACCCAGACCAACAGGCCCAGAGATTGTCGATCCTCGGTGTCTTTGACCCACAA
nucleotide
GTCTTCACCAGAATCGGTGTCAACTTGGGTTTGTTTGTTTCCTGTGTCCGTGGTAACGGTACCAACTCCCA
GTTCTTCTACTGGACCATAAATAAGGGTATCGACAAGTTGAGAGGTATCTATGGTTGTTTTGGTATGACT
GAGTTGGCCCACGGTTCCAACGTCCAAGGTATTGAAACCACCGCCACTTTTGACGAAGACACTGACGAGTT
TGTCATCAACACCCCACACATTGGTGCCACCAAGTGGTGGATCGGTGGTGCTGCGCACTCCGCCACCCA
CTGCTCCGTCTACGCCAGATTGAAGGTCAAAGGAAAGGACTACGGTGTCAAGACCTTTGTTGTCCCATTGAG-
A
GACTCCAACCACGACCTCGAGCCAGGTGTGACTGTTGGTGACATTGGTGCCAAGATGGGTAGAGACGGTAT
CGATAACGGTTGGATCCAGTTCTCCAACGTCAGAATCCCAAGATTCTTTATGTTGCAAAAGTACTGT
AAGGTTTCCCGTCTGGGTGAAGTCACCATGCCACCATCTGAACAATTGTCTTACTCGGCTTTGATTGGT
GGTAGAGTCACCATGATGATGGACTCCTACAGAATGACCAGTAGATTCATCACCATTGCCTTGAG
ATACGCCATCCACAGAAGACAATTCAAGAAGAAGGACACCGATACCATTGAAACCAAGTTGATTGACTACCC
ATTGCATCAAAAGAGATTGTTCCCATTCTTGGCTGCCGCTTACTTGTTCTCCCAAGGTGCCTTGTA
CTTAGAACAAACCATGAACGCAACCAACGACAAGTTGGACGAAGCTGTCAGTGCTGGTGAAAAGGAAGCC
ATTGACGCTGCCATTGTCGAATCCAAGAAATTGTTCGTCGCTTCCGGTTGTTTGAAGTCCACCTGTACCTG
GTTGACTGCTGAAGCCATTGACGAAGCTCGTCAAGCTTGTGGTGGTCACGGTTACTCGTCTTACAACGG
TTTCGGTAAAGCCTACTCCGACTGGGTTGTCCAATGTACCTGGGAAGGTGACAACAACATCTTGGCCA
TGAACGTTGCCAAGCCAATGGTTAGAGACTTGTTGAAGGAGCCAGAACAAAAGGGATTGGTTCTCTCCAGCG-
TT
GCCGACTTGGACGACCCAGCCAAGTTGGTTAAGGCTTTCGACCACGCCCTTTCCGGCTTGGCCAGAGAC
ATTGGTGCTGTTGCTGAAGACAAGGGTTTCGACATTACCGGTCCAAGTTTGGTTTTGGTTTCCA
AGTTGAACGCTCACAGATTCTTGATTGACGGTTTCTTCAAGCGTATCACCCCAGAATGGTCTGAAGTCT
TGAGACCTTTGGGTTTCTTGTATGCCGACTGGATCTTGACCAACTTTGGTGCCACCTTCTTGCAGTACGGT
ATCATTACCCCAGATGTCAGCAGAAAGATTTCCTCCGAGCACTTCCCAGCCTTGTGTGCCAAGGTTAGAC
CAAACGTTGTTGGTTTGACTGATGGTTTCAACTTGACTGACATGATGACCAATGCTGCTATTGGT
AGATATGATGGTAACGTCTACGAACACTACTTCGAAACTGTCAAGGCTTTGAACCCACCAGAAAACACCAA
GGCTCCATACTCCAAGGCTTTGGAAGACATGTTGAACCGTCCAGACCTTGAAGTCAGAGAAAGAGGTGAA
AAGTCCGAAGAAGCTGCTGAAATCTTGTCCAGTTAA SEQ ID NO: 32 acyl CoA
MPTELQKERELTKFNPKELNYFLEGSQERSEIISNMVEQMQKDPILKVDAS oxidase, POX5
YYNLTKDQQREVTAKKIARLSRYFEHEYPDQQAQRLSILGVFDPQVFTRIG (Candida strain
VNLGLFVSCVRGNGTNSQFFYWTINKGIDKLRGIYGCFGMTELAHGSNVQ ATCC20336)
GIETTATFDEDTDEFVINTPHIGATKWWIGGAAHSATHCSVYARLKVKGKD
amino acid YGVKTFVVPLRDSNHDLEPGVTVGDIGAKMGRDGIDNGWIQFSNVRIPRF
FMLQKYCKVSRSGEVTMPPSEQLSYSALIGGRVTMMMDSYRMTSRFITIA
LRYAIHRRQFKKKDTDTIETKLIDYPLHQKRLFPFLAAAYLFSQGALYLEQT
MNATNDKLDEAVSAGEKEAIDAAIVESKKLFVASGCLKSTCTWLTAEAIDE
ARQACGGHGYSSYNGFGKAYSDWVVQCTWEGDNNILAMNVAKPMVRD
LLKEPEQKGLVLSSVADLDDPAKLVKAFDHALSGLARDIGAVAEDKGFDIT
GPSLVLVSKLNAHRFLIDGFFKRITPEWSEVLRPLGFLYADWILTNFGATFL
QYGIITPDVSRKISSEHFPALCAKVRPNVVGLTDGFNLTDMMTNAAIGRYD
GNVYEHYFETVKALNPPENTKAPYSKALEDMLNRPDLEVRERGEKSEEAAEILSS* SEQ ID NO:
33 Acyl-CoA
atgatcagaaccgtccgttatcaatccctcaagaggttcagacctctggctttgtctcctgtttttcgtccac-
g Hydrolase
ctacaactcccagaaggccaatttccaccgtccagaccaccctgggtccgacgagccagctga-
agcc (ACHA)
gccgacgccgccgccacgatcctcgccgagttgcgagacaagcagacgaacccgaacaaggccac
Nucleotide Seq
ctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaagacgattgacagtttcagct
acgttgaccccaagacgaccgtcgtgggggagaagacacgcagtgactcgttctcgttcttgttgttgcc
gttcaaggacgacaagtggttgtgtgacgcgtacatcaatgcgtttggccggttgcgtgtagcgcagttgtt
ccaggacttggacgccttggcggggcgcatcgcgtacaggcactgttccccagcggagcccgtgaatg
tcacggcgagcgtggatagggtgtacatggtgaagaaagtggacgagattaacaattacaatttcgtgtt
gcggggtccgtgacgtggaccgggagatcgtcgatggagatcacggtgaaagggtatgcttttgaag
acgccgtgccggatataacgaacgaggagtccttgccggcagagaatgtgtttttggctgctaatttcacc
ttcgtggcacggaacccacttacacacaagtcctttgctattaacagattgttgcccgtgactgagaagga
ctgggtcgactatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaag
atcttggagcctaccgcggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacaga
acatcgagagggccaacgatgggatcgcgttcatgaaggacacgaccatgaagtccaccttgttcatg
cagccccagtaccgtaacagacactcatacatgattttcggagggtacttgttaagacaaactttcgaatt
ggcctactgtaccgcggcaacgttttccctggccgggccccgtttcgtcagcttggactccaccacgttca
agaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatctcgtacacggagcacgtgcac
gagggagtggaggagattgacgcggactcaccgttcaacttcagcttgcctgccacgaacaagatctc
gaagaaccccgaggcgttcttgtcggaacccggcacgttgattcaagtcaaggtcgacacatacatcc
aggagttagagcagagtgtgaagaagcccgcgggtacgttcatctactcgttctatgttgataaagaaag
cgttactgttgatggaaaggcgtcgttttgttcagttatcccgcagacgtactccgagatgatgacttatgt-
g
ggcgggagaagaagagcccaggatactgctaactacgtggagactttgccgtttagtggaagcggcaattaa
SEQ ID NO: 34 Acyl-CoA
MIRTVRYQSLKRFRPSALSPVFRPRYNSQKANFHRPDHPGSDEPAEAAD Hydrolase
AAATILAELRDKQTNPNKATWLDALTEREKLRAEGKTIDSFSYVDPKTTVV (ACHA) Amino
GEKTRSDSFSFLLLPFKDDKWLCDAYINAFGRLRVAQLFQDLDALAGRIAY Acid Seq
RHCSPAEPVNVTASVDRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEIT
VKGYAFEDAVPDITNEESLPAENVFLAANFTFVARNPLTHKSFAINRLLPVT
EKDWVDYRRAESHNAKKKLMAKNKKILEPTAEESKLIYDMWRSSKSLQNI
ERANDGIAFMKDTTMKSTLFMQPQYRNRHSYMIFGGYLLRQTFELAYCTA
ATFSSAGPRFVSLDSTTFKNPVPVGSVLTMDSSISYTEHVHEGVEEIDADS
PFNFSLPATNKISKNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYS
FYVDKESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVETLPFSGSGN SEQ ID NO:
35 Acyl-CoA
atgatcagaaccgtccgttatcaatccttcaagaggttcaaacctctgactttatcccccgttttccgtccac
Hydrolase
gctacaactcccagaaggccaatttccaccgtccagaccacgctgggtccgacgagccagccg-
aagc (ACHB)
cgccgacgccgctgccacgatcctcgccgagttgcgagacaagcagacgaacccgaacaaggcca
Nucleotide Seq
cctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaagacaatcgacagcttcag
ctacgttgaccccaagacaaccgtcgtgggggagaagacacgcagcgactcgttctcgttcttgttgttg
ccgttcaaggacgacaagtggttgtgtgacgcgtacatcaatgcgtttggccggttgcgtgtagcgcagtt
gttccaggacttggacgccttggcgggccgcatcgcgtacaggcactgttcccccgctgagcccgtgaa
tgtcacggcgagcgtggatagagtgtatatggtgaagaaagtggacgagattaataattacaatttcgtgt
tggcggggtccgtgacgtggaccgggagatcgtcgatggagatcacggtcaaagggtatgcttttgaag
acgccgtgccggagataactaacgaggagtccttgccggcagagaatgtgttcttggctgttaatttcacc
ttcgtggcacgtaacccactcacacacaagtccttcgctattaacagattgttgcccgtgactgagaagg
actgggtcgattatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaa
gatcttggagcctaccccggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacag
aacatcgagaaggccaacgacgggatcgcgttcatgaaggacacgataatgaagtccaccttgttcat
gcagccccagtaccgtaacagacactcatacatgattttcggtgggtatttgttaagacaaactttcgaatt
ggcctattgtaccgcagcaacgttttccctggcgggaccccgtttcgtcagcttggactccaccacgttca
agaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatctcgtacacggagcacgtccac
gatggcgttgaggagattgacgccgactccccgttcaacttcagcttgcctgccacgaacaagatctcga
agaaccccgaggcgttcttgtcggagcccggcacgttgatccaagtcaaggtcgacacgtacatccag
gagttagagcaaagtgtgaagaagcctgcgggaacgttcatctactcgttctatgttgataaagagagcg
ttactgtggatggaaaggcgtcgttttgttcagttatcccgcagacgtactccgagatgatgacttatgtgg-
g
cgggagaagaagagcccaggatactgctaattacgtggagactttgccgtttagtggaagcggcaattaa
SEQ ID NO: 36 Acyl-CoA
MIRTVRYQSFKRFKPLTLSPVFRPRYNSQKANFHRPDHAGSDEPAEAAD Hydrolase
AAATILAELRDKQTNPNKATWLDALTEREKLRAEGKTIDSFSYVDPKTTVV (ACHB)
GEKTRSDSFSFLLLPFKDDKWLCDAYINAFGRLRVAQLFQDLDALAGRIAY
RHCSPAEPVNVTASVDRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEIT
VKGYAFEDAVPEITNEESLPAENVFLAVNFTFVARNPLTHKSFAINRLLPVT
EKDWVDYRRAESHNAKKKLMAKNKKILEPTPEESKLIYDMWRSSKSLQNI
EKANDGIAFMKDTIMKSTLFMQPQYRNRHSYMIFGGYLLRQTFELAYCTA
ATFSLAGPRFVSLDSTTFKNPVPVGSVLTMDSSISYTEHVHDGVEEIDADS
PFNFSLPATNKISKNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYS
FYVDKESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVETLPFSGSGN SEQ ID NO:
37 E. coli Acyl-CoA
atggccgatacattgctcatcttgggtgactctttgtctgcagggtatcggatgtccgcatctgccgcatggc
Thioesterase
ctgcactcctcaatgacaaatggcaaagcaagacatcggtcgtgaatgcatctatctctggcgatacctc
(TESA) gene
gcagcaggggttggcccgtctcccagccttgttgaagcaacatcaaccacgttgggtcttggtcgaattg
without signal
ggcggcaatgatggtctcagaggttttcaacctcaacagaccgagcagacattgcgtcaaatcctccaa
peptide
gacgtgaaggcagcaaacgccgaacctctcttgatgcagataagattgcctgccaactatggtcg-
taga sequence
tacaatgaagccttttctgcaatctacccgaagcttgcaaaggagtttgacgtcccattgttgc-
cgtttttgat optimized for C.
ggaagaggtgtaccttaagcctcagtggatgcaagacgatggtatccatccgaaccgtgatg
tropicalis
cacaaccattcatcgcagattggatggccaaacaactccaacctttggtcaatcatgatagctaa
Nucleotide Seq SEQ ID NO: 38 E. coli Acyl-CoA
MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKTSVVNASISGDTSQ Thioesterase
QGLARLPALLKQHQPRWVLVELGGNDGLRGFQPQQTEQTLRQILQDVKA (TESA) without
ANAEPLLMQIRLPANYGRRYNEAFSAIYPKLAKEFDVPLLPFLMEEVYLKP signal peptide
QWMQDDGIHPNRDAQPFIADWMAKQLQPLVNHDS Amino Acid Seq SEQ ID NO: 39
Acyl-CoA
atgggtgcccctttaacagtcgccgttggcgaagcaaaaccaggcgaaaccgctccaagaagaaaa
Synthetase
gccgctcaaaaaatggcctctgtcgaacgcccaacagactcaaaggcaaccactttgccagacttcatt
(ACS1) Nuc. Seq
gaagagtgttttgccagaaacggcaccagagatgccatggcctggagagacttggtcgaaatccacgt
cgaaaccaaacaggttaccaaaatcattgacggcgaacagaaaaaggtcgataaggactggatcta
ctacgaaatgggtccttacaactacatatcctaccccaagttgttgacgttggtcaagaactactccaagg
gtttgttggagttgggcttggccccagatcaagaatccaagttgatgatctttgccagtacctcccacaagt
ggatgcagaccttcttagcctccagtttccaaggtatccccgttgtcaccgcctacgacaccttgggtgagt
cgggcttgacccactccttggtgcaaaccgaatccgatgccgtgttcaccgacaaccaattgttgtcctcc
ttgattcgtcctttggagaaggccacctccgtcaagtatgtcatccacggggaaaagattgaccctaacg
acaagagacagggcggcaaaatctaccaggatgcggaaaaggccaaggagaagattttacaaatta
gaccagatattaaatttatttctttcgacgaggttgttgcattgggtgaacaatcgtccaaagaattgcatt-
tc
ccaaaaccagaagacccaatctgtatcatgtacacctcgggttccaccggtgctccaaagggtgtggtt
atcaccaatgccaacattgttgccgccgtgggtggtatctccaccaatgctactagagacttggttagaac
tgtcgacagagtgattgcatttttgccattggcccacattttcgagttggcctttgagttggttaccttctg-
gt
ggggctccattgggttacgccaatgtcaagactttgaccgaagcctcctgcagaaactgtcagccagac
ttgattgaattcaaaccaaccatcatggttggtgttgctgccgtttgggaatcggtcagaaagggtgtcttg-
t
ctaaattgaaacaggcttctccaatccaacaaaagatcttctgggctgcattcaatgccaagtctactttga
accgttatggcttgccaggcggtgggttgtttgacgctgtcttcaagaaggttaaagccgccactggtggc
caattgcgttatgtgttgaatggtgggtccccaatctctgttgatgcccaagtgtttatctccaccttgctt-
gc
ccaatgttgttgggttacggtttgactgaaacctgtgccaataccaccattgtcgaacacacgcgcttcca
gattggtactttgggtaccttggttggatctgtcactgccaagttggttgatgttgctgatgctggatacta-
cg
caagaacaaccagggtgaaatctggttgaaaggcggtccagttgtcaaggaatactacaagaacgaa
gaagaaaccaaggctgcattcaccgaagatggctggttcaagactggtgatattggtgaatggaccgc
cgacggtggtttgaacatcattgaccgtaagaagaacttggtcaagactttgaatggtgaatacattgcttt
ggagaaattggaaagtatttacagatccaaccacttgattttgaacttgtgtgtttacgctgaccaaaccaa
ggtcaagccaattgctattgtcttgccaattgaagccaacttgaagtctatgttgaaggacgaaaagattat
cccagatgctgattcacaagaattgagcagcttggttcacaacaagaaggttgcccaagctgtcttgaga
cacttgctccaaaccggtaaacaacaaggtttgaaaggtattgaattgttgcagaatgttgtcttgttggat-
g
acgagtggaccccacagaatggttttgttacttctgcccaaaagttgcagagaaagaagattttagaaag
ttgtaaaaaagaagttgaagaggcatacaagtcgtcttag SEQ ID NO: 40 Acyl-CoA
MGAPLTVAVGEAKPGETAPRRKAAQKMASVERPTDSKATTLPDFIEECFA Synthetase
RNGTRDAMAWRDLVEIHVETKQVTKIIDGEQKKVDKDWIYYEMGPYNYIS (ACS1)
YPKLLTLVKNYSKGLLELGLAPDQESKLMIFASTSHKWMQTFLASSFQGIP A. A. Seq
VVTAYDTLGESGLTHSLVQTESDAVFTDNQLLSSLIRPLEKATSVKYVIHG
EKIDPNDKRQGGKIYQDAEKAKEKILQIRPDIKFISFDEVVALGEQSSKELH
FPKPEDPICIMYTSGSTGAPKGVVITNANIVAAVGGISTNATRDLVRTVDRV
IAFLPLAHIFELAFELVTFWWGAPLGYANVKTLTEASCRNCQPDLIEFKPTI
MVGVAAVWESVRKGVLSKLKQASPIQQKIFWAAFNAKSTLNRYGLPGGG
LFDAVFKKVKAATGGQLRYVLNGGSPISVDAQVFISTLLAPMLLGYGLTET
CANTTIVEHTRFQIGTLGTLVGSVTAKLVDVADAGYYAKNNQGEIWLKGG
PVVKEYYKNEEETKAAFTEDGWFKTGDIGEWTADGGLNIIDRKKNLVKTL
NGEYIALEKLESIYRSNHLILNLCVYADQTKVKPIAIVLPIEANLKSMLKDEKII
PDADSQELSSLVHNKKVAQAVLRHLLQTGKQQGLKGIELLQNVVLLDDEW
TPQNGFVTSAQKLQRKKILESCKKEVEEAYKSS SEQ ID NO: 41 Long-chain Acyl-
atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattgcc
CoA Synthetase
gacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccagcagaggt
(FAT1)
aaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaagcgttggcg-
ttcc Nuc. Seq
caagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgacga- t
cagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttgaa
gaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattgtctt
gtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagccattgat
ccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcagagatac-
c
gaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttgtttgaca
gattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaaccgatact
gactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcatgtcctgg
agaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgacc
gccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtc-
tc
cgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaat
acgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgtcag
aattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacattgaaggta
tcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacggtgagtacggtgt
cggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagcagaaattggccaagatgg
acccagaagacgagagtgaaatctacaaggaccccaagaccgggttctgtaccgaggccgcttaca
acgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcagaaatccttccagggttattat
ggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaaaggtgacgcgtggtacag
atccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcgacagattaggtgacactttccg-
t
tggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattgatgggctccaaggccttgaagc
agtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaa
ggacgagttgagccatgaagaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgc
tcaacctgcgttcatcaagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattc
aagaaccaaaagttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagt
accaggagttgactgaagacgattggtctttgatttgtaccggtaaagccaaattg SEQ ID NO:
42 Long-chain Acyl-
MSGLEIAAAAILGSQLLEAKYLIADDVSLAKTVAVNALPYLWKASRGKASY CoA Synthetase
WYFFEQSVFKNPNNKALAFPRPRKNAPTPKTDAEGFQIYDDQFDLEEYTY (FAT1)
KELYDMVLKYSYILKNEYGVTANDTIGVSCMNKPLFIVLWLALWNIGALPA A.A. Seq
FLNFNTKDKPLIHCLKIVNASQVFVDPDCDSPIRDTEAQIREELPHVQINYID
EFALFDRLRLKSTPKHRAEDKTRRPTDTDSSACALIYTSGTTGLPKAGIMS
WRKAFMASVFFGHIMKIDSKSNVLTAMPLYHSTAAMLGLCPTLIVGGCVS
VSQKFSATSFWTQARLCGATHVQYVGEVCRYLLNSKPHPDQDRHNVRIA
YGNGLRPDIWSEFKRRFHIEGIGEFYAATESPIATTNLQYGEYGVGACRKY
GSLISLLLSTQQKLAKMDPEDESEIYKDPKTGFCTEAAYNEPGELLMRILN
PNDVQKSFQGYYGNKSATNSKILTNVFKKGDAWYRSGDLLKMDEDKLLY
FVDRLGDTFRWKSENVSATEVENELMGSKALKQSVVVGVKVPNHEGRA
CFAVCEAKDELSHEEILKLIHSHVTKSLPVYAQPAFIKIGTIEASHNHKVPKN
QFKNQKLPKGEDGKDLIYWLNGDKYQELTEDDWSLICTGKAKL SEQ ID NO: 43 Acyl-CoA
Sterol
atgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatccacgtcttcgttcc
acyl transferase
aggaagaactagtcgaggttgaaatgtccaactcgtccattaacgaatcccagaccgatgagtcgtacc
(ARE1)
gtcctgaagaaacctcattgcattacaggaggaagtcccacaggaccccgtcagaggagtcgttcc-
ta Nuc. Seq
gagatcaccaagaacgtgaatgatccggatctagtttccaagattgagaacctaaggggcaaag-
taag
ccaacgggaagacaggttgaggaagcactaccttcacacctcccaggacgtcaagttcttgtcccggtt
caacgacatcaagttcaagctgaactccgcgacgattctagattcggatgcgttttacaagagtgaatact
ttggagtcttgaccatcttctgggtggttatcgcactctacatattgtcaacgttgtcagatgtttactttg-
gc
ggccaagcccttactggactggatcatcataggaatgttcaagcaggacttggtgaaagttgcactcgtt
gatcttgccatgtacctatcctcgtattttccttatttcttgcaggttgcatgcaaacggggtgatgtatct-
tg
atggtcttggatgggcaatacagggggtttacagcttggtgtttttgacgttctggacggtagttccgcagg-
a
gttggccatggatcttccttggattgcacgaattttcttgatcttgcattgcttggtgtttattatgaagat-
gc
cgtatgggcattacaatggatacctttgggatgtgtatcaggaaggattggcctctgaggctgatctcagg
gacctttctgagtatgatgaagatttccccctggatcacgtggaggttctagaacagagcttgtggtttgcc
aaacacgagttggagtttcaatcgaatggaactgctgagaggaaggaccaccatcaccatgtattcga
cgaaaaggatgtcaacaaaccaatacgtgtcttgcaagaagagggaattatcaagtttccggcaaaca
tcaacttcaaggattatttcgagtacagtatgttcccaacgctagtctacacgttgagcttcccccgaactc-
g
acagattagatggacgtatgtgttgcagaaggttttgggaacatttgccttagtgtttgccatgattatcgt-
cg
ccgaagagagtttctgccccttgatgcaagaagttgatcagtacacaaaattgccaaccaaccaaaggt
tcccaaaatacttcgtcgttctttcccacttgatattaccgctcggcaagcagtacttgctctcattcatcc-
tc
tctggaatgaaattctcaacggcatagcggagttaagcaggtttggcgaccggcatttctacggcgcttg
gtggtcgagcgtcgattacatggactattcaagaaaatggaacaccatcgtgcaccgattcctccgtcgg
cacgtttacaattcgagcattcacatcctcggtatttccaggacgcaagccgcgatagttacacttttgctt-
tc
tgccacaatccacgaactcgttatgtacgtcctatttggcaaattacgagggtacctattccttacgatgct-
t
gtccagatccccatgaccgtcacctccaagttcaacaaccgtgtttggggcaacatcatgttctggttgac
gtatttatctggccccagcttggttagtgcgttgtatttactcttctag SEQ ID NO: 44
Acyl-CoA Sterol
MSDDEIAGIVIEIDDDVKSTSSFQEELVEVEMSNSSINESQTDESYRPEETS acyl
transferase LHYRRKSHRTPSEESFLEITKNVNDPDLVSKIENLRGKVSQREDRLRKHYL
(ARE1) HTSQDVKFLSRFNDIKFKLNSATILDSDAFYKSEYFGVLTIFWVVIALYILST A.A.
Seq LSDVYFGMAKPLLDWIIIGMFKQDLVKVALVDLAMYLSSYFPYFLQVACKR
GDVSWHGLGWAIQGVYSLVFLTFWTVVPQELAMDLPWIARIFLILHCLVFI
MKMQSYGHYNGYLWDVYQEGLASEADLRDLSEYDEDFPLDHVEVLEQS
LWFAKHELEFQSNGTAERKDHHHHVFDEKDVNKPIRVLQEEGIIKFPANIN
FKDYFEYSMFPTLVYTLSFPRTRQIRWTYVLQKVLGTFALVFAMIIVAEESF
CPLMQEVDQYTKLPTNQRFPKYFVVLSHLILPLGKQYLLSFILIWNEILNGIA
ELSRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHVYNSSIHILGIS
RTQAAIVTLLLSATIHELVMYVLFGKLRGYLFLTMLVQIPMTVTSKFNNRVW
GNIMFWLTYLSGPSLVSALYLLF SEQ ID NO: 45 Acyl-CoA Sterol
atgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatctacgtcttcgttcc
acyl transferase
aggaagacctagtcgaggttgagatgtccaactcgtccattaacgaatcccagacggatgagttgtcgt
(ARE2)
accgtcctgaagaaatctcattgcattcgagaaggaagtcccacaagaccccgtcagatgagtcgt-
tcc Nuc. Seq
tagagatcaccaagaacgtgaatgatccggatctagtctccaagattgagaacttaaggggcaa-
agta
agccaacgggaagacaggttgaggaaacactacctccacacatcccaggacgtcaagttcttgtctcg
gttcaacgacatcaagttcaagctgaactccgcgacgattctagattcggatgcgttttacaagagcgag
cactttggagtcttgactatcttctgggtggttatcggactctacataatgtcaacgttgtcagacatgtat-
tt
gcatggccaagcccttactggactggataatcataggaatgttcaagaaggatttgatgcaagttgcact
cgttgatcttgtcatgtacttatcctcgtattttccttatttcctacaggttgcatgcaagaccggagctat-
at
ggcatggtcttggatgggccatacagggggtttacagcttggtgtttttaactttctgggcggtacttccgc-
tg
gagctggccatggatcttccttggattgcacgagttttcttgatcttgcattgcttggtgtttattatgaag-
at
aatcatatggacattacaatggatacctttgggatgtatatcaggaaggattggtctcggaagctgatctca
cggctgtttctgagtatgatgatgatttccccctggatcacggggaggttctagaacagagcttgtggttcg
ccaaacacgagttggagtttcaatctaatggaactacggagaggaaggatcaccatcatcatgtattcg
acgaaaaggatgtcaacaaaccaatgcgtgtcttgcaagaagagggaattatcaaatttccggcaaac
atcaatttcaaggattatttcgagtacagtatgttccccacgctagtctacacattgaacttccccagaatt-
c
gacatattagatgggcgtatgtgttgcagaaagttttgggaacatttgccttagtgtttgccatgattatcg-
tc
gccgaagagagtttctgtcccttgatgcaagaagttgaacagtacacaagattgccaaccaaccaaag
gttctcaaagtacttcgtcgttctttcccacttgatattgcccctcggcaaacagtacttgctctcgtttat-
cc
atttggaacgaaattctcaacgggatagcggagttaagcaggtttggggatcgccatttctacggcgcct
ggtggtcaagcgtcgactacatggactattcaagaaaatggaacacgatcgtgcaccgattcctccgcc
ggcacgtttacaattcgaccattcgcatcctcggtatttccaggacccaagccgcgataattacacttttgc-
t
ttcagccacaatccacgaactcgttatgtacatcctatttggaaaattacgagggtacctattccttacgat-
g
cttgtccagatccccatgacagtcaccgccaagttcaacaaccgtttgtggggcaacatcatgttctggttg
acgtatttatctggccccagcttggttagtgcgttgtatttactcttctga SEQ ID NO: 46
Acyl-CoA Sterol
MSDDEIAGIVIEIDDDVKSTSSFQEDLVEVEMSNSSINESQTDELSYRPEEI acyl
transferase SLHSRRKSHKTPSDESFLEITKNVNDPDLVSKIENLRGKVSQREDRLRKH
(ARE2) YLHTSQDVKFLSRFNDIKFKSNSATILDSDAFYKSEHFGVLTIFWVVIGLYI A.A.
Seq MSTLSDMYFGMAKPLSDWIIIGMFKKDLMQVALVDLVMYLSSYFPYFLQV
ACKTGAISWHGLGWAIQGVYSLVFLTFWAVLPSESAMDLPWIARVFLILHC
LVFIMKMQSYGHYNGYLWDVYQEGLVSEADLTAVSEYDDDFPSDHGEVL
EQSLWFAKHELEFQSNGTTERKDHHHHVFDEKDVNKPMRVLQEEGIIKFP
ANINFKDYFEYSMFPTLVYTLNFPRIRHIRWAYVLQKVLGTFALVFAMIIVA
EESFCPLMQEVEQYTRLPTNQRFSKYFVVLSHLILPLGKQYLLSFILIWNEI
LNGIAELSRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHVYNSTI
RILGISRTQAAIITLLLSATIHELVMYILFGKLRGYLFLTMLVQIPMTVTAKFN
NRLWGNIMFWLTYLSGPSLVSALYLLF SEQ ID NO: 47 Diacylglycerol
atgactcaggactataaagacgatagtcctacgtccactgagttggacactaacatagaagaggtgga
acyltransferase
aagcactgcaaccctagagtcggaactcagacagagaaaacagaccacggaaactccagcatcaa
(DGA1)
ccccaccaccacctccacaacaacagcaggcgcataagaaagccctgaagaatggcaagaggaa
Nuc. Seq
gagaccatttataaacgtggcgccgctcaacaccccgttggctcacaggctcgagactttggct-
gttgttt
ggcactgtgtcagtatcccgttctttatgtttttgttcttgcttacggtctccatggggttgcttggg
tggttctttatcattttgccatatttcatttggtggtacggtttcgacttgcacactcc
atcgaatggtaaagttgtctatcgtgtgc
gcaactcgttcaagaatttcatcatttgggactggtttgtcaagtatttcccgattgaagtgcacaagacgg-
t
cgagttggatcctacttttagcgaattgcctgtggaagagagcggcgacagttcggacgacgacgaac
aagacttggtgtctgagcacagcagaactttggttgatcaaatcttcaagtttttcgggttgaagaaacgct-
t
gaatgacacctccctgggcaaaccagagacattcaagaatgtgcctacgggtccaaggtatatttttggg
taccacccacacggagtgatttctatgggggcagtggggttgtttgccaacaacgccttgaggaacgaa
ccatatacgccaatttccaaatggttaaaaccattcttccacgacagctccaagggcgagagattgttccc
tggtattggcaatatcttcccattgacgcttaccacacagtttgcgctcccattttaccgtgactacttgat-
gg
tttggggatcactagtgcatcggctaaaaacattagaagcttgatcaacaatggagacaactctgtgtgtc
tcgtcgttggcggtgcacaagaatcgttgttgaacaatatgattgccaagcacgccagagtcgggtacg
gttacaaagagagcctagatattcatggcgaccagtccgaagaagaagaagaagaagaggatgata
ccaagcagctagagaacccaagtcctaaacgtgaagtgcaattggtcttgaacaaacgtaaaggttttg
tgaagttggctatcgaactaggaaatgtttccttggtgcctatttttgcattcggagaagctgatgtttaca-
ga
tggcccagccagcaccaggctcgttcttgtacaagttccagcaatggatgaaggcaacttttcaattcacc
atcccattgtttagtgctcgaggcgtgttcatctatgatttcggattgttgccattcagaaacccaataaac-
at
ttgcgtcggtagacccgtctacattccgcacaacgtcttgcaagaatacaagcaaaagcacccagagg
agtttgccgaagaggaacctgccagtaccccgatgaagaagtctggatctttcaccgatatgttcaaagc
tggtgaaaagaagcccaagacttcaagtatcaagactaaaatcccacctgcattactagacaagtacc
acaagctatacgtcgacgagttgaagaaggtctatgaagagaacaaggaaaggtttggctacggtgat
gttgaattaaacattgtagaatag SEQ ID NO: 48 Diacylglycerol
MTQDYKDDSPTSTELDTNIEEVESTATLESELRQRKQTTETPASTPPPPP acyltransferase
QQQQAHKKASKNGKRKRPFINVAPLNTPLAHRLETLAVVWHCVSIPFFMF (DGA1)
LFLLTVSMGLLGWFFIILPYFIWWYGFDLHIPSNGKVVYRVRNSFKNFIIW A.A. Seq
DWFVKYFPIEVHKTVELDPTFSELPVEESGDSSDDDEQDLVSEHSRTLVD
QIFKFFGLKKRLNDTSSGKPETFKNVPTGPRYIFGYHPHGVISMGAVGLFA
NNALRNEPYTPISKWLKPFFHDSSKGERLFPGIGNIFPLTLTTQFALPFYRD
YLMALGITSASAKNIRSLINNGDNSVCLVVGGAQESLLNNMIAKHARVGYG
YKESLDIHGDQSEEEEEEEDDTKQLENPSPKREVQLVLNKRKGFVKLAIEL
GNVSLVPIFAFGEADVYRLAQPAPGSFLYKFQQWMKATFQFTIPLFSARG
VFIYDFGLLPFRNPINICVGRPVYIPHNVLQEYKQKHPEEFAEEEPASTPM
KKSGSFTDMFKAGEKKPKTSSIKTKIPPALLDKYHKLYVDELKKVYEENKE RFGYGDVELNIVE
SEQ ID NO: 49 Diacylglycerol
atgtcgtctttaaagaacagaaaatccgcaagcgtcgccacaagcgatacagaagactcagaaacag
acyltransferase
aggcagtatcctcctcaattgatcccaacggcaccatattgcgaccagtcctacatgacgaaccccacc
(LRO1)
acagccatcaccaccacaacataactagaccagtattggaggacgatggcagcatcctggtgtcca-
g Nuc. Seq
aagatcgtcgatctccaaatccgacgacctgcaggcaaagcaaaagaagaagaaacccaagaag-
aaga
tcttggagtctcgtcgggtcatgtttatctttggtaccctcattgggttaatctttgcgtgggcgtttacca-
c
agacacgcatcctttcaatggcgacttggagaagtttatcaactttgaccagctcaacgggatctttgacg
actggaagaactggaaggatatcttgcccaacagcatccagacgtacttgcaggaatcgggcaaggg
cgaagataacgacgggttgcatggtctggccgattccttctccgtcgggctccgcttgaaagcccagaa
gaacttcactgacaaccacaatgtcgtgttggttcctggtgtggtgagcacggggttggaatcgtgggga
acaaccaccaccggtgattgtccatctatcggatacttcaggaagagattgtggggatcattttatatgtta
aggacaatgattttggagaaaacgtgctggttgaagcatatccagttggacgagaagacggggttggat
cctcccaatattaaggtccgtgcggcgcagggtttcgaagcggcagatttctttatggctgggtactggatc
tggaacaagatcttgcagaacttggcggttattgggtacggaccaaataacatggtgagtgctagttatg
actggagattggcttacattgacttggagagaagagatggatatttttcgaaacttaaagcgcagattgag
ttgaataacaagttgaacaacaagaagactgtgttgattggccactcgatggggacccagattattttcta
ctttttgaaatgggtcgaagccaccgggaaaccatactatggcaatggcggaccaaactgggtgaatg
atcatattgagtcgattattgacatcagtgggtcgactttgggtacccccaagagtattcctgtgttgatct-
ct
ggggaaatgaaagacaccgttcaattgaacgcgttggcggtttacgggttggagcaatttttcagcaggc
gtgaaagagtcgatatgttgcgtacatttggtggcgttgccagtatgttacccaaggggggagacaagat
atggggcaacttgacgcatgcgccagatgatccaatttccacattcagtgatgacgaagttacggacag
ccacgaacctaaagatcgttcttttggtacgtttatccaattcaagaaccaaactagcgacgctaagccat
acagggagatcaccatggctgaaggtatcgatgaattgttggacaaatcaccagactggtattccaaga
gagtccgtgagaactactcttacggcattacagacagcaaggcgcaattagagaagaacaacaatga
ccacctgaagtggtcgaacccattagaagctgccttgcctaaagcacccgacatgaagatctattgtttct
acggagttggaaatcctaccgaaagggcatacaagtatgtgactgccgataaaaaagccacgaaatt
ggactacataatagacgccgacgatgccaatggagtcatattaggagacggagacggcactgtttcgtt
attaacccactcgatgtgccatgagtgggccaagggagacaagtcgagatacaacccagccaactcg
aaggttaccattgttgaaatcaagcacgagccagacagatttgatttacgaggcggcgccaagactgc
ggaacatgttgatattttggggagtgccgagttgaacgagttgattttgactgtggttagcgggaacgggg
acgagattgagaatagatatgtcagcaacttaaaagaaatagtagaggccataaatttataa SEQ
ID NO: 50 Diacylglycerol
MSSLKNRKSASVATSDTEDSETEAVSSSIDPNGTILRPVLHDEPHHSHHH acyltransferase
HNITRPVLEDDGSISVSRRSSISKSDDSQAKQKKKKPKKKILESRRVMFIFG (LRO1)
TLIGLIFAWAFTTDTHPFNGDLEKFINFDQLNGIFDDWKNWKDILPNSIQTY A.A. Seq
LQESGKGEDNDGLHGSADSFSVGLRLKAQKNFTDNHNVVLVPGVVSTGL
ESWGTTTTGDCPSIGYFRKRLWGSFYMLRTMILEKTCWLKHIQLDEKTGL
DPPNIKVRAAQGFEAADFFMAGYWIWNKILQNLAVIGYGPNNMVSASYD
WRLAYIDLERRDGYFSKLKAQIELNNKLNNKKTVLIGHSMGTQIIFYFLKWV
EATGKPYYGNGGPNWVNDHIESIIDISGSTLGTPKSIPVLISGEMKDTVQLN
ALAVYGLEQFFSRRERVDMLRTFGGVASMLPKGGDKIWGNLTHAPDDPI
STFSDDEVTDSHEPKDRSFGTFIQFKNQTSDAKPYREITMAEGIDELLDKS
PDWYSKRVRENYSYGITDSKAQLEKNNNDHSKWSNPLEAALPKAPDMKI
YCFYGVGNPTERAYKYVTADKKATKLDYIIDADDANGVILGDGDGTVSLLT
HSMCHEWAKGDKSRYNPANSKVTIVEIKHEPDRFDLRGGAKTAEHVDILG
SAELNELILTVVSGNGDEIENRYVSNLKEIVEAINL SEQ ID NO: ECI1,
atgtccgacgaggaatcagatatcttatacgaggtcagagacagaaccgccatcatcaccttgaacatc
3674 ATCC20336
cccaagagattgaacgcattgaacggtgctcaatacttgaagttgggtaagttcttggagagagccaaca
(Nucleic
acgaagaggacaccgtcttgacattgatccaggccctgggcagattcttctccgccggtgcca-
atttcgcc Acid Seq.)
gacaacgatatggccaaggtcgaaatgtccaagttgttcagtcacgagtactggttggaaagattcgtcg
ccagaaacatctggttgaccaacttgttcaacgaccacaagaagatcttggctgctgctgtcaatggtcca
gttatcggtttgagcactggtttgttgttgttggtcgatttggtctacgtccacgacttgaacaagttctac-
ct
cttggccccatttgccaacttgggtttggttgccgaaggtgcttcctctgccactttgttcaacagattggg
ctggtcaaaggcttctgaggccttgttgttggccaagccaatcggcggccaagactgttacaacgcc
ggtttcatcaacaagcactacgacggtaagttttcctccactgaagagttcaacgaacacgtctacaa
ggagttgacggaagcttttgaaaacttgcacgatgactccattttgcagaacaag
caattgttgaagttgtccagagaccaggccatcaactag SEQ ID NO: ECI1,
MSDEESDILYEVRDRTAIITLNIPKRLNALNGAQYLKLGKFLERANNEEDTVL 3675
ATCC20336 TLIQASGRFFSAGANFADNDMAKVEMSKLFSHEYWLERFVARNIWLTNLF (Amino
Acid NDHKKILAAAVNGPVIGLSTGLLLLVDLVYVHDLNKFYLLAPFANLGLVAEG seq.)
ASSATLFNRLGWSKASEALLLAKPIGGQDCYNAGFINKHYDGKFSSTEEFN
EHVYKELTEAFENLHDDSILQNKQLLKLSRDQAIN* SEQ ID NO: ECI2,
atgtccgacgaccttatcacctacgaagtcaaagaccgagctgccgtgatcaccttgaacaaccccaag
3676 ATCC20336
aagctcaacgccttatcgatcccgcagtacgacaccatctgcaagctcttagaacgagccaacgccga
(Nucleic Acid
agaagacaccgtcatcaccttgctccagtccacgggccgggtgttctctgccggggccaacgccgactc
Seq.)
catcgtggggcaggatgccgagctcgagacctggttgaacatgtcggtggccaagcagacgttcttg-
gtg
cagacgttcctcgcacacaagaagatccttgccgtcgccttgaacggccccgtgattggcttatcggcgg
cgttcgtggcgctctgcgacttggtctacgtgcacaacgccgcaaagacgttcttcttgaccccgttcgcca
acatcgggatccttgccgagggcggcacctcagccacgttgcccatgcgcgtggggtggtccagggcc
gcggaagcgttgttgttgtcaaagaggatttcgggagatgacttgcagagagcggggttcttcaataagg
actacaaggggcagttcaagtccgcggaggagtttaacgaggtcgtcttgaaggagttgcttgacgccac
ggaaaacttgcatgaggactcgatcatccagaacaaggagttgttgaaggctattttcaagccaaagatc
agtgaggtcaactcgcaggaggtgtcaagaggtgtgtacaagtggacctctggggtgccaatggataga
tttaaaaaattgcttaatggtgagttgaaacataaattatag SEQ ID NO: ECI2,
MSDDLITYEVKDRAAVITLNNPKKLNALSIPQYDTICKLLERANAEEDTVITLL 3677
ATCC20336 QSTGRVFSAGANADSIVGQDAELETWLNMSVAKQTFLVQTFLAHKKILAVA
(Amino Acid LNGPVIGLSAAFVALCDLVYVHNAAKTFFLTPFANIGILAEGGTSATLPMRV
seq.) GWSRAAEALLLSKRISGDDLQRAGFFNKDYKGQFKSAEEFNEVVLKELLD
ATENLHEDSIIQNKELLKAIFKPKISEVNSQEVSRGVYKWTSGVPMDRFKKL LNGELKHKL* SEQ
ID NO: >gi|50550800|ref|
ATGTTGTCCATTCGATCCATTACCCGATCTCTCCCCATTGGCAGCCGAA 3678 XM_502873.1|
TCTGCCAGCAGAGTGCCATGAAGGCCTCTACTGTGCGACCTCTCGCCT Yarrowia
TGAGAGCTTACTCCACCCGACCCCCTGTCACTCACTTCTCCGAGGAGG lipolytica
AGGAGATGTTTCGTGACATGGTTAGCAAGTTTGCTGATGAGGTGATTGC
YALI0D15708p TCCCAAGGTCCGTGAGATGGACGAGGCCGAGCAGATGGACAAGACAA
(YALI0D15708g) TCATCCAGGACATGTTCGACAATGGCCTTATGGGCATCGAGACTCCCG
mRNA, complete AGGAGTTCGGTGGTGCAGGTGCCAACTTCACCTCTGCTATCATCGTCG cds
(similar to TTGAGGAGCTTGCCAAGGTGGACCCCTCAGTGTCTGTGATGAACGATG
uniprot|P45954 TCCACAATACCCTCGTCAACACCTGCATCCGATCCTGGGGATCCGACG
Homo sapiens CACTCCGAAACAAGTATCTCCCCCAGCTTGCTGCCCAGAAGGTCGGAT
Acyl-CoA CTTTCGCTCTTTCTGAGCCCTCTTCCGGATCTGATGCCTTCGCCATGAA
dehydrogenase GTCTCGAGCCACAAAGACTGACGATGGATACATTTTGAACGGTTCCAA
short/branched GATGTGGATCACCAACGCTGCCGAGGCTGAGCTTTTCATTGTTTTTGCT
chain specific AATCTCGATCCCAGCAAGGGCTACAAGGGTATTACTGCCTTTGTTGTCG
mitochondrial AGAAGGACATGGGAGTGCAGATTGCTAAGAAGGAGCAGAAGCTGGGT
precursor) ATCCGAGCCTCTTCTACCTGCGTTCTCAACTTCGAGGACGTTTTCATTC
CTAAGGAGAACCTTCTTGGCGAGGAGGGCAAGGGCTACAAGATTGCTA
TCGAGTGCTTGAACGAGGGCCGAATCGGAATTGCGGCCCAGATGCTTG
GCCTTGCTGGTGGAGCTTTCAAGAAGGCTACCGGCTATGCTTTCAACG
ACAGAAAGCAGTTCGGCCAGTACATCGGTGAGTTCCAGGGTATGCAGC
ACCAGATTGGCCAGGCCGCTACTGAGATCGAGGCTGCTCGACTCCTG
GTCTACAACGCTGCCCGACTCAAGGAGGCTGGCGTTCCTTTCACAAAG
GAGGCTGCTATGGCAAAGCTCTATGCTTCCCAGGTTGCAGGAAACGTC
GCATCCAAGGCTGTCGAATGGATGGGTGGTGTCGGATTCACTCGAGAG
GAGACTCTGGAGAAGTTCTTCCGAGATTCTAAGATCGGTGCCATTTACG
AGGGAACTTCCAACATCCAGCTGCAGACTATTGCCAAGATCATCCAGAA GGAGTCTGCCTAA SEQ
ID NO: >gi|49648741|emb|
MLSIRSITRSLPIGSRICQQSAMKASTVRPLALRAYSTRPPVTHFSEEEEMF 3679
CAG81061.1| RDMVSKFADEVIAPKVREMDEAEQMDKTIIQDMFDNGLMGIETPEEFGGA
YALI0D15708p GANFTSAIIVVEELAKVDPSVSVMNDVHNTLVNTCIRSWGSDALRNKYLPQ
[Yarrowia LAAQKVGSFALSEPSSGSDAFAMKSRATKTDDGYILNGSKMWITNAAEAE
lipolytica LFIVFANLDPSKGYKGITAFVVEKDMGVQIAKKEQKLGIRASSTCandidaLNF
CLIB122](similar
EDVFIPKENLLGEEGKGYKIAIECLNEGRIGIAAQMLGLAGGAFKKATGYAF to
NDRKQFGQYIGEFQGMQHQIGQAATEIEAARLLVYNAARLKEAGVPFTKE uniprot|P45954
AAMAKLYASQVAGNVASKAVEWMGGVGFTREETLEKFFRDSKIGAIYEGT Homo sapiens
SNIQLQTIAKIIQKESA Acyl-CoA dehydrogenase short/branched chain
specific mitochondrial precursor) SEQ ID NO: >gi|210075528|ref|
ATGAGCGAGCAGTACACCCCCGAACAAGTTGCGGAGCACAACTCTCCC 3680 XM_501919.2|
GAATCTCTGTGGATCATCATTGACGGTAACGTTTTCGACCTCACTGAAT Yarrowia
TCCAGAAAGAACACCCCGGCGGAAAAAAGATTCTCAAACGAGTCGCAG lipolytica
GAAAAGACGCTACCAAGTTTTTCCACAAATACCACGACGCCCCCAAGAT YALI0C16797p
TATGCGAAAGGTTGGACACAAGTTCAAGATCGGAACCCTTAAAGACGC (YALI0C16797g)
TGAAGCAAACCCCACTCGAGCCATGATTGCCCCTAACAAGACCACCGC mRNA, complete
CCTCGAGCCCTACGGAGACCTTGTCCCCTACGCCGACCCCAACTGGTA cds (similar to
CCACGGCTACCACAACCCCTACTACAAGGAGTCCCACGCCAAGCTGCG uniprot|Q96VP9
TGACGAGGTCCGACAGTGGGTTGAGGAGAAGATTGAGCCCTTCGTTGA Glomus
GGAATGGGATGAGGAGAAGGAGGTTCCCAAGGAGATCTTCCAGGAGA intraradices
TGGGCAAGCGAGGTTACCTTGCCGGCTCTCTCGGCACCCCCTACAAG Probable acyl-
GAGCTGGCCAAGTACACCAACGTCAAGCCCGCCTCTGTGCCCATTGAG CoA
GAGTACGACATGTTCCACGAGCTCATCATCACCGACGAGATCATGCGA dehydrogenase)
GCTGGCTCCGGAGGTCTCACCTGGAACCTGCTTGGTGGCTACTGTATT
GGTCTGCCTCCCGTGATCAAGTTCGCCAAGGAGCCCCTTAAGGAGCGA
ATCCTCCCCGGCCTGCTCGACGGTTCCAAGCGAATCTGTCTGTGTATC
ACTGAGCCCGACGCTGGCTCCGATGTTGCCAACATCACCACTACCGCC
GAGAAGACCCCCGACGGAAAGTTCTACATTGTCAACGGTATCAAGAAG
TGGATCACCAACGGTATCTGGGCTGACTACTTCACTGTTGCCGTCCGA
ACCGGTGGCCCCGGCTCTGGCATGAACGGTATCTCTGTTCTGCTGCTC
GAGCGAGGCATGGAGGGTCTTGAGACCCGACGAATGAACACTCAGGG
TATGCTGTCTTCCGGCTCTACCTGGGTCACCATGGAGGATGTCAAGGT
CCCCGTGGAGAACCTGCTCGGCAAGGAGAACAAGGGTTTCAAGGTCAT
CATGACCAACTTCAACCACGAGCGAGTTGGTATCATCATCCAGGCAAA
CCGAGCTTCTCGAGTTTGCTACGAGGAGGCCTGCAAGTACGCCCACAA
GCGAAAGACTTTCGGCAAGCCTCTGATTGAGCACCCCGTCATCCGAGC
CAAGCTCGCCAACATGGCCATTCGAATCGAGTCCACCCACGCCTGGCT
CGAGAACCTGGTCTTCCAGTGCCAGATGTTCCCCGAGGAGGAGGCCAT
GCTTCGACTTGGTGGTGCCATTGCTGGTTGCAAGGCCCAGGCCACCCA
GACCCTCGAGCTGTGTGCCCGAGAGGCTTCCCAGATCTTTGGTGGTCT
TTCCTACACCCGAGGCGGTCTCGGAGGTAAGGTTGAGCGACTGTACCG
AGAGGTCCGAGCCTACGCCATCCCCGGTGGATCCGAGGAGATTATGCT
GGATCTGGCCATGCGACAGGCCCTCAAGGTCCACAAGGCTGTTGGCG CCAAGCTTTAA SEQ ID
NO: >gi|199425292|emb|
MSEQYTPEQVAEHNSPESLWIIIDGNVFDLTEFQKEHPGGKKILKRVAGKD 3681
CAG82239.2| ATKFFHKYHDAPKIMRKVGHKFKIGTLKDAEANPTRAMIAPNKTTALEPYG
YALI0C16797p DLVPYADPNWYHGYHNPYYKESHAKLRDEVRQWVEEKIEPFVEEWDEEK
[Yarrowia EVPKEIFQEMGKRGYLAGSLGTPYKELAKYTNVKPASVPIEEYDMFHELIIT
lipolytica DEIMRAGSGGLTWNLLGGYCIGLPPVIKFAKEPLKERILPGLLDGSKRICLCI
CLIB122] (similar
TEPDAGSDVANITTTAEKTPDGKFYIVNGIKKWITNGIWADYFTVAVRTGGP to
GSGMNGISVLLLERGMEGLETRRMNTQGMLSSGSTWVTMEDVKVPVENL uniprot|Q96VP9
LGKENKGFKVIMTNFNHERVGIIIQANRASRVCYEEACKYAHKRKTFGKPLI Glomus
EHPVIRAKLANMAIRIESTHAWLENLVFQCQMFPEEEAMLRLGGAIAGCKA intraradices
QATQTLELCAREASQIFGGLSYTRGGLGGKVERLYREVRAYAIPGGSEEIM Probable acyl-
LDLAMRQALKVHKAVGAKL CoA dehydrogenase) SEQ ID NO:
>gi|50556785|ref|
ATGCTTACCAGAATCTCCCGTTTGGCACCTGCTGCCCGAGGCTTTGCT 3682 XM_505801.1|
ACCTCCTCCGTCAACCGATCCACAGCCGCCATGGACTGGCAGGATCCC Yarrowia
TTCCAGCTGGACTCTCTTCTCACCGAGGACGAGATTGCCGTGGCTGAG lipolytica
GCTGCTCGAGACTTCTGCCAGACAGAGCTCTACCCCAAGGTACTTGAG YALI0F23749p
GGCTACCGAACCGAGGAGTTCCCCCGAAGCATCATGAAGCAGATGGG (YALI0F23749g)
TGAGGTTGGTCTGCTCGGAACAACCGTCAAGAGCCACGGATGCCCCG mRNA, complete
GCATGTCTTCTGTCGCTTACGGTCTCGTGGCCCGAGAGGTCGAGAGG cds (highly
GTCGACTCCGGCTACCGATCTGCCATGTCTGTGCAGTCGTCGCTGGTC similar to
ATGCACCCCATTGAACAGTTTGGATCCCAGGAGCAGAAGGACCGGTTC uniprot|Q7S579
CTGCCCAAATTGGCCTCCGGCGAGATGATCGGCTGCTTCGGTCTCACC Neurospora
GAGCCTAACCACGGTTCCGACCCTGGATCCATGGAGACCGTCGCCAA crassa
GATGCACCCTACTAAGAAGGGCGTCATTGTGCTCAATGGAGCCAAGAA NCU02291.1
CTGGATCACTAACTCTCCTATTGCCGATCTCATGGTTGTGTGGGCCAAG hypothetical
TTGGACGGTAAGATCCGAGGCTTCCTTGTCGAGCGATCTCAGGTCGCC protein probable
TCCGGCCTCGCTACTCCCGCCATCAAGAACAAGACCGCTCTGCGAGCC Glutaryl-CoA
TCCATCACCGGTATGATCCAGATGGACGACGTTGAGATCCCTGTGGAG dehydrogenase)
AACATGTTCCCCGAGGTGACCGGTCTCAAGGGCCCCTTCACCTGCCTC
AACTCTGCCCGATACGGTATCGCCTGGGGAACCATGGGCGCTCTGTCC
GAGTCCATCAAGCTCGCTCGAGAGTACTCTCTGGACCGAAAGCAGTTT
AAGGGCCAGCCTCTGGCCAAGTACCAGCTCATCCAGAAGAAGCTCGCT
GACGCTCTGACCGATGCCACCTACGGACAGGTCGCTGCCATTCAGGTC
GGCCGGCTCAAGGATGCCGGCAATTGTCCTCCCGAGCTCATCTCCATG
ATTAAGAGACAGAACTGTGACCGAGCCCTCGCTGGCGCTCGAAACCTG
ATGGAGATCTTTGGCGGTAACGCTGCCTCTGACGAGTACCACATTGGC
CGAATTGCCGCCAACCTGTGGGTTGTCCAGACCTATGAGGGCCAGTCT
GATATCCATGCTCTCATCCTGGGAAGAGCCATGACCGGCGTCCAGGCT TTTGCTTAA SEQ ID
NO: >gi|50556786|ref|
MLTRISRLAPAARGFATSSVNRSTAAMDWQDPFQLDSLLTEDEIAVAEAAR 3683
XP_505801.1| DFCQTELYPKVLEGYRTEEFPRSIMKQMGEVGLLGTTVKSHGCPGMSSVA
YALI0F23749p YGLVAREVERVDSGYRSAMSVQSSLVMHPIEQFGSQEQKDRFLPKLASG
[Yarrowia EMIGCFGLTEPNHGSDPGSMETVAKMHPTKKGVIVLNGAKNWITNSPIADL
lipolytica] (highly
MVVWAKLDGKIRGFLVERSQVASGLATPAIKNKTALRASITGMIQMDDVEIP similar to
VENMFPEVTGLKGPFTCLNSARYGIAWGTMGALSESIKLAREYSLDRKQF uniprot|Q7S579
KGQPLAKYQLIQKKLADALTDATYGQVAAIQVGRLKDAGNCPPELISMIKR Neurospora
QNCDRALAGARNLMEIFGGNAASDEYHIGRIAANLWVVQTYEGQSDIHALI crassa
LGRAMTGVQAFA NCU02291.1 hypothetical protein probable Glutaryl-CoA
dehydrogenase) SEQ ID NO: >gi|255723091|ref|
ATGTCAGTCAAAGAAGATATCCCAGCTGTTTTTCTTTCCCAAATTTCTCC 3684
XM_002546434.1| TCGTGGTCTTGAAGCTATCCAGAAAACCAAAGACTTTGTCAATGACTAC
Candida TGTATTCCAGCCGATGAAATCTACTTCAAACAGGTCTCTACTGATCCTG
tropicalis MYA- CCAAAAGATGGAAAACAATCCCACCTATTATTGAGACATTGAAATCCAA
3404 conserved AGCCAAAGAACTTGGTTTATGGAATATGTTTTTATCCAAACATTATAAGG
hypothetical AAGGTCCACAATATACCAACTTAGAATATGGTTTGATGGCTAGATATTTG
protein, mRNA GGTCGTGCACACACTGCTCCTGAAGCTACTAATACTGCTGCTCCAGATA
(similar to C. albicans
CTGGTAACATGGAATTACTTGCTAAATACGGTACTCCATACCAAAAAGA ACD99)
AAAGTACTTACAACCATTGTTAGATGGAAAGATCAGATCTGCTTTCTTGA
TGACCGAAAAGGGCACATCATCTTCCAATGCATTAAATATCTCCACTAG
TGCCAAAAAGAATGCCAGTGGTAACTATGTTCTTGATGGTGTAAAGTGG
TTTGCTTCAGGTGCTGGTGATCCAAGGTGTTCTGTTTGGTTGGTCATGT
GTAAAACTGAAGACAATAAGAAGAACCCATATGCAAACCACACCGTGTT
GGTTCTTGATGCCAAGAGAGCATTGGCTAGCGGCAGGGCCAAATTAGT
CAGACCTTTGCATGTTATTGGATATGATGATGCTCCTCATGGTCATTGT
GAAATTTCTTTTGAAAACTACGAAGTTCCTGCTGACGAAATGCCAAATG
CTGTTTTGGCCGGTATTGGAAGAGGATTTGAGTTGATTCAGTCTAGATT
AGGACCTGGTAGAATTCATCATTGTATGAGAGCTATTGGTACAGGTGAA
ATTGCATTGTTGATCATTGCTCATAGAGCTAACCACAGAATGATTTTTGG
AAAACCAATGAAAGACAGAGAAGGATTTTTGTCTAAGTTCGGTCAGAGC
AGAATTGATATTACCAGATGCTTGTTATTGGTATTAAATGCTGCTCATAA
AATTGATATTTCCAACGCAAAGGCTGCTCAGAAAGAGATTGCCATGGCT
AAGATTGAAACACCAAGAACCATCTCTGATATCCTTGACTGGGGTATCC
AAGTTTTTGGCGCAGAAGGGGTCTCACAAGACACAGACTTAGCTAGAA
TGTATGCTCTCAACAGAACCTTGAGAATTGCTGATGGTCCTGATGAAGC
TCACTTGGCACAATTGGCAAGAAATGAGGCCAAAAAATTCCCAGAGGT
CGATATCTTCTTTGAACATGTTGCTAGTCAACGTAATAAATTATAG SEQ ID NO:
>gi|240130997|gb|
MSVKEDIPAVFLSQISPRGLEAIQKTKDFVNDYCIPADEIYFKQVSTDPAKR 3685
EER30559.1| WKTIPPIIETLKSKAKELGLWNMFLSKHYKEGPQYTNLEYGLMARYLGRAH
conserved TAPEATNTAAPDTGNMELLAKYGTPYQKEKYLQPLLDGKIRSAFLMTEKGT
hypothetical SSSNALNISTSAKKNASGNYVLDGVKWFASGAGDPRCSVWLVMCKTEDN
protein [Candida
KKNPYANHTVLVLDAKRALASGRAKLVRPLHVIGYDDAPHGHCEISFENYE tropicalis MYA-
VPADEMPNAVLAGIGRGFELIQSRLGPGRIHHCMRAIGTGEIALLIIAHRANH 3404]
(similar to RMIFGKPMKDREGFLSKFGQSRIDITRCLLLVLNAAHKIDISNAKAAQKEIAM
C. albicans AKIETPRTISDILDWGIQVFGAEGVSQDTDLARMYALNRTLRIADGPDEAHL
ACD99) AQLARNEAKKFPEVDIFFEHVASQRNKL SEQ ID NO: XP_716423.1|
MSVKEDIPAVFLEKVSPRGLEAIQKTKDFVNDYCLPADQIYFEQLSDIPSER 3686 probable
acyl- WKSVPPVIETLKKKAKELGLWNMFLSKHYKEGPQYTNLEYGLMARYLGRS CoA
YTAPEATNTAAPDTGNMELFAKYGTTYQKDRYLKPLLNGEIRSAFLMTEKG dehydrogenase
VSSSNALNISTSAVKNSNGNYVLNGVKWFASGAGDPRCSVWLVMCKTDN [Candida
NKQNPYQNHTVLIIDAKKALATGKAKLIRPLQVIGFDDAPHGHCEIQFQDYE albicans
VPADEMPNVVMAGVGRGFELIQSRLGPGRIHHCMRAIGAGEFALLRIAHR SC5314]
ANHRLIFGKPMNQREGFLSRYGQSKIDIERCLLLVLNAAHKIDISNAKEAQK
EIAMAKIETPRTISDILDWGIQVYGAEGMSQDTELARMYAHNRTLRIADGPD
EAHLAQLARNEAKKFAKVDDFFANMETQRSKL SEQ ID NO: XM_711330.1|
ATGTCAGTTAAAGAAGACATTCCTGCTGTTTTCCTTGAAAAGGTTTCTCC 3687 Candida
albicans TCGTGGTCTCGAAGCCATCCAGAAAACCAAAGATTTCGTTAACGATTAT SC5314
probable TGTCTTCCAGCCGATCAAATTTATTTTGAACAACTTTCAGACATCCCATC
acyl-CoA AGAAAGATGGAAGAGTGTTCCTCCTGTCATTGAGACATTGAAGAAGAAA
dehydrogenase GCCAAGGAACTTGGTTTATGGAACATGTTTTTGTCAAAGCATTATAAGG
(ACD99) mRNA, AAGGTCCACAATATACAAACTTAGAGTATGGATTGATGGCCAGATACTT
complete cds GGGTCGTTCATACACAGCACCAGAGGCTACCAACACAGCTGCTCCAGA
TACCGGTAATATGGAATTGTTTGCCAAATATGGAACCACTTATCAGAAA
GATAGATACTTGAAACCCTTGTTAAATGGGGAAATTAGATCAGCATTCTT
GATGACAGAAAAAGGTGTTTCATCATCTAATGCTCTCAATATTTCTACAA
GTGCTGTCAAGAATTCGAATGGAAATTACGTGCTCAATGGTGTCAAATG
GTTTGCTTCAGGTGCAGGAGATCCAAGATGTTCCGTCTGGTTGGTGAT
GTGCAAGACAGACAACAACAAGCAAAATCCATATCAAAACCACACAGTT
TTGATCATCGATGCCAAAAAGGCTTTGGCTACTGGAAAAGCCAAATTGA
TCAGACCATTGCAGGTCATTGGTTTTGATGATGCTCCTCATGGACATTG
TGAGATTCAATTTCAAGATTACGAAGTTCCTGCCGATGAAATGCCTAAT
GTTGTTATGGCTGGTGTTGGTAGAGGATTTGAGTTGATTCAATCCAGAT
TGGGTCCAGGTAGAATCCACCATTGTATGAGAGCTATTGGTGCTGGTG
AATTTGCATTATTGAGAATTGCTCACAGAGCAAATCACAGATTGATTTTT
GGTAAACCTATGAACCAGAGGGAAGGATTCTTATCCAGATACGGACAA
AGCAAAATCGACATTGAAAGATGTTTATTGTTGGTGTTGAATGCTGCTC
ACAAAATTGATATTTCCAATGCCAAAGAAGCACAAAAGGAAATTGCTAT
GGCTAAGATTGAGACCCCGAGAACTATCTCTGATATTCTCGATTGGGGT
ATTCAAGTTTATGGTGCTGAGGGTATGTCACAAGATACCGAGTTGGCCA
GAATGTATGCTCATAACAGAACATTGAGAATAGCTGATGGACCTGATGA
AGCTCATTTGGCCCAATTGGCTAGAAATGAAGCTAAAAAGTTTGCAAAA
GTTGACGACTTTTTCGCCAACATGGAAACTCAACGTAGCAAATTATAA SEQ ID NO:
acyl-CoA ATGACAGACCTTGACATTCCAGCAGTATTCCTTGATAAGATCTCACCAC 3688
dehydrogenase GTGGCCTCGAGGCGATCCGCAAGACCTACGACTTTGTGCATAACTACT NM
domain-like GTATTCCTGCGGATGCTCTCTACTTTGACCAAATTTCCCAGGATCCCGA
protein [Candida ACAAAGGTGGAAAACCACTCCTGAAGTCACTGAAAAATTGAAACAAAAG
tenuis ATCC GCCAAACAATTAGGTTTGTGGAACATGTTCCTCTCTAAGCACTATACCG
10573] (similar to
ATGGACCTGGCTACACAAACTTGGAGTATGGCCTTATGGCGCAATTCTT C. albicans
GGGCCGGTCGTTCGTGGCACCCGAGGCCACCAATACAGGTGCACCCG ACD99)
ATACAGGTAACATGGAGATTCTCGCCAAGTTCGGCTCGGCCTATCACC
GGGAGCAGTACCTCCTTCCATTGCTCCGCGGTGAGATCCGCTCGGCGT
TCTTGATGACAGAAAAAGGCACTTCTTCATCCAATGCCTTGAACATCTC
ATGCTCGGCCCAGAAGAATTCACACGGCAACTACGTTCTCAATGGAGT
CAAGTGGTTTGCCTCTGGTGCAGGTGATCCTCGGTGTCGCGTGTGGTT
GGTGATGTGCAAAACCGAGTCTCTGGACAACATCTACCGTAATCACAGT
GTGTTGGTGTTGGATGCGAAAAAGGCTTTAGCTTCAGGAAAAGCCAAAT
TGATCCGACCACTCAGCGTGTTTGGCTATGACGATGCTCCTCATGGAC
ACTGTGAGGTGGAGTTCAACGACTTTGAGGTGCCAGCCGAGGATATGG
ATAATTCTATCCTTGGTAAGGTGGGTATGGGATTTGAGATCATCCAGTC
TCGTTTAGGCCCTGGGCGTATTCACCACTGTATGCGTCTTATTGGTGCC
GGAGAATATGCCTTAATGAGGGCGGTGCTGAGGGCTGCCGGCAGAGACA
CATTTTCGGCAAGCCCATGGTGAAGAGAGAATCATTTCTCAATGCTTAT
GGAGAGCATAAGCTTTCACTTTCAGAAATGCCGTCTTTTGGTGCTTAATG
CAGCTCATCAAATCGATATTTCGAATGCTAAGACTGCCAAAAGAGATAT
AGCCATGGCCAAAATCGAGACTCCCAGAGCAGTATTGAAGATTCTTGA
CTGGTGTATTCAGGTTTATGGGGCTGAAGGAGTGTCTCAAGACACAGA
GCTTGCAAAGATGTATGCTCACGCTCGGACTTTGAGAATCGCAGATGG
ACCAGATGAAGCACACCTTGGACAGCTTGCACGGGACGAGTCAAAGAA
GTTTGCGGAGGTGGTGAAGTACTTTGAGGGACACAAGGCACGTCAAGA
CCAAGTCCTGAAGTTGTGA SEQ ID NO: >gi|344233800|gb|
MTDLDIPAVFLDKISPRGLEAIRKTYDFVHNYCIPADALYFDQISQDPEQRW 3689
EGV65670.1| KTTPEVTEKLKQKAKQLGLWNMFLSKHYTDGPGYTNLEYGLMAQFLGRSF
acyl-CoA VAPEATNTGAPDTGNMEILAKFGSAYHREQYLLPLLRGEIRSAFLMTEKGT
dehydrogenase SSSNALNISCSAQKNSHGNYVLNGVKWFASGAGDPRCRVWLVMCKTESS NM
domain-like DNIYRNHSVLVLDAKKALASGKAKLIRPLSVFGYDDAPHGHCEVEFNDFEV
protein [Candida
PAEDMDNSILGKVGMGFEIIQSRLGPGRIHHCMRLIGAGEYALMRAVSRAA tenuis ATCC
GRDIFGKPMVKRESFLNAYGEHKLSLQKCRLLVLNAAHQIDISNAKTAKRDI 10573](similar
to AMAKIETPRAVLKILDWCIQVYGAEGVSQDTELAKMYAHARTLRIADGPDE C. albicans
AHLGQLARDESKKFAEWKYFEGHKARQDQVSKL ACD99) SEQ ID NO:
>gi|50309254|ref|
ATGCCTAATGTCAGTGATAGACCGCGGACATATAAGAAACCTGCTTTAG 3690 XM_454634.1|
AAGATGTTGATCCCATCACAAACTATATACCTGCCAGTGTTAGGGATAA Kluyveromyces
ATTTGATGAGAGGCAGATGGATCGGTTCAAGAAGTTGCGGAAATTTGTT lactis NRRL Y-
GAGTTTGAATGTTTGCCATTAGATACGGTGTATTTGCAAGAGAGTACCC 1140
TATTTGAGCATGAAAGCGATTTAGAGACGTGCCCAGTCATTATTAATTTA hypothetical
AGGAAGAAATTGGAGGCATACCAGTTGCATAAAATGTTTGTTCCAATGG protein partial
ATCAACGTGGGTACGACCATAGTTTCAACGATAATTGGGAAGTGGTGA mRNA (similar to
GTATGGTTGAATTTGCTATGATCGCTTTCCTTGCTGGAAGATCTGTCATT C. albicans
GCCAGTTATTTGTTCCATTTGGATGATTTGATCGATTTAGGAACTATACA ACD99)
AGTTTTGTTGAGAAATGGTTGTTCGAACCATGATTTGTGGGTACAAGTG
ATAGATGAGTTAGTTTCTAATAATATGAAATCGTGTTTGATGGTAAGTGA
AAGAGATGTGTCTGGTTCTGATGCGTTGAACGTTCAAACCACCTGTAAA
ATTGAAGGGGATGATCTAAACGAAGAGGAGGCTACTATGACACTTAAC
GGTACTAAATGGTTTATCAAAGATGCAGGAGACTCAGATATTTGGTTAG
TTTTATGTGTCACTGAATTTGATGAGGGCAACATTTATAGAAAACATACA
TTATGCCTTGTTAACAGGAATGATTTACCACCAAATTCAACAAGAATTGA
ACCTATAGAAACAAATGAAGCGATTGGTAAATTTTATGAAGTACAATTTA
AAGATTGTAAAGTACCGTTAAATATTATTGGTGAAAGAGGTGAAGGTTA
TCAAATTTTACAAATGAAATCCTCTGTTACAAAATTATTTCAATGCTTAAA
ACTTTGTGGTATGGGACAAGAATCCTTGAGACTTTCCAATAAGAGAGCT
GCTGAAAGGAAAGTGTTTGGTTCCAAATTACAGAAGAGTGAGTATTTCA
AATTTGATCTTGCTCATTGGAGGATTAAGATTGAAACCTGTAAGCTGCTT
TGTTTCAACGCGGCAATCAAATGTGATTACGAAGGTGTAAAAGCGGCAA
GAGAAGAAATTGGGATGGTGAAAGCCGTGACACCAAAGGAAATCTCGT
CACTGGTGGATTGGTCTATCCAGTTGCATGGATGTTACGGACTCTGTTC
AACACAAACACCCTTGTCACATATGTGGCAAGTGAGTCGATCGCTAAGA
ATTAATGATACGCCGGACGAATCATTAATATCACAACTGGGGAGGTTGG
AAATCAGTAATTATAACAAATTTCAAAAGACATACGATCAAGAATTAACG
ACGCTCGCTGGCAAATGA SEQ ID NO: >gi|49643769|emb|
MPNVSDRPRTYKKPALEDVDPITNYIPASVRDKFDERQMDRFKKLRKFVEF 3691
CAG99721.1| ECLPLDTVYLQESTLFEHESDLETCPVIINLRKKLEAYQLHKMFVPMDQRG
KLLA0E15181p YDHSFNDNWEVVSMVEFAMIAFLAGRSVIASYLFHLDDLIDLGTIQVLLRNG
[Kluyveromyces CSNHDLWVQVIDELVSNNMKSCLMVSERDVSGSDALNVQTTCKIEGDDLN
lactis] (similar
EEEATMTLNGTKWFIKDAGDSDIWLVLCandidaTEFDEGNIYRKHTLCLVN to
RNDLPPNSTRIEPIETNEAIGKFYEVQFKDCKVPLNIIGERGEGYQILQMKS C. albicans
SVTKLFQCLKLCGMGQESLRLSNKRAAERKVFGSKLQKSEYFKFDLAHWR ACD99)
IKIETCKLLCFNAAIKCDYEGVKAAREEIGMVKAVTPKEISSLVDWSIQLHGC
YGLCSTQTPLSHMWQVSRSLRINDTPDESLISQLGRLEISNYNKFQKTYDQELTTLAGK SEQ ID
NO: >gi|301507715|gb|
ATGTCGATTAAGGACGACATCCCTGCCATCTTTTACGAAAAACTTTCCC 3692 GU338397.1|
CCCGCGGGCTTGAGGCTATCGCCAAAACCAAGGAATTCGTCGACACTT Candida rugosa
ACTGCTCCCCCGCCGACGAGATCTACTTCCAACAGGTGAGAACTGACG propionyl-CoA
ACCGCCGGTGGAAGGAAACGCCCCCCATCACCGAGCACTTGAAGAAG dehydrogenase
AAAGCTAAAGAGCTCGGGTTATGGAACATGTTCTTGCTGAAGCACTACG mRNA, complete
CCGAGGGCGCCGGCTACACCAACTTGGAGTATGGGCTTATGGCCCAG cds (similar to C.
TACCTTGGCCGCAGTCACATCGCCCCTGAAGCTACCAACACCAATGCT albicans
CCTGACACCGGCAACATGGAGATCCTTGCCAAGTACGGCAACGACTAC ACD99)
CACAAGCAGCGCTACCTCCAGCCGCTTCTCGACGGTAAAATCCGCCTG
GCGTTCTTAATGACGGAAAAGGGGACGTCGCTGTCCAACGCCCTTAAC
ATCTCGTGCCTGGCAAAACTTAACCAAAATGGCAACTACGTCATCAACG
GCGTCAAGTGGTTCGCCCTGGGTGCCGGCGACCCCCGGTGCAAGGTG
TGGTTGACGATGTGCAAGACCAGCGACGACGACGCCAACCCATATTTC
AACCACTCGTTGCTTGTGCTTGATGTCGACAAGGCCCTCGCCCTGGGA
CAGGCTCGTGTTGTCCGCCCGTTGCACGTGTTTGGCTACGACGACGCT
CCTCACGGTCACTGTGAAATTGAATTTAACAACTACGAAGTGTCCAAAG
AGGAAATGGCCAACGTCATCCTCGGCCAGGTGGGCCAAGGATTTGCCA
TCATCCAGCTGAGATTGGGGCCGGGGCGCATCCACCACTGCATGCGG
ATGATTGGCGTCGGCGAATTCGCCTTGATGAGAGTGGCTCAGCGGGCT
AACCACCGTATCATCTTCGGTAAGCCCATGGCCAAGCGCGAACTGTTTT
TGAACGCCTACGCTCAGGCAAAGATCGACATCCAAAAGTGCCGCTTGT
TTGTTCTTAATGCCGCCCACCACATCGACATTGCCGGAGCCAAAGCGG
CGCAAGCCGACATCGCCATGGCCAAGATCGAGACCCCGAGAACCATC
CTTCGCATCTTGGACTGGGGGATCCAGATGTTTGGCGCCGAAGGGGT
GTCTCAAGACACCGAGCTCTCGCGCATGTACGCGTTGGGGCGGACGT
TACGCATTGCCGACGGCCCCGATGAAGCTCACTTGGGCCAATTGGCCC
GTAAGGAGCTGAAGAAGTTCCCTTACGTCGATGAGTACTTTAAGCGGTT
TGAAGAAAATAAGGCGAAGTTGGCCAAGTTGTAA SEQ ID NO: >gi|301507716|gb|
MSIKDDIPAIFYEKLSPRGLEAIAKTKEFVDTYCSPADEIYFQQVRTDDRRW 3693
ADK77878.1| KETPPITEHLKKKAKELGLWNMFLSKHYAEGAGYTNLEYGLMAQYLGRSHI
propionyl-CoA APEATNTNAPDTGNMEILAKYGNDYHKQRYLQPLLDGKIRSAFLMTEKGTS
dehydrogenase SSNALNISCSAKLNQNGNYVINGVKWFASGAGDPRCKVWLTMCKTSDDD
[Candida ANPYFNHSLLVLDVDKALASGQARVVRPLHVFGYDDAPHGHCEIEFNNYE rugosa]
(similar VSKEEMANVILGQVGQGFAIIQSRLGPGRIHHCMRMIGVGEFALMRVAQR to C.
albicans ANHRIIFGKPMAKRESFLNAYAQAKIDIQKCRLFVLNAAHHIDIAGAKAAQA
ACD99) DIAMAKIETPRTILRILDWGIQMFGAEGVSQDTELSRMYALGRTLRIADGPD
EAHLGQLARKESKKFPYVDEYFKRFEENKAKLAKL SEQ ID NO:
>gi|380353348:2148
ATGTCAGTTAAAGACGATATCCCAGCTATCTTTTTAGATAAGGTTTCTCC 3694 09-216140
AAGAGGTCTTGAAGCAATTCAAAAGACAAAGGACTTTGTCGACCAATAT Candida
TGTATCCCTGCTGATAAGATTTTCAAGGAGCAAATTTCGCAAGACCCAA orthopsilosis Co
AAATAAGATGGAAACAATATCCAGCTATCATTGAACCATTGAAGAAAAA 90-125,
GGCTAGAGAGTTGGGTTTGTGGAACATGTTTTTGTCCAAGCATTACAAA chromosome 4
GAGGGTCCTCAATTTACCAATTTGGAATACGGATTAATGGCTAGGTATT draft sequence
TGGGAAGATGTCACACTGGACCAGAAGCAACCAACACCAGTGCCCCAG (similar to C.
ACACAGGTAATATGGAATTGTTTGCTAAATATGGTACAAAGGCGCAAAA albicans ACD99)
GGATAAGTATTTAGTGCCCTTGATGGATGGTAAGATCAGATCGGCATTC
TTGATGACCGAAAAGGGGATTTCATCGTCGAATGCATTAAACATTTCAA
CCACTGCCATTAAGAATGCCCGTGGTAACTATGTGTTGAATGGAACAAA
GTGGTTTGCCTCTGGTGCTGGAGATCCAAGAACTGCTGTTTGGTTGGT
TATGTGCAAAACAGACAATGATGAAAGTAATATGTTCAGAAACCACTCC
GTGTTAGTCATTGATGTCAAGCATGCATTAGCATCAGGTAAGGCTGAAG
TTATCAGGCCTTTGAGTATTTTTGGCTACGATGATGCACCCCATGGTCA
TTGTGAAATCGTTTTCAAGGATTATGAAGTTTCATCTGAATTGATGCCAG
AAACGATTTTGGCCGGTGTCGGTAGGGGATTTGAATTGATTCAATCCCG
TTTGGGTCCAGGTAGAATCCATCATTGTATGAGAGCCATAGGTGCTGGT
GAATTTGCCTTGTTGCGTATTGCTCACAGAGCAAATCACAGAACCATCT
TTGGTAGGCCAATGAATAGAAGAGAAGGCTTCTTGATGCAGTATGCCAA
GTACAGAATTGAAATTCAAAAATGTTTATTATTGGTTTTGAATGCTGCTC
ACAAGATTGACATCACTAATGCCAAACATGCACAAAGAGAAATTGCCAT
GGCTAAAATTGAGACTCCAAAAACAATTTGCGATATTCTCGACTGGGGT
ATTCAAGTCTTTGGAGCCGAAGGATTCTCTCAAGATACAGAATTGGCAC
AAATGTATGCTTGGAATAGAACTTTGAGAATCGCTGATGGTCCTGATGA
AGCACATTTGGCTCAATTGTCAAGAAGAGAAGCTGCCAAGTTTCCAGAA
GTTGATGAGTTTTTCAAGAGTGTTGAATCAAGAGTTGAAGCTATTAGTAA GTTATAA SEQ ID
NO: >gi|380353467|emb|
MSVKDDIPAIFLDKVSPRGLEAIQKTKDFVDQYCIPADKIFKEQISQDPKIRW 3695
CCG22977.1| KQYPAIIEPLKKKARELGLWNMFLSKHYKEGPQFTNLEYGLMARYLGRCH
hypothetical TGPEATNTSAPDTGNMELFAKYGTKAQKDKYLVPLMDGKIRSAFLMTEKGI
protein SSSNALNISTTAIKNARGNYVLNGTKWFASGAGDPRTAVWLVMCKTDNDE
CORT_0D01290 SNMFRNHSVLVIDVKHALASGKAEVIRPLSIFGYDDAPHGHCEIVFKDYEVS
[Candida SELMPETILAGVGRGFELIQSRLGPGRIHHCMRAIGAGEFALLRIAHRANHR
orthopsilosis] TIFGRPMNRREGFLMQYAKYRIEIQKCLLLVLNAAHKIDITNAKHAQREIAM
(similar to C. AKIETPKTICDILDWGIQVFGAEGFSQDTELAQMYAWNRTLRIADGPDEAH
albicans ACD99) LAQLSRREAAKFPEVDEFFKSVESRVEAISKL SEQ ID NO:
>gi|354545630:
ATGTCAGTTAAGGACGATATTCCAGCAATCTTTTTAGATAAGGTTTCCCC 3696
225012-226343 AAGAGGTCTTGAAGCTATTCAAAAGACAAAAGACTTTGTTGAGCAATAC
Candida TGTATTCCTGCCGATAAAGTTTTCAAGAAACAGATTTCGACAGACCCAG
parapsilosis CGGTAAGATGGAAACAATACCCTGCTATTATTGAACCATTGAAGAAAAA
strain CDC317 GGCTAGGGAATTGGGATTGTGGAACATGTTTTTGTCCAAGCATTACAAA
annotated contig GAGGGTCCTCAATTTACCAACTTGGAATATGGATTGATGGCTAGGTATC
005809 (similar to TAGGAAGATGCCACACTGGTCCTGAAGCCACTAACACTAGTGCACCAG
C. albicans ACACGGGTAATATGGAGTTGTTTGCAAAATATGGTACAAAGGCGCAAAA
ACD99) AGACAAATATTTGGTGCCCTTGATGGATGGTAAGATTAGATCAGCATTT
TTGATGACTGAAAAGGGGATCTCATCGTCCAATGCGTTGAACATTTCCA
CCACTGCAATTAAAAACTCACGTGGAAACTATGTCTTGAATGGTACCAA
GTGGTTTGCATCAGGCGCTGGTGATCCTAGAACTGCCGTTTGGTTGGT
TATGTGTAAGACTGCCAACGATGAAAAGAATGCATTTAAAAACCACTCA
GTATTAGTGATTAATGTTAAGCATGCATTAGCATCAGGCAAGGCTGAAG
TTATTAGACCTTTGGGAATTTTCGGATACGACGATGCTCCTCATGGACA
TTGTGAAATTGTTTTCAAAGATTATGAAGTTTCATCAGAGTTGATGCCAG
ATACCATTTTGGCTGGTGTTGGTAAAGGATTCGAATTGATTCAATCTAG
ATTGGGCCCGGGTAGAATCCATCATTGTATGAGAGCTATTGGTGCTGG
TGAATTTGCATTGTTGCGTATCGCCCACAGAGCTAATCACAGAATTATTT
TTGGTAAACCAATGAATAGAAGAGAAGGCTTTTTGATGCAGTATGCCAA
GTACAGAATCGAGATTCAAAAATGTTTATTGTTGGTTTTAAATGCTGCCC
ACAAGATAGATATCACTAATGCCAAAGAAGCTCAAAGAGAAATTGCAAT
GGCCAAGATTGAAACTCCAAAAACCATTTGTGATATTCTTGATTGGGGT
ATTCAAGTGTTTGGAGCTGAGGGTTTCTCTCAGGATACAGAATTGGCGC
AAATGTACGCTTGGAACAGAACTTTGAGAATTGCAGATGGACCAGATGA
AGCACATTTGGCTCAATTAGCAAGAAGAGAAGCCGCAAAGTTCCCTGA
CGTTGACGTGTTTTTTAAAGATGTTGATTCAAGAGTTGAGGCTGTTAGT AAATTATAA SEQ ID
NO: >gi|354545753|emb|
MSVKDDIPAIFLDKVSPRGLEAIQKTKDFVEQYCIPADKVFKKQISTDPAVR 3697
CCE42481.1| WKQYPAIIEPLKKKARELGLWNMFLSKHYKEGPQFTNLEYGLMARYLGRC
hypothetical HTGPEATNTSAPDTGNMELFAKYGTKAQKDKYLVPLMDGKIRSAFLMTEK
protein GISSSNALNISTTAIKNSRGNYVLNGTKWFASGAGDPRTAVWLVMCKTAN
CPAR2_201240 DEKNAFKNHSVLVINVKHALASGKAEVIRPLGIFGYDDAPHGHCEIVFKDYE
[Candida VSSELMPDTILAGVGKGFELIQSRLGPGRIHHCMRAIGAGEFALLRIAHRAN
parapsilosis] HRIIFGKPMNRREGFLMQYAKYRIEIQKCLLLVLNAAHKIDITNAKEAQREIA
(similar to C. MAKIETPKTICDILDWGIQVFGAEGFSQDTELAQMYAWNRTLRIADGPDEA
albicans ACD99) HLAQLARREAAKFPDVDVFFKDVDSRVEAVSKL SEQ ID NO:
>gi|241959309|ref|
ATGTCAGTTAAAGAAGATATTCCTGCTATTTTCCTTGAAAAGATTTCCCC 3698
XM_002422329.1| TCGTGGTCTTGATGCTATCCAGAAAACCAAAGATTTCGTAAACGATTATT
Candida GTCTTCCAGCAGATCAGATCTATTTTGAGCAGCTCTCTGACATCCCTTC
dubliniensis AGAAAGATGGAAAAGTGTTCCTCCTGTCATTGAGACATTGAAGAAGAAA CD36
acyl-coa GCCAAGGAACTTGGTTTATGGAACATGTTTTTGTCAAAGCATTATAAAG
dehydrogenase, AAGGTCCACAATACACAAACTTAGAGTATGGGTTGATGGCCAGATACTT
putative GGGTCGTTCATACACTGCGCCAGAGGCTACCAATACTGCTGCTCCAGA
(CD36_34410) TACCGGTAATATGGAATTGTTTGCCAAATATGGTACCACTTATCAGAAA
mRNA, complete GATAGATACTTGAAACCCTTGTTAAATGGGGAAATCAGATCGGCATTCT
cds TGATGACCGAAAAGGGTGTTTCATCATCCAATGCTCTCAATATTTCTACA
AGCGCTATCAAGAACTCTAATGGTAATTACGTGCTCAATGGTGTCAAAT
GGTTTGCTTCAGGGGCAGGAGATCCAAGATGCTCTGTATGGTTGGTAA
TGTGCAAGACCGACAACAATAAGCAAAACCCTTATCAGAACCACACTGT
TTTGATTATCGATGCAAAAAAGGCTTTGGCTACCGGAAAAGCCAAATTG
ATCAGACCATTGCAAGTCATTGGTTTTGATGATGCTCCCCATGGACATT
GTGAAATCCAATTCAAAGACTACGAAGTTCCTGCTGATGAAATGCCTAA
TGTTGTAATGGCAGGTGTTGGTAGAGGATTTGAGTTGATTCAATCCAGA
TTGGGTCCAGGTAGAATCCACCATTGTATGAGAGCTATTGGTTCTGGTG
AATTTGCTTTATTAAGAATTGCTCATAGAGCAAATCACAGATTAATTTTT
GGTAAGCCCATGAACCAAAGAGAGGGGTTCTTATCCAGATACGGACAA
AGCAAAATAGATATTGAAAGATGTTTGTTATTGGTGTTGAATGCCGCTC
ACAAAATCGATATTTCCAATGCCAAAGAGGCACAAAGGGAAATTGCTAT
GGCCAAGATTGAAACCCCAAGAACTATTTCTGATATTCTCGATTGGGGT
ATTCAAGTTTATGGAGCTGAAGGTATGTCTCAAGACACTGAGTTGGCCA
GAATGTATGCCCATAACAGAACATTGAGAATAGCTGATGGACCTGATGA
AGCTCATTTGGCTCAATTGGCTAGAAACGAAGCTAAGAAGTTTCCAAAA
GTTGACGCCTTCTTTACCAACATGGAAACACAACGTAGCAAATTATAA SEQ ID NO:
>gi|241959310|ref|
MSVKEDIPAIFLEKISPRGLDAIQKTKDFVNDYCLPADQIYFEQLSDIPSERW 3699
XP_002422374.1| KSVPPVIETLKKKAKELGLWNMFLSKHYKEGPQYTNLEYGLMARYLGRSY
acyl-coa TAPEATNTAAPDTGNMELFAKYGTTYQKDRYLKPLLNGEIRSAFLMTEKGV
dehydrogenase, SSSNALNISTSAIKNSNGNYVLNGVKWFASGAGDPRCSVWLVMCKTDNN
putative [Candida
KQNPYQNHTVLIIDAKKALATGKAKLIRPLQVIGFDDAPHGHCEIQFKDYEV dubliniensis
PADEMPNVVMAGVGRGFELIQSRLGPGRIHHCMRAIGSGEFALLRIAHRA CD36]
NHRLIFGKPMNQREGFLSRYGQSKIDIERCLLLVLNAAHKIDISNAKEAQREI
AMAKIETPRTISDILDWGIQVYGAEGMSQDTELARMYAHNRTLRIADGPDE
AHLAQLARNEAKKFPKVDAFFTNMETQRSKL SEQ ID NO: >gi|126138209|ref|
ATGTCCGCCAAAGACGATATCCCTGCCATTTTCTTGGACAAGATCTCTC 3700
XM_001385591.1| CCAGAGGTCTTGAGGCCATTGAGAAGACCAAACGTTTCGTGGAAGACT
Scheffersomyces ACTGTTTGCCAGCTGACGATATCTACTTCAAGCAGATCAAGACCGATCC
stipitis CBS 6054 CGCAGTTAGATGGAAATATACTCCCGAAATCACGGAAAAGTTGAAGAAG
acetyl- AAAGCAAAGGAACTCGGGCTCTGGAACATGTTCTTGTCTAAGCACTACA
coenzyme-A AGGAAGGACCCCAGTTCACTAACTTGGAGTACGGGTTGATGGCTGAGT
dehydrogenase ACTTGGGCAAATCCTTTGTTGCTCCAGAGGCTACCAACACTGCAGCTC
partial mRNA CAGATACCGGAAACATGGAACTTTTTGCCAAATACGGAACTCCATACCA
AAAGGAGAAGTGGCTCAAGCCATTGTTGAACGGAGAAATCAGATCAGC
TTTCTTGATGACAGAGAAGGGTGTTTCTTCATCGAATGCCTTGAACATTT
CGACTAGTGCCATTAAGAACGCCCAAGGCAACTACGTTCTTAACGGTG
TCAAGTGGTTTGCTTCTGGAGCTGGAGATCCCAGATGTTCAGTCTGGC
TTGTCATGTGTAAAACCACCGACGACTCCAGCAAGCCATACTTCAACCA
TTCTGTCTTGATTTTAGATCCCAAAGTCGCTATTGCTTCTGGAAAAGCCA
GGGTGGTCAGACCTTTGCATGTGATTGGGTACGACGATGCGCCCCATG
GCCATTGTGAAATCGAGTTCACCAACTACGAGGTTTCAGCTGAAGAAAT
GAAGAACACCATTCTTGCTGGTGTTGGCCGTGGTTTTGAGCTCATCCA
GTCCCGTTTGGGACCAGGCAGAATCCATCACTGTATGAGACTGATTGG
TTCTGGCGAGTTTGCTTTGCTCAAGACAGCACACAGAGCCAACAACAG
AATCATCTTTGGCAAGCCCTTGGCCAATAGAGAGTCCTTTATCACAGCT
TTTGCTCAACATAAGATCGACATTCAGAAGTGTCGTTTGTTGGTGTTGA
ACGCGGCCCACAAGATTGACATCACCAATGCCAAGGGTGCCCAGAAG
GAAATTGCCATGGCAAAGATCGAGACTCCAAGGACAGTGTGCAAGATC
ATAGATTGGGGCATGCAAATGTTTGGTGCCGAAGGGTTATCTCAAGAC
ACTGAGCTTGCCAGAATTTATGCCATGACCAGAATATTGAGAATTGCCG
ACGGTCCAGATGAAGCTCATTTGAACCAGTTAGGTAGAAACGAAGCAA
AGAAATTCAACGAGGCTGATGCCTTCTTTGCTACCTATGAGGCAAGCAG
AGCCAGATTGGAAAAATTGTAG SEQ ID NO: >gi|150866135|ref|
MSAKDDIPAIFLDKISPRGLEAIEKTKRFVEDYCLPADDIYFKQIKTDPAVRW 3701
XP_001385628.2| KYTPEITEKLKKKAKELGLWNMFLSKHYKEGPQFTNLEYGLMAEYLGKSFV
acetyl- APEATNTAAPDTGNMELFAKYGTPYQKEKWLKPLLNGEIRSAFLMTEKGV
coenzyme-A SSSNALNISTSAIKNAQGNYVLNGVKWFASGAGDPRCSVWLVMCKTTDDS
dehydrogenase SKPYFNHSVLILDPKVAIASGKARVVRPLHVIGYDDAPHGHCEIEFTNYEVS
[Scheffersomyces
AEEMKNTILAGVGRGFELIQSRLGPGRIHHCMRSIGSGEFALLKTAHRANN stipitis CBS
RIIFGKPLANRESFITAFAQHKIDIQKCRLLVLNAAHKIDITNAKGAQKEIAMA
6054] KIETPRTVCKIIDWGMQMFGAEGLSQDTELARIYAMTRILRIADGPDEAHLN
QLGRNEAKKFNEADAFFATYEASRARLEKL SEQ ID NO: <gi|146422929|ref|
ATGTCTGTTAAAGAGGATATTCCGGCTATTTTTCTCGACAAGATTTCGC 3702
XM_001487349.1| CCAAAGGATTGGACGCGATCCAGAAATGTAAGGATTTTGTCGAGCAATA
Meyerozyma CTGTCTTCCGGCGGATAAAATATACCTAGAGCAGCTTAGCCCTGACCC
guilliermondii CACAAAAAGATGGAAATCTACCCCACAAATCACTGAAAAATTGAAGAAA
ATCC 6260 AAAGCCCAAGAATTGGGACTTTGGAACATGTTCTTGTCAAAACACTATG
hypothetical CTGAGGGTGCAGGGTACACCAACTTGGAATATGGGCTCATGGCAGGTT
protein ATTTAGGGCGGTCGTTGGTGGCCCCAGAAGCAACCAATACCAATGCAC
(PGUG_00776) CCGACACGGGCAATATGGAATTGCTTGCCAAATACGGCACTCAGTACC
partial mRNA ATAAAGAACGTTGGCTCAAGCCATTGTTGAACGGAGAGATTCGGTCGG
CTTTTTTGATGACGGAAAAGGGTACTTCTTCGTCTAATGCGTTGAACATT
TCTGTTTCGGCCAAGAAAAATGCCAATGGGAATTGGGTATTGAATGGTA
TTAAGTGGTTCGCTTCTGGATCAGGAGACCCACGGTGTTCAGTTTGGTT
GGTAATGTGCAAAACAGCCGAAACTAAGGCGATTTATGAAAACCACTCG
GTTCTCGTTATCGATGCCAAAAAGGCATTGGCTACAGGAAATGCCAAAT
TGATCCGGCCATTACATGTTTTTGGCTATGACGATGCTCCTCACGGACA
CTTTGAGGTGGAATTCAACAACTATGAGATTCCAAGTGAAGATATGCCC
CATTCCATATTGGCTTCTGAAGGTAGAGGATTCGAGCTCATTCAGTCGA
GACTTGGTCCTGGTCGTATCCACCACTGTATGAGACTGATTGGTGCTG
GAGAACAAGCGTTGTTGCGCGTGAGCCATCGTGCCAACAATCGGCTCA
TTTTCGGTACACTTATGGCAAAGAGAGAATCATTTATTACTGCATTTGCC
CAGCACAAGATCAACCTTCAGAAATGTAGATTGCTCGTTTTGAATGCTG
CCCACAAAATTGACATCAGTAATGCCAAACAGGCACAACGGGAGATTG
CTATGGCCAAAATTGAGACTCCAAGAACCGTTGGTAGGGTACTTGACT
GGGGTATCCAAATGTTTGGAGCAGAGGGAGTTTCGCAAGACACCGAAT
TGGCTCGTCTGTATGCTATCAACCGGACACTCCAGATTGCTGATGGCC
CCGACGAAGCTCATTTGAACCAATTGGGATTGAAAGAGGCCAAGAAATT
TGCACTTGCAAGTGAATTCTTTGCTCAACAAGAAGAATACCGCAAACGA TTATCTAACCTCTAG
SEQ ID NO: >gi|146422930|ref|
MSVKEDIPAIFLDKISPKGLDAIQKCKDFVEQYCLPADKIYLEQLSPDPTKR 3703
XP_001487399.1| WKSTPQITEKLKKKAQELGLWNMFLSKHYAEGAGYTNLEYGLMAGYLGR
hypothetical SLVAPEATNTNAPDTGNMELLAKYGTQYHKERWLKPLLNGEIRSAFLMTE
protein KGTSSSNALNISVSAKKNANGNWVLNGIKWFASGSGDPRCSVWLVMCKT
PGUG_00776 AETKAIYENHSVLVIDAKKALATGNAKLIRPLHVFGYDDAPHGHFEVEFNNY
[Meyerozyma EIPSEDMPHSILASEGRGFELIQSRLGPGRIHHCMRLIGAGEQALLRVSHRA
guilliermondii
NNRLIFGTLMAKRESFITAFAQHKINLQKCRLLVLNAAHKIDISNAKQAQREI ATCC 6260]
AMAKIETPRTVGRVLDWGIQMFGAEGVSQDTELARLYAINRTLQIADGPDE
AHLNQLGLKEAKKFALASEFFAQQEEYRKRLSNL SEQ ID NO: oAA2835
CACACAGCTCTTCCATAATGTCCGACGAGGAATCAGA 3704 SEQ ID NO: oAA2836
CACACAGCTCTTCCCTCTCTTCTATTCCTAGTTGATGGCCTGGTCTC 3705 SEQ ID NO:
oAA2837 CACACAGCTCTTCCATAATGTCCGACGACCTTATCAC 3706 SEQ ID NO:
oAA2838 CACACAGCTCTTCCCTCTCTTCTATTCCTATAATTTATGTTTCAACTCACC 3707
SEQ ID NO: oAA3085 ATCGTTACCACCATCCCTACAAT 3708 SEQ ID NO: oAA3086
CCGAAACAACCGTAGATACCTTTAAGCTACAACACTATACACGATAATTCCC 3709 SEQ ID
NO: oAA3087 GGGAATTATCGTGTATAGTGTTGTAGCTTAAAGGTATCTACGGTTGTTTCGG
3710 SEQ ID NO: oAA3088 CTTGGACATTTCGACCTTGGCGGTACCGAGCTCTGCGAATT
3711 SEQ ID NO: oAA3089 AATTCGCAGAGCTCGGTACCGCCAAGGTCGAAATGTCCAAG
3712 SEQ ID NO: oAA3090 GCTTGTTCTGCAAAATGGAGTCA 3713 SEQ ID NO:
oAA3212 GGGGGAGATCGTTACCACCA 3714 SEQ ID NO: oAA3213
AATTCGCAGAGCTCGGTACCGCTGCTGCTGCTGCTGTTTT 3715 SEQ ID NO: oAA3214
AAAACAGCAGCAGCAGCAGCGGTACCGAGCTCTGCGAATT 3716 SEQ ID NO: oAA3215
TTCGTTGTTGGCTCTCTCCATTAAAGGTATCTACGGTTGTTTCGG 3717 SEQ ID NO:
oAA3216 CCGAAACAACCGTAGATACCTTTAATGGAGAGAGCCAACAACGAA 3718 SEQ ID
NO: oAA3217 CAAAGGCATCGGTCAACTCC 3719 SEQ ID NO: Candida strain
ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGGTCCTGACCCTA 3720 ATCC20336
GATCATCCATCCAAAAGGAAAGAGACAGCTCCAAATGGAACCCTCAAC POX4
AAATGAACTACTTCTTGGAAGGCTCCGTCGAAAGAAGTGAGTTGATGAA
GGCTTTGGCCCAACAAATGGAAAGAGACCCAATCTTGTTCACAGACGG
CTCCTACTACGACTTGACCAAGGACCAACAAAGAGAATTGACCGCCGT
CAAGATCAACAGAATCGCCAGATACAGAGAACAAGAATCCATCGACACT
TTCAACAAGAGATTGTCCTTGATTGGTATCTTTGACCCACAGGTCGGTA
CCAGAATTGGTGTCAACCTCGGTTTGTTCCTTTCTTGTATCAGAGGTAA
CGGTACCACTTCCCAATTGAACTACTGGGCTAACGAAAAGGAAACCGC
TGACGTTAAAGGTATCTACGGTTGTTTCGGTATGACCGAATTGGCCCAC
GGTTCCAACGTTGCTGGTTTGGAAACCACCGCCACATTTGACAAGGAA
TCTGACGAGTTTGTCATCAACACCCCACACATTGGTGCCACCAAGTGGT
GGATTGGTGGTGCTGCTCACTCCGCCACCCACTGTTCTGTCTACGCCA
GATTGATTGTTGACGGTCAAGATTACGGTGTCAAGACTTTTGTTGTCCC
ATTGAGAGACTCCAACCACGACCTCATGCCAGGTGTCACTGTTGGTGA
CATTGGTGCCAAGATGGGTAGAGATGGTATCGATAACGGTTGGATCCA
ATTCTCCAACGTCAGAATCCCAAGATTCTTTATGTTGCAAAAGTTCTGTA
AGGTTTCTGCTGAAGGTGAAGTCACCTTGCCACCTTTGGAACAATTGTC
TTACTCCGCCTTGTTGGGTGGTAGAGTCATGATGGTTTTGGACTCCTAC
AGAATGTTGGCTAGAATGTCCACCATTGCCTTGAGATACGCCATTGGTA
GAAGACAATTCAAGGGTGACAATGTCGATCCAAAAGATCCAAACGCTTT
GGAAACCCAATTGATAGATTACCCATTGCACCAAAAGAGATTGTTCCCA
TACTTGGCTGCTGCCTACGTCATCTCCGCTGGTGCCCTCAAGGTTGAA
GACACCATCCATAACACCTTGGCTGAATTGGACGCTGCCGTTGAAAAG
AACGACACCAAGGCTATCTTTAAGTCTATTGACGACATGAAGTCATTGT
TTGTTGACTCTGGTTCCTTGAAGTCCACTGCCACTTGGTTGGGTGCTGA
AGCCATTGACCAATGTAGACAAGCCTGTGGTGGTCACGGTTACTCGTC
CTACAACGGCTTCGGTAAAGCCTACAACGATTGGGTTGTCCAATGTACT
TGGGAAGGTGACAACAATGTCTTGGCCATGAGTGTTGGTAAGCCAATT
GTCAAGCAAGTTATCAGCATTGAAGATGCCGGCAAGACCGTCAGAGGT
TCCACCGCTTTCTTGAACCAATTGAAGGACTACACTGGTTCCAACAGCT
CCAAGGTTGTTTTGAACACTGTTGCTGACTTGGACGACATCAAGACTGT
CATCAAGGCTATTGAAGTTGCCATCATCAGATTGTCCCAAGAAGCTGCT
TCTATTGTCAAGAAGGAATCTTTCGACTATGTCGGCGCTGAATTGGTTC
AACTCTCCAAGTTGAAGGCTCACCACTACTTGTTGACTGAATACATCAG
AAGAATTGACACCTTTGACCAAAAGGACTTGGTTCCATACTTGATCACC
CTCGGTAAGTTGTACGCTGCCACTATTGTCTTGGACAGATTTGCCGGTG
TCTTCTTGACTTTCAACGTTGCCTCCACCGAAGCCATCACTGCTTTGGC
CTCTGTGCAAATTCCAAAGTTGTGTGCTGAAGTCAGACCAAACGTTGTT
GCTTACACCGACTCCTTCCAACAATCCGACATGATTGTCAATTCTGCTA
TTGGTAGATACGATGGTGACATCTATGAGAACTACTTTGACTTGGTCAA
GTTGCAGAACCCACCATCCAAGACCAAGGCTCCTTACTCTGATGCTTTG
GAAGCCATGTTGAACAGACCAACCTTGGACGAAAGAGAAAGATTTGAA
AAGTCTGATGAAACCGCTGCTATCTTGTCCAAGTAA SEQ ID NO: POX4
GTTCACTGCCATATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGG 3721 Candida
strain ATCC20336, Fwd. Primer, NdeI SEQ ID NO: POX4
CTTCGAGATGCGGCCGCTTACTTGGACAAGATAGCAGCGGTTTCATC 3722 Candida strain
ATCC20336, Rev. Primer, NotI SEQ ID NO: Candida strain
ATGCCTACCGAACTTCAAAAAGAAAGAGAACTCACCAAGTTCAACCCAA 3723 ATCC20336
AGGAGTTGAACTACTTCTTGGAAGGTTCCCAAGAAAGATCCGAGATCAT POX5
CAGCAACATGGTCGAACAAATGCAAAAAGACCCTATCTTGAAGGTCGA
CGCTTCATACTACAACTTGACCAAAGACCAACAAAGAGAAGTCACCGCC
AAGAAGATTGCCAGACTCTCCAGATACTTTGAGCACGAGTACCCAGAC
CAACAGGCCCAGAGATTGTCGATCCTCGGTGTCTTTGACCCACAAGTC
TTCACCAGAATCGGTGTCAACTTGGGTTTGTTTGTTTCCTGTGTCCGTG
GTAACGGTACCAACTCCCAGTTCTTCTACTGGACCATAAATAAGGGTAT
CGACAAGTTGAGAGGTATCTATGGTTGTTTTGGTATGACTGAGTTGGCC
CACGGTTCCAACGTCCAAGGTATTGAAACCACCGCCACTTTTGACGAA
GACACTGACGAGTTTGTCATCAACACCCCACACATTGGTGCCACCAAG
TGGTGGATCGGTGGTGCTGCGCACTCCGCCACCCACTGCTCCGTCTAC
GCCAGATTGAAGGTCAAAGGAAAGGACTACGGTGTCAAGACCTTTGTT
GTCCCATTGAGAGACTCCAACCACGACCTCGAGCCAGGTGTGACTGTT
GGTGACATTGGTGCCAAGATGGGTAGAGACGGTATCGATAACGGTTGG
ATCCAGTTCTCCAACGTCAGAATCCCAAGATTCTTTATGTTGCAAAAGTA
CTGTAAGGTTTCCCGTCTGGGTGAAGTCACCATGCCACCATCTGAACA
ATTGTCTTACTCGGCTTTGATTGGTGGTAGAGTCACCATGATGATGGAC
TCCTACAGAATGACCAGTAGATTCATCACCATTGCCTTGAGATACGCCA
TCCACAGAAGACAATTCAAGAAGAAGGACACCGATACCATTGAAACCAA
GTTGATTGACTACCCATTGCATCAAAAGAGATTGTTCCCATTCTTGGCT
GCCGCTTACTTGTTCTCCCAAGGTGCCTTGTACTTAGAACAAACCATGA
ACGCAACCAACGACAAGTTGGACGAAGCTGTCAGTGCTGGTGAAAAGG
AAGCCATTGACGCTGCCATTGTCGAATCCAAGAAATTGTTCGTCGCTTC
CGGTTGTTTGAAGTCCACCTGTACCTGGTTGACTGCTGAAGCCATTGAC
GAAGCTCGTCAAGCTTGTGGTGGTCACGGTTACTCGTCTTACAACGGT
TTCGGTAAAGCCTACTCCGACTGGGTTGTCCAATGTACCTGGGAAGGT
GACAACAACATCTTGGCCATGAACGTTGCCAAGCCAATGGTTAGAGAC
TTGTTGAAGGAGCCAGAACAAAAGGGATTGGTTCTCTCCAGCGTTGCC
GACTTGGACGACCCAGCCAAGTTGGTTAAGGCTTTCGACCACGCCCTT
TCCGGCTTGGCCAGAGACATTGGTGCTGTTGCTGAAGACAAGGGTTTC
GACATTACCGGTCCAAGTTTGGTTTTGGTTTCCAAGTTGAACGCTCACA
GATTCTTGATTGACGGTTTCTTCAAGCGTATCACCCCAGAATGGTCTGA
AGTCTTGAGACCTTTGGGTTTCTTGTATGCCGACTGGATCTTGACCAAC
TTTGGTGCCACCTTCTTGCAGTACGGTATCATTACCCCAGATGTCAGCA
GAAAGATTTCCTCCGAGCACTTCCCAGCCTTGTGTGCCAAGGTTAGAC
CAAACGTTGTTGGTTTGACTGATGGTTTCAACTTGACTGACATGATGAC
CAATGCTGCTATTGGTAGATATGATGGTAACGTCTACGAACACTACTTC
GAAACTGTCAAGGCTTTGAACCCACCAGAAAACACCAAGGCTCCATACT
CCAAGGCTTTGGAAGACATGTTGAACCGTCCAGACCTTGAAGTCAGAG
AAAGAGGTGAAAAGTCCGAAGAAGCTGCTGAAATCTTGTCCAGTTAA SEQ ID NO: POX5
GTTCACTGCCATATGCCTACCGAACTTCAAAAAGAAAGAGAACTC 3724 Candida strain
ATCC20336, Fwd. Primer, NdeI SEQ ID NO: POX5
CTTCGAGATGCGGCCGCTTAACTGGACAAGATTTCAGCAGCTTCTTCG 3725 Candida
strain ATCC20336, Rev. Primer, NotI SEQ ID NO: Aco1
GTTCACTGCCATATGACAACCAACACATTCACCGATCCTC 3726 (AJ001299.1) Yarrowia
lipolytica, Fwd. Primer, NdeI SEQ ID NO: Aco1
CTTCGAGATCTCGAGTCACTCATCGAGATCGCAAATTTCATCGTC 3727 (AJ001299.1)
Yarrowia lipolytica, Rev. Primer, XhoI SEQ ID NO: Aco2
GTTCACTGCCATATGAACCCCAACAACACTGGCACC 3728 (XM_505264) Yarrowia
lipolytica, Fwd. Primer, NdeI SEQ ID NO: Aco2
CTTCGAGATGCGGCCGCCTATTCCTCATCAAGCTCGCAAATGTCATC 3729 (XM_505264)
Yarrowia lipolytica, Rev. Primer, NotI SEQ ID NO: Aco3
GTTCACTGCCATATGATCTCCCCCAACCTCACAGCTAAC 3730 (XM_503244) Yarrowia
lipolytica, Fwd. Primer, NdeI SEQ ID NO: Aco3
CTTCGAGATGCGGCCGCCTATTCCTCGTCCAGCTCGCAAATG 3731 (XM_503244)
Yarrowia lipolytica, Rev. Primer, NotI SEQ ID NO: Aco4
GTTCACTGCCATATGATCACCCCAAACCCCGCTAAC 3732 (XM_504475) Yarrowia
lipolytica, Fwd. Primer, NdeI SEQ ID NO: Aco4
CTTCGAGATCTCGAGTTACTGAATATCCTCGGGCTCCATGG 3733 (XM_504475) Yarrowia
lipolytica, Rev. Primer, XhoI SEQ ID NO: Aco5
GTTCACTGCCATATGAACAACAACCCCACCAACGTGATC 3734 (XM_502199) Yarrowia
lipolytica, Fwd. Primer, NdeI SEQ ID NO: Aco5
CTTCGAGATGCGGCCGCCTACTCGTCCAGGTCGCAAATCTC 3735 (XM_502199) Yarrowia
lipolytica, Rev. Primer, NotI SEQ ID NO: Aco6
GTTCACTGCCATATGCTCTCTCAACAGTCCCTCAACAC 3736 (XM_503632) Yarrowia
lipolytica, Fwd. Primer, NdeI/NcoI SEQ ID NO: Aco6
CTTCGAGATCTCGAGCTACTCATCCTCAAGAGAGCAAATTTCCTC 3737 (XM_503632)
Yarrowia lipolytica, Rev. Primer, NcoI/XhoI SEQ ID NO: Aco1
GTTCACTGCCATATGGACGCATCGGCGGAGGTGG 3738 (NM_001136902) Zea mays,
Fwd. Primer, NdeI/EarI SEQ ID NO: Aco1
CTTCGAGATCTCGAGCTAGAGCCTGGAGAGCTTGAGCTGC 3739 (NM_001136902) Zea
mays, Rev. Primer, EarI/XhoI SEQ ID NO: Aco1b
GTTCACTGCCATATGGCGGAAGTGGACCACCTCGC 3740 (NM_001175167) Zea mays,
Fwd. Primer, NdeI/BstXI SEQ ID NO: Aco1b
CTTCGAGATCTCGAGCTAGAGCCTGGAGAGCTTGAGCTGC 3741 (NM_001175167) Zea
mays, Rev. Primer, BstXI/XhoI SEQ ID NO: Aco2
GTTCACTGCCATATGGACCTCACCTCGCCGTCGCC 3742 (NM_001158552) Zea mays,
Fwd. Primer, SEQ ID NO: Aco2 CTTGCGGCCGCTCAGTGGCTCCCGGTTGACAGTGCA
3743 (NM_001158552) Zea mays, Rev. Primer, SEQ ID NO: Aco4
GTTCACTGCCATATGATGGCCGGGAAACGAGTTACGGG 3744 (NM_001156834) Zea
mays, Fwd. Primer, SEQ ID NO: Aco4
CTTCGAGATCTCGAGTCACAGCCGGGCTTTCGCTGG
3745 (NM_001156834) Zea mays, Rev. Primer, SEQ ID NO: ACOX2
GTTCACTGCCATATGATCCTGTTGCCCAAAGAGCTCC 3746 (XM_001386762)
Scheffersomyces stipitis, Fwd. Primer, NdeI/SalI SEQ ID NO: ACOX2
GTTCACTGCGCGGCCGCCTAGCGGGACAATATCTTGGCAGCTTCG 3747 (XM_001386762)
Scheffersomyces stipitis, Rev. Primer, SalI/NotI SEQ ID NO:
DEHA2D17248p GTTCACTGCCATATGGTTAGTGCTACTAATACAGTGAATTCAGG 3748
(XM_459235) Debaryomyces hansenii, Fwd. Primer, NdeI SEQ ID NO:
DEHA2D17248p CTTCGAGATCTCGAGTTATTTGGATAAGATCTTAGCAGTTTCAGTAGACTTTTC
3749 (XM_459235) Debaryomyces hansenii, Rev. Primer, XhoI SEQ ID
NO: ACX1 GTTCACTGCATTAATATGGAAGGAATTGATCACCTCGCCG 3750 (NM_117778)
Arabidopsis thaliana, Fwd. Primer, AseI SEQ ID NO: ACX1
CTTCGAGATGTCGACTCAGAGCCTAGCGGTACGAAGTTGC 3751 (NM_117778)
Arabidopsis thaliana, Rev. Primer, SalI SEQ ID NO: ACX2
GTTCACTGCCATATGGAATCGCGGCGAGAGAAGAATCC 3752 (NM_001037068)
Arabidopsis thaliana, Fwd. Primer, NdeI SEQ ID NO: ACX2
CTTCGAGATGTCGACTTATACAAGAAAACAAACCTTAGCTTTGTTAGGCGC 3753
(NM_001037068) Arabidopsis thaliana, Rev. Primer, SalI SEQ ID NO:
ACX2b GTTCACTGCCATATGGAATCGCGGCGAGAGAAGAATCC 3754 (NM_125910)
Arabidopsis thaliana, Fwd. Primer, NdeI SEQ ID NO: ACX2b
CTTCGAGATGTCGACTTAGAATCCAACAACTTGAGTATACTGGGAATAAG 3755 (NM_125910)
Arabidopsis thaliana, Rev. Primer, SslI SEQ ID NO: ACX3
GTTCACTGCATTAATATGTCGGATAATCGTGCACTCCGACG 3756 (NM_100511)
Arabidopsis thaliana, Fwd. Primer, AseI SEQ ID NO: ACX3
CTTCGAGATGTCGACCTAAACTGAAGACCAAGCATTGGCTTCG 3757 (NM_100511)
Arabidopsis thaliana, Rev. Primer, SALI SEQ ID NO: ACX5
GTTCACTGCCATATGGAGAGAGTTGATCACCTTGCCGATG 3758 (NM_129124)
Arabidopsis thaliana, Fwd. Primer, NdeI/EarI SEQ ID NO: ACX5
GTTCACTGCGCGGCCGCTTAGAGTTTGGCAGAGCGGAAGCGTTG 3759 (NM_129124)
Arabidopsis thaliana, Rev. Primer, EarI/NotI SEQ ID NO: aco2
GTTCACTGCCATATGCAAACTCCGAACTGTGAAGCA 3760 (XM_003525015) Glycine
max, Fwd. Primer, NdeI SEQ ID NO: aco2
CTTGCGGCCGCTCAAAAACCGACGTATTGAGTGTAT 3761 (XM_003525015) Glycine
max, Rev. Primer, NotI SEQ ID NO: aoxA
GTTCACTGCCATATGCCAAATCCACCTCCCGCCTGG 3762 (XM_659264) Aspergillus
nidulans, Fwd. Primer, NdeI SEQ ID NO: aoxA
CTTCGAGATCTCGAGTCACAGCTTGCTCTTAATCTCCCCCG 3763 (XM_659264)
Aspergillus nidulans, Rev. Primer, XhoI SEQ ID NO: AcoI
GTTCACTGCCATATGAACCCAGACTTGAGAAAGGAAAGAGC 3764 (NM_017340) Rattus
norvegicus, Fwd. Primer, NdeI SEQ ID NO: AcoI
CTTCGAGATCTCGAGCTACAACTTGGATTGCAATGGCTTCAAGTGC 3765 (NM_017340)
Rattus norvegicus, Rev. Primer, XhoI SEQ ID NO: AcolI (1IS2_A)
GTTCACTGCCATATGAACCCAGACTTGAGAAAGGAAAGAGC 3766 Rattus norvegicus,
Fwd. Primer, NdeI SEQ ID NO: AcolI (1IS2_A)
CTTCGAGATCTCGAGCTACAACTTGGATTGCAATGGCTTCAAGTGC 3767 Rattus
norvegicus, Rev. Primer, XhoI SEQ ID NO: Aco
GTTCACTGCCATATGGCTTCGCCGCGCGAGTC 3768 (Cucsa.029560.1) Cucumis
sativus, Fwd. Primer, NdeI SEQ ID NO: Aco
CTTCGAGATCTCGAGTTAGAAGCCAACATACTGCGTATACTGCG 3769 (Cucsa.029560.1)
Cucumis sativus, Rev. Primer, XhoI SEQ ID NO: Aco (BAE47462)
GTTCACTGCCATATGACAGAAGTAGTGGACCGCGCATC 3770 Arthrobacter
ureafaciens, Fwd. Primer, NdeI SEQ ID NO: Aco (BAE47462)
CTTCGAGATCTCGAGCTAGCGGGACTTGCCGGCC 3771 Arthrobacter ureafaciens,
Rev. Primer, XhoI SEQ ID NO: Aco
GTTCACTGCCATATGCTCGATACCGACTCGCCACG 3772 (YP_003571780)
Salinobacter rubber, Fwd. Primer, NdeI SEQ ID NO: Aco
CTTCGAGATCTCGAGCTCGAGTCATTTCGGGCCGGG 3773 (YP_003571780)
Salinobacter rubber, Rev. Primer, XhoI SEQ ID NO: Aco
GTTCACTGCCATATGCCCCAGCACGGCGATACAG 3774 (YP_290295.1) Thermobifida
fusca, Fwd. Primer, NdeI SEQ ID NO: Aco
CTTCGAGATCTCGAGCTCGAGTCACGTCTCCGCGC 3775 (YP_290295.1) Thermobifida
fusca, Rev. Primer, XhoI SEQ ID NO: Aco
GTTCACTGCCATATGAAACCAGCTAAACTTCAAGCCTTTACTCC 3776 (NC_009441.1)
Flavobacterium johnsoniae, Fwd. Primer, NdeI SEQ Aco
CTTCGAGATCTCGAGCTAAACTGCAATTGGCGCTGCTAAACAG ID (NC_009441.1) NO:
Flavobacterium johnsoniae, Rev. Primer, XhoI 3777 SEQ ID NO: POX1
MAKERGKTQFTVRDVTNFLNGGEEETQIVEKIMSSIERDPVLSVTADYDCN 3778
>gi|50554589|ref|
LQQARKQTMERVAALSPYLVTDTEKLSLWRAQLHGMVDMSTRTRLSIHNN XP_504703.1|
LFIGSIRGSGTPEQFKYWVKKGAVAVKQFYGCFAMTELGHGSNLKGLETT YALI0E32835p
ATYDQDSDQFIINTPHIGATKWWIGGAAHTSTHCVCFAKLIVHGKDYGTRN [Yarrowia
FVVPLRNVHDHSLKVGVSIGDIGKKMGRDGVDNGWIQFTNVRIPRQNMLM lipolytica]
RYAKVSDTGVVTKPALDQLTYGALIRGRVSMIADSFHVSKRFLTIALRYACV
RRQFGTSGDTKETKIIDYPYHQRRLLPLLAYCYAMKMGADEAQKTWIETTD
RILALNPNDPAQKNDLEKAVTDTKELFAASAGMKAFTTWGCAKIIDECRQA
CGGHGYSGYNGFGQGYADWVVQCTWEGDNNVLCLSMGRGLVQSALQIL
AGKHVGASIQYVGDKSKISQNGQGTPREQLLSPEFLVEAFRTASRNNILRT
TDKYQELVKTLNPDQAFEELSQQRFQCARIHTRQHLISSFYARIATAKDDIK
PHLLKLANLFALWSIEEDTGIFLRENILTPGDIDLINSLVDELCVAVRDQVIGL
TDAFGLSDFFINAPIGSYDGNVYEKYFAKVNQQNPATNPRPPYYESTLKPF LFREEEDDEICDLDE
SEQ ID NO: POX2 MNPNNTGTIEINGKEYNTFTEPPVAMAQERAKTSFPVREMTYFLDGGEKN
3779 >gi|50555712|ref|
TLKNEQIMEEIERDPLFNNDNYYDLNKEQIRELTMERVAKLSLFVRDQPED XP_505264.1|
DIKKRFALIGIADMGTYTRLGVHYGLFFGAVRGTGTAEQFGHWISKGAGDL YALI0F10857p
RKFYGCFSMTELGHGSNLAGLETTAIYDEETDEFIINTPHIAATKWWIGGAA [Yarrowia
HTATHTVVFARLIVKGKDYGVKTFVVQLRNINDHSLKVGISIGDIGKKMGRD lipolytica]
GIDNGWIQFTNVRIPRQNLLMKYTKVDREGNVTQPPLAQLTYGSLITGRVS
MASDSHQVGKRFITIALRYACIRRQFSTTPGQPETKIIDYPYHQRRLLPLLA
YVYALKMTADEVGALFSRTMLKMDDLKPDDKAGLNEVVSDVKELFSVSAG
LKAFSTWACADVIDKTRQACGGHGYSGYNGFGQAYADWVVQCTWEGDN
NILTLSAGRALIQSAVALRKGEPVGNAVSYLKRYKDLANAKLNGRSLTDPK
VLVEAWEVAAGNIINRATDQYEKLIGEGLNADQAFEVLSQQRFQAAKVHTR
RHLIAAFFSRIDTEAGEAIKQPLLNLALLFALWSIEEDSGLFLREGFLEPKDID
TVTELVNKYCTTVREEVIGYTDAFNLSDYFINAPIGCYDGDAYRHYFQKVN
EQNPARDPRPPYYASTLKPFLFREEEDDDICELDEE SEQ ID NO: POX3
MISPNLTANVEIDGKQYNTFTEPPKALAGERAKVKFPIKDMTEFLHGGEEN 3780
>gi|50551539|ref|
VTMIERLMTELERDPVLNVSGDYDMPKEQLRETAVARIAALSGHWKKDTE XP_503244.1|
KEALLRSQLHGIVDMGTRIRLGVHTGLFMGAIRGSGTKEQYDYWVRKGAA YALI0D24750p
DVKGFYGCFAMTELGHGSNVAGLETTATYIQDTDEFIINTPNTGATKWWIG [Yarrowia
GAAHSATHTACFARLLVDGKDYGVKIFVVQLRDVSSHSLMPGIALGDIGKK lipolytica]
MGRDAIDNGWIQFTNVRIPRQNMLMKYAKVSSTGKVSQPPLAQLTYGALI
GGRVTMIADSFFVSQRFITIALRYACVRRQFGTTPGQPETKIIDYPYHQRRL
LPLLAFTYAMKMAADQSQIQYDQTTDLLQTIDPKDKGALGKAIVDLKELFAS
SAGLKAFTTWTCANIIDQCRQACGGHGYSGYNGFGQAYADWVVQCTWE
GDNNVLCLSMGRGLIQSCLGHRKGKPLGSSVGYLANKGLEQATLSGRDLK
DPKVLIEAWEKVANGAIQRATDKFVELTKGGLSPDQAFEELSQQRFQCAKI
HTRKHLVTAFYERINASAKADVKPYLINLANLFTLWSIEEDSGLFLREGFLQ
PKDIDQVTELVNHYCKEVRDQVAGYTDAFGLSDWFINAPIGNYDGDVYKH
YFAKVNQQNPAQNPRPPYYESTLRPFLFREDEDDDICELDEE SEQ ID NO: POX4
MITPNPANDIVHDGKLYDTFTEPPKLMAQERAQLDFDPRDITYFLDGSKEE 3781
>gi|50554133|ref|
TELLESLMLMYERDPLFNNQNEYDESFETLRERSVKRIFQLSKSIAMDPEP XP_504475.1|
MSFRKIGFLGILDMGTYARLGVHYALFCNSIRGQGTPDQLMYWLDQGAMV YALI0E27654p
IKGFYGCFAMTEMGHGSNLSRLETIATFDKETDEFIINTPHVGATKWWIGG [Yarrowia
AAHTATHTLAFARLQVDGKDYGVKSFVVPLRNLDDHSLRPGIATGDIGKKM lipolytica]
GRDAVDNGWIQFTNVRVPRNYMLMKHTKVLRDGTVKQPPLAQLTYGSLIT
GRVQMTTDSHNVSKKFLTIALRYATIRRQFSSTPGEPETRLIDYLYHQRRLL
PLMAYSYAMKLAGDHVRELFFASQEKAESLKEDDKAGVESYVQDIKELFS
VSAGLKAATTWACADIIDKARQACGGHGYSAYNGFGQAFQDWVVQCTWE
GDNTVLTLSAGRALIQSALVYRKEGKLGNATKYLSRSKELANAKRNGRSLE
DPKLLVEAWEAVSAGAINAATDAYEELSKQGVSVDECFEQVSQERFQAAR
IHTRRALIEAFYSRIATADEKVKPHLIPLANLFALWSIEEDSALFLAEGYFEPE
DIIEVTSLVNKYCGIVRKNVIGYTDAFNLSDYFINAAIGRYDGDVYKNYFEKV
KQQYPPEGGKPHYYEDVMKPFLHRERIPDVPMEPEDIQ SEQ ID NO: POX5
MNNNPTNVILGGKEYDTFTEPPAQMELERAKTQFKVRDVTNFLTGSEQET 3782
>gi|50549457|ref|
LLTERIMREIERDPVLNVAGDYDADLPTKRRQAVERIGALARYLPKDSEKE XP_502199.1|
AILRGQLHGIVDMGTRTRIAVHYGLFMGAIRGSGTKEQYDYWVAKGAATLH YALI0C23859p
KFYGCFAMTELGHGSNVAGLETTATLDKDTDEFIINTPNSGATKWWIGGAA [Yarrowia
HSATHTACLARLIVDGKDYGVKIFIVQLRDLNSHSLLNGIAIGDIGKKMGRDA lipolytica]
IDNGWIQFTDVRIPRQNMLMRYDRVSRDGEVTTSELAQLTYGALLSGRVT
MIAESHLLSARFLTIALRYACIRRQFGAVPDKPETKLIDYPYHQRRLLPLLAY
TYAMKMGADEAQQQYNSSFGALLKLNPVKDAEKFAVATADLKALFASSAG
MKAFTTWAAAKIIDECRQACGGHGYSGYNGFGQAYADWVVQCTWEGDN
NVLCLSMGRSLIQSCIAMRKKKGHVGKSVEYLQRRDELQNARVDNKPLTD
PAVLITAWEKVACEAINRATDSFIKLTQEGLSPDQAFEELSQQRFECARIHT
RKHLITSFYARISKAKARVKPHLTVLANLFAVWSIEEDSGLFLREGCFEPAE
MDEITALVDELCCEAREQVIGFTDAFNLSDFFINAPIGRFDGDAYKHYMDEV
KAANNPRNTHAPYYETKLRPFLFRPDEDEEICDLDE SEQ ID NO: POX6
MLSQQSLNTFTEPPVEMARERNQTSFNPRLLTYFLDGGEKNTLLMDRLMQ 3783
>gi|50552444|ref|
EYERDPVFRNEGDYDITDVAQSRELAFKRIAKLIEYVHTDDEETYLYRCMLL XP_503632.1|
GQIDMGAFARYAIHHGVWGGAIRGAGTPEQYEFWVKKGSLSVKKFYGSF YALI0E06567p
SMTELGHGSNLVGLETTATLDKNADEFVINTPNVAATKWWIGGAADTATH [Yarrowia
TAVFARLIVDGEDHGVKTFVVQLRDVETHNLMPGIAIGDCGKKMGRQGTD lipolytica]
NGWIQFTHVRIPRQNMLMRYCHVDSDGNVTEPMMAQMAYGALLAGRVG
MAMDSYFTSRKFLTIALRYATIRRAFAAGGGQETKLIDYPYHQRRLLPLMA
QTYAIKCTADKVRDQFVKVTDMLLNLDVSDQEAVPKAIAEAKELFSVSAGV
KATTTWACAHTIDQCRQACGGHGYSAYNGFGRAYSDWVIQCTWEGDNNI
LCLSAGRALVQSNRAVRAGKPIGGPTAYLAAPAGSPKLAGRNLYDPKVMI
GAWETVSRALINRTTDEFEVLAKKGLSTAQAYEELSQQRFLCTRIHTRLYM
VKNFYERIAEEGTEFTKEPLTRLANLYAFWSVEEEAGIFLREGYITPQELKYI
SAEIRKQLLEVRKDVIGYTDAFNVPDFFLNSAIGRADGDVYKNYFKVVNTQ
NPPQDPRPPYYESVIRPFLFRKDEDEEICSLEDE SEQ ID NO: pAA298
gatctggaatccctcggcgtcggtcttgggggtgggggcattctttcttggtcttgggaacgc
3784 (Nucleic Acid
caacgctttgttgtttgggttcttgaacacggactgctcgaaaaagtaccagtatgatgcctt
Seq.)
acctctgctggctttccacaagtatgggagggcattgacagcgactgtcttggctaacagcac
gtcgtcggcaattaaatatttggcttccaataactgactaccaaggatggcagcagcggctat
ttctaatcctgacatgtttctcgtacgtagtagtgaatgaagggaaggtggaataatatcaag
ggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaat
tcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaa
aaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaata
gcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggttta
cacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggc
gacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtg
gtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcg
gggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggg
gaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagt
ccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaa
gcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatgga
cagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaa
ctggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatg
aggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctat
tcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggg
gcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcgg
ctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaaggg
actggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtat
ccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagc
gaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacga
agagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcga
ggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggatt-
c
atcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctga
agagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcat-
cg
ccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctcc-
t tacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtg
cgcggaacccctatttgtt
tatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagc-
ac
gtgaggagggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccgg
agcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtg
gtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggc
ctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccg
ggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcg
acccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctaaaacttcatttttaa
tttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc
cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc-
t
gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttc
cgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggcca
ccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag-
t
ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggct
gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctaca
gcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcag
ggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgg
gtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgc
cagcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatc-
cc
ctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgag
cgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgt
tggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaat
taatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg
aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctatttaggtga
cactatagaatactcaagctatgcatcaagcttggtaccgagctcggatccactagtaacggccgccagt
gtgctggaattcgcccttccgttaaacaaaaatcagtctgtaaaaaaggttctaaataaatattctgtctag-
t
gtacacattctcccaaaatagtgaaatccagctctacaatttggctttaccggtacaaatcaaagaccaatc-
g
tcttcagtcaactcctggtacttgtcgccattcaaccagtagatcaaatccttgccgtcttcaccctttggc-
aa
cttttggttcttgaattggttcttaggaaccttgtggttgtgcgaagcctcaatggtgccaatcttgatgaa-
cg
caggttgagcatacacaggcaaagacttggtcacgtgagagtgaatcaatttcaagatttcttcatggctc
aactcgtccttggcttcacagacggcaaaacaggctctaccttcgtggtttggcaccttgacaccgacaac
gacggactgcttcaaggccttggagcccatcaattcgttctcgacctcggtggcggagacgttttcggactt
ccaacggaaagtgtcacctaatctgtcgacaaagtacaacaatttgtcctcgtccatcttcaacaagtcacc-
g
gatctgtaccacgcgtcaccttttttgaaaacattggtgaggattttgctgttggtggcggacttgttacca-
t
aataaccctggaaggatttctgcacgtcgttagggttcaagattctcatcaacaactcacctggctcgttgt-
a
agcggcctcggtacagaacccggtcttggggtccttgtagatttcactctcgtcttctgggtccatcttggc-
ca
atttctgctgggtagacaataacaagctgatgagggacccgtacttacgacaggcgccgacaccgtact
caccgtactgcaagttggtggtggcgataggggactcggtggcggcgtagaactcaccgataccttcaat
gtggaatctgcgcttgaactcagaccatatatctggacgcaacccgttaccgtaggcaattctgacattgtg
tctgtcttggtctggatgaggcttggagttcaacaagtaacgacagacctcaccgacgtattgcacgtgggt
ggcaccacataatctggcctgggtccagaacgaagtagcggagaatttctgggacacggagacacag
ccaccgacaatcaaagtaggacacaaccccaacatggccgcggtggagtggtacaagggcatggcg
gtcaagacgttcgatttcgagtcaatcttcatgatgtggccaaagaaaaccgaggccatgaaggcttttctc
caggacatgataccggcttttggcaaaccggtggtacccgaggtgtaaatcaatgcacaagcggaggagtc
agtatcggttggtcttctggtcttgtcctcggctctgtgttttggagtcgacttgagtctcaatctgtcaaa-
ca
aggcaaactcgtcaatgtagtttatttgcacatgtggcaattcctctctgatctgagcctcggtatctctga-
tt
ggggaatcacagtccgggtcaacgaaaacttgcgaagcgttgacaatcttaagacagtggatcaatggct
tgtccttggtgttgaagttcaagaacgcaggcaaggcaccaatgttccacaatgccaaccacaagacaa
tgaaaagcggcttgttcatacaagaaacaccgatggtgtcgttggcagtgacgccgtactcgttcttcaaga-
tg
tatgagtacttcaaaaccatgtcgtacaattccttgtaggtgtattcttctaggtcaaactgatcgtcgtag
atccactagtaacggccgccagtgtgctggaattcgcccttgggctaacgaaaaggaaaccgctgacgt
taaaggtatctacggttgtttcggtatgacccccggggatctgacgggtacaacgagaattgtattgaattg
atcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactgagaatgcacagatataca
aggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtttatggaaagtggtcaacc
agaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaagaaggtaaataagtattttg
tattatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatct
catccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacct
ccccatcatcatcgtcatcaaacccccagctcaattcgcaatggttagcacaaaaacatacacagaaag
ggcatcagcacacccctccnaggttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctc
tgcgcctcgatcgacgtgaccacaaccgccgagttcctttcgctcatcgacaagctcggtccccacatctg
tctcgtgaagacgcacatcgatntcatctcagacttcagctacgagggcacgattgagccgttgcttgtgct
tgcagagcgccacgggttcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgc
agtacacctcgggggtataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgg
gaagggcgtcgttgaagggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatg
tnggcggagttgtcgagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcga
agagtgatcgggagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaagggtttg
attggatcatcatgacgcctggtgtggggttggatgataaaggcgatgcgttgggccagcagtataggact
gttgatgaggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaaaggaagagac
cctgaggtggagggaaagagatacagggatgctggatggaaggcatacttgaagagaactggtcagtt
agaataaatattgtaataaataggtctatatacatacactaagcttctaggacgtcattgtagtcttcgaag-
tt
gtctgctagtttagttctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgc-
g
ttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcacttgc
ttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtctacggtcgacgacgggtaca
acgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactg
agaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtt
tatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaa
gaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccat
actagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatc
aaacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcagagctcggtacccg-
gg SEQ ID NO: Candida strain
MRGMEKPHSLFRRMSTAPFAIIQPPISILSATLITSEFFFVYSLYNFFHFIIDLFYYYYTHIYPHRAMIEN
3785 ATCC20336
ISGNGNYPQNHEVDLEKEFGVEKIGINLYRDKSPIPKPDRRSRGAYGGYLAGQALLVAMKSTPPEY
Thioesterase
RPHSFHSYFIKAVNDKETLEWRVEETSNGRNYANRSLQAFQAGNLVYTANVSLTKKNSAKKAEEA
PTE1
TGVKPFEFQGKPHEWFEKHKRDDLPLATPSSSLLIYHKFFPEVVSLEASKEEESKPAADRELSWYFKW-
GINNE
EGHHQPLVNLNSDYQYVGMAALTDAVYLNRLLRILRVEDADHTQLVHYFSVSLDHTMYFHDDDFDVTKWMG
FTFKVTRFSHNRALCQGEVYNDKGVHVCTIVQEGLMMLNGLEEGAKL* SEQ ID NO: Candida
strain
MICVFFPTSTFTTAHKFVSNLQSFFLSQQPHTTSYTMPTFNYKDGETIDVQKEFGVVETAPNKYVGVKPLVKP-
M 3786 ATCC20336
PHVKGVFGGNLAGQALLVAMKSVGPDFSPHSLHSYFIRAGSDQTPVEWTVQAISDGNSFCNRFIKGVQNGQVI-
Y Thioesterase
IANVSLTKRNSAADAMKKYEEYHAQIRQKGKDGDADEEDEDDDDEDDNAPAKPFGFQTPSHKWIKDRDLDKLP-
V PTE2
SDMESNLLLYYKLPPEFVSLKSSTEEESLPVSERRMGALAKWGIENEQGFNQPLTNLDKSFQYVGLAN-
ITD
GLYLGTLNRILRIDDLTLDERATNYFSVSLDHVIYFHDDDFDVTKWMGFTFRCSRYSHNRVIFEGEIYSD
KGVQVASIIQEGLVRFKDGYLKNAKL
Example 44
Examples of Certain Non-Limiting Embodiments
[0505] A1. A genetically modified yeast, comprising an active,
modified endogenous acyl-coA oxidase polypeptide or an active,
modified endogenous acyl-coA dehydrogenase polypeptide, which yeast
is capable of producing a diacid from a feedstock comprising one or
more components from a vegetable oil.
[0506] A2. The genetically modified yeast of embodiment A1, wherein
the yeast is a genetically modified Candida spp. yeast.
[0507] A2.1. The genetically modified yeast of embodiment A2,
wherein the Candida spp. yeast is chosen from C. tropicalis and C.
viswanathii.
[0508] A2.2. The genetically modified yeast of embodiment A1,
wherein the Candida spp. yeast is a genetically modified ATCC20336
yeast.
[0509] A2.3. The genetically modified yeast of any one of
embodiments A2 to A2.2, wherein the endogenous acyl-coA oxidase
polypeptide is a POX4 polypeptide.
[0510] A2.4. The genetically modified yeast of embodiment A2.3,
wherein the POX4 polypeptide comprises a modified amino acid
sequence of SEQ ID NO: 30.
[0511] A2.5. The genetically modified yeast of embodiment A2.3 or
A2.4, wherein the POX4 polypeptide comprises an amino acid
modification at one or more amino acid positions chosen from 88,
90, 96, 98, 99, 100, 102, 103, 302, 309, 310, 473, 474, 475, 476,
477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,
503, 504 and 505.
[0512] A2.6. The genetically modified yeast of any one of
embodiments A2 to A2.2, wherein the endogenous acyl-coA oxidase
polypeptide is a POX5 polypeptide.
[0513] A2.7. The genetically modified yeast of embodiment A2.6,
wherein the POX5 polypeptide comprises a modified amino acid
sequence of SEQ ID NO: 32.
[0514] A2.8. The genetically modified yeast of embodiment A2.6 or
A2.7, wherein the POX5 polypeptide comprises an amino acid
modification at one or more amino acid positions chosen from 81,
82, 83, 84, 85, 86, 88, 93, 94, 95, 96, 98, 102, 284, 287, 290,
291, 292, 294, 295, 436, 453, 454, 455, 456, 457, 458, 459, 460,
461, 462 and 463.
[0515] A2.9. The genetically modified yeast of any one of
embodiments A2 to A2.2, wherein the acyl-coA dehydrogenase
polypeptide is chosen from ACAD, VLCAD, LOAD, MCAD and SCAD
polypeptides.
[0516] A2.10. The genetically modified yeast of embodiment A2.9,
wherein the acyl-coA dehydrogenase polypeptide comprises a modified
amino acid sequence of SEQ ID NO: 3685.
[0517] A2.11. The genetically modified yeast of embodiment A2.9 or
A2.10, wherein the acyl-coA dehydrogenase polypeptide comprises an
amino acid modification at VLCAD position 461.
[0518] A2.12. The genetically modified yeast of any one of
embodiments 2.5, 2.8 and 2.11, wherein at least one of the amino
acid modifications is an amino acid substitution.
[0519] A2.13. The genetically modified yeast of embodiment A2.12,
wherein at least one of the one or more amino acid substitutions is
conservative.
[0520] A2.14. The genetically modified yeast of embodiment A2.12,
wherein at least one of the one or more amino acid substitutions is
not conservative.
[0521] A3. The genetically modified yeast of embodiment A1, wherein
the yeast is a genetically modified Yarrowia spp. yeast.
[0522] A3.1. The genetically modified yeast of embodiment A3.1,
wherein the Yarrowia spp. yeast is Y. lipolytica.
[0523] A3.2. The genetically modified yeast of embodiment A3 or
A3.1, wherein the endogenous acyl-coA oxidase polypeptide is chosen
from a POX1 polypeptide, POX2 polypeptide, POX3 polypeptide, POX4
polypeptide, POX5 polypeptide or POX6 polypeptide.
[0524] A3.3. The genetically modified yeast of embodiment 3.2,
wherein the endogenous acyl-coA oxidase polypeptide is chosen from
SEQ ID NOs: 3778 to 3783.
[0525] A4. The genetically modified yeast of embodiment A1, wherein
the yeast is a genetically modified Pichia spp. yeast.
[0526] A4.1. The genetically modified yeast of embodiment A4.1,
wherein the Pichia spp. yeast is chosen from P. pastoris, P.
membranifaciens, P. kluyveri, P. guilliermondii, P. heedii and P.
subpelliculosa.
[0527] A5. The genetically modified yeast of embodiment A1, wherein
the yeast is a genetically modified Saccharomyces spp. yeast.
[0528] A5.1. The genetically modified yeast of embodiment A5.1,
wherein the Saccharomyces spp. yeast is chosen from S. cerevisiae,
S. bayanus, S. pastorianus and S. carlsbergensis.
[0529] A6. The genetically modified yeast of embodiment A1, wherein
the yeast is a genetically modified Kluyveromyces spp. yeast.
[0530] A6.1. The genetically modified yeast of embodiment A6.1,
wherein the Kluyveromyces spp. yeast is chosen from K. lactis and
K. marxianus.
[0531] A7. The genetically modified yeast of any one of embodiments
A1 to A6.1, wherein the modified endogenous acyl-coA oxidase
polypeptide comprises an amino acid modification in the N-terminal
loop.
[0532] A8. The genetically modified yeast of any one of embodiments
A1 to A7, wherein the modified endogenous acyl-coA oxidase
polypeptide comprises an amino acid modification in the D alpha
helix.
[0533] A9. The genetically modified yeast of any one of embodiments
A1 to A8, wherein the modified endogenous acyl-coA oxidase
polypeptide comprises an amino acid modification in the loop
between the D alpha helix and the E' alpha helix.
[0534] A10. The genetically modified yeast of any one of
embodiments A1 to A9, wherein the modified endogenous acyl-coA
oxidase polypeptide comprises an amino acid modification to an
amino acid in effective contact with carbons 6 to 9 in a feedstock
component.
[0535] A11. The genetically modified yeast of any one of
embodiments A1 to A10, wherein the modified endogenous acyl-coA
oxidase polypeptide comprises an amino acid modification to an
amino acid in effective contact with carbons 10 to 12 in a
feedstock component.
[0536] A12. The genetically modified yeast of any one of
embodiments A1 to A11, wherein the modified endogenous acyl-coA
oxidase polypeptide comprises an amino acid modification in the L
alpha helix.
[0537] A13. The genetically modified yeast of any one of
embodiments A1 to A12, wherein the modified endogenous acyl-coA
oxidase polypeptide comprises an amino acid modification in the
loop C-terminal to the L alpha helix.
[0538] A14. The genetically modified yeast of any one of
embodiments A1 to A13, wherein the modified endogenous acyl-coA
oxidase polypeptide comprises an amino acid modification in the
loop between the L alpha helix and the M alpha helix.
[0539] A15. The genetically modified yeast of any one of
embodiments A7 to A14, wherein the amino acid modification
comprises an amino acid substitution.
[0540] A16. The genetically modified yeast of embodiment A15,
wherein the amino acid substitution is conservative.
[0541] A17. The genetically modified yeast of embodiment A15,
wherein the amino acid substitution is not conservative.
[0542] A18. The genetically modified yeast of any one of
embodiments A1 to A17, which comprises a genetic modification that
reduces the activity of an enoyl coA isomerase polypeptide.
[0543] A19. The genetically modified yeast of embodiment A18,
wherein the genetic modification disrupts a polynucleotide that
encodes the enoyl coA isomerase polypeptide.
[0544] A20. The genetically modified yeast of embodiment A18 or
A19, wherein the enoyl coA isomerase polypeptide is a polypeptide
native to the yeast.
[0545] A21. The genetically modified yeast of embodiment A20,
wherein the yeast is a Candida spp. yeast.
[0546] A22. The genetically modified yeast of embodiment A21,
wherein the enoyl coA isomerase polypeptide comprises the amino
acid sequence of SEQ ID NO: 3675 or 3677.
[0547] A23. The genetically modified yeast of any one of
embodiments A1 to A22, which comprises a genetic modification that
reduces the cytoplasmic activity of an acyl-CoA synthetase (ACS)
polypeptide.
[0548] A24. The genetically modified yeast of any one of
embodiments A1 to A23, which comprises a genetic modification that
reduces the peroxisomal activity of an acyl-CoA synthetase (ACS)
polypeptide.
[0549] A25. The genetically modified yeast of embodiment A23 or
A24, wherein the genetic modification disrupts a polynucleotide
that encodes the acyl-CoA synthetase (ACS) polypeptide.
[0550] A26. The genetically modified yeast of embodiment A25,
wherein the genetic modification disrupts an ACS1 polypeptide or
ACS2 polypeptide.
[0551] A27. The genetically modified yeast of any one of
embodiments A23 to A26, wherein the genetic modification disrupts a
polynucleotide that encodes a long-chain acyl-CoA synthetase
polypeptide.
[0552] A28. The genetically modified yeast of embodiment A27,
wherein the genetic modification disrupts a FAT1 polypeptide.
[0553] A29. The genetically modified yeast of any one of
embodiments A23 to A28, wherein the acyl-CoA synthetase (ACS)
polypeptide is a polypeptide native to the yeast.
[0554] A30. The genetically modified yeast of embodiment A29,
wherein the yeast is a Candida spp. yeast.
[0555] A31. The genetically modified yeast of embodiment A30,
wherein the acyl-CoA synthetase (ACS) polypeptide comprises an
amino acid sequence chosen from SEQ ID NOs: 80, 82, 84, 158 and
159.
[0556] A32. The genetically modified yeast of embodiment A30,
wherein the FAT1 polypeptide comprises the amino acid sequence of
SEQ ID NO: 90.
[0557] A33. The genetically modified yeast of any one of
embodiments A1 to A32, which comprises a genetic modification that
reduces the activity of a PXA polypeptide.
[0558] A34. The genetically modified yeast of embodiment A33,
wherein the genetic modification disrupts a polynucleotide that
encodes the PXA polypeptide.
[0559] A35. The genetically modified yeast of embodiment A33 or
A34, wherein the PXA polypeptide is a PXA1 polypeptide or a PXA2
polypeptide, or a PXA1 polypeptide and a PXA2 polypeptide.
[0560] A36. The genetically modified yeast of any one of
embodiments A33 to A35, wherein the PXA polypeptide is native to
the yeast.
[0561] A37. The genetically modified yeast of embodiment A36,
wherein the yeast is a Candida spp. yeast.
[0562] A38. The genetically modified yeast of embodiment A37,
wherein the PXA1 polypeptide comprises the amino acid sequence of
SEQ ID NO: 92.
[0563] A39. The genetically modified yeast of embodiment A37,
wherein the PXA2 polypeptide comprises the amino acid sequence of
SEQ ID NO: 94.
[0564] A40. The genetically modified yeast of any one of
embodiments A1 to A39, comprising an active, modified endogenous
acyl-coA oxidase polypeptide and no active, modified endogenous
acyl-coA dehydrogenase polypeptide.
[0565] A41. The genetically modified yeast of any one of
embodiments A1 to A39, comprising no active, modified endogenous
acyl-coA oxidase polypeptide and an active, modified endogenous
acyl-coA dehydrogenase polypeptide.
[0566] B1. A genetically modified yeast, comprising a heterologous
acyl-coA oxidase polypeptide or a heterologous acyl-coA
dehydrogenase polypeptide, which yeast is capable of producing a
diacid from a feedstock comprising one or more components from a
vegetable oil.
[0567] B2. The genetically modified yeast of embodiment B1, wherein
the heterologous acyl-coA oxidase polypeptide is a native
polypeptide.
[0568] B3. The genetically modified yeast of embodiment B1, wherein
the heterologous acyl-coA oxidase polypeptide is an active,
modified polypeptide.
[0569] B4. The genetically modified yeast of embodiment B1, wherein
the heterologous acyl-coA dehydrogenase polypeptide is a native
polypeptide.
[0570] B5. The genetically modified yeast of embodiment B1, wherein
the heterologous acyl-coA dehydrogenase polypeptide is an active,
modified polypeptide.
[0571] B6. The genetically modified yeast of embodiment B1 or B2,
wherein the heterologous acyl-coA oxidase polypeptide is chosen
from a polypeptide having an amino acid sequence set forth in SEQ
ID NO: 51 to SEQ ID NO: 3673.
[0572] B7. The genetically modified yeast of embodiment B1 or B4,
wherein the heterologous acyl-coA dehydrogenase polypeptide is
chosen from SEQ ID NOs: 3679 to 3683, 3686, 3689, 3691, 3693, 3695,
3697, 3699, 3701 and 3703.
[0573] B8. The genetically modified yeast of any one of embodiments
B1 to B7, which is chosen from a Candida spp. yeast, Yarrowia spp.
yeast, Pichia spp. yeast, Saccharomyces spp. yeast and
Kluyveromyces spp. yeast.
[0574] B9. The genetically modified yeast of embodiment B8, which
is chosen from C. tropicalis, C. viswanathii, Y. lipolytica, P.
pastoris, P. membranifaciens, P. kluyveri, P. guilliermondii, P.
heedii, P. subpelliculosa, S. cerevisiae, S. bayanus, S.
pastorianus, S. carlsbergensis, K. lactis and K. marxianus.
[0575] B10. The genetically modified yeast of any one of
embodiments B1 to B9, which comprises a genetic modification that
reduces the activity of an enoyl coA isomerase polypeptide.
[0576] B11. The genetically modified yeast of embodiment B10,
wherein the genetic modification disrupts a polynucleotide that
encodes the enoyl coA isomerase polypeptide.
[0577] B12. The genetically modified yeast of embodiment B10 or
B11, wherein the enoyl coA isomerase polypeptide is a polypeptide
native to the yeast.
[0578] B13. The genetically modified yeast of embodiment B12,
wherein the yeast is a Candida spp. yeast.
[0579] B14. The genetically modified yeast of embodiment B13,
wherein the enoyl coA isomerase polypeptide comprises the amino
acid sequence of SEQ ID NO: 3675 or 3677.
[0580] B15. The genetically modified yeast of any one of
embodiments B1 to B14, which comprises a genetic modification that
reduces the cytoplasmic activity of an acyl-CoA synthetase (ACS)
polypeptide.
[0581] B16. The genetically modified yeast of any one of
embodiments B1 to B15, which comprises a genetic modification that
reduces the peroxisomal activity of an acyl-CoA synthetase (ACS)
polypeptide.
[0582] B17. The genetically modified yeast of embodiment B15 or
B16, wherein the genetic modification disrupts a polynucleotide
that encodes the acyl-CoA synthetase (ACS) polypeptide.
[0583] B18. The genetically modified yeast of embodiment B17,
wherein the genetic modification disrupts an ACS1 polypeptide or
ACS2 polypeptide.
[0584] B19. The genetically modified yeast of any one of
embodiments B15 to B18, wherein the genetic modification disrupts a
polynucleotide that encodes a long-chain acyl-CoA synthetase
polypeptide.
[0585] B20. The genetically modified yeast of embodiment B19,
wherein the genetic modification disrupts a FAT1 polypeptide.
[0586] B21. The genetically modified yeast of any one of
embodiments B15 to B20, wherein the acyl-CoA synthetase (ACS)
polypeptide is a polypeptide native to the yeast.
[0587] B22. The genetically modified yeast of embodiment B21,
wherein the yeast is a Candida spp. yeast.
[0588] B23. The genetically modified yeast of embodiment B22,
wherein the acyl-CoA synthetase (ACS) polypeptide comprises an
amino acid sequence chosen from SEQ ID NOs: 80, 82, 84, 158 and
159.
[0589] B24. The genetically modified yeast of embodiment B21,
wherein the FAT1 polypeptide comprises the amino acid sequence of
SEQ ID NO: 90.
[0590] B25. The genetically modified yeast of any one of
embodiments B1 to B24, which comprises a genetic modification that
reduces the activity of a PXA polypeptide.
[0591] B26. The genetically modified yeast of embodiment B25,
wherein the genetic modification disrupts a polynucleotide that
encodes the PXA polypeptide.
[0592] B27. The genetically modified yeast of embodiment B25 or
B26, wherein the PXA polypeptide is a PXA1 polypeptide or a PXA2
polypeptide, or a PXA1 polypeptide and a PXA2 polypeptide.
[0593] B28. The genetically modified yeast of any one of
embodiments B25 to B27, wherein the PXA polypeptide is native to
the yeast.
[0594] B29. The genetically modified yeast of embodiment B28,
wherein the yeast is a Candida spp. yeast.
[0595] B30. The genetically modified yeast of embodiment B29,
wherein the PXA1 polypeptide comprises the amino acid sequence of
SEQ ID NO: 92.
[0596] B31. The genetically modified yeast of embodiment B29,
wherein the PXA2 polypeptide comprises the amino acid sequence of
SEQ ID NO: 94.
[0597] B32. The genetically modified yeast of any one of
embodiments B1 to B31, comprising an active, modified endogenous
acyl-coA oxidase polypeptide and no active, modified endogenous
acyl-coA dehydrogenase polypeptide.
[0598] B33. The genetically modified yeast of any one of
embodiments B1 to B31, comprising no active, modified endogenous
acyl-coA oxidase polypeptide and an active, modified endogenous
acyl-coA dehydrogenase polypeptide.
[0599] C1. The genetically modified yeast of any one of embodiments
A1 to A41 and B1 to B33, comprising one or more genetic
modifications that reduce the activity of one or more native
endogenous acyl-coA oxidase polypeptides.
[0600] C2. The genetically modified yeast of embodiment C1,
comprising genetic modifications that reduce the activity of all
native endogenous acyl-coA oxidase polypeptides.
[0601] C3. The genetically modified yeast of embodiment C1 or C2,
wherein the genetic modifications partially block beta oxidation
activity.
[0602] C4. The genetically modified yeast of any one of embodiments
A1 to A41, B1 to B33, and C1 to C3, wherein the diacid is a C4 to
C24 diacid.
[0603] C5. The genetically modified yeast of embodiment C4, wherein
the diacid is a 010, C12, C14, C16, C18 or C20 diacid.
[0604] C6. The genetically modified yeast of embodiment C5, wherein
the diacid is a 010 diacid.
[0605] C7. The genetically modified yeast of embodiment C5, wherein
the diacid is a C12 diacid.
[0606] C8. The genetically modified yeast of embodiment C5, wherein
the diacid is a C18 diacid.
[0607] C9. The genetically modified yeast of any one of embodiments
C4 to C8, wherein the diacid contains no unsaturation.
[0608] C10. The genetically modified yeast of any one of
embodiments C4 to C8, wherein the diacid contains one or more
unsaturations.
[0609] C10.1. The genetically modified yeast of any one of
embodiments C4 to C10, wherein the diacid is the predominant diacid
in a mixture of diacids.
[0610] C11. The genetically modified yeast of any one of
embodiments A1 to A41, B1 to B33, and C1 to C10.1, wherein the
feedstock comprises a substantially pure oil.
[0611] C12. The genetically modified yeast of any one of
embodiments A1 to A41, B1 to B33, and C1 to 010, wherein the
feedstock comprises a plurality of fatty acids.
[0612] C13. The genetically modified yeast of embodiment C12,
wherein the feedstock comprises a soapstock.
[0613] C14. The genetically modified yeast of embodiment C12,
wherein the feedstock comprises a fatty acid distillate.
[0614] C15. The genetically modified yeast of any one of
embodiments A1 to A41, B1 to B33, and C1 to C14, wherein the
vegetable oil is from a plant chosen from palm, palm kernel,
coconut, soy, safflower, canola, palm, palm kernel or combination
thereof.
[0615] D1. A method for producing a diacid, comprising: [0616]
contacting a genetically modified yeast of any one of embodiments 1
to A41, B1 to B33, and C1 to C15 with a feedstock comprising one or
more components from a vegetable oil capable of being converted by
the yeast to a diacid; and [0617] culturing the yeast under
conditions in which the diacid is produced from the feedstock.
[0618] D2. The method of embodiment D1, wherein the diacid is a C4
to C24 diacid.
[0619] D3. The method of embodiment D2, wherein the diacid is a
010, C12, C14, C16, C18 or C20 diacid.
[0620] D4. The method of embodiment D3, wherein the diacid is a 010
diacid.
[0621] D5. The method of embodiment D3, wherein the diacid is a C12
diacid.
[0622] D6. The method of embodiment D3, wherein the diacid is a C18
diacid.
[0623] D7. The method of any one of embodiments D1 to D6, wherein
the diacid contains no unsaturation.
[0624] D8. The method of any one of embodiments D1 to D6, wherein
the diacid contains one or more unsaturations.
[0625] D8.1. The method of any one of embodiments D2 to D8, wherein
the diacid is the predominant diacid in a mixture of diacids.
[0626] D9. The method of any one of embodiments D1 to D8.1, wherein
the feedstock comprises a substantially pure oil.
[0627] D10. The method of any one of embodiments D1 to D8, wherein
the feedstock comprises a plurality of fatty acids.
[0628] D11. The method of embodiment D10, wherein the feedstock
comprises a soapstock.
[0629] D12. The method of embodiment D10, wherein the feedstock
comprises a fatty acid distillate.
[0630] D13. The method of any one of embodiments D1 to D12, wherein
the vegetable oil is from a plant chosen from palm, palm kernel,
coconut, soy, safflower, canola, palm, palm kernel or combination
thereof.
[0631] E1. An isolated nucleic acid, comprising a polynucleotide
that encodes a modified acyl-coA oxidase polypeptide from a
yeast.
[0632] E2. The isolated nucleic acid of embodiment E1, wherein the
modified acyl-coA oxidase polypeptide comprises an amino acid
modification in the N-terminal loop.
[0633] E3. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification in the D alpha helix.
[0634] E4. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification in the loop between the D alpha helix and the E'
alpha helix.
[0635] E5. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification to an amino acid in effective contact with
carbons 6 to 9 in a feedstock component.
[0636] E6. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification to an amino acid in effective contact with
carbons 10 to 12 in a feedstock component.
[0637] E7. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification in the L alpha helix.
[0638] E8. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification in the loop C-terminal to the L alpha helix.
[0639] E9. The isolated nucleic acid of embodiment E1, wherein the
modified endogenous acyl-coA oxidase polypeptide comprises an amino
acid modification in the loop between the L alpha helix and the M
alpha helix.
[0640] E10. The isolated nucleic acid of any one of embodiments E2
to E9, wherein the amino acid modification comprises an amino acid
substitution.
[0641] E11. The isolated nucleic acid of embodiment E10, wherein
the amino acid substitution is conservative.
[0642] E12. The isolated nucleic acid of embodiment E10, wherein
the amino acid substitution is not conservative.
[0643] E13. The isolated nucleic acid of any one of embodiments E1
to E12, wherein the yeast is chosen from a Candida spp. yeast,
Yarrowia spp. yeast, Pichia spp. yeast, Saccharomyces spp. yeast
and Kluyveromyces spp. yeast.
[0644] E14. The isolated nucleic acid of embodiment E13, wherein
the yeast is chosen from C. tropicalis, C. viswanathii, Y.
lipolytica, P. pastoris, P. membranifaciens, P. kluyveri, P.
guilliermondii, P. heedii, P. subpelliculosa, S. cerevisiae, S.
bayanus, S. pastorianus, S. carlsbergensis, K. lactis and K.
marxianus.
[0645] E15. The isolated nucleic acid of embodiment E13, wherein
the yeast is a Candida spp. yeast.
[0646] E16. The isolated nucleic acid of embodiment E15, wherein
the yeast is chosen from C. tropicalis and C. viswanathii.
[0647] E17. The isolated nucleic acid of embodiment E16, wherein
the yeast is a genetically modified ATCC20336 yeast.
[0648] E18. The isolated nucleic acid of any one of embodiments E15
to E17, wherein the endogenous acyl-coA oxidase polypeptide is a
POX4 polypeptide.
[0649] E19. The isolated nucleic acid of embodiment E18, wherein
the POX4 polypeptide comprises a modified amino acid sequence of
SEQ ID NO: 30.
[0650] E20. The isolated nucleic acid of embodiment E18 or E19,
wherein the POX4 polypeptide comprises amino acid modifications at
one or more amino acid positions chosen from 88, 90, 96, 98, 99,
100, 102, 103, 302, 309, 310, 473, 474, 475, 476, 477, 478, 479,
480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,
493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504 and
505.
[0651] E21. The isolated nucleic acid of any one of embodiments E15
to E17, wherein the endogenous acyl-coA oxidase polypeptide is a
POX5 polypeptide.
[0652] E22. The isolated nucleic acid of embodiment E21, wherein
the POX5 polypeptide comprises a modified amino acid sequence of
SEQ ID NO: 32.
[0653] E23. The isolated nucleic acid of embodiment E21 or E22,
wherein the POX5 polypeptide comprises amino acid modifications at
one or more amino acid positions chosen from 81, 82, 83, 84, 85,
86, 88, 93, 94, 95, 96, 98, 102, 284, 287, 290, 291, 292, 294, 295,
436, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 and 463.
[0654] E24. The isolated nucleic acid of embodiment E20 or E23,
wherein at least one of the amino acid modifications is an amino
acid substitution.
[0655] E25. The isolated nucleic acid of embodiment E24, wherein at
least one of the amino acid substitutions is conservative.
[0656] E26. The isolated nucleic acid of embodiment E24, wherein at
least one of the amino acid substitutions is non-conservative.
[0657] F1. The isolated nucleic acid of any one of embodiments E1
to E26, which is an expression vector.
[0658] F2. A cell comprising a nucleic acid of any one of
embodiments E1 to F1.
[0659] F3. The cell of embodiment F2, which is a bacterium.
[0660] F4. The cell of embodiment F2, which is a yeast.
[0661] F5. The cell of embodiment F4, which is a Candida spp.
yeast.
[0662] F6. The cell of embodiment F5, wherein the Candida spp.
yeast is chosen from C. tropicalis and C. viswanathii.
[0663] F7. The cell of embodiment F6, wherein the Candida spp.
yeast is a genetically modified ATCC20336 yeast.
[0664] F8. The cell of embodiment F4, which is chosen from a
Yarrowia spp. yeast, Pichia spp. yeast, Saccharomyces spp. yeast
and Kluyveromyces spp. yeast.
[0665] F9. The cell of embodiment F8, which is chosen from Y.
lipolytica, P. pastoris, P. membranifaciens, P. kluyveri, P.
guilliermondii, P. heedii, P. subpelliculosa, S. cerevisiae, S.
bayanus, S. pastorianus, S. carlsbergensis, K. lactis and K.
marxianus.
[0666] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[0667] Modifications may be made to the foregoing without departing
from the basic aspects of the technology. Although the technology
has been described in substantial detail with reference to one or
more specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, yet these modifications and
improvements are within the scope and spirit of the technology.
[0668] The technology illustratively described herein suitably may
be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising," "consisting essentially of," and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and use of such terms
and expressions do not exclude any equivalents of the features
shown and described or portions thereof, and various modifications
are possible within the scope of the technology claimed. The term
"a" or "an" can refer to one of or a plurality of the elements it
modifies (e.g., "a reagent" can mean one or more reagents) unless
it is contextually clear either one of the elements or more than
one of the elements is described. The term "about" as used herein
refers to a value within 10% of the underlying parameter (i.e.,
plus or minus 10%), and use of the term "about" at the beginning of
a string of values modifies each of the values (i.e., "about 1, 2
and 3" refers to about 1, about 2 and about 3). For example, a
weight of "about 100 grams" can include weights between 90 grams
and 110 grams. Further, when a listing of values is described
herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing
includes all intermediate and fractional values thereof (e.g., 54%,
85.4%). Thus, it should be understood that although the present
technology has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this technology.
[0669] Certain embodiments of the technology are set forth in the
claim(s) that follow(s).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140228587A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140228587A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References