U.S. patent application number 15/374811 was filed with the patent office on 2017-03-30 for genetically modified fungi and their use in lipid production.
This patent application is currently assigned to TEKNOLOGIAN TUTKIMUSKESKUS VTT. The applicant listed for this patent is TEKNOLOGIAN TUTKIMUSKESKUS VTT. Invention is credited to Kari KOIVURANTA, Merja PENTTILA, Laura RUOHONEN.
Application Number | 20170088842 15/374811 |
Document ID | / |
Family ID | 42308185 |
Filed Date | 2017-03-30 |
United States Patent
Application |
20170088842 |
Kind Code |
A1 |
KOIVURANTA; Kari ; et
al. |
March 30, 2017 |
GENETICALLY MODIFIED FUNGI AND THEIR USE IN LIPID PRODUCTION
Abstract
The invention refers to fungal cells, and especially to
oleaginous fungal cells that have been genetically modified to
produce enzymes of the pyruvate dehydrogenase bypass route to
enhance their lipid production. Especially the cells are modified
to overexpress genes encoding pyruvate decarboxylase (PDC),
acetaldehyde dehydrogenase (ALD) and/or acetyl-CoA synthetase
(ACS), optionally together with a gene encoding diacylglycerol
acyltransferase (DAT), or to express genes encoding PDC together
with ALD and/or ACS. Methods of producing lipids, biofuels and
lubricants using the modified fungi are also disclosed as well as
expression cassettes useful therein. A new enzyme having
phosholipid:diacylglycerol acyltransferase (PDAT) activity and a
polynucleotide encoding it are also disclosed, which are useful in
the lipid production. A recombinant Cryptococcus cell and its
construction is described.
Inventors: |
KOIVURANTA; Kari; (Espoo,
FI) ; RUOHONEN; Laura; (Espoo, FI) ; PENTTILA;
Merja; (Espoo, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TEKNOLOGIAN TUTKIMUSKESKUS VTT |
VTT |
|
FI |
|
|
Assignee: |
TEKNOLOGIAN TUTKIMUSKESKUS
VTT
VTT
FI
|
Family ID: |
42308185 |
Appl. No.: |
15/374811 |
Filed: |
December 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13806281 |
Dec 21, 2012 |
9518276 |
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PCT/FI2011/050594 |
Jun 21, 2011 |
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15374811 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/88 20130101; C12N
9/0008 20130101; C12Y 602/01001 20130101; C12Y 203/0102 20130101;
C12N 9/93 20130101; C12N 15/80 20130101; C12Y 401/01001 20130101;
C12P 7/649 20130101; Y02E 50/13 20130101; C12N 9/1029 20130101;
C12P 7/6463 20130101; Y02E 50/10 20130101; C12N 15/52 20130101;
C12Y 102/0101 20130101; Y02P 20/52 20151101 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12N 9/00 20060101 C12N009/00; C12N 9/10 20060101
C12N009/10; C12N 15/80 20060101 C12N015/80; C12P 7/64 20060101
C12P007/64; C12N 9/02 20060101 C12N009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2010 |
FI |
20105733 |
Claims
1. A genetically modified oleaginous fungal cell comprising: a) a
nucleic acid with enhanced expression encoding an acetaldehyde
dehydrogenase (ALD), and b) a nucleic acid with enhanced expression
encoding a diacylglycerol acyltransferase (DAT), wherein said
genetically modified oleaginous fungal cell has enhanced lipid
production in an aerobic bioreactor compared to a genetically
unmodified oleaginous fungal cell.
2. The genetically modified oleaginous fungal cell of claim 1,
wherein the encoded ALD is a cytosolic ALD.
3. The genetically modified oleaginous fungal cell of claim 2,
wherein the encoded ALD is a fungal ALD, preferably Saccharomyces
cerevisiae ALD6.
4. The genetically modified oleaginous fungal cell of claim 1,
wherein the DAT encoding nucleic acid encodes a
phosholipid:diacylglycerol acyltransferase (PDAT).
5. The genetically modified oleaginous fungal cell of claim 4,
wherein the encoded DAT is a fungal DAT, preferably of Rhizopus
oryzae, and most preferably it encodes a phosholipid:diacylglycerol
acyltransferase (PDAT) having at least 40% sequence identity to SEQ
ID NO:52, or an enzymatically active fragment or variant
thereof.
6. The genetically modified oleaginous fungal cell of claim 1,
further comprising a nucleic acid with enhanced expression encoding
acetyl-CoA synthetase (ACS).
7. The genetically modified oleaginous fungal cell of claim 6,
wherein the nucleic acid encoding ACS is a gene that is not under
glucose repression or its gene product is not subject to
post-translational regulation.
8. The genetically modified oleaginous fungal cell of claim 7,
wherein the encoded ACS is a fungal ACS, preferably Saccharomyces
cerevisiae ACS2.
9. The genetically modified oleaginous fungal cell of claim 1,
which is a yeast cell selected from the genera Cryptococcus,
Candida, Galactomyces, Hansenula, Lipomyces, Rhodosporidium,
Rhodotorula, Trichosporon and Yarrowia, preferably from the group
consisting of Candida sp., Cryptococcus curvatus, Cryptococcus
albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces
lipofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus,
Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon
pullulans and Yarrowia lipolytica, or a filamentous fungal cell
selected from the genera Aspergillus, Cunninghamella, Fusarium,
Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and
Rhizopus, preferably from the group consisting of Aspergillus
nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus
niger, Cuninghamella japonica, Fusarium oxysporum, Glomus
caledonius, Humicola lanuginose, Mortierella isabellina,
Mortierella pusilla, Mortierella vinacea, Mucor circinelloides,
Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum,
Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae.
10. The genetically modified oleaginous fungal cell of claim 9,
which is from the genera Cryptococcus or Mucor, preferably it is a
cell of Cryptococcus curvatus or Mucor circinelloides.
11. A method of producing lipids, comprising cultivating a
genetically modified oleaginous fungal cell according to claim 1 in
a medium containing carbon and nitrogen sources, and recovering the
lipids produced.
12. The method of claim 11, wherein the lipids are recovered from
the culture medium.
13. The method of claim 11, wherein lipids comprising
acylglycerols, preferably triacylglycerols (TAG) are produced.
14. The method of claim 11, wherein the carbon source is a hexose
or pentose sugars containing material.
15. The method of claim 11, comprising producing precursors for
functional fatty acids.
16. A method of producing biofuel, or lubricant, said method
comprising cultivating a genetically modified oleaginous fungal
cell according to claim 1 in a medium containing carbon and
nitrogen sources, and recovering the lipids produced, and
optionally esterifying said lipids to obtain biodiesel or
lubricant, or hydrogenizing the lipids to obtain renewable diesel
or lubricant.
17. A method of preparing an oleaginous fungal cell of claim 1,
said method comprising transforming a fungal cell with a) a nucleic
acid with enhanced expression encoding an acetaldehyde
dehydrogenase (ALD) enzyme, and b) a nucleic acid with enhanced
expression encoding a diacylglycerol acyltransferase (DAT).
18. Use of a genetically modified fungal cell of claim 1 for
producing lipids, precursors of functional fatty acids, functional
fatty acids, biofuels, biodiesel, renewable diesel or lubricants.
Description
[0001] This application is a Divisional of copending application
Ser. No. 13/806,281, filed on Dec. 21, 2012, which was filed as PCT
International Application No. PCT/FI2011/050594 on Jun. 21, 2011,
which claims the benefit under 35 U.S.C. .sctn.119(a) to Patent
Application No. 20105733, filed in FINLAND on Jun. 24, 2010, all of
which are hereby expressly incorporated by reference into the
present application.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] This application includes an electronically submitted
sequence listing in .txt format. The .txt file contains a sequence
listing entitled "0837_0322PUS2_SequenceListing.txt" created on
Dec. 21, 2012 and is 58,075 bytes in size. The sequence listing
contained in this .txt file is part of the specification and is
hereby incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to fungi, which have been
genetically modified to enhance their lipid production. The
invention also relates to a method for preparing the fungi, and to
expression cassettes for genetic modification of the fungi. The
invention further relates to a method of producing lipids by these
fungi. The lipids produced are useful in manufacturing biofuels,
lubricants and functional fatty acids. The invention thus also
provides a method for producing biofuels and lubricants. Still
further the invention provides a new enzyme protein that is useful
in the methods, and a nucleic acid encoding it. Even further the
invention relates to a recombinant Cryptococcus cell, and a method
for its construction.
BACKGROUND OF THE INVENTION
[0004] Biofuels are current favorites to be the next generation
transportation fuels. They are produced from renewable biological
sources such as vegetable oils and animal fats. They are
biodegradable, non-toxic and have a low emission profile. Due to
the limited sources of biodiesel raw materials such as rape seed
oil, soy bean oil or palm oil, it is of importance to expand
biodiesel raw materials to non-food materials like microbes. The
benefits of using microbes for production of oils are: they are
affected neither by seasons nor by climates, they are able to
produce high lipid contents, and the oils can be produced from a
wide variety of sources with short production times, especially
from residues with abundant nutrition. Microbiologically produced
lipids may also be used e.g. for the production of functional fatty
acids.
[0005] A few fungal species accumulate remarkable amounts of lipid
in the cells. It has been observed that lipids accumulate in these
so called oleaginous fungi under nitrogen limited conditions, which
has resulted in a hypothesis for effective lipid accumulation
(Review Ratledge and Wynn 2002 and references thenceforth).
Nitrogen limitation causes activation of the AMP deaminase which
utilizes AMP to produce NH.sub.4. The decrease in AMP concentration
inhibits the activity of mitochondrial isocitrate dehydrogenase
(IDH) which is part of the mitochondrial tricarboxylic (TCA) cycle.
Reduction in IDH activity results in equilibration of isocitrate to
citrate by aconitase. Produced citrate is transferred to the
cytosol where it is converted with Coenzyme A (CoA) to acetyl-CoA
by ATP:citrate lyase (ACL) with ATP hydrolysis.
[0006] Cytosolic acetyl-CoA can be further used in fatty acid
synthesis. A comprehensive review on fatty acid synthesis and
elongation in yeast, especially in Saccharomyces cerevisiae, is
that of Tehlivets et al., 1997. In the first step of fatty acid
synthesis acetyl-CoA is carboxylated by the addition of carbon
dioxide to malonyl-CoA by the enzyme acetyl-CoA carboxylase in an
ATP demanding reaction. In the following reactions by the fatty
acid synthase systems acyl and malonyl moieties from acyl-CoA and
malonyl-CoA, respectively, are transferred to acyl carrier proteins
(ACPs), after the acyl chain, typically initiated by acetyl-CoA, is
condensated with malonyl-ACP followed by reduction of the
3-ketoacyl-ACP to 3-hydroxyacyl-ACP, dehydration to enoyl-ACP, and
a second reduction to a saturated acyl-chain that is extended by
two carbon atoms. These synthesis steps are usually repeated seven
times resulting in palmitoyl ACP (C16:0). Palmitic acid and
intermediates of the fatty acid synthesis after hydrolysed to
acyl-CoAs by hydrolase/thioesterase, can be further modified by
different elongases and desaturases to different length acyl-chains
with or without double bonds. In one cycle of fatty acid synthesis
two NADPHs are required in the reduction steps. Acyl-CoAs can be
further synthesised to triacylglycerols.
[0007] Triacylglycerol synthesis starts from glycerol-3-phosphate
or dihydroxyacetone phosphate which is acylated
(dihydroxyacetone-phosphate also reduced) to
1-acyl-glycerol-3-phosphate which is further acylated to
phosphatidic acid. Phosphatidic acid can be further
dephosphorylated to diacylglycerol. Diacylglycerol is further
acylated to triacylglycerol mainly by acyl-CoA:diacylglycerol
acyltransferase (DGAT) and phospholipid:diacylglycerol
acyltransferase (PDAT) utilizing acyl-CoA or phosphatidylcholine,
respectively, as acyl donors. The triacylglycerol pathway in yeast
S. cerevisiae is described in more detail in a mini-review of
Sorger and Daum 2003.
[0008] Phospholipid:diacylglycerol acyltransferase (PDAT) encoding
genes originating from S. cerevisiae and Yarrowia lipolytica have
been expressed in yeasts S cerevisiae and Y. lipolytica to enhance
their triacylglycerol production (WO00/60095 and WO2005/003322,
respectively). WO2009/126890 provides systems for producing
engineered oleaginous yeast or fungi that express caroteinoids.
Oleaginy is promoted e.g. by increased or heterologous expression
of DGAT or PDAT, whereas reducing the activity of PDC is expected
to promote oleaginy.
[0009] Methods of manufacturing biodiesel and other oil-based
compounds using glycerol as an energy source in fermentation of
oil-bearing microorganisms have been described e.g. in
US2009/0004715. Methods of producing lipid-based biofuels from
cellulose containing feedstock by heterotrophic fermentation of
microorganisms have been described in US2009/0064567. Both
publications focus on the use of algae as lipid producers. No
details are given. WO2007/136762 provides genetically engineered
microorganisms that produce desired products from the fatty acid
biosynthetic pathway.
[0010] With the above-described triacylglycerol production pathway
high triglyceride yields indicated as triglyceride production per
used carbon source cannot be achieved or triacylglycerol production
per cell biomass cannot be significantly enhanced. In general,
lipids especially triglycerides are produced when nitrogen becomes
a growth limiting factor at the late logarithmic or early
stationary growth phase resulting in a low triglyceride production
rate compared e.g. to yeast ethanol production. Additionally, the
need of several carbons and reduced cofactors in synthesis of
triacylglycerol result in low yield per used carbon. The present
invention uses another route for microbial lipid production. In the
present invention microbial lipid production rate and yields are
enhanced, and the need of reduced cofactors from the outside of the
lipid pathway is decreased. The present invention further provides
lipid production that is not linked to nitrogen limitation.
SUMMARY OF THE INVENTION
[0011] The present invention is based on the use of a pyruvate
dehydrogenase bypass route for producing cytosolic acetyl-CoA. The
invention makes use of an active cytosolic pathway for acetyl-CoA
production, which proceeds via the enzymatic reaction catalyzed by
pyruvate decarboxylase (PDC). This pathway is known in
Crabtree-positive yeast S. cerevisiae, where it is essential for
cytosolic acetyl-CoA production, but it has not been characterized
in oleaginous yeasts and oleaginous filamentous fungi. In
oleaginous fungi another cytosolic pathway for acetyl-CoA
production, proceeding via the reaction catalyzed by pyruvate
dehydrogenase (PDH) is well known and which has been shown to be
essential for cytosolic acetyl-CoA production from pyruvate. This
pathway operates via mitochondria and in this pathway a higher
fraction of carbon is lost than in the pathway via pyruvate
decarboxylase. In the pyruvate decarboxylase pathway, exploited in
this invention, a higher fraction of carbon is directed to
triacylglycerol and it is not dependent on mitochondrial enzyme
activities. Further, it produces NAD(P)H which is needed in the
following fatty acid synthesis. The pyruvate dehydrogenase bypass
route optionally together with an enhanced diacylglycerol
acyltransferase activity as provided by the invention removes many
bottlenecks in microbial lipid production.
[0012] The invention is directed to genetically modified fungal
cells that have been modified to enhance the expression of a
nucleic acid encoding PDC, ALD, ACS and/or DAT, and to methods of
constructing them.
[0013] In particular the invention is directed to a genetically
modified oleaginous fungal cell comprising at least one nucleic
acid with enhanced expression encoding an enzyme selected from the
group consisting of pyruvate decarboxylase (PDC), acetaldehyde
dehydrogenase (ALD) and acetyl-CoA synthetase (ACS).
[0014] The invention is further directed to a genetically modified
fungal cell comprising:
[0015] a) a nucleic acid with modified expression encoding a
pyruvate decarboxylase (PDC) enzyme, and
[0016] b) at least one nucleic acid with modified expression
encoding an enzyme selected from the group consisting of
acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and
diacylglycerol acyltransferase (DAT).
[0017] The invention is also directed to a genetically modified
fungal cell comprising:
[0018] a) at least one nucleic acid with modified expression
encoding an enzyme selected from the group consisting of pyruvate
decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and
acetyl-CoA synthetase (ACS), and
[0019] b) a nucleic acid with modified expression encoding a
diacylglycerol acyltransferase (DAT) enzyme.
[0020] The invention is also directed to a method of producing
lipids comprising cultivating the genetically modified fungal cell
according to the invention in a medium containing carbon and
nitrogen sources, and recovering the lipids produced.
[0021] The invention is further directed to a method of producing
biofuel, or lubricant said method comprising cultivating the
genetically modified fungal cell according to the invention in a
medium containing carbon and nitrogen sources, and recovering the
lipids produced, and optionally esterifying said lipids to obtain
biodiesel or lubricant, or hydrogenizing the lipids to obtain
renewable diesel or lubricant.
[0022] The invention is still further directed to methods of
preparing i.e. constructing a genetically modified fungal cell of
the invention, said methods comprising transforming a fungal cell
with
[0023] at least one nucleic acid with enhanced expression encoding
an enzyme selected from the group consisting of pyruvate
decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and
acetyl-CoA synthetase (ACS); or with
[0024] a) a nucleic acid with enhanced expression encoding a
pyruvate decarboxylase (PDC) enzyme, and
[0025] b) at least one nucleic acid with enhanced expression
encoding an enzyme selected from the group consisting of
acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and
diacylglycerol acyltransferase (DAT); or with
[0026] a) at least one nucleic acid with modified expression
encoding an enzyme selected from the group consisting of pyruvate
decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and
acetyl-CoA synthetase (ACS), and
[0027] b) a nucleic acid with modified expression encoding a
diacylglycerol acyltransferase (DAT) enzyme.
[0028] In addition the invention is directed to an expression
cassette comprising
[0029] a) at least one nucleic acid with modified expression
encoding an enzyme selected from the group consisting of pyruvate
decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and
acetyl-CoA synthetase (ACS), and
[0030] b) a nucleic acid with modified expression encoding a
diacylglycerol acyltransferase (DAT) enzyme.
[0031] Conveniently the genetically modified fungal cells are
constructed according to the invention by transforming the fungal
cell with the nucleic acid(s) encoding said enzyme(s) resulting in
enhanced enzyme activity of said enzyme.
[0032] Still further the invention is directed to an enzyme protein
having phosholipid:diacylglycerol acyltransferase (PDAT) activity
and at least 40% sequence identity to SEQ ID NO:52, or an
enzymatically active fragment or variant thereof, and to an
isolated nucleic acid molecule selected from the group consisting
of: a) a nucleic acid encoding said protein, b) a nucleic acid
comprising the nucleotide sequence of SEQ ID NO:53 or SEQ ID NO:93,
c) a complementary strand of a) or b), and d) a sequence that is
degenerate as a result of the genetic code to anyone of sequences
a)-c).
[0033] The invention is additionally directed to a genetically
modified fungal cell comprising a nucleic acid with modified
expression encoding said PDAT, and to a method of producing lipids,
comprising cultivating said genetically modified fungal cell in a
medium containing carbon and nitrogen sources, and recovering the
lipids produced.
[0034] Still further the invention is directed to the use of a
genetically modified fungal cell of the invention for producing
lipids, biofuels, biodiesel, renewable diesel or lubricants. The
use for producing lipids includes e.g. the use for producing
precursors of fatty acids e.g. of functional fatty acids, and for
producing the fatty acids or functional fatty acids.
[0035] As a still further aspect, the invention is directed to a
genetically modified Cryptococcus cell, which has been modified to
enhance the expression of a heterologous nucleic acid, and to a
method of constructing the cell, said method comprising
transforming a Cryptococcus cell with a nucleic acid encoding a
heterologous protein.
[0036] Specific embodiments of the invention are set forth in the
dependent claims. Other objects, details and advantages of the
present invention will become apparent from the following drawings,
detailed description and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows probable metabolic routes for cytosolic
acetyl-CoA production via the pyruvate dehydrogenase route in grey,
and via the pyruvate dehydrogenase bypass route i.e. the pyruvate
decarboxylase route in black.
[0038] FIG. 2 shows the metabolic route for triacylglycerol
production.
[0039] FIG. 3 is a diagram depicting plasmid pKK81.
[0040] FIG. 4 is a diagram depicting plasmid pKK82.
[0041] FIG. 5 is a diagram depicting plasmid pKK86.
[0042] FIG. 6 is a diagram depicting plasmid pKK95.
[0043] FIG. 7 is a diagram depicting plasmid pKK85.
[0044] FIG. 8 is a diagram depicting plasmid pKK75.
[0045] FIG. 9 is a diagram depicting plasmid pKK94.
[0046] FIG. 10 is a diagram depicting plasmid pKK96.
[0047] FIG. 11 is a diagram depicting plasmid pKK98.
[0048] FIG. 12 is a diagram depicting plasmid pKK101.
[0049] FIG. 13 is a diagram depicting plasmid pKK102.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The presumed pyruvate dehydrogenase (PDH) pathway for
producing cytosolic acetyl-CoA is shown in grey in FIG. 1. First
cytosolic pyruvate is transported into the mitochondria where it is
oxidatively decarboxylated to acetyl-CoA and carbon dioxide by the
pyruvate dehydrogenase complex. The resulting acetyl-CoA is then
entering the tricarboxylic (TCA) cycle by citrate synthase (CS)
which catalyses the condensation reaction of the two-carbon acetate
residue from acetyl-CoA and a molecule of four-carbon oxaloacetate
(OAA) to form the six-carbon citrate. Citrate is then isomerised to
isocitrate which is the substrate for mitochondrial isocitrate
dehydrogenase (IDH). Under limited nitrogen supply AMP deaminase
activity increases leading to production of IMP and ammonium from
AMP. The decrease in the amount of AMP results in decrease in
mitochondrial isocitrate dehydrogenase (IDH) activity, whereby the
amount of citrate in the mitochondria increases. The mitochondrial
citrate is transported into the cytosol, where ATP:citrate lyase
(ACL) converts citrate, ATP and CoA into acetyl-CoA and
oxaloacetate. The oxaloacetate is degraded by malate dehydrogenase
(MDH) to malate, which in turn is converted to pyruvate and carbon
dioxide by malic enzyme (MAE) under production of NADPH, which is
an important cofactor in fatty acid synthesis. In this invention
cytosolic acetyl-CoA is produced via a pyruvate dehydrogenase
bypass pathway, which is shown in black in FIG. 1. This pathway is
also called the pyruvate decarboxylase pathway. In this pathway
pyruvate is decarboxylated to acetaldehyde and carbon dioxide by
pyruvate decarboxylase (PDC). Acetaldehyde is further oxidised to
acetate by NADP.sup.+ (or NAD.sup.+)-dependent acetaldehyde
dehydrogenase (ALD). Acetate is then converted to acetyl-CoA by
acetyl-CoA synthetase (ACS) with ATP and Coenzyme A. In this
cytosolic pathway
[pyruvate+CoA+ATP+NAD(P).sup.+=Acetyl-CoA+CO.sub.2+NAD(P)H+AMP+PPi+H.sup.-
+] the overall acetyl-CoA yield is higher than in the pyruvate
dehydrogenase-ATP:citrate lyase pathway [pyruvate+CoA+ATP+NAD.sup.+
(in mitochondria)+oxaloacetate (in
mitochondria)=Acetyl-CoA+CO.sub.2+ADP+Pi+NAD.sup.+ (in
mitochondria)+H.sup.++oxaloacetate (in cytosol)].
[0051] Cytosolic acetyl-CoA is used by NADPH-dependent fatty acid
synthase (FAS) and other enzymes for the production of acyl-CoA
esters of different length, which then are attached to for example
glycerol through the triglyceride metabolic pathway shown in FIG.
2. In the last step of this pathway an acyl group from acyl-CoA or
from a phospholipid is attached to the diacylglycerol by
acyl-CoA:diacylglycerol acyltransferase (DGAT) or
phospholipid:diacylglycerol acyltransferase (PDAT).
[0052] In the present invention triacylglycerol production of fungi
will be enhanced by overexpressing at least one gene, which encodes
an enzyme involved in the pyruvate decarboxylate pathway for
converting pyruvate to acetyl-CoA as shown in FIG. 1, together with
a gene, which encodes an enzyme that catalyses the acylation of
diacylglycerol to triacylglycerol as shown in FIG. 2.
[0053] Lipid production including triacylglycerol production in the
cell is a highly NADPH demanding process. E.g. in production of 1
mole of oleic acid (9-octadecenoic acid) 17 mole of NADPH is
needed. NADPH produced by malic enzyme has been proposed to be the
main source for NADPH needed in fatty acid synthesis. Said NADPH
production occurs totally outside the cytosolic acetyl-CoA
production pathway resulting in the consumption of extra carbons in
NADPH production, even though the reaction of the malic enzyme is
linked to the degradation of the oxaloacetate produced by
ATP:citrate lyase. In the present invention fatty acid and further
triacylglycerol production is connected directly to NADPH cofactor
production by NADP-dependent acetaldehyde dehydrogenase thus
reducing the need to produce NADPH outside the triglyceride
production pathway resulting in an increased triacylglycerol yield.
The NADP.sup.+-dependent acetaldehyde dehydrogenase produces
simultaneously one NADPH and one acetate molecule from NADP+ and
acetaldehyde resulting in production of one mole of NADPH per one
mole of pyruvate. This means that half of the NADPH molecules
needed in the fatty acid synthesis are produced simultaneously with
the cytosolic acetyl-CoA production. This simultaneous NADPH
production with lower carbon loss during production of cytosolic
acetyl-CoA results in a better yield in fatty acid production
following also better yield in triacylglycerol production. In this
invention only one carbon is lost from the carbon skeleton
downstream of pyruvate prior to cytosolic acetyl-CoA. Additionally,
no side reactions are needed to cleave metabolites further outside
the triglyceride production pathway. The production of cytosolic
acetyl-CoA via the pyruvate dehydrogenase bypass completes the
existing pyruvate dehydrogenase pathway for cytosolic acetyl-CoA
production.
[0054] Triacylglycerols and other lipids are naturally produced in
fungi via the pyruvate dehydrogenase pathway during growth, but the
main triacylglycerol and lipid accumulation occurs when excess
citrate will be available after nitrogen limitation in the late
stage of cultivation. This triacylglycerol production in late
logarithmic or stationary phases of cultivation results in low
triacylglycerol production rates, especially at the early stage of
cultivation. In this invention expression of the pyruvate
dehydrogenase bypass catalyzed by PDC, ALD, and ACS results in a
situation where triacylglycerol accumulation is not linked to
nitrogen limitation thus allowing enhanced triacylglycerol
production during cultivation resulting in a better triacylglycerol
production rate. The earlier triacylglycerol production is further
enhanced by expressing an acyltransferase such as a
phospholipid:diacylglycerol acyltransferase (PDAT) encoding gene
e.g. under a constitutive promoter thus increasing triacylglycerol
concentration at the expense of phospholipids.
[0055] Contrary to oleaginous yeasts and moulds like Cryptococcus
curvatus and Mucor circinelloides, S. cerevisiae lacks the pyruvate
dehydrogenase route for acetyl-CoA production. In this
Crabtree-positive yeast cytosolic acetyl-CoA, and further fatty
acids and triacylglycerols, are produced only via pyruvate
dehydrogenase bypass. The essential role of the pyruvate
dehydrogenase bypass including the enzymes pyruvate decarboxylate
(PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase
(ACS) in cytosolic acetyl-CoA and further in lipid production and
the role of ALD in the generation of reducing equivalents (NADH and
NADPH) in S. cerevisiae has been described for example in Flikweert
et al. 1996, Pronk et al 1996, Saint-Prix et al 2004). In
US2009/0053797 expression of endogenous NADP-dependent acetaldehyde
dehydrogenase (ALD6) gene and native or modified endogenous ACS1
gene or Salmonella enterica acetyl-CoA synthetase (ACS1) gene in S.
cerevisiae resulted in an increased concentration of cytosolic
acetyl-CoA in the production of isoprenoids. Shiba et al., 2007
found that overexpression of ALD6 and ACS1 in S. cerevisiae
increased cytosolic acetyl-CoA derived amorphadiene overproduction,
whereas overexpression of ACS2 with ALD6 did not. The acetyl-CoA
synthetase isoforms ACS1 and ACS2 behave differently in S.
cerevisiae: ACS1 gene has been shown to be under glucose repression
whereas ACS2 gene has been shown to be constitutively expressed and
co-regulated with structural genes of fatty acid biosynthesis (van
den Berg et al 1996, Hiesinger et al. 1997).
[0056] The essential role of the PDH bypass in S. cerevisiae has
been described: Deletion of three structural genes for pyruvate
decarboxylase (PDC1, PDC5 and PDC6) results in loss of growth in a
defined glucose medium (Flikweert et al 1996, Pronk et al. 1996),
and deletion of two acetyl-CoA synthetase encoding genes ACS1 and
ACS2 is lethal on all carbon sources (Takahashi et at 2006 and van
den Berg et at 1996), indicating that there is no alternative route
for cytosolic acetyl-CoA production in S. cerevisiae. The enhanced
expression of this pathway has been found to induce cytosolic
acetyl-CoA production in S. cerevisiae (e.g. US2009/0053797 and
WO2008/080124). However, the existence and functionality of the
pyruvate dehydrogenase bypass pathway in oleaginous yeasts or
moulds has not been described in the literature.
[0057] PDC has been characterised e.g. from the Rhizopus oryzae,
which in addition to lipids produced ethanol (Skory 2003). Also,
ALD and ACS have been characterised from some of the oleaginous
fungi e.g. A. nidulans (Flipphi et al. 2001, Connerton et al.
1990). Still, it has also been shown that deletion of the only
cytoplasmic ACS encoding gene from A. nidulans had no effect on
growth on glucose (Sandeman et al. 1989). Instead, it has been
shown in several articles that the cytosolic acetyl-CoA for lipid
production will be produced via pyruvate dehydrogenase and
ATP:citrate lyase (ACL) in oleaginous fungi (Wynn et al 2001,
Boulton and Ratledge 1981). ATP:citrate lyase has been shown to be
absent from the non-oleaginous yeasts (Boulton and Ratledge 1981).
E.g. ACL is absent from the sequenced members of the
Saccharomycotina with the exception of Y. lipolytica which is
oleaginous yeast. The essential role of ACL for cytosolic
acetyl-CoA production has also been shown at a functional level by
deteting the acl gene from A. nidulans. This deletion strain could
not grow in the absence of external sources of cytoplasmid
acetyl-CoA, which strongly suggests that ACL activity is required
to generate cytoplasmic acetyl-CoA. This also indicates the absence
of any pyruvate dehydrogenase bypass pathway, which could
compensate acl deletion (Hynes and Murray 2010). The production of
cytosolic acetyl-CoA via ATP:citrate lyase in oleaginous fungi is
also suggested in the patent applications WO2005/003322 and
US2006/094087, where diacylglycerol transferase encoding genes have
been expressed to enhance triacylglycerol production.
[0058] The term "pyruvate dehydrogenase bypass" refers to an
alternative route to the pyruvate dehydrogenase reaction for the
conversion of pyruvate to acetyl-CoA. The pyruvate dehydrogenase
bypass comprises the enzymes pyruvate decarboxylase (PDC),
acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS).
It has been shown in literature that the pyruvate dehydrogenase
bypass is not essential in Crabtree-negative yeasts.
[0059] In the present context the corresponding genes encoding the
enzyme proteins are indicated in italics.
[0060] The term "PDC" refers to pyruvate decarboxylase enzyme (EC
4.1.1.1). This enzyme catalyses the thiamine pyrophosphate- and
Mg.sup.2+-dependent decarboxylation of pyruvate to acetaldehyde and
carbon dioxide. A preferred PDC is one of a Crabtree-positive
organism. Preferably the PDC is a fungal PDC, especially of a
Crabtree-positive fungus, such as S. cerevisiae e.g. PDC1 of S.
cerevisiae (GenBank accession number CAA54522, version number
CAA54522.1). According to one embodiment of the invention the PDC1
contains the amino acid sequence of SEQ ID NO:95, and/or is encoded
for example by a polynucleotide containing the nucleotide sequence
of SEQ ID NO:94, SEQ ID NO:96 or SEQ ID NO:97.
[0061] The term "ALD" refers to acetaldehyde dehydrogenase enzyme
(EC 1.2.1.5 and EC 1.2.1.4). This enzyme catalyses the reaction
where acetaldehyde is oxidized to acetate, and NAD or NADP-cofactor
is reduced to NADH or NADPH, respectively. NADP-specific ALDs are
preferred. In the present invention the ALD is preferably a fungal
ALD, more preferably of S. cerevisiae, and most preferably it is S.
cerevisiae ALD6, which is encoded for example by a polynucleotide
of SEQ ID NO:48 or 49, and/or comprises the amino acid sequence of
SEQ ID NO:47. Further, the ALD is preferably a cytosolic ALD. ALD6
is cytosolic. Cytosolic ALD can be modified from mitochondrial
isoforms of ALD by removing the mitochondrial targeting signal from
the originally mitochondrial ALD by genetic engineering. The
cleavage site of the mitochondrial targeting signal can be decided
e.g. with programs designed for this purpose such as MITOPROT.
Examples of mitochondrial ALD, which can be modified to be
cytosolic are S. cerevisiae ALD4 and ALD5 encoding genes. Suitable
ALD encoding genes can be found from databases e.g. KEGG Enzyme
database and Brenda with EC numbers 1.2.1.4 and 1.2.1.5. Table 1
contains examples of NAD(P).sup.+ dependent acetaldehyde
dehydrogenases.
TABLE-US-00001 TABLE 1 NAD(P).sup.+ -dependent acetaldehyde
dehydrogenase enzymes Accession Version number of the number of the
amino acid amino acid Organism sequence sequence Database
Saccharomyces cerevisiae AAB68304 AAB68304.1 GenBank Saccharomyces
cerevisiae DAA07732 DAA07732.1 GenBank Saccharomyces cerevisiae
DAA11133 DAA11133.1 GenBank Aspergillus niger A2QMA4 A2QMA4.1
TrEMBL Aspergillus niger A2QiG1 A2QiG1.1 TrEMBL Aspergillus niger
A5AAZ8 A5AAZ8.1 TrEMBL Aspergillus niger A2Q9V7 A2Q9V7.1 TrEMBL
Aspergillus fumigatus Q4WQP1 Q4WQP1.1 TrEMBL Pichia angusta Q12648
Q12648 Swiss-Prot Pichia stipitis A3M013 A3M013.2 TrEMBL Candida
dubliniensis B9W6J2 B9W6J2.1 TrEMBL Candida glabrata CAG59952
CAG59952.1 GenBank Kluyveromyces lactis CAH00079 CAH00079.1 Genbank
Lachancea thermotolerans CAR23570 CAR23570.1 Genbank Burkholderia
xenovorans Q13WK4 Q13WK4.1 TrEMBL Vibrio harveyi Q56694 Q56694.1
Swiss-Prot Mus musculus P47739 P47739.1 Swiss-Prot Mus musculus
Q80VQ0 Q80VQ0.1 Swiss-Prot Rattus norvegicus P11883 P11883.3
Swiss-Prot Rattus norvegicus Q5XI42 Q5XI42.1 Swiss-Prot Canis lupus
familiaris A3RF36 A3RF36.1 Swiss-Prot Bos taurus P30907 P30907.2
Swiss-Prot Bos taurus Q1JPA0 Q1JPA0.1 Swiss-Prot Homo sapiens
P30838 P30838.2 Swiss-Prot Homo sapiens P43353 P43353.1
Swiss-Prot
[0062] The term "cytosolic" refers to a fluid component of the
cytoplasm excluding organelle and other suspended intracellular
structures.
[0063] The term "ACS" refers to acetyl-CoA synthetase enzyme (EC
6.2.1.1). The enzyme catalyses the reaction where acetyl-CoA is
formed from acetate and CoA with hydrolysis of ATP. Preferably the
ACS is a fungal ACS, more preferably S. cerevisiae ACS, especially
S. cerevisiae ACS2. Preferably the ACS encoding gene to be
expressed is a gene, which is not under glucose repression and/or
which gene product is not subject to post-translational regulation
e.g. acetylation, in the original species. In a particular
embodiment the ACS contains the sequence of SEQ ID NO:50. Several
yeasts such as Candida albicans have genes similar to S. cerevisiae
ACS2, which most likely are not under post-translational
regulation. This abolishes the need to modify the existing gene.
According to one embodiment ACS2 is encoded by a polynucleotide
having the sequence of SEQ ID NO:51 or 92.
[0064] The term "DAT" refers to diacylglycerol acyltransferase
enzyme (EC 2.3.1.X). The enzyme catalyses a reaction where an acyl
group is transferred to 1,2-diacylglycerol to the position sn-3.
DAT includes both acyl-CoA:diacylglycerol acyltransferase (DGAT, EC
2.3.1.20) and phospholipid:diacylglycerol acyltransferase (PDAT).
Preferably the DAT is PDAT.
[0065] The term "PDAT" refers to phospholipid:diacylglycerol
acyltransferase enzyme (EC 2.3.1.158). The enzyme catalyses a
reaction where an acyl group from a phospholipid is transferred to
1,2-diacylglycerol to the position sn-3 via an acyl-CoA-independent
mechanism. Preferably the PDAT is of fungal origin, and more
preferably of an oleaginous fungus. In one preferred embodiment the
PDAT is a Rhizopus oryzae PDAT, and most preferably it is encoded
by a polynucleotide that contains the sequence of SEQ ID NO:53 or
93. Preferably the PDAT contains an amino acid sequence having at
least 40% identity to SEQ ID NO:52, or an enzymatically active
fragment or variant thereof.
[0066] In particular preferred embodiments of the invention the
encoded enzyme comprises an amino acid sequence with a sequence
identity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,
99% or 100% to SEQ ID NO:95, 47, 50, or 52, or an enzymatically
active fragment or variant thereof.
[0067] Percent identity of amino acid sequences can conveniently be
computed using BLASTP version 2.2.23 software with default
parameters. Sequences having an identities score and a positives
score of a given percentage, using the BLASTP version 2.2.23
algorithm with default parameters, are considered to be that
percent identical or homologous (Altschul et al. 1997).
[0068] It is well known that deletion, addition or substitution of
one or a few amino acids does not necessarily change the catalytic
properties of an enzyme protein. Therefore the invention also
encompasses variants and fragments of the given amino acid
sequences having the stipulated enzyme activity. The term "variant"
as used herein refers to a sequence having minor changes in the
amino acid sequence as compared to a given sequence. Such a variant
may occur naturally e.g. as an allelic variant within the same
strain, species or genus, or it may be generated by mutagenesis or
other gene modification. It may comprise amino acid substitutions,
deletions or insertions, but it still functions in substantially
the same manner as the given enzymes, in particular it retains its
catalytic function as an enzyme.
[0069] A "fragment" of a given protein sequence means part of that
sequence, i.e. a sequence that has been truncated at the N- and/or
C-terminal end. It may for example be the mature part of a protein
comprising a signal sequence, or it may be only an enzymatically
active fragment of the mature protein.
[0070] The term "lipid" refers to a group of organic compounds that
are relatively or completely insoluble in water but soluble in
nonpolar organic solvents. These properties are a result of long
hydrocarbon tails, which are hydrophobic in nature. The term thus
encompasses fats, oils, waxes, fatty acids, fatty acid derivatives,
like phospholipids, glycolipids, acylglycerids such as
monoglycerides, diglycerides, and triglycerides and terpenoids such
as carotenoids and steroids.
[0071] The term "fatty acid" refers to a compound obtainable via
condensation of malonyl coenzyme A units by a fatty acid synthase
system. They may be saturated or unsaturated. "Functional fatty
acid" refers to a fatty acid compound having at least one
functional group e.g. a hydroxyl (--OH) or carboxyl (--COOH) group
within the fatty acid and being responsible for the characteristic
chemical reactions of those molecules.
[0072] The term "fatty acid derivative" refers to a compound having
at least one esterified fatty acyl group. Fatty acid derivatives
include e.g. phospholipids, glycolipids and acylglycerides.
[0073] The term "acylglyceride" is synonymous with "acylglycerol"
and refers to a compound having a glycerol moiety with one or
several hydroxyl groups esterified to a fatty acid.
[0074] The terms "monoglyceride" and "monoacylglycerol" refer to a
glyceride where one fatty acid residue has been esterified to a
glycerol molecule. The fatty acid residue in the monoacylglycerol
can be a short or long chain fatty acid with or without double
bonds.
[0075] The terms "diglyceride" and "diacylglycerol" and "DAG" refer
to glyceride where two fatty acid residues have been esterified to
a glycerol molecule. Fatty acid residues in diacylglycerol can be
short or long chain fatty acids with or without double bonds.
[0076] The terms "triglyceride" and "triacylglycerol" and "TAG"
refer to a glyceride where three fatty acid residues have been
esterified to a glycerol molecule. Fatty acid residues in
triacylglycerol can be short or long chain fatty acids with or
without double bonds. Triacylglycerol is the major acylglycerol
group in oleaginous fungi.
[0077] The term "acyl-CoA" refers to a fatty acid residue, which
has been esterified to a CoA molecule. Fatty acid residues in
acyl-CoA can be short or long chain fatty acids with or without
double bonds.
[0078] The term "phospholipids" refers to any lipid containing a
diglyceride combined with a phosphate group and a simple organic
molecule such as choline or ethanolamine.
[0079] The term "glycolipid" refers to a lipid attached with a
carbohydrate.
[0080] The term "fat" refers to a group of organic compounds that
are relatively or completely insoluble in water but soluble in
nonpolar organic solvents and which are solids at normal room
temperature.
[0081] The term "oil" refers to a group of organic compounds that
are relatively or completely insoluble in water but soluble in
nonpolar organic solvents and which are liquids at normal room
temperature.
[0082] Generally fats and oils are triesters of glycerol and fatty
acids.
[0083] The term "wax" refers to a compound that may contain
long-chain alkanes, esters, polyesters and hydroxyl esters of
long-chain primary alcohols and fatty acids.
[0084] The term "terpenoid" refers to a compound formally derived
from hydrocarbon isoprene.
[0085] The term "steroid" refers to a terpenoid lipid compound
having a sterane core and additional functional groups. Sterols are
special forms of steroids, with a hydroxyl group at the atom C-3
and a skeleton derived from cholestane.
[0086] The term "carotenoids" refers to a compound belonging to the
category of tetraterpenoids. Structurally they are in the form of a
polyene chain, which is sometimes terminated by rings.
[0087] In the present invention fungal cells are genetically
modified to express particular enzymes. The cells can be
genetically modified to produce increased levels of lipid by
transforming them with nucleic acids that have been modified to
enhance the expression of nucleic acids encoding at least one of
PDC, ALD and ACS, together with a nucleic acid that has been
modified to enhance the expression of a nucleic acid encoding DAT
so as to allow overexpression of the enzymes. A "genetically
modified" organism or cell is an organism or cell that comprises an
expression modified nucleic acid. It may be a recombinant organism
or cell, or a host organism or cell, or a mutant.
[0088] "Nucleic acid" is a macromolecule comprising a chain of
monomeric nucleotides i.e. a polynucleotide. It can be e.g. DNA
such as cDNA or genomic DNA or mRNA, and it can be e.g.
recombinantly or synthetically produced, double or single stranded,
encompassing both sense and antisense strands.
[0089] "Recombinant" nucleic acid refers to an artificial
combination of at least two otherwise separated sequences, i.e. to
a not naturally occurring combination of nucleic acids.
[0090] "Nucleic acid with modified expression" as used herein
denotes nucleic acids that are foreign or exogenous to the host
meaning that they are not naturally found in said host. The term
also includes nucleic acids that are endogenous i.e. naturally
found in the host, but which are produced in an unnatural amount
e.g. as multiple copies, or nucleic acids that differ in sequence
from the naturally occurring nucleic acids but encode the same type
of protein. Further, the term includes nucleic acids comprising at
least two nucleotide sequences that do not occur in the same
relationship to each other in nature, such as e.g. an endogenous
protein encoding sequence that is operably linked to a
transcriptional control element e.g. a promotor and/or terminator
in a way that does not occur in nature. Said promotor and/or
terminator can be of endogenous or exogenous origin. High copy
number plasmids comprising the protein encoding nucleotide sequence
are also considered nucleic acids with modified expression. The
above mentioned expression modified nucleic acids encompass
recombinant nucleic acids, herein also called heterologous nucleic
acids. Alternatively the expression modified nucleic acid can be a
mutated nucleic acid. "Modified expression" in this context is used
only in the meaning of over-expression i.e. "enhanced expression".
The enhanced expression results in overproduction of the expressed
protein in the modified organism compared to that in an unmodified
organism.
[0091] In one embodiment of the invention the nucleic acid with
modified expression contains an exogenous gene encoding PDC, ALD,
ACS or DAT derived from another organism. The genes encoding PDC,
ALD or ACS can be obtained from yeast such as Saccharomyces
cerevisiae, Candida glabrata, Dekkera bruxellensis, Kluyvemmyces
lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia angusta,
Pichia stipitis, Zygosaccharomyces rouxii, Issatchenkia terricola,
Debaryomyces hansenii, Candida angusta and Pichia guilliermondii,
and the DAT can be obtained from yeast or filamentous fungi such as
Saccharomyces cerevisiae, Candida glabrata, Zygosaccharornyces
rouxii, Lachancea thermotolerans, Ashbya gossypii, Cryptococcus
curvatus, Cryptococcus albidus, Lipomyces lipofer, Lipomyces
starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides,
Rhodotorula Rhodotorula graminis, Yarrowia lipolytica, Aspergillus
nidulans, Aspergillus oryzae, Fusarium oxysporum, Humicola
lanuginose, Mortierella alpina, Mortierella vinacea, Mucor
circinelloides, Mucor plumbeus, Penicillium spinulosum, and
Rhizopus oryzae. In another embodiment the enzyme genes to be
expressed are endogenous genes, the promotor of which is replaced
with another promotor, preferably a constitutive promotor, or the
existing promotor is modified to become a constitutive
promotor.
[0092] Proteins or polynucleotides "derived from", "originated
from" or "obtained from" a particular organism encompass products
isolated from said organism, as well as modifications thereof. A
protein derived from a particular organism may be a recombinantly
produced product, which is identical to, or a modification of the
naturally occurring protein. The protein may also be modified e.g.
by glycosylation, phosphorylation or other chemical modification.
Products derived from the particular organism also encompass
mutants and natural variants of the products, where one or more
nucleic acid and/or amino acid is deleted, inserted and/or
substituted.
[0093] Expression of any combination of the genes of the pyruvate
dehydrogenase bypass route may be linked to expression of the DAT
encoding gene. The expression of the DAT encoding gene may for
example be combined to the expression of an ALD encoding gene, or
an ACS encoding gene, or a PDC encoding gene. In another embodiment
both ASC and ALD, or both PDC and ACS, or PDC and ALD, are
overexpressed, and in still another all PDC, ALD and ACS are
overexpressed together with the DAT encoding gene. In one specific
embodiment of the invention the expression of S. cerevisiae ALD
encoding gene ALD6 is linked to the expression of a PDAT encoding
gene.
[0094] Alternatively expression of PDC may be combined with
expression of ACS and/or ALD thus providing the combinations
PDC+ALD, PDC+ACS and PDC+ALD+ACS. These combinations may be
expressed with or without further expressing a gene encoding DAT,
such as PDAT.
[0095] "Fungal" "fungus" and "fungi" as used herein refers to yeast
and filamentous fungi i.e. moulds. A genetically modified fungal
cell is also referred to as host cell.
[0096] The yeast host cell may be selected for example from the
genera Cryptococcus, Candida, Galactomyces, Hansenula, Lipomyces,
Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia. Preferably
the yeast host cell is selected from the group consisting of
Candida sp., Cryptococcus curvatus, Cryptococcus albidus,
Galactomyces geotrichum, Hansenula ciferri, Lipomyces hpofer,
Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus,
Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon
pullulans and Yarrowia lipolytica. In one embodiment the yeast is
selected from the phylum Basidiomycota, which includes
Cryptococcus, Rhodotorula and Rhodosporidium. Most preferably it is
Cryptococcus curvatus.
[0097] The filamentous fungus host cell may be selected from the
genera Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola,
Mortierella, Mucor, Penicillium, Pythium and Rhizopus. Preferably
the filamentous fungus is selected from the group consisting of
Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus,
Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum,
Glomus caledonius, Humicola lanuginose, Mortierella isabellina,
Mortierella pusilla, Mortierella vinacea, Mucor circinelloides,
Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum,
Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae.
According to one embodiment the filamentous fungus belongs to
subphylum Mucoromycotina, which includes Mucor and Mortierella.
Most preferably it is Mucor circinelloides.
[0098] According to one preferred embodiment the fungal host cell
is an oleaginous fungus. The term "Oleaginous fungi" refers to
yeasts or filamentous fungi, which accumulate at least 10%, 12.5%,
15%, 17.5%, preferably at least 20% or even at least 25% (w/w) of
their biomass as lipid. They may even accumulate at least 30%, 40%,
50%, 60%, 70%, 80% (w/w) or more of their biomass as lipids. The
biomass is usually measured as cell dry weight (CDW). Oleaginous
fungi are found e.g. in genera Cryptococcus, Candida, Galactomyces,
Hanseluna, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon,
Yarrowia, Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola,
Mortierella, Mucor, Penicillium, Pythium and Rhizopus, and
especially in species Candida sp., Cryptococcus curvatus,
Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri,
Lipomyces lipofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces
tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis,
Trichosporon pullulans, Yarrowia lipolytica, Aspergillus nidulans,
Aspergillus oryzae, Aspergillus terreus, Aspergillus niger,
Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius,
Humicola lanuginose, Mortierella isabellina, Mortierella pusilla,
Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor
ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium
ultimum and Rhizopus oryzae. In one embodiment it is a
Crabtree-negative oleaginous yeast, and in another embodiment it is
a Crabtree-positive filamentous fungus. In still another embodiment
the filamentous fungus is Crabtree-negative, or the yeast is
Crabtree-positive. Saccharomyces yeasts including S. cerevisiae are
not oleaginous fungi. A "Crabtree-positive" organism is one that is
capable of producing ethanol in the presence of oxygen, whereas a
"Crabtree-negative" organism is not.
[0099] According to one preferred embodiment the host cell is a
Cryptococcus, and especially Cryptococcus curvatus. Genetically
modified Cryptococcus curvatus strains have not been described in
literature (Meesters et al. 1997). Routinely, in yeast expression
systems S. cerevisiae promoters and terminators, original genes
without codon optimisation or codon-optimised for S. cerevisiae are
used. In this invention we showed that it is possible to express
genes successfully in C. curvatus when using endogenous promoters
and terminators i.e. which originate from the species wherein the
expression cassette will be transformed, and an expressed gene that
is codon-optimised according to codon usage of the species wherein
the expression cassette will be transformed, or its close relative,
which has a genome that is known at a level where codonoptimisation
is possible. Such a close relative is e.g. Ustilago maydis. In a
preferred embodiment the promotors are constitutive promotors,
especially from the glycolysis pathway.
[0100] Promoters and terminators of oleaginous fungi might contain
sites for different regulatory elements and transcription factors
than the promoters and terminators of S. cerevisiae due to the
different nature of the strains: S. cerevisiae can grow and produce
ethanol under anaerobic conditions whereas C. curvatus does not
produce ethanol at all, and lipids it produces under aerobic
conditions. Also the codon usage differs in S. cerevisiae and in C.
curvatus: E.g. S. cerevisiae codons and genome are adenine and
thymine rich, whereas the ratio of guanine and cytosine is much
higher in C. curvatus (Meesters et al. 1997).
[0101] It is also important to use endogenous promoters and
terminators with resistance markers to detect transformants after
transformations. Due to the fact that the genome of C. curvatus is
not known different procedures described in this invention can be
carried out to clone endogenous promoters and terminators.
Preferably constitutive promoters are used.
[0102] The fungal cell can be genetically modified by transforming
it with a heterologous nucleic acid that encodes a heterologous
protein. "Heterologous" in this context means not naturally
occurring. In one embodiment, the cell is transformed with a
heterologous nucleic acid that encodes at least one of PDC, ALD and
ACS, and a heterologous nucleic acid comprising a nucleic acid that
encodes DAT, operably linked to allow expression of the genes
encoding said enzymes. DNA isolation, enzymatic treatment and
genetical modifications may be carried out using standard molecular
biology methods as described e.g. Sambrook and Russell (2001). The
promoter and terminator regions of the genes of interest can be
cloned e.g. from a yeast or filamentous fungus strain of interest
by polymerase chain reaction (PCR) using gene specific
oligonucleotides designed based on known published gene sequences
of strains of the same species or other species or gene sequences
of the strain of interest. Oligonucleotides designed based on the
sequence of the strain of interest are preferred.
[0103] New gene fragments from yeast and filamentous fungus species
or strains with unknown genomic sequences can be cloned by PCR by
using degenerative primers. The term "degenerative primer" refers
to mixtures of similar kinds of synthesized primers differing from
each other by one nucleotide. Degenerative primers for cloning of
specific gene fragments from desired species or strains are
designed based on known characterised or putative gene
sequences.
[0104] A person skilled in art can use known characterised gene
sequences as templates in a Blast search to find out other
characterised or putative gene sequences. Another possibility is to
search specific gene sequences by enzymes names from databases
containing genomic sequences of species from different genome
sequencing projects. These kinds of databases are found for
example, but not excluding, in Broad Institute's Fungal Genome
Initiative sequence projects. In the searches of specific gene
sequences species that are closely related to the yeast and
filamentous fungus species of interest are preferred. After a set
of specific gene sequences has been found nucleotide sequence
alignments are carried out with appropriate programs e.g. Clustal
W, resulting in a consensus sequence, which is used in designing
degenerative primers. Designed degenerative primers are used in a
PCR reaction with DNA of the yeast or filamentous fungus strain of
interest as a template. Resulting PCR fragments are gel isolated
and sequenced directly or after being cloned into plasmids.
Detected sequences are used in Blast searches to confirm that right
gene fragments have been cloned.
[0105] An unknown promoter and/or terminator region of a gene of
interest can be cloned by a chromosome walking method. The term
"chromosome walking" refers to sequential isolation of clones
carrying overlapping sequences of a known gene region and an
unknown sequence of an adjacent gene region produced by
ligation-mediated PCR amplification method (Mueller and Wold 1989).
Gene specific oligonucleotides corresponding to a known sequence of
a gene of interest are designed and used in PCR reactions with
linker specific oligonucleotides. The known sequence of the gene of
interest may originate e.g. from a sequence of a gene fragment
generated in a PCR reaction with degenerative oligos or from a gene
sequence published in sequence databases. The resulting PCR
fragments are gel isolated and sequenced directly or after being
cloned into plasmids. Detected sequences are used in Blast searches
to confirm that right gene fragments have been cloned. If needed,
the chromosome walking experiment will be repeated so many times
that desired length of promoter or terminator region has been
cloned. The existence of the sequence of the promoter or terminator
region of the desired gene is confirmed by usual bioinformatics
methods e.g. with multiple sequence alignment.
[0106] A strain specific gene fragment containing the promoter
and/or terminator region of the gene of interest can also be cloned
by conventional library screening methods described e.g. in
Sambrook and Russell (2001). The sequences of the oligonucleotides
used in PCR reactions to clone desired promoter or terminator
regions can also contain sequences of restriction sites of specific
restriction enzymes in addition to the gene specific sequence. The
PCR fragment containing the desired promoter or terminator will be
cloned into plasmid e.g. pBluescript and sequenced.
[0107] Promoters used in expression cassettes can be promoters of
constitutively expressed genes e.g. of 3-phosphoglycerate
dehydrogenase (PGK), triose phosphate isomerase (TPI) or enolase
(ENO). Alternatively, the promoter used in the expression cassette
can be a promoter of a gene, which is expressed under specific
cultivation conditions.
[0108] "Expression cassette" as used herein refers to a nucleic
acid construct that comprises a transcription initiation or
transcription control sequence, e.g. a promotor, operably linked to
a coding region for the protein to be transcribed, and preferably a
transcription termination region. In addition it conveniently
comprises one or more marker regions, i.e. regions encoding a
selection marker.
[0109] Genes to be expressed can be cloned directly from the
desired species or strains by conventional molecular biology
methods e.g. by using PCR. More preferably the genes to be
expressed are synthesised using a codon optimised nucleotide
sequence based on the known codon usage of the host strain. If the
codon usage of the host strain or host species is not known, the
gene to be expressed can be codon optimised based on the known
codon usage of a closely related species of the host strain.
Alternatively, the gene to be expressed can be synthesised
according to a known amino acid sequence by translating the amino
acid sequence into a DNA sequence, preferably into a
codon-optimised DNA sequence. The term "codon optimization" refers
to an optimization of a synthetic nucleotide sequence encoding
expressed genes to enhance gene product production in a host
strain. Codon optimization can occur by replacing existing codons
of the original gene by the codons used more often in the host
strain. In codon optimisation also internal TATA-boxes, chi-sites,
ribosomal entry sites, AT-rich or GC-rich sequence stretches,
repeated sequences, RNA secondary structures and cryptic splice
donor and acceptor sites can be avoided. Additionally in codon
optimisation sequences of restriction sites of specific restriction
enzymes can be avoided. Preferably, in a synthesised gene sequence
the CTG codon will be avoided due to its different coding in
different fungi (leucine or serine).
[0110] A nucleic acid that is "degenerate as a result of the
genetic code" to a given sequence, means that it contains one or
more different codons, but encodes for the same amino acids. A
"polynucleotide" as used herein may be a single or double stranded
polynucleic acid. The term encompasses genome DNA, cDNA and
RNA.
[0111] A "synthetic gene" or "synthetic nucleotide sequence" is an
artificially designed gene or sequence, which has been synthesised
into a physical DNA sequence.
[0112] The gene to be expressed is cloned between a promoter and
terminator. An expression cassette of the gene to be expressed with
promoter and terminator, and a marker gene can be transformed. The
marker gene can be under its own promoter and terminator, but
preferably it is under a functional promoter and terminator from
another species, or more preferably under a functional promoter and
terminator of the host strain. Markers to be used can be antibiotic
markers like genes for hygromycin, geneticin and cerulenin
resistances or other dominant marker like the melibiase gene.
Additionally genes of the amino acid synthesis can be used as
markers with auxotrophic fungi. The gene to be expressed can be
transformed into the yeast or filamentous fungus as a plasmid to
produce epitopic transformants, or as a DNA fragment containing the
expression cassette to produce transformants with the expression
cassette integrated into the genome of the host strain.
[0113] Expression cassettes containing the marker gene and gene to
be expressed can be transformed in the same DNA fragment, or the
expression cassettes of marker gene and gene to be expressed can be
in separate DNA fragments. Transformation methods contain chemical,
protoplast, electroporation methods. Transformants can be selected
based on their ability to grow on a medium (solid or liquid)
containing antibiotics, or a medium lacking some essential
component e.g. an amino acid, or transformants can be selected
based on different phenotype such as colour reaction of the
transformants in specific conditions.
[0114] The DNA level of the transformants can be characterised by
PCR or by Southern analysis using conventional molecular biology
methods. Additionally enzyme activities of the expressed gene can
be assayed as indicated in the example 27 or as described e.g. in
Dahlqvist et al. 2000 and Postma et al. 1989,
[0115] The gene to be expressed can be an endogenous gene, whereby
the promoter region can be replaced with a promoter of a
constitutively expressed gene, or with a promoter of a gene, which
is expressed under specific cultivation conditions. Additionally,
expression of an endogenous gene can be enhanced by classical
mutagenesis.
[0116] The genetically modified fungi of the present invention are
capable of producing increased levels of lipids, and especially of
triacylglycerols. The increase may be at least a 1.5, 3, 5 or 10
fold increase in lipid or triacylglycerol concentration in
transformants compared to the unmodified host strain during
cultivation. Alternatively, it may be at least a 1.5, 3, 5 or 10
fold increase in lipid or triacylglycerol yield per used carbon
source (e.g. glucose) in transformants compared to the unmodified
host strain. It may also refer to a 1.5, 3, 5 or 10 fold increase
in lipid or triacylglycerol production rate (mg/l/h) compared to
the unmodified host strain. This increase in lipid or
triacylglycerol production can be detected either intracellularly
or in the amount of lipids and triacylglycerols in culture
medium.
[0117] The genetically modified fungi are cultivated in a medium
containing appropriate carbon and nitrogen sources together with
other optional ingredients like yeast extract, peptone, minerals
and vitamins, such as KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4,
MgSO.sub.4, CaCl.sub.2, FeCl.sub.3, ZnSO.sub.4, citric acid,
MnSO.sub.4, CoCl.sub.2, CuSO.sub.4, Na.sub.2MoO.sub.4, FeSO.sub.4,
H.sub.3BO.sub.4, D-biotin, CaPantothenate, nicotinic acid,
myoinositol, thiamine, pyridoxine, p-aminobenzoic acid. The
invention works at a wide range of C/N ratios about from 20 to 160
under microaerobic (50 ml medium in 250 ml flask with 100 rpm
shaking) and aerobic (50 ml medium in 250 ml flask with 250 rpm
shaking or 1-2 vvm in bioreactors) conditions from the beginning of
the cultivation to the end of cultivation as far as up to at least
8 days. The host cells used are preferably such that are able not
only to use hexoses, such as glucose, but also pentoses such as
xylose, and arabinose, or even glycerol as carbon source.
Preferably the carbon source is a hexose and/or pentose sugars
containing material such as cellulose or hemicellulose. The
genetically modified host cells are preferably grown on
agricultural or industrial waste materials e.g. cellulose or
hemicellulose containing materials, which makes the lipid
production economically and environmentally beneficial.
[0118] After cultivation, the yeast or filamentous fungal cells are
normally separated from the culture medium. Lipids are recovered
from the cells typically by using non-polar organic solvents, such
as hexane. Prior to extraction, cells can be dried and/or
disrupted. Alternatively, the lipids can be recovered directly from
the culture medium, which of course is an advantage. This is
especially convenient when the host cell is Cryptococcus, such as
C. curvatus. Preferably lipid extraction is carried out to obtain
lipids which mainly contain triacylglycerol (TAG). The lipid
fraction can also contain mono- and diglycerides, free fatty acids,
phospholipids, glycolipids and other lipids.
[0119] The lipids, and especially the TAG and the fatty acids,
obtained can be used for preparing biofuels and lubricants. Said
lipids may be directly used as biofuel or lubricant, but usually
they are further processed to e.g. biodiesel or renewable diesel
and/or lubricant formulations. "Biofuel" as used herein refers to
fuel that has been at least partially biologically produced.
[0120] "Biodiesel" consists essentially of fatty acid methyl esters
and is typically produced by transesterification in which the
acylglycerides are converted to fatty acid methyl esters. According
to EU directive 2003/30/EU biodiesel refers to a methyl-ester
produced from vegetable oil or animal oil, of diesel quality to be
used as biofuel. More broadly, biodiesel refers to long-chain alkyl
esters, such as methyl, ethyl or propyl esters, from vegetable oil
or animal fat of diesel quality. In the present content biodiesel
can also be produced from fungal lipids.
[0121] "Renewable diesel" refers to fuel which is produced by
hydrogen treatment (hydrogenation or hydroprocessing) of lipids of
animal, vegetable or fungal origin, or their mixtures. Renewable
diesel can be produced also from waxes derived from biomass by
gasification and Fischer-Tropsch synthesis. Optionally, in addition
to hydrogen treatment, isomerization or other processing steps can
be performed. In hydrogen treatment, acylglycerides are converted
to corresponding alkanes (paraffins). The alkanes (paraffins) can
be further modified by isomerization or by other process
alternatives. These processes can also produce hydrocarbons which
are suitable for jet fuel or gasoline applications.
[0122] "Lubricant" refers to a substance, such as grease, lipid or
oil that reduces friction when applied as a surface coating to
moving parts. Two other main functions of a lubricant are heat
removal and dissolving impurities. Applications of lubricants
include, but are not limited to uses in internal combustion engines
as engine oils, additives in fuels, in oil-driven devices such as
pumps and hydraulic equipment, or in different types of bearings.
Typically lubricants contain 75-100% base oil and the rest is
additives. In the present invention at least part of the base oil
is of fungal origin. Viscosity index is used to characterise base
oil. Typically high viscosity index is preferred.
[0123] Lubricants or at least the base oil for lubricants may be
prepared in the same way as described above for the biofuels.
Usually conventional additives such as viscosity modifyers,
antioxidants, pour point modifyers etc. are added to the base oil
to obtain the lubricant.
[0124] The lipids, and especially the TAG and the fatty acids
obtained can also be used for precursors for functional fatty
acids. Said lipids are further processed to release fatty acyls
which can be used in the production of functional fatty acids, like
dicarboxylic acids and epoxides, which can be further converted
into products like polyesters, polyurethane, coatings and
resins.
[0125] The following examples are provided to illustrate the
invention, but are not intended to limit the scope thereof. The
enzyme names used are based on sequence homology.
Example 1A: Cloning of Cryptococcus curvatus TEF (CcTEF1) Promoter
and Terminator Region
[0126] A genomic .about.800 bp fragment of the C. curvatus TEF gene
was amplified by PCR with degenerative primers identified as SEQ ID
NO:1 (Yeast TEF1), and SEQ ID NO:2 (Yeast TEF4), using C. curvatus
(C-01440, VTT Culture Collection) genomic DNA as the template. The
degenerative primers were designed based on a consensus sequence of
the putative TEF1 genes of Ustilago maydis, Candida guilliermondii
and Candida tropicalis. The detected genomic fragment was
sequenced.
[0127] Genomic fragments containing the CcTEF1 promoter region were
obtained with ligation-mediated PCR amplification (Mueller, P. R.
and Wold, B. 1989). A mixture of a linker identified as SEQ ID NO:3
(PCR linker I), and a linker identified as SEQ ID NO:4 (PCR linker
II) was ligated to PvuII digested C. curvatus genomic DNA with T4
DNA ligase (New England BioLabs). Samples of the ligation mixtures
were used as templates for 50 .mu.l PCR reactions containing 0.1
.mu.M of a primer identified as SEQ ID NO: 3 (PCR linker I), and 1
.mu.M of a primer identified as SEQ ID NO:5 (CC_TEF2). The reaction
mixture was heated at 94.degree. C. for 3 minutes after 2 U of
Dynazyme EXT was added. The reactions were cycled 30 times as
follows: 1 minute at 94.degree. C., 2 minutes at 60.degree. C. and
2 minutes at 72.degree. C., with final extension of 10 minutes at
72.degree. C. A diluted sample of this first PCR-amplification was
used as the template in a nested PCR reaction (50 .mu.l) containing
0.05 .mu.M of a primer identified as SEQ ID NO:3 (PCR Linker I),
and 0.5 .mu.M of a primer identified as SEQ ID NO:6 (CC_TEF1). The
reaction mixture was heated at 94.degree. C. for 3 minutes after 2
U of Dynazyme EXT was added. The reactions were then cycled 30
times as follows: 1 minute at 94.degree. C., 2 minutes at
60.degree. C. and 2 minutes at 72.degree. C., with final extension
of 10 minutes at 72.degree. C.
[0128] A .about.800 bp fragment was isolated and sequenced. Nested
primers identified as SEQ ID NO:7 (CC_TEF6), and SEQ ID NO:8
(CC_TEF5) were designed and used in a ligation-mediated PCR
amplification together with oligonucleotides identified as SEQ ID
NO:3 (PCR linker I), and a linker identified as SEQ ID NO:4 (PCR
linker II) similarly as above except that NruI-digested C. curvatus
DNA was used. A .about.2000 bp PCR fragment was isolated and
sequenced.
[0129] The C. curvatus TEF1 promoter was PCR amplified using
primers identified as SEQ ID NO:9 (CC_TEF10), and SEQ ID NO:10
(CC_TEF11) and the C. curvatus genomic DNA as the template. A PCR
fragment was digested with SacII and XbaI. A 1276 bp fragment was
gel isolated and ligated to a SacII and XbaI-digested pBluescript
KS-plasmid (Stratagene). The resulting plasmid was designated
pKK58. Plasmid pKK58 contains C. curvatus TEF1 promoter.
[0130] A genomic fragment containing the CcTEF1 terminator region
was obtained with a ligation-mediated PCR amplification with C.
curvatus TEF1 gene specific oligonucleotides identified as SEQ ID
NO:11 (CC_TEF3), and SEQ ID NO:12 (CC_TEF4) together with
oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ
ID NO:4 (PCR Linker II) similarly as above except that
NruI-digested C. curvatus DNA was used. A .about.1600 bp PCR
fragment was isolated and sequenced.
[0131] The C. curvatus TEF1 terminator was PCR amplified using
primers identified as SEQ ID NO:13 (CC_TEF7) and SEQ ID NO:14 and
the C. curvatus genomic DNA as the template. A PCR fragment was
digested with XmaI and EcoRI. A 358 bp fragment was gel isolated
and ligated to XmaI and EcoRI-digested pBluescript KS-plasmid. The
resulting plasmid was designated pKK55. Plasmid pKK55 contains the
C. curvatus TEF1 terminator.
Example 1B: Cloning of Cryptococcus curvatus TPI (CcTPI1) Promoter
and Terminator Region
[0132] A genomic fragment of the C. curvatus TPI1 gene was
amplified by PCR from genomic C. curvatus (C-01440, VTT Culture
Collection) DNA with degenerative primers identified as SEQ ID
NO:15 (Yeast TPI5), and SEQ ID NO:16 (Yeast TPI8). The degenerative
primers were designed based on a consensus sequence of the TPI1
genes of Ustilago maydis and Cryptococcus neoformans. A .about.800
bp genomic fragment was isolated and sequenced.
[0133] A genomic fragment containing the CcTPI1 promoter region was
obtained with a ligation-mediated PCR amplification with TPI1 gene
specific oligonucleotides identified as SEQ ID NO:17 (CC_TPI2), and
SEQ ID NO:18 (CC_TPI1), together with oligonucleotides identified
as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II),
similarly as in Example 1A except that EcoRV-digested C. curvatus
DNA was used. A .about.1300 bp PCR fragment was isolated and
sequenced.
[0134] The C. curvatus TPI1 promoter was PCR amplified by using
primers identified as SEQ ID NO:19 (CC_TPI7) and SEQ ID NO:20
(CC_TPI_9) and the C. curvatus DNA as the template. A PCR fragment
was digested with BamHI and SbfI. A 851 bp fragment was gel
isolated and ligated to a BamHI and PstI-digested pBluescript
KS-plasmid. The resulting plasmid was designated pKK63. Plasmid
pKK63 contains the C. curvatus TPI1 promoter.
[0135] A genomic fragment containing the CcTPI1 terminator region
was obtained with a ligation-mediated PCR amplification with TPI1
gene specific oligonucleotides identified as SEQ ID NO:21 (CC_TPI
4) and SEQ ID NO:22 (CC_TPI3), together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as in Example 1A except that EcoRV-digested
C. curvatus DNA was used. A .about.1200 bp PCR fragment was
isolated and sequenced.
[0136] The C. curvatus TPI1 terminator was PCR amplified by using
primers identified as SEQ ID NO:23 (CC_TPI5) and SEQ ID NO:24
(CC_TPI6) and the C. curvatus genomic DNA as the template. A PCR
fragment was digested with XbaI and BamHI. A 361 bp fragment was
gel isolated and ligated to a XbaI and BamHI-digested pBluescript
KS-plasmid. The resulting plasmid was designated pKK61. Plasmid
pKK61 contains C. curvatus TPI1 terminator.
Example 1C: Cloning of Cryptococcus curvatus ENO (CcENO1) Promoter
and Terminator Region
[0137] A genomic fragment of the C. curvatus ENO1 gene was
amplified by PCR from genomic C. curvatus (C-01440, VTT Culture
Collection) DNA with degenerative primers identified as SEQ ID
NO:25 (YeastENO5) and SEQ ID NO:26 (YeastENO10). The degenerative
primers were designed based on a consensus sequence of ENO1 genes
of Ustilago maydis and Cryptococcus neoformans. A .about.1000 bp
genomic fragment was isolated and sequenced.
[0138] A genomic fragment containing the CcENO1 promoter region was
obtained with a ligation-mediated PCR amplification with ENO1 gene
specific oligonucleotides identified as SEQ ID NO:27 (CC_ENO2) and
SEQ ID NO:28 (CC_ENO1), together with oligonucleotides identified
as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II),
similarly as in Example 1A except that PvuII-digested C. curvatus
DNA was used.
[0139] A .about.600 bp fragment was isolated and sequenced. Nested
primers identified as SEQ ID NO:29 (CC_ENO5) and SEQ ID NO:30
(CC_ENO6) were designed and used in a ligation-mediated PCR
amplification together with oligonucleotides identified as SEQ ID
NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II) similarly as
above except that SspI-digested C. curvatus DNA was used. A 2000 bp
PCR fragment was isolated and sequenced.
[0140] The C. curvatus ENO1 promoter was PCR amplified by using
primers identified as SEQ ID NO:31 (CC_ENO9) and SEQ ID NO:32
(CC_ENO10) and the C. curvatus genomic DNA as the template. A PCR
fragment was digested with EcoRI. A 1214 bp fragment was gel
isolated and ligated to a EcoRI-digested pBluescript KS-plasmid.
The resulting plasmid was designated pKK74. Plasmid pKK74 contains
the C. curvatus ENO1 promoter.
[0141] A genomic fragment containing the CcENO1 terminator region
was obtained with a ligation-mediated PCR amplification with ENO1
gene specific oligonucleotides identified as SEQ ID NO:33 (CC_ENO4)
and SEQ ID NO:34 (CC_ENO3), together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as in Example 1A except that NruI-digested C.
curvatus DNA was used. A .about.1100 bp PCR fragment was isolated
and sequenced.
[0142] The C. curvatus ENO1 terminator was PCR amplified by using
primers identified as SEQ ID NO:35 (CC_ENO7) and SEQ ID NO:36
(CC_ENO8), and the C. curvatus genomic DNA as the template. A PCR
fragment was digested with HindIII. A 375 bp fragment was gel
isolated and ligated to a HindIII-digested pBluescript KS-plasmid.
The resulting plasmid was designated pKK60. Plasmid pKK60 contains
the C. curvatus ENO1 terminator.
Example 1D: Cloning of C. curvatus GPD (CcGPD1) Terminator
Region
[0143] A genomic fragment containing the CcGPD1 terminator region
was obtained with a ligation-mediated PCR amplification with GPD1
gene specific oligonucleotides identified as SEQ ID NO:37 (CC_GPD3)
and SEQ ID NO:38 (CC_GPD4), together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as in Example 1A except that SspI-digested C.
curvatus DNA was used. A .about.1800 bp fragment was isolated and
partially sequenced. GPD1 gene specific oligonucleotides were
designed according to the C. curvatus GPD1 gene (GenBank Accession
number AF126158, version number AF126158.1) sequence.
[0144] The C. curvatus GPD1 terminator was PCR amplified by using
primers identified as SEQ ID NO:39 (CC_GPD6) and SEQ ID NO:40
(CC_GPD7), and the C. curvatus genomic DNA as the template. A PCR
fragment was digested with XbaI and BamHI. A 336 bp fragment was
gel isolated and ligated to a XbaI and BamHI-digested pBluescript
KS-plasmid. The resulting plasmid was designated pKK54. Plasmid
pKK54 contains C. curvatus GPD1 terminator.
Example 2A: Cloning of the E. coli Hygromycin Resistance Gene;
Construction of a Plasmid (pKK76) Having the E. Con Hygromycin
Resistance Gene Under the Control of the CcTEF1 Promoter and the
CcTPI1 Terminator
[0145] The E. coli hygromycin (hph) gene, that confers resistance
to hygromycin B, was PCR amplified using primers identified as SEQ
ID NO:41 (Hph 5) and SEQ ID NO:42 (Hph 3), and the plasmid pRLMEX30
(Mach et al. 1994) DNA as the template. A PCR fragment was digested
with SpeI. A 1048 bp fragment was gel isolated and ligated to
SpeI-digested pBluescript KS-plasmid and sequenced. The resulting
plasmid was designated pKK52.
[0146] Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp
fragment was gel isolated. Plasmid pKK52 was digested with SbfI. A
1034 bp fragment was gel isolated. Plasmid pKK58 contains the C.
curvatus TEF1 promoter and plasmid pKK61 contains the C. curvatus
TPI1 terminator. The 1272 bp fragment originating from plasmid
pKK58 and the 1034 bp fragment originating from the pKK52 plasmid
were ligated to a 3285 bp fragment obtained by digesting a plasmid
designated as pKK61 with SacII and SbfI. The resulting plasmid was
designated pKK76. Plasmid pKK76 contains the E. coli hygromycin
gene under the control of the C. curvatus TEF1 promoter and the C.
curvatus TPI1 terminator.
Example 2B: Cloning of the E. coli G418 Resistance Gene;
Construction of a Plasmid (pKK67) Having the E. coli Geneticin
Resistance Gene Under the Control of the CcTEF1 Promoter and the
CcTPI1 Terminator
[0147] The E. coli G418 resistance gene was PCR amplified using
primers identified as SEQ ID NO:43 (Kan 5) and SEQ ID NO:44 (Kan
3), and the plasmid pPIC9K (Invitrogen) DNA as the template. A PCR
fragment was digested with SpeI. A 838 bp fragment was gel isolated
and ligated to SpeI-digested pBluescript KS-plasmid and sequenced.
The resulting plasmid was designated pKK51.
[0148] Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp
fragment was gel isolated. Plasmid pKK51 was digested with SbfI. A
824 bp fragment was gel isolated. The 1272 bp fragment originating
from pKK58 plasmid and the 824 bp fragment originating from pKK51
plasmid were ligated to a 3285 bp fragment obtained by digesting a
plasmid designated pKK61 with SacII and SbfI. Plasmid pKK58
contains the C. curvatus TEF1 promoter and plasmid pKK61 contains
the C. curvatus TPI1 terminator. The resulting plasmid was
designated pKK67. Plasmid pKK67 contains the E. coli G418
resistance gene under the control of the C. curvatus TEF1 promoter
and the C. curvatus TPI1 terminator.
Example 2C: Cloning of S. cerevisiae Cerulenin Resistance Gene;
Construction of a Plasmid (pKK91) Having the S. cerevisiae
Cerulenin Resistance Gene Under the Control of the CcTEF1 Promoter
and the CcTPI1 Terminator
[0149] The S. cerevisiae cerulenin resistance gene was PCR
amplified using primers identified as SEQ ID NO:45 (CERR 5) and SEQ
ID NO:46 (CERR 3), and the plasmid pSH47Y DNA as the template.
Plasmid pSH47Y contains the cerulenin resistance gene from the
plasmid pCR1 (Nakazawa et al. 1993). A PCR fragment was digested
with SbfI. A 1685 bp fragment was gel isolated and ligated to
PstI-digested pBluescript KS-plasmid and sequenced. The resulting
plasmid was designated pKK80.
[0150] Plasmid pKK80 was digested with PstI. A 1685 bp fragment was
gel isolated and ligated to a 4557 bp fragment obtained by
digesting a plasmid designated as pKK67 with SbfI. Plasmid pKK67
contains the E. coli G418 resistance gene linked to a C. curvatus
TEF1 promoter and C. curvatus TEF1 terminator. The resulting
plasmid was designated pKK91. Plasmid pKK91 contains the S.
cerevisiae cerulenin resistance gene under the control of the C.
curvatus TEF1 promoter and the C. curvatus TPI1 terminator.
Example 3A. Construction of a Plasmid (pKK81, FIG. 3) Containing
the Hygromycin Resistance Gene Under the Control of the CcTEF1
Promoter and the CcTPI1 Terminator and the S. cerevisiae ALD6
Encoding Gene Under the Control of the CcENO1 Promoter and the
CcTEF1 Terminator
[0151] The plasmid UmALD (Geneart AG, Germany) contains a S.
cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codon
optimized according to Ustilago maydis yeast codon usage (SEQ ID
NO:48) with flanking SbfI restriction sites. The plasmid RoALD
(Geneart AG, Germany) contains a S. cerevisiae ALD6 (SEQ ID NO:47)
encoding gene which has been codon optimized according to Rhizopus
oryzae filamentous fungus codon usage (SEQ ID NO:49) with flanking
SbfI restriction sites and an E. coli kanamycin resistance gene.
Plasmid UmALD was digested with SfiI. A 1554 bp fragment was gel
isolated and ligated to a 2258 bp fragment obtained by digesting a
plasmid RoALD with SfiI. The resulting plasmid was designated as
pKK50. The plasmid pKK50 contains a S. cerevisiae ALD6 (SEQ ID
NO:47) encoding gene which has been codon optimized according to
Ustilago maydis yeast codon usage (SEQ ID NO:48) with flanking SbfI
restriction sites and an E. coli kanamycin resistance gene.
[0152] Plasmid pKK74 was digested with EcoRI and SbfI. A 1204 bp
fragment was gel isolated. Plasmid pKK55 was digested with EcoRI
and SbfI. A 347 bp fragment was gel isolated. The 1204 bp fragment
originating from plasmid pKK74 and the 347 bp fragment originating
from plasmid pKK55 were ligated to a 2961 bp fragment obtained by
digesting a plasmid designated pKK74 with EcoRI. Plasmid pKK74
contains C. curvatus ENO1 promoter and plasmid pKK55 contains C.
curvatus TEF1 terminator. The resulting plasmid was designated as
pKK77pre.
[0153] Plasmid pKK50 was digested with SbfI. A 1514 bp fragment was
gel isolated and ligated to a 4517 bp fragment obtained by
digesting plasmid pKK77pre with SbfI. The resulting plasmid was
designated as pKK77. The plasmid pKK77 contains the S. cerevisiae
ALD6 encoding gene under the control of the CcENO1 promoter and the
CcTEF1 terminator.
[0154] Plasmid pKK76 was digested with BamHI and XmnI. A 2652 bp
fragment was gel isolated and ligated to a 6031 bp fragment
obtained by digesting plasmid pKK77 with BamHI. The plasmid pKK76
contains the E. coli hygromycin resistance gene under the control
of the CcTEF1 promoter and the CcTPI1 terminator. The resulting
plasmid was designated as pKK81 (FIG. 3).
Example 3B. Generation of a Genetically Modified C. curvatus
(Y23/81) with an Integrated ALD6 Encoding Gene and Hygromycin
Resistance Gene by Transforming Wild-Type C. curvatus with Digested
Plasmid pKK81 (FIG. 3, Ex. 3A)
[0155] Plasmid pKK81 was restricted with NotI and PspOMI, and the
resulting linear DNA was used to transform a wild-type C. curvatus
strain ATCC 20509 designated as Y23 by electroporation using a
standard electroporation method.
[0156] The transformed cells were screened for hygromycin
resistance. Several hygromycin-resistant colonies were analysed at
DNA level by PCR. The transformants originating from the
transformation of C. curvatus with NotI and PspOMI cut pKK81 and
containing a S. cerevisiae ALD6 encoding gene under the control of
the CcENO1 promoter and the CcTEF1 terminator were designated as
Y23/81-8, Y23/81-51, Y23/81-59, Y23/81-66 and Y23/81-69.
Example 4A. Construction of a Plasmid (pKK82, FIG. 4) Containing
the G418 Resistance Gene Under the Control of the CcTEF1 Promoter
and the CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene
Under the Control of the CcENO1 Promoter and the CcTEF1
Terminator
[0157] Plasmid pKK67 was digested with BamHI and XmnI. A 2442 bp
fragment was gel isolated and ligated to a 6031 bp fragment
obtained by digesting a plasmid pKK77 with BamHI. The plasmid pKK67
contains the E. coli G418 resistance gene under the control of the
CcTEF1 promoter and the CcTPI1 terminator and the plasmid pKK77
contains S. cerevisiae ALD6 encoding gene under the control of the
CcENO1 promoter and the CcTEF1 terminator. The resulting plasmid
was designated as pKK82 (FIG. 4).
Example 4B. Generation of a Genetically Modified C. curvatus
(Y23/82) with an Integrated ALD6 Encoding Gene and G418 Resistance
Gene by Transforming Wild-type C. curvatus with Digested Plasmid
pKK82 (FIG. 4, Ex. 4A)
[0158] Plasmid pKK82 was restricted with NotI and PspOMI, and the
resulting linear DNA was used to transform wild-type C. curvatus
strain ATCC 20509 designated as Y23 by electroporation. The
transformed cells were screened for G418 resistance. Several
G418-resistant colonies were analysed at DNA level by PCR. The
transformants originating from the transformation of C. curvatus
with NotI and PspOM1 cut pKK82 and containing S. cerevisiae ALD6
encoding gene under the control of the CcENO1 promoter and the
CcTEF1 terminator were designated as Y23/82-1, Y23/82-2, Y23/82-4
and Y23/82-13.
Example 5A. Construction of a Plasmid (pKK86, FIG. 5) Containing
the Hygromycin Resistance Gene Under the Control of the CcTEF1
Promoter and the CcTPI1 Terminator and the S. cerevisiae ACS2
Encoding Gene Under the Control of the CcTPI1 Promoter and the
CcENO1 Terminator
[0159] Plasmid pKK63 was digested with BamHI and SbfI. A 851 bp
fragment was gel isolated and ligated to a 3295 bp fragment
obtained by digesting plasmid pKK60 with Ban-II and SbfI. The
plasmid pKK63 contains the CcTPI1 promoter and the plasmid pKK60
contains the CcENO1 terminator. The resulting plasmid was
designated as pKK78pre.
[0160] The plasmid UmACS contains the S. cerevisiae ACS2 (SEQ ID
NO:50) encoding gene which has been codon optimized according to
Ustilago maydis yeast codon usage (SEQ ID NO:51) with flanking SbfI
restriction sites. Plasmid UmACS was digested with SbfI and DraI. A
2060 bp fragment was gel isolated and ligated to a 4146 bp fragment
obtained by digesting plasmid pKK78pre with SbfI. The resulting
plasmid was designated as pKK78.
[0161] Plasmid pKK76 was digested with BamHI and DraI. A 2652 bp
fragment was gel isolated and ligated to a 6206 bp fragment
obtained by digesting plasmid pKK78 with BamHI. The plasmid pKK76
contains the E. coli hygromycin resistance gene under the control
of the CcTEF1 promoter and the CcTPI1 terminator. The resulting
plasmid was designated as pKK86 (FIG. 5).
Example 5B. Generation of a Genetically Modified C. curvatus
(Y23/86) with an Integrated ACS2 Encoding Gene and Hygromycin
Resistance Gene by Transforming Wild-Type C. curvatus with Digested
Plasmid pKK86 (FIG. 5, Ex. 5A)
[0162] Plasmid pKK86 was restricted with NotI and PspOMI, and the
resulting linear DNA was used to transform a wild-type C. curvatus
strain ATCC 20509 designated as Y23 by electroporation. The
transformed cells were screened for hygromycin resistance. Several
hygromycin-resistant colonies were analysed at DNA level by PCR.
The transformants originating from the transformation of C.
curvatus with Moil and PspOMI cut pKK86 and containing S.
cerevisiae ACS2 encoding gene under the control of the CcTPI1
promoter and the CcENO1 terminator were designated as Y23/86-86,
Y23/86-92, Y23/86-93, Y23/86-98 and Y23/86-100.
Example 6A. Construction of a Plasmid (pKK95, FIG. 6) Containing
the Cerulenin Resistance Gene Under the Control of the CcTEF1
Promoter and the CcTPI1 Terminator and the R. oryzae PDAT Encoding
Gene Under the Control of the CcTPI1 Promoter and the CcGPD1
Terminator
[0163] Plasmid pKK54 was digested with KpnI and SbfI. A 394 bp
fragment was gel isolated and ligated to a 3742 bp fragment
obtained by digesting plasmid designated as pKK63 with KpnI and SOL
The plasmid pKK54 contains the CcGPD1 terminator and the plasmid
pKK63 contains the CcTPI1 promoter. The resulting plasmid was
designated as pKK93pre. Plasmid UmPDAT was digested with SbfI. A
1844 bp fragment was gel isolated and ligated to a 4136 bp fragment
obtained by digesting plasmid pKK93pre with SbfI. The plasmid
UmPDAT contains a R. oryzae PDAT (SEQ ID NO:52) encoding gene which
has been codon optimized according to U. maydis yeast codon usage
(SEQ ID NO:53) with flanking SbfI restriction sites. The resulting
plasmid was designated as pKK93.
[0164] The plasmid pKK93 was digested with EcoRI and DraI. A 3029
bp fragment was gel isolated and ligated to a 6242 bp fragment
obtained by digesting plasmid pKK91 with EcoRI. The plasmid pKK91
contains the S. cerevisiae cerulenin resistance gene under the
control of the CcTEF1 promoter and the CcTPI1 terminator. The
resulting plasmid was designated as pKK95 (FIG. 6).
Example 6B. Generation of a Genetically Modified C. curvatus
(Y23/95) with an Integrated PDAT Encoding Gene and Cerulenin
Resistance Gene by Transforming Wild-Type C. curvatus with Digested
Plasmid pKK95 (FIG. 6, Ex. 6A)
[0165] Plasmid pKK95 was restricted with EcoRV, and the resulting
linear DNA was used to transform a wild-type C. curvatus strain
ATCC 20509 designated as Y23 by electroporation. The transformed
cells were screened for cerulenin resistance. Several
cerulenin-resistant colonies were analysed at DNA level by PCR. The
transformants originating from the transformation of C. curvatus
with EcoRV cut pKK95 and containing the R. oryzae PDAT encoding
gene under the control of the CcTPI1 promoter and the CcGPD1
terminator were designated as Y23/95-87, Y23/95-98, Y23/95-99,
Y23/95-104 and Y23/95-109.
Example 7A. Construction of a Plasmid (pKK85, FIG. 7) Containing
the Hygromycin Resistance Gene Under the Control of the CcTEF1
Promoter and the CcTPI1 Terminator and the S. cerevisiae ALD6
Encoding Gene Under the Control of the CcENO1 Promoter and the
CcTEF1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under
the Control of the CcTPI1 Promoter and the CcENO1 Terminator
[0166] Plasmid pKK77 was digested with EcoRI and XmnI. A 3073 bp
fragment was gel isolated and ligated to a 6206 bp fragment
obtained by digesting plasmid pKK78 with EcoRI. The plasmid pKK77
contains the S. cerevisiae ALD6 encoding gene under the control of
the CcENO1 promoter and the CcTEF1 terminator and the plasmid pKK78
contains the S. cerevisiae ACS2 encoding gene under the control of
the CcTPI1 promoter and the CcENO1 terminator. The resulting
plasmid was designated as pKK84. The plasmid pKK76 was digested
with BamHI and XmnI. A 2652 bp fragment was gel isolated and
ligated to a 9279 bp fragment obtained by digesting pKK84 with
BamHI. The plasmid pKK76 contains the hygromycin resistance gene
under the control of the CcTEF1 promoter and the CcTPI1 terminator.
The resulting plasmid was designated as pKK85 (FIG. 7).
Example 7B. Generation of a Genetically Modified C. curvatus
(Y23/85) with an Integrated ALD6 Encoding and ACS2 Encoding Genes
and Hygromycin Resistance Gene by Transforming Wild-Type C.
curvatus with Digested Plasmid pKK85 (FIG. 7, Ex. 7A)
[0167] Plasmid pKK85 was restricted with NotI and PspOMI, and the
resulting linear DNA was used to transform wild-type C. curvatus
strain ATCC 20509 designated as Y23 by electroporation. The
transformed cells were screened for hygromycin resistance. Several
hygromycin-resistant colonies were analysed at DNA level by PCR.
The transformants originating from the transformation of C.
curvatus with NotI and PspOMI cut pKK85 and containing the S.
cerevisiae ALD6 encoding gene under the control of the CcENO1
promoter and the CcTEF1 terminator and the S. cerevisiae ACS2
encoding gene under the control of the CcTPI1 promoter and the
CcENO1 terminator were designated as Y23/85-119, Y23/85-125,
Y23/85-128 Y23/85-129 and Y23/85-139.
Example 8. Generation of a Genetically Modified C. curvatus
(Y23/81/95) with an Integrated ALD6 Encoding and PDAT Encoding
Genes and Hygromycin and Cerulenin Resistance Genes by Transforming
Genetically Modified Strain Y23/81-51 (Ex. 3B) with Plasmid pKK95
(FIG. 6, Ex. 6A)
[0168] Plasmid pKK95 was restricted with EcoRV and the resulting
linear DNA was used to transform a genetically modified strain
Y23/81-51 by electroporation. The transformed cells were screened
for cerulenin and hygromycin resistance. Several cerulenin and
hygromycin resistance colonies were analysed at DNA level by PCR.
The transformants originating from the transformation of
genetically modified strain Y23/81-51 with EcoRV cut pKK95 and
containing the S. cerevisiae ALD6 encoding gene under the control
of the CcENO1 promoter and the CcTEF1 terminator and the R. oryzae
PDAT encoding gene under the control of the CcTPI1 promoter and the
CcGPD1 terminator were designated as Y23/81/95-18 and
Y23/81/95-42.
Example 9. Generation of a Genetically Modified C. curvatus
(Y23/85/95) with an Integrated ALD6, ACS2 and PDAT Encoding Genes
and Hygromycin and Cerulenin Resistance Genes by Transforming
Genetically Modified Strain Y23/85-128 (Ex. 7B) with Plasmid pKK95
(FIG. 6, Ex. 6A)
[0169] Plasmid pKK95 was restricted with EcoRV and the resulting
linear DNA was used to transform a genetically modified strain
Y23/85-128 by electroporation. The transformed cells were screened
for cerulenin and hygromycin resistance. Several cerulenin and
hygromycin resistance colonies were analysed at DNA level by PCR.
The transformants originating from the transformation of
genetically modified strain Y23/85-128 with EcoRV cut pKK95 and
containing the S. cerevisiae ALD6 encoding gene under the control
of the CcENO1 promoter and the CcTEF1 terminator, the S. cerevisiae
ACS2 encoding gene under the control of the CcTPI1 promoter and the
CcENO1 terminator and R. oryzae PDAT encoding gene under the
control of the CcTPI1 promoter and the CcGPD1 terminator were
designated as Y23/85/95-4 and Y23/85/95-68.
Example 10. Cloning of Mucor circinelloides TPI (McTPI1) Promoter
and Terminator Region
[0170] A genomic fragment of the M. circinelloides TPI1 gene was
amplified by PCR from genomic M. circinelloides f. griseocyanus
(D-82202, VTT Culture Collection) DNA with degenerative primers
identified as SEQ ID NO:54 (Mould TPI1) and SEQ ID NO:55 (Mould
TPI3). The degenerative primers were designed based on a consensus
sequence of TPI1 genes of Rhizopus oryzae, Fusarium oxysporum,
Aspergillus fumigatus, A. terreus and A. nidulans. A .about.400 bp
genomic fragment was isolated and sequenced.
[0171] A genomic fragment containing the McTPI1 promoter region was
obtained with a ligation-mediated PCR amplification with TPI1 gene
specific oligonucleotides identified as SEQ ID NO:56 (MC_TPI2) and
SEQ ID NO:57 (MC_TPI1), together with oligonucleotides identified
as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II),
similarly as in Example 1A except that SspI-digested M.
circinelloides DNA was used. A .about.1500 bp PCR fragment was
isolated and sequenced.
[0172] The M. circinelloides TPI1 promoter was PCR amplified by
using primers identified as SEQ ID NO:58 (MC_TPI1) and SEQ ID NO:59
(MC_TPI_8), and the M. circinelloides genomic DNA as the template.
A PCR fragment was digested with PstI and BamHI. A 1251 bp fragment
was gel isolated and ligated to a PstI and BamHI-digested
pBluescript KS-plasmid. The resulting plasmid was designated pKK56.
Plasmid pKK56 contains the M. circinelloides TPI1 promoter.
[0173] A genomic fragment containing the McTPI1 terminator region
was obtained with a ligation-mediated PCR amplification with TPI1
gene specific oligonucleotides identified as SEQ ID NO:60 (MC_TPI4)
and SEQ ID NO:61 (MC_TPI3), together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as in Example 1A except that NruI-digested M.
circinelloides DNA was used. A .about.1500 bp PCR fragment was
isolated and partially sequenced.
[0174] The M. circinelloides TPI1 terminator was PCR amplified by
using primers identified as SEQ ID NO:62 (MC_TPI5) and SEQ ID NO:63
(MC_TPI6), and the M. circinelloides genomic DNA as the template. A
PCR fragment was digested with XbaI and BamHI. A 347 bp fragment
was gel isolated and ligated to a XbaI and BamHI-digested
pBluescript KS-plasmid. The resulting plasmid was designated pKK57.
Plasmid pKK57 contains the M. circinelloides TPI1 terminator.
Example 11. Cloning of Mucor circinelloides TEF (McTEF1) Promoter
and Terminator Region
[0175] A genomic fragment of the M. circinelloides TEF1 gene was
amplified by PCR from genomic M. circinelloides f. griseocyanus
(D-82202, VTT Culture Collection) DNA with degenerative primers
identified as SEQ ID NO:64 (Mould TEF1) and SEQ ID NO:65 (Mould
TEF4). The degenerative primers were designed based on a consensus
sequence of TEF1 genes of Rhizopus oryzae, Fusarium oxysporum,
Aspergillus terreus and A. nidulans. A .about.600 bp genomic
fragment was isolated and sequenced.
[0176] A genomic fragment containing the McTEF1 promoter region was
obtained with a ligation-mediated PCR amplification with TEF1 gene
specific oligonucleotides identified as SEQ ID NO:66 (MC_TEF2) and
SEQ ID NO:67 (MC_TEF1), together with oligonucleotides identified
as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II),
similarly as in Example 1A except that SspI-digested M.
circinelloides DNA was used. A .about.500 bp PCR fragment was
isolated and sequenced. Nested primers identified as SEQ ID NO:68
(MC_TEF6) and SEQ ID NO:69 (MC_TEF5) were designed and used in a
ligation-mediated PCR amplification together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as above except that HaeIII-digested M.
circinelloides DNA was used. A .about.1500 bp PCR fragment was
isolated and sequenced.
[0177] The M. circinelloides TEF1 promoter was PCR amplified by
using primers identified as SEQ ID NO:70 (MC_TEF9) and SEQ ID NO:71
(MC_TEF10), and the M. circinelloides genomic DNA as the template.
A PCR fragment was digested with HindIII and PstI and a 1387 bp
fragment was gel isolated and ligated to a HindIII and
PstI-digested pBluescript KS-plasmid. The resulting plasmid was
designated pKK64. Plasmid pKK64 contains the M. circinelloides TEF1
promoter.
[0178] A genomic fragment containing the McTEF1 terminator region
was obtained with a ligation-mediated PCR amplification with TPI1
gene specific oligonucleotides identified as SEQ ID NO:72 (MC_TEF4)
and SEQ ID NO:73 (MC_TEF3), together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as in Example 1A except that SspI-digested M.
circinelloides DNA was used. A .about.1100 bp PCR fragment was
isolated and sequenced. Nested primers identified as SEQ ID NO:74
(MC_TEF8) and SEQ ID NO:75 (MC_TEF7) were designed and used in a
ligation-mediated PCR amplification together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as above except that HpaI-digested M.
circinelloides DNA was used. A .about.1200 bp PCR fragment was
isolated and sequenced.
[0179] The M. circinelloides TEF1 terminator was PCR amplified by
using primers identified as SEQ ID NO:76 (MC_TEF11) and SEQ ID
NO:77 (MC_TEF12), and the M. circinelloides genomic DNA as the
template. A PCR fragment was digested with XmaI and EcoRI and a 389
bp fragment was gel isolated and ligated to a XmaI and
EcoRI-digested pBluescript KS-plasmid. The resulting plasmid was
designated pKK65. Plasmid pKK65 contains the M. circinelloides TEF1
terminator.
Example 12. Cloning of Mucor circinelloides PGK (McPGK1) Promoter
Region
[0180] A genomic fragment of the M. circinelloides PGK1 gene was
amplified by PCR from genomic M. circinelloides griseocyanus
(D-82202, VTT Culture Collection) DNA with degenerative primers
identified as SEQ ID NO:78 (Mould PGK4) and SEQ ID NO:79 (Mould
PGK2). The degenerative primers were designed based on a consensus
sequence of PGK1 genes of Rhizopus oryzae, Fusarium oxysporum,
Aspergillus fumigatus, A. oryzae and A. nidulans. A .about.250 bp
genomic fragment was isolated and sequenced.
[0181] A genomic fragment containing the McPGK1 promoter region was
obtained with a ligation-mediated PCR amplification with PGK1 gene
specific oligonucleotides identified as SEQ ID NO:80 (MC_PGK2) and
SEQ ID NO:81 (MC_PGK1), together with oligonucleotides identified
as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II),
similarly as in Example 1A except that SspI-digested M.
circinelloides DNA was used. A .about.1300 bp PCR fragment was
isolated and sequenced. Nested primers identified as SEQ ID NO:82
(MC_PGK4) and SEQ ID NO:83 (MC_PGK3) were designed and used in a
ligation-mediated PCR amplification together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as above except that HaeIII-digested M.
circinelloides DNA was used. A .about.600 bp PCR fragment was
isolated and sequenced.
[0182] The M. circinelloides PGK1 promoter was PCR amplified by
using primers identified as SEQ ID NO:84 (MC_PGK5) and SEQ ID NO:85
(MC_PGK6), and the M. circinelloides genomic DNA as the template. A
PCR fragment was digested with SacII and XbaI and a 1291 bp
fragment was gel isolated and ligated to a SacII and XbaI-digested
pBluescript KS-plasmid. The resulting plasmid was designated pKK62.
Plasmid pKK62 contains the M. circinelloides PGK1 promoter.
Example 13. Cloning of Mucor circinelloides GPD (McGPD1) Promoter
Region
[0183] A genomic fragment containing the McGPD1 promoter region was
obtained with a ligation-mediated PCR amplification with Mucor
circinelloides (Syn. racemosus) GPD1 gene (GenBank accession number
AJ293012, version number AJ293012.1) specific oligonucleotides
identified as SEQ ID NO:86 (MC_GPD2) and SEQ ID NO:87 (MC_GPD1),
together with oligonucleotides identified as SEQ ID NO:3 (PCR
Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example
1A except that EcoRV-digested M. circinelloides (D-82202, VTT
Culture Collection) DNA was used. A .about.500 bp PCR fragment was
isolated and sequenced. Nested primers identified as SEQ ID NO:88
(MC_GPD10) and SEQ ID NO:89 (MC_GPD9) were designed and used in a
ligation-mediated PCR amplification together with oligonucleotides
identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR
Linker II), similarly as above except that StuI-digested M.
circinelloides DNA was used. A .about.1400 bp PCR fragment was
isolated and sequenced.
[0184] The M. circinelloides GPD1 promoter was PCR amplified by
using primers identified as SEQ ID NO:90 (MC_GPD11) and SEQ ID
NO:91 (MC_GPD12), and the M. circinelloides genomic DNA as the
template. A PCR fragment was digested with EcoRI and HindIII and a
1440 bp fragment was gel isolated and ligated to a EcoRI and
HindIII-digested pBluescript KS-plasmid. The resulting plasmid was
designated pKK59. Plasmid pKK59 contains the M. circinelloides GPD1
promoter.
Example 14A: Cloning of E. coli Hygromycin Resistance Gene;
Construction of a Plasmid (pKK69) Having the E. coli Hygromycin
Resistance Gene Under the Control of the McPGK1 Promoter and the
McTPI1 Terminator
[0185] Plasmid pKK62 was digested with SacII and SbfI. A 1286 bp
fragment was gel isolated. Plasmid pKK52 was digested with SbfI. A
1034 bp fragment was gel isolated. The 1286 bp fragment originated
from plasmid pKK62 and the 1034 bp fragment originated from plasmid
pKK52 were ligated to a 3268 bp fragment obtained by digesting
plasmid pKK57 with SacII and W. Plasmid pKK52 contains the E. coli
hygromycin resistance gene, plasmid pKK62 contains the M.
circinelloides PGK1 promoter and plasmid pKK57 contains the M.
circinelloides TPI1 terminator. The resulting plasmid was
designated pKK69. Plasmid pKK69 contains the E. coli hygromycin
resistance gene under the control of the M. circinelloides PGK1
promoter and the M. circinelloides TPI1 terminator.
Example 14B: Cloning of S. cerevisiae Cerulenin Resistance Gene;
Construction of a Plasmid (pKK92) Having the S. cerevisiae
Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter
and the McTPI1 Terminator
[0186] Plasmid pKK80 was digested with PstI. A 1685 bp fragment was
gel isolated. Plasmid pKK80 contains the S. cerevisiae cerulenin
resistance gene. The 1685 bp fragment was ligated to a 4554 bp
fragment obtained by digesting plasmid pKK69 with SbfI. Plasmid
pKK69 contains the M. circinelloides PGK1 promoter and the M
circinelloides TRI1 terminator. The resulting plasmid was
designated pKK92. Plasmid pKK92 contains the S. cerevisiae
cerulenin resistance gene under the control of the M.
circinelloides PGK1 promoter and the M. circinelloides TPI1
terminator.
Example 15A. Construction of a Plasmid (pKK75, FIG. 8) Containing
the Hygromycin Resistance Gene Under the Control of the McPGK1
Promoter and the McTPI1 Terminator and the S. cerevisiae ALD6 Gene
Under the Control of the McTPI1 Promoter and the McTEF1
Terminator
[0187] Plasmid pKK56 was digested with BamHI and PstI. A 1251 bp
fragment was gel isolated. Plasmid pKK56 contains the McTPI1
promoter. Plasmid RoALD was digested with SbfI. A 1514 bp fragment
was gel isolated. The plasmid RoALD contains the S. cerevisiae ALD6
(SEQ ID NO:47) encoding gene which has been codon optimized
according to Rhizopus oryzae filamentous fungus codon usage (SEQ ID
NO:49) with flanking SbfI restriction sites. The 1251 bp fragment
originating from the plasmid pKK56 and the 1514 bp fragment
originating from the plasmid RoALD were ligated to a 3321 bp
fragment obtained by digesting plasmid pKK65 with BamHI and SbfI.
Plasmid pKK65 contains the McTEF1 terminator. The resulting plasmid
was designated as pKK73. Plasmid pKK73 contains the S. cerevisiae
ALD6 encoding gene under the control of the M. circinelloides TPI1
promoter and the M. circinelloides TEF1 terminator.
[0188] Plasmid pKK69 was digested with BamHI and XmnI. A 2652 bp
fragment was gel isolated and ligated to a 6086 bp fragment
obtained by digesting plasmid pKK73 with BamHI. Plasmid pKK69
contains the E. coli hygromycin resistance gene under the control
of the M. circinelloides PGK1 promoter and the M. circinelloides
TPI1 terminator. The resulting plasmid was designated as pKK75
(FIG. 8).
Example 15B. Generation of a Genetically Modified Mucor
circinelloides (M22/75) with an Integrated ALD6 Encoding Gene and a
Hygromycin Resistance Gene by Transforming Wild-Type M.
circinelloides with Digested Plasmid pKK75 (FIG. 8, Ex. 15A)
[0189] Plasmid pKK75 was restricted with KpnI and NotI. A 5866 bp
fragment was gel isolated and used to transform a wild-type M.
circinelloides strain (D-82202, VTT Culture Collection) designated
as M22, using a Mucor protoplast transformation method (Wolff et.
al. 2002). The transformed cells were screened for hygromycin
resistance. Several hygromycin-resistant colonies were analysed at
DNA level by PCR. The transformants originating from the
transformation of the wild-type M. circinelloides strain with KpnI
and NotI cut pKK75 and containing the S. cerevisiae ALD6 encoding
gene under the control of the McTPI1 promoter and the McTEF1
terminator were designated as M22/75-80 and M22/75-86.
Example 16A. Construction of a Plasmid (pKK94, FIG. 9) Containing
the Hygromycin Resistance Gene Under the Control of the McPGK1
Promoter and the McTPI1 Terminator and the S. cerevisiae ALD6
Encoding Gene Under the Control of the McTPI1 Promoter and the
McTEF1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under
the Control of the McTEF1 Promoter and the McTPI1 Terminator
[0190] Plasmid pKK57 was digested with PstI. A 351 bp fragment was
gel isolated and ligated to a 4326 bp fragment obtained by
digesting plasmid pKK64 with PstI. The plasmid pKK57 contains the
McTPI terminator and the plasmid pKK64 contains the McTEF promoter.
The resulting plasmid was designated pKK90Pre. Plasmid RoACS was
digested with SbfI. A 2060 bp fragment was gel isolated and ligated
to a 4677 bp fragment obtained by digesting plasmid pKK90Pre with
SbfI. The plasmid RoACS contains the S. cerevisiae ACS2 (SEQ ID
NO:50) encoding gene which has been codon optimized according to
Rhizopus oryzae filamentous fungus codon usage (SEQ ID NO:92) with
flanking SbfI restriction sites. The resulting plasmid was
designated as pKK90.
[0191] Plasmid pKK75 was digested with KpnI followed by removal of
the 3' overhangs by T4 DNA polymerase and each of the 4 dNTPs. KpnI
(blunt)-digested plasmid pKK75 was digested with NotI and a 5866 bp
fragment was gel isolated. The plasmid pKK75 contains the E. coli
hygromycin resistance gene under the control of the M.
circinelloides PGK1 promoter and the M. circinelloides TPI1
terminator and S. cerevisiae ALD6 encoding gene under the control
of the M. circinelloides TPI1 promotor and the M. circinelloides
TEF1 terminator. The 5866 bp fragment originating from plasmid
pKK75 was ligated to a 6698 bp fragment obtained by digesting
plasmid pKK90 with SmaI and NotI. The resulting plasmid was
designated as pKK94 (FIG. 9).
Example 16B. Generation of a Genetically Modified Mucor
circinelloides (M22/94) with an Integrated ALD6 and ACS2 Encoding
Genes and a Hygromycin Resistance Gene by Transforming Wild-Type M.
circinelloides with Digested Plasmid pKK94 (FIG. 9, Ex. 16A)
[0192] Plasmid pKK94 was restricted with KpnI and SacI. A 9701 bp
fragment was gel isolated and used to transform a wild-type M.
circinelloides strain (D-82202, VTT Culture Collection) designated
as M22, using the transformation method described in Example 15B.
The transformed cells were screened for hygromycin resistance.
Several hygromycin resistant colonies were analysed at DNA level by
PCR. The transformants originating from the transformation of a
wild-type M. circinelloides strain with KpnI and SacI cut pKK94 and
containing the S. cerevisiae ALD6 encoding gene under the control
of the McTPI1 promoter and the McTEF1 terminator and S. cerevisiae
ACS2 encoding gene under the control of the McTEF1 promoter and the
McTPI1 terminator were designated as M22/94-12, M22/94-16 and
M22/94-24.
Example 17A. Construction of a Plasmid (pKK96, FIG. 10) Containing
the Hygromycin Gene Under the Control of the McPGK1 Promoter and
the McTPI1 Terminator and the S. cerevisiae ACS2 Encoding Gene
Under the Control of the McTEF1 Promoter and the McTPI1
Terminator
[0193] Plasmid pKK94 was digested with BsrGI and a 9683 bp fragment
was gel isolated and self ligated. The resulting plasmid was
designated as pKK96 (FIG. 10). The plasmid pKK96D contains the S.
cerevisiae ACS2 encoding gene under the control of the McTEF1
promoter and the McTPI1 terminator.
Example 17B. Generation of a Genetically Modified Mucor
circinelloides (M22/96) with an Integrated ACS2 Encoding Gene and
Hygromycin Resistance Gene by Transforming Wild-Type M.
circinelloides with Digested Plasmid pKK96 (FIG. 10, Ex. 17A)
[0194] Plasmid pKK96 was restricted with KpnI and SacI. A 6824 bp
fragment was gel isolated and used to transform the wild-type M.
circinelloides strain (D-82202, VTT Culture Collection) designated
as M22, using the transformation method described in Example 15B.
The transformed cells were screened for hygromycin resistance.
Several hygromycin resistant colonies were analysed at DNA level by
PCR. The transformants originating from the transformation of the
wild-type M. circinelloides strain with KpnI and SacI cut pKK96D
and containing the S. cerevisiae ACS2 encoding gene under the
control of the McTEF1 promoter and the McTPI1 terminator were
designated as M22/96-1 and M22/96-6.
Example 18A. Construction of a Plasmid (pKK98) Containing the
Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter
and the McTPI1 Terminator and the R. oryzae PDAT Gene Under the
Control of the McGPD1 Promoter and the McTEF1 Terminator
[0195] Plasmid pKK59 was digested with SbfI and XbaI. A 1467 bp
fragment was gel isolated and ligated to a 3309 bp fragment
obtained by digesting plasmid pKK65 with SbfI and XbaI. The plasmid
pKK59 contains the McGPD1 promoter and the plasmid pKK65 contains
the McTEF1 terminator. The resulting plasmid was designated as
pKK88. Plasmid RoPDAT was digested with SbfI. A 1844 bp fragment
was gel isolated and ligated to a 4776 bp fragment obtained by
digesting plasmid pKK88 with SbfI. The plasmid RoPDAT contains the
R. oryzae PDAT (SEQ ID NO:52) encoding gene, which has been codon
optimized according to R. oryzae codon usage (SEQ. ID. NO 93) with
flanking SbfI restriction sites. The resulting plasmid was
designated as pKK97.
[0196] Plasmid pKK97 was digested with EcoRI. A 3659 bp fragment
was gel isolated and ligated to a 6239 bp fragment obtained by
digesting plasmid pKK92 with EcoRI. The plasmid pKK92 contains the
S. cerevisiae cerulenin resistance gene under the control of the
McPGK1 promoter and the McTPI1 terminator. The resulting plasmid
was designated as pKK98 (FIG. 11).
Example 18B. Generation of a Genetically Modified Mucor
circinelloides (M22/98) with an Integrated PDAT Encoding Gene and
Cerulenin Resistance Gene by Transforming Wild-Type M.
circinelloides with Digested Plasmid pKK98 (FIG. 11, Ex. 18A)
[0197] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp
fragment was gel isolated and used to transform the wild-type M.
circinelloides strain (D-82202, VTT Culture Collection) designated
as M22, using the transformation method described in Example 15B.
The transformed cells were screened for cerulenin resistance.
Several cerulenin resistant colonies were analysed at DNA level by
PCR. The transformants originating from the transformation of the
wild-type M. circinelloides strain with ApaI and SacII cut pKK98
and containing the R. oryzae PDAT encoding gene under the control
of the McGPD1 promoter and the McTEF1 terminator were designated as
M22/98-16.
Example 19. Generation of a Genetically Modified Mucor
circinelloides (M22/75/98) with Integrated ALD6 and PDAT Encoding
Genes and Hygromycin and Cerulenin Resistance Genes by Transforming
Genetically Modified Strain M22/75-86 (Ex 15B) with Digested
Plasmid pKK98 (FIG. 11, Ex. 18A)
[0198] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp
fragment was gel isolated and used to transform the genetically
modified strain M22/75-86, using the transformation method
described in Example 15B. The transformed cells were screened for
cerulenin resistance. Several cerulenin resistant colonies were
analysed at DNA level by PCR. The transformants originating from
the transformation of the recombinant M22/75-86 strain with ApaI
and SacII cut pKK98 and containing the R. oryzae PDAT encoding gene
under the control of the McGPD1 promoter and the McTEF1 terminator
and the S. cerevisiae ALD6 encoding gene under the control of the
McTPI1 promoter and the McTEF1 terminator were designated as
M22/75/98-7 and M22/75/98-9.
Example 20. Generation of a Genetically Modified Mucor
circinelloides (M22/94/98) with Integrated ALD6, ACS2 and PDAT
Encoding Genes and Hygromycin and Cerulenin Resistance Genes by
Transforming Genetically Modified Strain M22/94-31 (Ex 16B) with
Digested Plasmid pKK98 (FIG. 11, Ex. 18A)
[0199] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp
fragment was gel isolated and used to transform the genetically
modified strain M22/94-31, using the transformation method
described in Example 15B. The transformed cells were screened for
cerulenin resistance. Several cerulenin resistant colonies were
analysed at DNA level by PCR. The transformants originating from
the transformation of the recombinant M22/94-31 strain with ApaI
and SacII cut pKK98 and containing the R. oryzae PDAT encoding gene
under the control of the McGPD1 promoter and the McTEF1 terminator,
the S. cerevisiae ALD6 encoding gene under the control of the
McTPI1 promoter and the McTEF1 terminator and the S. cerevisiae
ACS2 encoding gene under the control of the McTEF1 promoter and the
McTPI1 terminator were designated as M22/94/98-19 and
M22/94/98-22.
Example 21. Lipid Extraction and Total Lipid and Triglyceride
Concentration Measurements
[0200] A lipid extraction method was modified from the protocol of
Folch et al., 1957. 0.5 to 2 ml of cell culture was taken into an
Eppendorf tube. The sample was centrifuged and the supernatant
discarded. The pellet was placed rapidly in liquid nitrogen and
stored at -80.degree. C. Alternatively, filamentous fungal cells of
2 to 12 ml culture broth were collected by vacuum filtration
through disks of glass microfiber filters (Whatman, England). After
washing twice with distilled water, biomass was removed from the
filter using a clean spatula and put into 2 ml microfuge tubes,
which were placed rapidly in liquid nitrogen and stored at
-80.degree. C. In a homogenisation step the frozen pellet was
suspended in 500 .mu.l of ice-cold methanol with 0.1% BHT
(2,6-Di-tertbutyl-4-methylphenol) and homogenised with a Mixer Mill
homogenizator with 5-mm zirconium oxide and 3-mm yttrium stabilized
zirconium oxide balls (Retsch) at 25 Hz for 5 min. After
homogenisation 1000 .mu.l of chloroform was added and
homogenisation repeated. After re-homogenisation 300 .mu.l of 20 mM
acetic acid was added and the sample vortexed for 10 min. After
vortexing the sample was centrifuged 13 000 rpm for 5 min at RT.
The lower phase was recovered and 1000 .mu.l of chloroform was
added to the remaining phase, vortexed and recentrifuged. The lower
phases were combined into pre-weighed 2 ml microfuge tubes, and
dried, after which the total lipid content of the sample was
determined by gravimetry. Then the lipid sample was redissolved in
1.5 ml of chloroform:methanol (2:1)+0.1% BHT and stored at
-20.degree. C. For triacylglycerol analysis 100 to 1500 .mu.l of
chloroform:methanol extracted lipids were evaporated and
re-dissolved in 200-1000 .mu.l of isopropanol. Triacylglycerols
were measured enzymatically from the samples by using the Konelab
Triglycerides Kit (Thermo Scientific, Finland) and Cobas Mira
automated analyser (Roche) or a microtitre-plate reader (Varioskan,
Thermo Electron Corporation). This lipid extraction and total lipid
and triglyceride concentration measurements methods were used in
the following examples if not otherwise indicated.
Example 22. Microaerobic Shake Flask Characterization of Strains
Y23/81-51 and Y23/81-66 (Ex. 3B), Y23/86-86 and Y23/86-92 (Ex. 5B),
Y23/95-87 and Y23/95-109 (Ex. 6B) and Y23/85/95-4 (Ex. 9), in
Glucose Medium with C/N Ratio of 20
[0201] Transformants were separately cultivated in 50 ml of culture
medium "glucose CN20" (pH 5.5, 20 g glucose, 0.3 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 100 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control.
[0202] Lipid extraction and triacylglycerol concentration
measurement were carried out as described in Example 21. Cell dry
weight was determined by centrifuging 1 ml of the culture broth in
pre-dried, pre-weighed Eppendorf tubes. After washing with 1 ml of
distilled water the cell pellet was dried at 100.degree. C. for 24
hours and weighed again. HPLC analyses for sugars were conducted
with a Waters 2690 Separation Module and Water System Interfase
Module liquid chromatography coupled with a Waters 2414
differential refractometer and Waters 2487 dual absorbance
detector. The liquid chromatography columns were a 100.times.7.8 mm
Fast Acid Analysis column from Bio-Rad and a 300.times.7.8 mm
Aminex HPX-87H column from Bio-Rad. The columns were equilibrated
with 2.5 mM H.sub.2SO.sub.4 in water at 55.degree. C. and samples
were eluted with 2.5 mM H.sub.2SO.sub.4 in water at 0.5 ml/min flow
rate. Data acquisition was done using Waters Millennium software.
This HPLC method was used in all appropriate Examples.
[0203] After 48 hours cultivation (Table 2A), when 4 to 8 g/l
glucose was left, Y23/81, Y23/86, Y23/95 and Y23/85/95
transformants produced 12, 22, 15 and 24% more triacylglycerols
with higher rate, respectively, than the control strain in glucose
medium. Triacylglycerol yields per used glucose were also better up
to 10% with the transformants compared to the control strain.
Additionally Y23/85/95 transformant had 13% higher triacylglycerol
yield on biomass than the control strain (12.9 and 11.4% TAG yield
on biomass, respectively). In particular, after 24 hours of
cultivation (Table 2B), the strains expressing ALD or ACS alone, or
ALD and ACS together, had enhanced production of triacylglycerols,
measured as concentration (g/l) and as yield per biomass and used
glucose, with higher rate (mg/l/h) compared to the control
strain.
TABLE-US-00002 TABLE 2A Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per used glucose after 48 hours
microaerobic cultivation in glucose medium with C/N ratio of 20
Yield TAG TAG (% used TAG Strain (g/l) glucose) mg/l/h Control 0.72
5.88 15.0 Y23/81 (ALD) 0.81 5.90 16.8 Y23/86 (ACS) 0.88 6.00 18.3
Y23/95 (PDAT) 0.82 6.02 17.2 Y23/85/95 0.89 6.46 18.5 (ALD + ACS +
PDAT)
TABLE-US-00003 TABLE 2B Triacylglycerol (TAG) concentration (g/l)
and yield (%) per biomass (CDW) and used glucose after 24 hours
microaerobic cultivation in glucose medium with C/N ratio of 20 TAG
Yield TAG Yield TAG TAG Strain (g/l) (% CDW) (% used glucose)
mg/l/h Control 0.22 5.15 4.29 9.1 Y23/81-66 (ALD) 0.26 6.09 4.86
10.8 Y23/85-125 (ALD + 0.31 7.01 5.49 12.8 ACS) Y23/86-86 (ACS)
0.30 6.76 5.26 12.5
[0204] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol
concentrations and rates of production, and triacylglycerol yields
from used glucose or per dry weight in cultivation with low C/N
ratios.
Example 23. Aerobic Shake Flask Characterization of Strains
Y23/81-8, 51, 59, 66 and 69 (Ex. 3B), Y23/85-119, 125, 128, 129 and
139 (Ex. 7B), Y23/86-86, 92, 93, 98 and 100 (Ex. 5B), Y23/95-99 and
104 (Ex. 6B), Y23/81/95-42 (Ex. 8) and Y23/85/95-4 and 68 (Ex. 9),
in Glucose Medium with C/N Ratio of 65
[0205] Transformants were separately cultivated in 50 ml of Yeast
culture medium II (pH 5.5, 20 g glucose, 0.3 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.6 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction and triacylglycerol
measurements as described in Example 21.
[0206] After 44 hours cultivation (Table 3A) transformants Y23/81,
Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had 4, 3, 6, 1, 12
and 4% better triacylglycerol yields per used glucose,
respectively, than the control strain. Additionally, transformants
Y23/81/95 and Y23/85/95 had 7-8% higher triacylglycerol yield on
biomass than the control strain (42.8, 43.0 and 40.0% TAG yield
[/CDW], respectively). After 25 hours cultivation (Table 3B), the
strains expressing ALD or ACS alone, or ALD and ACS together, had
enhanced production of triacylglycerols, measured as concentration
(g/l) and as yield per biomass and used glucose.
TABLE-US-00004 TABLE 3A Maximal triacylglycerol (TAG) yield (%) per
used glucose after 44 hours cultivation in glucose medium with C/N
ratio of 65 Yield TAG Strain (% used glucose) Control 16.6 Y23/81
(ALD) 17.3 Y23/85 (ALD + ACS) 17.2 Y23/86 (ACS) 17.6 Y23/95 (PDAT)
16.9 Y23/81/95 (ALD + PDAT) 18.6 Y23/85/95 (ALD + ACS + PDAT)
17.4
TABLE-US-00005 TABLE 3B Triacylglycerol (TAG) concentration (g/l)
and yield (%) per biomass (CDW) and used glucose after 25 hours
cultivation in glucose medium with C/N ratio of 65 TAG Yield TAG
Yield TAG TAG Strain (g/l) (% CDW) (% used glucose) mg/l/h Control
1.01 21.7 10.3 40.5 Y23/81-51, 66 (ALD) 1.15 26.4 12.5 46.0
Y23/85-125, 129 1.14 27.2 13.3 45.4 (ALD + ACS) Y23/86-92, 100
(ACS) 1.18 22.4 12.0 47.1
[0207] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yields
from used glucose or per dry weight.
Example 24. Aerobic Shake Flask Characterization of Strains
Y23/81-66 (Ex. 3B), Y23/85-125 (Ex. 7B), Y23/86-92 (Ex. 5B),
Y23/95-98 (Ex. 6B) and Y23/81/95-18 (Ex. 8) in Glucose Medium with
C/N Ratio of 103
[0208] Transformants were separately cultivated in 50 ml of Yeast
culture medium IV (pH 5.5, 20 g glucose, 0.15 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction and triacylglycerol
measurements as described in Example 21.
[0209] After 35 hours cultivation, when 6-8 g/l glucose was left,
transformants Y23/81-66, Y23/85-125, Y23/86-92, Y23/95-98 and
Y23/81/95-18 had 3, 6, 9, 11 and 11% higher triacylglycerol titre
and rate than the control strain, respectively. The transformants
Y23/81-66, Y23/85-125, Y23/86-92, Y23/95-98 and Y23/81/95-18 had
also 11, 19, 7, 20 and 30% better triacylglycerol yields per used
glucose, respectively, than the control strain. Additionally
Y23/81/95-18 had 24% higher yield on biomass than the control
strain (49.4 and 40.0% TAG yields on biomass, respectively).
TABLE-US-00006 TABLE 4 Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per used glucose after 35 hours
cultivation in glucose medium with C/N ratio of 103 Yield TAG (%
used Strain TAG (g/l) glucose) TAG mg/l/h Control 2.00 16.2 57.1
Y23/81-66 (ALD) 2.05 18.0 58.7 Y23/85-125 (ALD + ACS) 2.11 19.3
60.3 Y23/86-92 (ACS) 2.17 17.4 61.9 Y23/95-98 (PDAT) 2.22 19.4 63.5
Y23/81/95-18 (ALD + PDAT) 2.22 21.1 63.5
[0210] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yield
from used glucose or per dry weight with high C/N ratios.
Example 25. Aerobic Shake Flask Characterization of Strains
M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B),
M22/75/98-9 (Ex. 19) and M22/94/98-19 (Ex. 20), in Glucose Medium
with C/N Ratio of 40
[0211] Transformants were separately cultivated in 50 ml of mould
C/N 40 medium (pH 5.5, 20 g glucose, 1.4 g yeast extract, 2.5 g
KH.sub.2PO.sub.4, 0.3 g (NH.sub.4).sub.2SO.sub.4, 10 mg
ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4,
0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg
FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated
with 1*10.sup.7 spores. The cultivations were maintained at a
temperature of 25.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Mucor
circinelloides wild type strain M22 was used as a control. Cell dry
weight was determined by vacuum filtration through disks of glass
microfiber filters (Whatman, England) of 2 to 24 ml culture broth.
After washing twice with distilled water, biomass was removed from
the cloth using clean spatula and transferred to pre-dried,
pre-weighed 2 ml microfuge tubes in which the mycelia were dried at
100.degree. C. for 48 hours and weighed after cooling in a
dessicator. HPLC analysis was carried out as described in Example
22. Lipid extraction and total lipid and triacylglycerol
measurements as described in Example 21.
[0212] After 46 hours cultivation (Table 5) transformants
M22/75-86, M22/96-1, M22/94-24, M22/75/98-9 and M22/94/98-19
produced 21, 9, 18, 91 and 55% more triacylglycerol with higher
rate than the control strain, respectively. The transformant
M22/75-86, M22/96-1., M22/94-24, M22/75/98-9 and M22/94/98-19 had
also 39 (20), 23 (10), 18 (13), 127 (57) and 80 (25)% higher
triacylglycerol yield on used glucose (on biomass) than the control
strain, respectively. Additionally the transformants M22/75/98-9
and M22/94/98-19 had higher total lipid concentration (0.93-1.09
g/l) and rate (20-24 mg/l/h) with yields on biomass (26.1-31.4%)
and on used glucose (6.29-7.52%) than the control strain (0.71 g/l,
15 mg/l/h, 24.6% and 4.09%, respectively).
TABLE-US-00007 TABLE 5 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW; cell dry weight) and used glucose after 46 hours cultivation
in glucose medium with C/N ratio of 40 Yield Yield TAG TAG (% used
TAG Strain TAG g/l (% CDW) glucose) mg/l/h Control 0.33 11.5 1.92
7.2 M22/75-86 (ALD) 0.40 13.8 2.67 8.8 M22/96-1 (ACS) 0.36 12.6
2.36 7.9 M22/94-24 (ALD + ACS) 0.39 13.0 2.27 8.5 M22/75/98-9 (ALD
+ 0.63 18.1 4.35 13.7 PDAT) M22/94/98-19 0.51 14.4 3.46 11.1 (ALD +
ACS + PDAT) Yield lipid Yield lipid (% used Lipid Strain Lipid g/l
(% CDW) glucose) mg/l/h Control 0.71 24.6 4.09 15 M22/75-86 (ALD)
0.76 26.1 5.04 17 M22/96-1 (ACS) 0.70 24.2 4.53 15 M22/94-24 (ALD +
ACS) 0.71 23.5 4.10 15 M22/75/98-9 (ALD + 1.09 31.4 7.52 24 PDAT)
M22/94/98-19 (ALD + 0.93 26.1 6.29 20 ACS + PDAT)
[0213] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and rates of production and triacylglycerol
and total lipid yields from used glucose or per dry weight.
Example 26. Aerobic Shake Flask Characterization of Strains
M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B),
M22/75/98-9 (Ex. 19) and M22/94/98-19 (Ex. 20), in Glucose Medium
with C/N Ratio of 66
[0214] Transformants were separately cultivated in 50 ml of mould
C/N 66 medium (pH 5.5, 20 g glucose, 1.0 g yeast extract, 2.5 g
KH.sub.2PO.sub.4, 0.1 g (NH.sub.4).sub.2SO.sub.4, 10 mg
ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4,
0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg
FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated
with 1*10.sup.7 spores. The cultivations were maintained at a
temperature of 25.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction, enzyme activity
measurement and HPLC analysis were withdrawn periodically during
cultivation. Mucor circinelloides wild type strain M22 was used as
a control. Cell dry weight was determined as described in Example
25 and HPLC analysis was carried out as described in Example 22.
Lipid extraction and total lipid and triacylglycerol measurements
as described in Example 21.
[0215] After 93 hours cultivation, when 2-6 g/l glucose was left,
transformants M22/75-86, M22/96-1, M22/94-24, M22/75+98-9 and
M22/94+98-19 had produced 34, 30, 45, 186 and 214% more
triacylglycerols with higher rate than the control strain. The
transformants M22/75-86, M22/96-1, M22/94-24, M22/75+98-9 and
M22/94+98-19 also had 63 (24), 68 (30), 62 (43), 229 (72) and 102
(104)% higher triacylglycerol yield on used glucose (on biomass)
than the control strain. Additionally, the transformants produced
more lipids (0.89-2.29 g/l) with higher rates (9.6-24.6 mg/l/h) and
with higher yields on used glucose (6.90-17.68%) and on biomass
(37.2-57.7%) than the control strain (0.71 g/l, 7.6 mg/l/h, 4.82%
and 30.2%, respectively).
TABLE-US-00008 TABLE 6 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW; cell dry weight) and used glucose after 93 hours cultivation
in glucose medium with C/N ratio of 66. Yield Yield TAG TAG TAG (%
used TAG Strain g/l (% CDW) glucose) mg/l/h Control 0.44 18.6 2.97
4.7 M22/75-86 (ALD) 0.59 23.0 4.84 6.4 M22/96-1 (ACS) 0.57 24.1
4.99 6.1 M22/94-24 (ALD + ACS) 0.64 26.6 4.82 6.9 M22/75/98-9 1.26
31.9 9.76 13.6 (ALD + PDAT) M22/94/98-19 1.38 37.9 9.00 14.8 (ALD +
ACS + PDAT) Yield lipid Yield lipid (% used Lipid Strain Lipid g/l
(% CDW) glucose) mg/l/h Control 0.71 30.2 4.8 7.6 M22/75-86 (ALD)
0.96 37.2 7.8 10.3 M22/96-1 (ACS) 0.89 37.3 7.7 9.6 M22/94-24 (ALD
+ ACS) 0.91 37.6 6.9 9.8 M22/75/98-9 (ALD + 2.29 57.7 17.7 24.6
PDAT) M22/94/98-19 2.00 55.1 13.1 21.5 (ALD + ACS + PDAT)
[0216] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and rates of production and triacylglycerol
and total lipid yields from used glucose or per dry weight with
high C/N ratios.
Example 27. Microaerobic Shake Flask Characterization of Strains
Y23/81-51 and Y23/81-66 (Ex. 3B), Y23/85-125 and Y23/85-128 (Ex.
7B), Y23/86-86 and Y23/86-92 (Ex. 5B), Y23/95-87 and Y23/95-109
(Ex. 6B) and Y23/85/95-4 (Ex. 9), in Xylose Medium with C/N Ratio
of 20
[0217] Transformants were separately cultivated in 50 ml of culture
medium "xylose CN20" (pH 5.5, 20 g xylose, 0.3 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 100 rpm. Samples for
cell dry weight measurement, lipid extraction, enzyme activity
measurement and HPLC analysis were withdrawn periodically during
cultivation. Cryptococcus curvatus wild type strain Y23 was used as
a control.
[0218] Lipid extraction and triacylglycerol concentration
measurements were carried out as described in Example 21. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22.
[0219] At the end of cultivation samples for acetaldehyde
dehydrogenase and acetyl-CoA synthetase activity measurements were
taken. Cells were harvested by centrifugation and washed once with
100 mM Tris-HCl pH 7.0. Washed cells were stored at -20.degree. C.
Frozen cells were melted and washed once with 100 mM Tris-HCl pH
7.0 and suspended in 100 mM Tris-HCl pH 7.0, 1 mM DTT buffer
containing EDTA-free protease inhibitors (Roche, USA). Cell
disruption was carried out with 0.5 mm diameter glass beads (Sigma
Chemicals Co, USA) in a Fast Prep homogenizer (Thermo Scientific,
USA). Cell debris was removed by centrifugation and supernatant was
used in enzyme activity measurements. Acetaldehyde dehydrogenase
and acetyl-CoA synthetase activity measurements were carried out
with a Konelab Arena automatic analyzer (Thermo Scientific,
Finland). The acetaldehyde dehydrogenase reaction mixture contained
(final concentration) 50 mM potassium phosphate pH 7.0, 15 mM
pyrazole, 0.4 mM DTT, 10 mM MgCl.sub.2, 0.4 mM NADP and cell
extract. The reaction was started with 0.1 mM acetaldehyde. The
formation of NADPH was followed at 340 nm. One unit was defined as
the amount of formation of 1 .mu.mol of NADPH per min. The
acetyl-CoA synthetase reaction mixture contained (final
concentration) 100 mM Tris-HCl pH 7.5, 10 mM L-malate pH 7.5, 0.2
mM Coenzyme A, 8 mM ATP, 1 mM NAD, 10 mM MgCl.sub.2, 3 U/ml malate
dehydrogenase, 0.4 U/ml citrate synthase and cell extract. The
reaction was started with 100 mM potassium acetate. The formation
of NADH was followed at 340 nm. One unit was defined as the amount
of formation of 1 .mu.mol of NADH per min.
[0220] After 24 hours cultivation the transformants Y23/81, Y23/85,
Y23/86, Y23/95 and Y23/85/95 produced 13-31% more triacylglycerol
with 5-38% higher yield on biomass than the control strain. The
transformants Y23/81, Y23/86, Y23/95 and Y23/85/95 also had 4-12%
higher yields on used xylose than the control strain. The
transformants having ALD (ACS) encoding gene expressed had 21.5 to
42.7 (1.5 to 1.9) times higher ALD (ACS) activity than the control
strain.
TABLE-US-00009 TABLE 7 Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and
used xylose after 24 hours microaerobic cultivation in xylose
medium with C/N ratio of 20 TAG Yield TAG Yield TAG (% TAG Strain
(g/l) (% CDW) used xylose) mg/l/h Control 0.16 4.33 3.46 6.8 Y23/81
(ALD) 0.20 5.98 3.61 8.2 Y23/85 (ALD + ACS) 0.18 4.54 3.46 7.3
Y23/86 (ACS) 0.21 5.33 3.86 8.9 Y23/95 (PDAT) 0.20 5.55 3.68 8.4
Y23/85/95 0.20 5.55 3.72 8.2 (ALD + ACS + PDAT)
TABLE-US-00010 TABLE 8 Relative acetaldehyde dehydrogenase (ALD)
and acetyl-CoA synthetase (ACS) activities in microaerobic
cultivation in xylose with C/N ratio of 20 compared to the control
strain ALD and ACS activities Strain ALD ACS Control 1 1 Y23/81
(ALD) 42.7 1.2 Y23/85 (ALD + ACS) 27.3 1.9 Y23/86 (ACS) 4.3 1.5
Y23/95 (PDAT) 2.3 1.2 Y23/85/95 (ALD + ACS + PDAT) 21.5 1.5
[0221] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yields
from used xylose or per dry weight with low C/N ratios.
Additionally, the example shows that acetaldehyde dehydrogenase
(ALD) and acetyl-CoA synthetase (ACS) enzymes are expressed in
active forms.
Example 28. Aerobic Shake Flask Characterization of Strains
Y23/81-8 and 59 (Ex. 3B), Y23/85-119 and 128 (Ex. 7B), Y23/86-86
and 92 (Ex. 5B), Y23/95-99 and 109 (Ex. 6B), Y23/81/95-18 and 42
(Ex. 8) and Y23/85/95-4 and 68 (Ex. 9), in Xylose Medium with C/N
Ratio of 103
[0222] Transformants were separately cultivated in 50 ml of Yeast
xylose culture medium IV (pH 5.5, 20 g xylose, 0.15 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction and triacylglycerol
measurements as described in Example 21.
[0223] After 52 hours cultivation the transformants Y23/81, Y23/85,
Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had produced 23, 19, 50,
52, 40 and 75% more triacylglycerol than the control strain. The
transformants Y23/81, Y23/85, Y23/86, Y23/95, Y23/81/95 and
Y23/85/95 had also 35, 47, 59, 60, 103 and 111% higher
triacylglycerol yield on biomass and 11, 11, 15, 28, 66 and 63%
higher triacylglycerol yield on used xylose than the control
strain. Additionally the transformants Y23/81, Y23/85, Y23/86,
Y23/95, Y23/81/95 and Y23/85/95 had higher lipid concentration
(2.40-2.90 g/l) and rate (46-56 mg/l/h) with higher yield on
biomass (56.5-78.8%) than the control strain (2.35 g/l, 45 mg/l/h
and 52.2%, respectively). The transformants Y23/81/95 and Y23/85/95
had also higher lipid yield on used xylose (29.0-31.6%) than the
control strain (25.6%).
TABLE-US-00011 TABLE 9 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW; cell dry weight) after 52 hours cultivation in xylose medium
with C/N ratio of 103. Triacylglycerol yield (%) from used xylose
is also indicated. Yield TAG Yield TAG (% used TAG Strain TAG (g/l)
(% CDW) xylose) mg/l/h Control 0.60 13.3 6.52 19.0 Y23/81 (ALD)
0.83 17.9 7.23 26.4 Y23/85 (ALD + ACS) 0.79 19.6 7.26 25.0 Y23/86
(ACS) 0.90 21.1 7.52 28.5 Y23/95 (PDAT) 0.91 21.3 8.37 28.8
Y23/81/95 0.84 27.0 10.8 26.6 (ALD + PDAT) Y23/85/95 1.05 28.0 10.7
33.3 (ALD + ACS + PDAT) Yield lipid Strain Lipid (g/l) (% CDW)
Lipid mg/l/h Control 2.35 52.2 45 Y23/81 (ALD) 2.90 62.3 56 Y23/85
(ALD + ACS) 2.53 62.9 49 Y23/86 (ACS) 2.58 60.6 50 Y23/95 (PDAT)
2.40 56.5 46 Y23/81/95 (ALD + PDAT) 2.45 78.8 47 Y23/85/95 (ALD +
ACS + PDAT) 2.85 76.0 55
[0224] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and rates of production and triacylglycerol
and total lipid yields per dry weight and triacylglycerol yields
from used xylose with high C/N ratios.
Example 29. Aerobic Shake Flask Characterization of Strains
M22/75-80 (Ex. 15B), M22/96-6 (Ex. 17B), M22/98-16 (Ex. 18B),
M22/94-16 (Ex. 16B), M22/75/98-7 (Ex. 19) and M22/94/98-22 (Ex.
20), in Xylose Medium with C/N Ratio of 66
[0225] Transformants were separately cultivated in 50 ml of mould
xylose C/N 66 medium (pH 5.5, 20 g xylose, 1.0 g yeast extract, 2.5
g KH.sub.2PO.sub.4, 0.1 g (NH.sub.4).sub.2SO.sub.4, 10 mg
ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO4, 0.5 g
MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg FeCl.sub.3*6H.sub.2O
per litre). Each flask (250 ml) was inoculated with 1*10.sup.7
spores. The cultivations were maintained at a temperature of
25.degree. C. with shaking at 250 rpm. Samples for cell dry weight
measurement, lipid extraction, enzyme activity measurement and HPLC
analysis were withdrawn periodically during cultivation. Mucor
circinelloides wild type strain M22 was used as a control. Cell dry
weight was determined as described in Example 25, and HPLC analysis
was carried out as described in Example 22. Lipid extraction and
total lipid and triacylglycerol measurements as described in
Example 21.
[0226] After 143 hours cultivation the transformants M22/75-80,
M22/96-6, M22/98-16, M22/75/98-7 and M22/94/98-22 had higher TAG
concentration (0.33-0.48 g/l TAG) compared to the control strain
(0.28 g/l TAG). The all transformants had also higher TAG yield (%)
per biomass (13.71-17.78%) than the control strain (12.15%) and the
transformants M22/96-6, M22/98-16, M22/75/98-7 and M22/94/98-22 had
also higher TAG yield (%) per used xylose (4.83-6.23%) than the
control strain (3.97%). Additionally the transformants M22/75/98-7
and M22/94/98-22 had higher lipid concentration (0.79-1.13 g/l),
rate (9.2-10.8 mg/l/h) and lipid yields on biomass (48.9-55.6%) and
on used xylose (17.1-18.3%) than the control strain (0.92 g/l, 6.4
mg/l/h, 40.0% and 13.0%, respectively).
TABLE-US-00012 TABLE 10 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW; cell dry weight) and used xylose after 143 hours cultivation
in xylose medium with C/N ratio of 66 Yield TAG TAG Yield TAG (%
used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 0.28 12.2 3.97
1.9 M22/75-80 (ALD) 0.33 14.8 3.45 2.3 M22/96-6 (ACS) 0.33 15.3
5.40 2.3 M22/94-16 (ALD + ACS) 0.27 13.7 3.67 1.9 M22/98-16 (PDAT)
0.48 13.7 4.83 3.3 M22/75/98-7 0.48 17.8 6.23 3.3 (ALD + PDAT)
M22/94/98-22 0.45 16.1 5.31 3.1 (ALD + ACS + PDAT) Yield lipid
Lipid Yield lipid (% used Lipid Strain (g/l) (% CDW) xylose) mg/l/h
Control 0.92 40.0 13.0 6.4 M22/75-80 (ALD) 1.00 44.8 10.5 7.0
M22/96-6 (ACS) 0.90 41.5 14.7 6.3 M22/94-16 (ALD + ACS) 0.82 41.6
11.1 5.7 M22/98-16 (PDAT) 1.62 46.4 16.4 11.3 M22/75/98-7 (ALD +
PDAT) 1.32 48.9 17.1 9.2 M22/94/98-22 (ALD + 1.54 55.6 18.3 10.8
ACS + PDAT)
[0227] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and rates of production and triacylglycerol
and total lipid yields from used xylose or per dry weight with high
C/N ratios.
Example 30. Production of Triacylglycerol or Lipid by Strains of C.
curvatus Modified by Addition of Genes Encoding ALD and PDAT
(Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4 Ex. 9)
in High Cell Density Cultures Grown on Glucose with C/N Ratio of
80
[0228] Transformants (Y23/85/95-4 and Y23/81/95-18) were separately
cultivated in Multifors bioreactors (max. working volume 500 ml,
Infors HT, Switzerland) at pH 4.0, 30.degree. C., in 500 ml medium
containing 90 to 96 g glucose, 2.56 g (NH.sub.4).sub.2SO.sub.4, 1.2
g KH.sub.2PO.sub.4, 0.3 g Na.sub.2HPO.sub.4.2H.sub.2O, 1.5 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.6H.sub.2O, 5.26 mg citric
acid.H.sub.2O, 5.26 mg ZnSO.sub.4.7H.sub.2O, 0.1 mg
MnSO.sub.4.4H.sub.2O, 0.5 mg CoCl.sub.2.6H.sub.2O, 0.26 mg
CuSO.sub.4.5H.sub.2O, 0.1 mg Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg
FeSO.sub.4.7H.sub.2O, 0.1 mg H.sub.3BO.sub.4, 0.05 mg D-biotin, 1.0
mg CaPantothenate, 5.0 mg nicotinic acid, 25 mg myoinositol, 1.0 mg
thiamine.HCl, 1.0 mg pyridoxine.HCl and 0.2 mg p-aminobenzoic acid
per litre. The pH was maintained constant by addition of 1 M KOH or
1 M H.sub.2PO.sub.4. Cultures were agitated at 1000 rpm (2 Rushton
turbine impellors) and aerated at 2 volumes air per volume culture
per minute (vvm). Clerol FBA 3107 antifoaming agent (Cognis,
SaintFargeau-Ponthierry Cedex France, 1 ml 1.sup.-1) was added to
prevent foam accumulation. Bioreactors were inoculated to initial
OD.sub.600 of 0.5 to 4.0 with cells grown in the same medium
(substituting 1.5 g urea per litre for (NH.sub.4).sub.2SO.sub.4 and
omitting the CaCl.sub.2.6H.sub.2O) in 50 ml volumes in 250 ml
flasks at 30.degree. C. with shaking at 200 rpm for 24 to 42 h.
Samples for cell dry weight measurement, lipid extraction and HPLC
analysis were withdrawn periodically during cultivation.
Cryptococcus curvatus wild type strain Y23 was used as the control.
For measurement of the yield of triacylglycerol on glucose or
biomass, some control cultures contained 58 to 134 g glucose
1.sup.-1.
[0229] Lipid extraction and triacylglycerol concentration
measurements were carried out as described in Example 21. Cell dry
weight was determined by centrifuging 0.5 to 2.0 ml culture broth
in pre-dried, pre-weighed 2 ml microfuge tubes. After washing twice
with 1.8 ml distilled water, the cell pellet was dried at
100.degree. C. for 48 h and weighed after cooling in a dessicator.
HPLC analyses were carried out as described in Example 22.
[0230] Table 11 shows that a transformant containing the genes for
ALD and PDAT produced 23% more triacylglycerol than Y23, with a 24%
increase in the yield on glucose consumed when cells were
cultivated to high cell density in bioreactor cultures. A
transformant containing the genes for ALD, ACS and PDAT produced
15% more triacylglycerol than Y23, with a 12% increase in the yield
on glucose consumed.
TABLE-US-00013 TABLE 11 Triacylglycerol produced in pH controlled
bioreactor culture of Y23 and transformants of Y23 expressing ALD +
PDAT or ALD + ACS + PDAT, with glucose as carbon source and C/N 80.
Data is the average of 2 (transformants) or 4 to 9 (Y23) cultures
.+-. standard error of the mean. Percentage increase is shown in
parenthesis Yield TAG Strain TAG (g/l) (% glucose consumed) Y23
17.3 .+-. 0.3 18.8 .+-. 1.0 Y23/81/95-18 (ALD + PDAT) 21.3 .+-. 0.8
23.3 .+-. 1.0 (23%) (24%) Y23/85/95-4 19.9 .+-. 0.1 21.1 .+-. 0.1
(ALD + ACS + PDAT) (15%) (12%)
[0231] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol
concentrations and triacylglycerol yields from used glucose in high
cell density cultures.
Example 31. Production of Triacylglycerol or Lipid by Strains of C.
curvatus Modified by Addition of Genes Encoding ALD and PDAT
(Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4, Ex. 9)
in High Cell Density Cultures Grown on Xylose with C/N Ratio of
80
[0232] Transformants (Y23/85/95-4 and Y23/81/95-18) were separately
cultivated in Multifors bioreactors as described in Example 30. The
medium contained 92 to 118 g xylose per litre, instead of glucose.
Bioreactors were inoculated to initial OD.sub.600 of 17 to 24 with
cells grown in low nitrogen medium with glucose as carbon source in
the Multifors bioreactors at 30.degree. C., as described for lipid
production in Example 30. Alternatively, some cultures of Y23 were
inoculated with cells grown in flasks, as described in Example 30,
to initial OD.sub.600 0.2 to 0.5. Samples for cell dry weight
measurement, lipid extraction and HPLC analysis were withdrawn
periodically during cultivation. Cryptococcus curvatus wild type
strain Y23 was used as the control.
[0233] Lipid extraction and total lipid and triacylglycerol
concentration measurements were carried out as described in Example
21. Cell dry weight was determined as described in Example 30. HPLC
analyses were carried out as described in Example 22.
[0234] Table 12 shows that a transformant containing the genes for
ALD and PDAT produced 7% more triacylglycerol than Y23, with a 17%
increase in the yield on xylose consumed and a 3% increase in the
yield on biomass when cells were cultivated to high cell density in
bioreactor cultures. A transformant containing the genes for ALD,
ACS and PDAT produced only 1% more triacylglycerol than Y23, but
showed 13% increase in the yield on xylose consumed and 4% increase
in yield on biomass.
TABLE-US-00014 TABLE 12 Triacylglycerol produced in pH controlled
bioreactor culture of Y23 and transformants of Y23 expressing ALD +
PDAT or ALD + ACS + PDAT, with lose as carbon source and C/N 80.
Data is the average of 2 (transformants) or 4 (Y23) cultures, .+-.
standard error of the mean. Percentage increase is shown in
parenthesis. Yield TAG Yield TAG (% Strain TAG (g/l) (% CDW) xylose
consumed) Y23 17.9 .+-. 1.4 46.9 .+-. 4.0 17.2 .+-. 1.2
Y23/81/95-18 19.1 .+-. 0.3 48.4 .+-. 5.7 20.2 .+-. 0.5 (ALD + PDAT)
(7%) (3%) (17%) Y23/85/95-4 18.1 .+-. 0.3 49.0 .+-. 3.4 19.5 .+-.
0.0 (ALD + ACS + PDAT) (1%) (4%) (13%)
[0235] Table 13 shows that a transformant containing the genes for
ALD and PDAT produced 10% more lipid than Y23, with a 21% increase
in the yield on xylose consumed and a 2% increase in the yield on
biomass when cells were cultivated to high cell density in
bioreactor cultures. A transformant containing the genes for ALD,
ACS and PDAT produced 3% more lipid than Y23, with 16% higher yield
on xylose consumed and a 3% increase in yield on biomass.
TABLE-US-00015 TABLE 13 Lipid produced in pH controlled bioreactor
culture of Y23 and transformants of Y23 expressing ALD + PDAT or
ALD + ACS + PDAT, with xylose as carbon source and C/N 80. Data is
the average of 2 (transformants) or 4 (Y23) cultures, .+-. standard
error of the mean. Percentage increase is shown in parenthesis.
Yield lipid Yield lipid (% Strain Lipid (g/l) (% CDW) xylose
consumed) Y23 20.5 .+-. 1.3 55.9 .+-. 7.2 19.8 .+-. 2.5
Y23/81/95-18 (ALD + PDAT) 22.5 .+-. 0.6 57.0 .+-. 1.9 23.8 .+-. 0.8
(10%) (2%) (21%) Y23/85/95-4 21.2 .+-. 0.1 57.5 .+-. 1.0 22.9 .+-.
0.3 (ALD + ACS + PDAT) (3%) (3%) (16%)
[0236] This example shows that expression of ALD6, ACS2 and PDAT
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and triacylglycerol and total lipid yields
from used xylose or per dry weight in high cell density
cultures.
Example 32. Production of Triacylglycerol or Lipid by Strain of M.
circinelloides Modified by Addition of Genes Encoding ALD and PDAT
and ACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures
Grown on Glucose with C/N Ratio of 60
[0237] Transformant (M22/94/98-19) was cultivated in Braun
Biostat.RTM. CT bioreactors (2.5 max working volume, B. Braun
Biotech International, Sartorius AG, Germany) at pH 5.0, 30.degree.
C., in 1.0 to 1.2 l medium containing 53 g glucose, 1.57 g
(NH.sub.4).sub.2SO.sub.4, 2.5 g KH.sub.2PO.sub.4, 0.2 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.6H.sub.2O, 5.26 mg citric
acid.H.sub.2O, 5.26 mg ZnSO.sub.4.7H.sub.2O, 0.1 mg
MnSO.sub.4.4H.sub.2O, 0.5 mg CoCl.sub.2.6H.sub.2O, 0.26 mg
CuSO.sub.4.5H.sub.2O, 0.1 mg Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg
FeSO.sub.4.7H.sub.2O, 0.1 mg H.sub.3BO.sub.4, 0.005 mg D-biotin,
and 0.05 mg thiamine.HCl per litre. The pH was maintained constant
by addition of 1 M KOH or 1 M H.sub.2PO.sub.4. Cultures were
agitated at 600 rpm (2 Rushton turbine impellors) and aerated at 1
volume air per volume culture per minute (vvm). Polypropylene
glycol (mixed molecular weights containing Fluka P1200, Fluka P2000
and Henkel Performance Chemicals Foamaster in a ratio of 4:4; 1, 1
ml 1.sup.-1) was added to prevent foam accumulation. Bioreactors
were inoculated to an initial biomass concentration of
approximately 100 mg 1.sup.-1 with mycelia grown in the same medium
with the following modifications: 15 g glucose 1.sup.-1, enough
(NH.sub.4).sub.2SO.sub.4 to provide a C/N ratio of 16.2, and
additionally 4.0 g agar 1.sup.-1. Pre-cultures were grown in 50 ml
volumes in 250 ml flasks at 30.degree. C. with shaking at 200 rpm
for 42 to 72 h. Samples for cell dry weight measurement, lipid
extraction and HPLC analysis were withdrawn periodically during
cultivation. Biomass was separated from the culture supernatant by
filtration under vacuum. Mucor circinelloides wild type strain M22
was used as the control.
[0238] Lipid extraction and total lipid and triacylglycerol
concentration measurements were carried out as described in Example
21. Cell dry weight was determined as described in Example 25,
except that disposable cleaning cloth (X-tra, 100% viscose
household cleaning cloth, Inex Partners Oy, Helsinki) was used in
vacuum filtration. HPLC analyses were carried out as described in
Example 22.
[0239] Table 14 shows that a transformant containing the genes for
ALD, ACS and PDAT produced 17% more triacylglycerol than Y23, with
8% increase in the yield on glucose consumed, when cells were
cultivated in bioreactor cultures.
TABLE-US-00016 TABLE 14 Triacylglycerol produced in pH controlled
bioreactor cultures of M22 and transformant of M22 expressing ALD +
ACS + PDAT, with glucose as carbon source and C/N 60. Percentage
increase is shown in parenthesis. Yield TAG (% Strain TAG (g/l)
glucose consumed) M22 9.8 21.9 M22/94/98-19 11.5 (17%) 23.6 (8%)
(ALD + ACS + PDAT)
[0240] Table 15 shows that a transformant containing the genes for
ALD, ACS and PDAT produced 44% more lipid than M22, with 55% higher
yield on glucose consumed and 26% increase in yield of lipid on
biomass, when cells were cultivated in bioreactor cultures.
TABLE-US-00017 TABLE 15 Lipid produced in pH controlled bioreactor
cultures of M22 and transformant of M22 expressing ALD + ACS +
PDAT, with glucose as carbon source and C/N 60. Percentage increase
is shown in parenthesis. Yield lipid Yield lipid (% Strain Lipid
(g/l) (% CDW) glucose consumed) M22 10.4 59.4 .+-. 2.8 19.7
M22/94/98-19 14.9 (44%) 75.4 .+-. 3.5 30.5 (55%) (ALD + ACS + PDAT)
(26%)
[0241] This example shows that expression of ALD6, ACS2 and PDAT
genes enhanced triacylglycerol and total lipid concentrations and
triacylglycerol and total lipid yields from used glucose or total
lipid yield per dry weight in high cell density cultures.
Example 33. Production of Triacylglycerol or Lipid by Strain of M.
circinelloides Modified by Addition of Genes Encoding ALD and PDAT
and ACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures
Grown on Xylose with C/N Ratio of 60
[0242] Transformant (M22/94/98-19) was cultivated in Braun Biostat
CT bioreactors as described in Example 32. The medium contained 44
to 56 g xylose per litre, instead of glucose cultures were
supplemented with 1 g peptone per litre and the
(NH.sub.4).sub.2SO.sub.4 concentration was reduced to 1.12 g per
litre. Samples for cell dry weight measurement, lipid extraction
and HPLC analysis were withdrawn periodically during cultivation.
Mucor circinelloides wild type strain M22 was used as the
control.
[0243] Lipid extraction and total lipid and triacylglycerol
concentration measurements were carried out as described in Example
21. Cell dry weight was determined as described in Example 32. HPLC
analyses were carried out as described in Example 22.
[0244] Table 16 shows that a transformant containing genes for ALD,
ACS and PDAT produced 11% more triacylglycerol than M22, with 10%
increased yield on xylose consumed and 9% increased yield on
biomass in pH controlled bioreactor cultures.
TABLE-US-00018 TABLE 16 Triacylglycerol produced in pH controlled
bioreactor cultures of M22 and transformant of M22 expressing ALD +
ACS + PDAT, with xylose as carbon source and C/N 60. Percentage
increase is shown in parenthesis. Yield TAG TAG Yield TAG (% xylose
Strain (g/l) (% CDW) consumed) M22 5.6 46.5 .+-. 2.7 22.9
M22/94/98-19 6.2 50.9 .+-. 1.4 25.1 (ALD + ACS + PDAT) (11%) (9%)
(10%)
[0245] Table 17 shows that a transformant containing the genes for
ALD and ACS and PDAT produced 24% more lipid than M22 and the yield
of lipid on biomass was 22% higher than in M22, when mycelia were
grown on xylose. The yield of lipid on xylose consumed was
increased 18%.
TABLE-US-00019 TABLE 17 Lipid produced in pH controlled bioreactor
culture of M22 and transformant of M22 expressing ALD + ACS + PDAT,
with xylose as carbon source and C/N 60. Percentage increase is
shown in parenthesis. Yield lipid Lipid Yield lipid (% xylose
Strain (g/l) (% CDW) consumed) M22 5.8 49.2 .+-. 4.6 24.1
M22/94/98-19 7.1 58.2 .+-. 0.6 28.7 (ALD + ACS + PDAT) (22%) (18%)
(19%)
[0246] This example shows that expression of ALD6, ACS2 and PDAT
genes enhanced triacylglycerol and total lipid concentrations and
triacylglycerol and total lipid yields from used xylose or per dry
weight in high cell density cultures.
Example 34. Construction of a Plasmid Containing a Marker Gene
Under the Control of an Endogenous Promoter and Terminator and a
Pyruvate Decarboxylase (PDC) Encoding Gene Under the Control of an
Endogenous Promoter and Terminator
[0247] A pyruvate decarboxylase (PDC) encoding gene, such as PDC1
from S. cerevisiae (SEQ ID NO:94) which encodes the amino acid
sequence of SEQ ID NO:95 is codon optimised. The codon optimised
PDC encoding gene with flanking SbfI restriction sites is digested
with SbfI and ligated to a plasmid containing an endogenous
promoter and terminator, such as plasmid pKK77pre. The resulting
plasmid which contains the PDC encoding gene under the control of
the endogenous promoter and terminator will be linearised e.g. with
BamHI and ligated with a fragment containing the marker gene under
the control of the endogenous promoter and terminator. Such
fragment can be obtained e.g. by digesting a plasmid pKK67 with
BamHI and XmnI. The resulting plasmid contains the PDC encoding
gene under the control of the endogenous promoter and terminator
and the marker gene under the control of the endogenous promoter
and terminator.
Example 35. Generation of Genetically Modified Strain with an
Integrated PDC Together with ALD6 and/or ACS2 Encoding Genes and
Marker Genes by Transforming Genetically Modified Strains with
Plasmid Containing PDC Encoding Gene (Ex 34)
[0248] The plasmid containing the PDC encoding gene (Ex. 34) is
restricted e.g. with NotI and PspOMI, and the resulting linear DNA
containing the PDC encoding gene under the control of the
endogenous promoter and terminator and the marker gene under the
control of the endogenous promoter and terminator is used to
transform e.g. a genetically modified strain Y23/81-51 (Ex. 3B),
Y23/86-92 (Ex. 5B) or Y23/85-128 (Ex. 7B) by electroporation or a
genetically modified strain M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B)
or M22/94-31 (Ex 16B) using the transformation method described in
Example 15B. The transformed cells are screened e.g. for antibiotic
resistance. Several transformed colonies are analysed at DNA level
by PCR.
Example 36. Aerobic Shake Flask Characterization of Strains
Harbouring PDC Together with ALD6 and/or ACS2 Encoding Genes and
Marker Genes in Glucose or Xylose Medium with Different C/N
Ratios
[0249] Transformants are separately cultivated in 50 ml of culture
medium such as described in Examples 22, 23, 24, 27 or 28. Each
flask (250 ml) is inoculated to an OD.sub.600 of 0.3 with cells
grown on yeast peptone plus glucose plates. The cultivations are
maintained at a temperature of 30.degree. C. with shaking at 250
rpm. Samples for cell dry weight measurement, lipid extraction and
HPLC analysis are withdrawn periodically during cultivation.
Cryptococcus curvatus wild type strain Y23 is used as a control.
Cell dry weight is determined and HPLC analysis is carried out as
described in Example 22. Lipid extraction and triacylglycerol
measurements as described in Example 21. Alternatively
transformants are separately cultivated in 50 ml of culture medium
such as described in Examples 25, 26 or 29. Each flask (250 ml) is
inoculated with 1*10.sup.7 spores. The cultivations are maintained
at a temperature of 25.degree. C. with shaking at 250 rpm. Samples
for cell dry weight measurement, lipid extraction and HPLC analysis
are withdrawn periodically during cultivation. Mucor circinelloides
wild type strain M22 is used as a control. Cell dry weight is
determined as described in Example 25 and HPLC analysis is carried
out as described in Example 22. Lipid extraction and
triacylglycerol measurements as described in Example 21.
[0250] The transformants harbouring PDC together with ALD6 and/or
ACS2 encoding genes produce more triacylglycerol than the control
strain. Additionally the transformants harbouring PDC together with
ALD6 and/or ACS2 encoding gene have higher triacylglycerol yield on
used carbon than the control strain.
Example 37A. Construction of a Plasmid (pKK101) Containing the G418
Resistance Gene Under the Control of the CcTEF1 Promoter and the
CcTPI1 Terminator and the S. cerevisiae PDC1 Encoding Gene Under
the Control of the CcTPI1 Promoter and the CcTEF1 Terminator
[0251] The plasmid Y4_TPIp-PDC-TEFt (Geneart AG, Germany) contains
a S. cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been
codon optimized according to Ustilago maydis yeast codon usage (SEQ
ID NO:96) with the CcTPI1 promoter and the CcTEF1 terminator.
Plasmid Y4_TPIp-PDC-TEFt was digested with EcoRI. A 2893 bp
fragment was gel isolated and ligated to a 5381 bp fragment
obtained by digesting a plasmid designated pKK67 (Ex. 2B) with
EcoRI. The resulting plasmid was designated as pKK101 (FIG. 12).
The plasmid pKK101 contains a S. cerevisiae PDC1 encoding gene
under the control of the CcTPI1 promoter and the CcTEF1 terminator
and the E. coli G418 resistance gene under the control of the
CcTEF1 promoter and the CcTPI1 terminator.
Example 37B. Generation of Genetically Modified Strains with an
Integrated PDC Encoding Gene and G418 Resistance Gene by
Transforming Wild-Type C. curvatus and Genetically Modified Strains
Y23/81-66 (Ex. 3B), Y23/85-125 (Ex. 7B), Y23/86-86 (Ex. 5B),
Y23/95-99 (Ex. 6B), Y23/81/95-18 (Ex. 8) and Y23/85/95-4 (Ex. 9)
with Digested Plasmid pKK101 (FIG. 12, Ex. 37A)
[0252] Plasmid pKK101 was restricted with SacII and PspOMI, and the
resulting linear DNA was used to transform wild-type C. curvatus
strain ATCC20509 designated as Y23 and genetically modified strains
Y23/81-66, Y23/85-125, Y23/86-86, Y23/95-99, Y23/81/95-18 and
Y23/85/95-4 by electroporation. The transformed cells were screened
for G418 resistance. Several G418 resistance colonies were analysed
at DNA level by PCR. The transformants originating from the
transformation of wild-type C. curvatus strain ATCC20509 designated
as Y23 and genetically modified strains Y23/81-66, Y23/85-125,
Y23/86-86, Y23/95-99, Y23/81/95-18 and Y23/85/95-4 with SacII and
PspOMI cut pKK101 and containing the S. cerevisiae PDC1 encoding
gene under the control of the CcTPI1 promoter and the CcTEF1
terminator were designated as Y23/101-55, Y23/101-57, Y23/101-59,
Y23/81/101-4, Y23/85/101-13, Y23/85/101-14, Y23/85/101-19,
Y23/86/101-23, Y23/95/101-1, Y23/95/101-2, Y23/81/95/101-20,
Y23/85/95/101-7 and Y23/85/95/101-8.
Example 38A. Construction of a Plasmid (pKK102) Containing the
Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter
and the McTPI1 Terminator and the S. cerevisiae PDC1 Encoding Gene
Under the Control of the McPGK1 Promoter and the McTEF1
Terminator
[0253] The plasmid M22_PGKp-PDC-TEFt (Geneart AG, Germany) contains
a S. cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been
codon optimized according to Rhizopus oryzae filamentous fungus
codon usage (SEQ ID NO:97) with the McPGK1 promoter and the McTEF1
terminator. Plasmid M22_PGKp-PDC-TEFt was digested with SalI. A
3363 bp fragment was gel isolated and ligated to a 6239 bp fragment
obtained by digesting a plasmid designated pKK92 (Ex. 14B) with
SalI. The resulting plasmid was designated as pKK102 (FIG. 13). The
plasmid pKK102 contains a S. cerevisiae PDC1 encoding gene under
the control of the McPGK1 promoter and the McTEF1 terminator and
the g cerevisiae cerulenin resistance gene under the control of the
McPGK1 promoter and the McTPI1 terminator.
Example 38B. Generation of a Genetically Modified Mucor
Circinelloides (M22/94/102) with Integrated ALD6, ACS1 and PDC1
Encoding Genes and Hygromycin and Cerulenin Resistance Genes by
Transforming Genetically Modified Strain M22/94-12 (Ex. 16B) with
Digested Plasmid pKK102 (FIG. 13, Ex. 38A)
[0254] Plasmid pKK102 was restricted with SacII and PspOMI, and the
resulting linear DNA was used to transform the genetically modified
strain M22/94-12, using the transformation method described in
Example 15B. The transformed cells were screened for cerulenin
resistance. Several cerulenin resistant colonies were analysed at
DNA level by PCR. The transformant originating from the
transformation of the genetically modified strain M22/94-12 with
SacII and PspOMI cut pKK102 and containing the S. cerevisiae PDC1
encoding gene under the control of the McPGK1 promoter and the
McTEF1 terminator was designated as M22/94/102-31.
Example 39. Aerobic Shake Flask Characterization of Strains
Y23/101-59 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-14 (Ex.
37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B),
Y23/81/95/101-20 (Ex. 37B) and Y23/85/95/101-9 (Ex. 37B) in Glucose
Medium with C/N Ratio of 153
[0255] Transformants were separately cultivated in 50 ml of
Glucose-CN153 medium (pH 5.5, 30 g glucose, 0.15 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction and triacylglycerol
measurements as described in Example 21. Lipid extractions from the
culture medium i.e. from the supernatant samples recovered after
centrifugation of the cell lipid extraction samples or dry weight
measurement samples were carried out as follows. To 150 .mu.l of
supernatant sample 150 .mu.l of 0.9% NaCl and 150-1500 .mu.l of
chloroform:methanol (2:1) was added. The sample was vortexed 2
minutes and sample was incubated at room temperature at 30 min.
After incubation sample was centrifuged 10 000 rpm for 3 min at RT.
The lower phase was recovered into microfuge tubes, dried and
redissolved in 1.5 ml of chloroform:methanol (2:1) and stored at
-20.degree. C. prior triacylglycerol measurements as described in
Example 21. Alternatively, dried lipid pellet was redissolved
directly in isopropanol and triacylglycerol was measured as
described in Example 21.
[0256] After 47 hours cultivation (Table 18), when 17 to 18 g/l
glucose was left, Y23/101-59, Y23/85/101-14, Y23/86/101-23,
Y23/95/101-2, Y23/81/95/101-20 and Y23/85/95/101-8 transformants
produced 52, 78, 9, 27, 32 and 48% more triacylglycerol with higher
rate, respectively, than the control strain in glucose medium. Also
triacylglycerol yields on biomass and per used glucose were 33 to
83% and 35 to 85% higher in transformants Y23/101-59,
Y23/85/101-14, Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 and
Y23/85/95/101-8 than the control strain, respectively.
TABLE-US-00020 TABLE 18 Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in
the yeast cells after 47 hours cultivation in glucose medium with
C/N ratio of 153 Yield TAG TAG Yield TAG (% used TAG Strain (g/l)
(% CDW) glucose) mg/l/h Control 1.30 22.5 10.9 28 Y23/101-59 (PDC)
1.98 37.1 16.3 42 Y23/85/101-14 2.31 41.1 20.2 49 (PDC + ALD + ACS)
Y23/86/101-23 1.42 29.8 15.4 30 (PDC + ACS) Y23/95/101-2 1.65 30.9
14.7 35 (PDC + PDAT) Y23/81/95/101-20 1.72 36.4 16.4 37 (PDC + ALD
+ PDAT) Y23/85/95/101-8 1.93 36.6 18.1 41 (PDC + ALD + ACS +
PDAT)
[0257] After 94 hours cultivation (Table 19A), when 6 to 11 g/l
glucose was left, Y23/101-59, Y23/85/101-14, Y23/81/95/101-20 and
Y23/85/95/101-8 transformants produced 5, 27, 8 and 12% more
triacylglycerol with higher rate, respectively, than the control
strain in glucose medium. Also triacylglycerol yields on biomass
and per used glucose were 9 to 33% and 7 to 38% higher in
transformants Y23/101-59, Y23/81/101-4, Y23/85/101-14,
Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 and Y23/85/95/101-8
than the control strain, respectively. Additionally,
triacylglycerol concentration in the culture medium in the
cultivations with the transformants Y23/81/101-4 and Y23/86/101-23
was 525 and 350% higher, respectively, than in the cultivation with
the control strain (Table 19B). Additionally, the total
triacylglycerol yields per used glucose calculated from the
intracellular triacylglycerol concentration and the triacylglycerol
concentration detected from the culture medium were 13 to 38%
higher with the transformants than with the control strain.
TABLE-US-00021 TABLE 19A Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in
the yeast cells after 94 hours cultivation in glucose medium with
C/N ratio of 153 Yield TAG TAG Yield TAG (% used TAG Strain (g/l)
(% CDW) glucose) mg/l/h Control 4.06 43.4 17.4 43 Y23/101-59 (PDC)
4.28 51.6 19.9 46 Y23/81/101-4 3.39 55.7 18.6 36 (PDC + ALD
Y23/85/101-14 5.17 57.9 22.7 55 (PDC + ALD + ACS) Y23/86/101-23
4.00 53.4 21.1 43 (PDC + ACS) Y23/95/101-2 3.50 47.5 20.1 37 (PDC +
PDAT) Y23/81/95/101-20 4.38 62.0 24.0 47 (PDC + ALD + PDAT)
Y23/85/95/101-8 4.55 54.4 21.5 48 (PDC + ALD + ACS + PDAT)
TABLE-US-00022 TABLE 19B Triacylglycerol (TAG) concentration (g/l)
in the culture medium and calculated total TAG concentration (g/l),
rate (mg/l/h) and yield (%) per used glucose in cultivation after
94 hours cultivation in glucose medium with C/N ratio of 153 Yield
total TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l)
glucose) mg/l/h Control 0.04 4.10 17.6 44 Y23/101-59 (PDC) 0.01
4.29 20.0 46 Y23/81/101-4 0.25 3.63 19.9 39 (PDC + ALD
Y23/85/101-14 0.03 5.20 22.8 55 (PDC + ALD + ACS) Y23/86/101-23
0.18 4.18 22.1 44 (PDC + ACS) Y23/95/101-2 0.01 3.51 20.2 37 (PDC +
PDAT) Y23/81/95/101-20 0.03 4.42 24.2 47 (PDC + ALD + PDAT)
Y23/85/95/101-8 0.04 4.6 21.7 49 (PDC + ALD + ACS + PDAT)
[0258] This example shows that expression of PDC1, ALD6, ACS2 and
PDAT genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yields
from used glucose or per dry weight in the yeast cell and/or in the
culture medium with high C/N ratio in different stages of the
cultivation.
Example 40. Aerobic Shake Flask Characterization of Strains
Y23/101-55 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-19 (Ex.
37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B) and
Y23/85/95/101-7 (Ex. 37B) in Glucose Medium with C/N Ratio of
28
[0259] Transformants were separately cultivated in 50 ml of
Glucose-CN28 medium (pH 5.5, 30 g glucose, 0.3 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus glucose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction from the yeast cells and
triacylglycerol measurements were carried out as described in
Example 21 and lipid extraction from the culture medium as
described in Example 39.
[0260] After 46 hours cultivation (Table 20A), when 12 to 14 g/l
glucose was left, Y23/101-55, Y23/81/101-4, Y23/85/101-19,
Y23/86/101-23, Y23/95/101-2 and Y23/85/95/101-7 transformants
produced 18, 17, 20, 24, 7 and 17% more triacylglycerol with higher
rate, respectively, than the control strain in glucose medium. Also
triacylglycerol yields on biomass and per used glucose were 10 to
35% and 7 to 26% higher with the transformants than the control
strain, respectively. After 72 and 94 hours cultivation
triacylglycerol concentration in the culture medium with the
transformants Y23/81/101-4 and Y23/86/101-23 was 195 and 56% and 35
and 30% higher, respectively, than in the cultivations with the
control strain (Table 20B). Additionally, yield of triacylglycerol
detected in culture medium per used glucose was 57 to 206% and 36
to 56% higher with the transformants Y23/81/101-4 and Y23/86/101-23
than with the control after 72 and 94 hours cultivation,
respectively.
TABLE-US-00023 TABLE 20A Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in
the yeast cells after 46 hours cultivation in glucose medium with
C/N ratio of 28 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (%
CDW) glucose) mg/l/h Control 3.10 46.6 20.6 67 Y23/101-55 (PDC)
3.67 51.2 24.4 80 Y23/81/101-4 3.64 53.5 22.1 79 (PDC + ALD
Y23/85/101-19 3.71 53.8 23.2 81 (PDC + ALD + ACS) Y23/86/101-23
3.84 63.1 26.0 83 (PDC + ACS) Y23/95/101-2 3.31 54.7 23.3 72 (PDC +
PDAT) Y23/85/95/101-7 3.64 58.5 26.3 79 (PDC + ALD + ACS +
PDAT)
TABLE-US-00024 TABLE 20B Triacylglycerol (TAG) concentration (g/l)
and yield (%) per used glucose in the culture medium after 72 and
94 hours cultivation in glucose medium with C/N ratio of 28 72 h
Yield TAG % 94 h Yield TAG TAG (/used TAG (% used Strain (g/l)
glucose) (g/l) glucose) Control 0.85 3.19 2.79 9.84 Y23/81/101-4
2.51 9.75 4.35 15.4 (PDC + ALD) Y23/86/101-23 1.15 5.00 3.62 13.4
(PDC + ACS)
[0261] This example shows that expression of PDC1, ALD6, ACS2 and
PDAT genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yields
from used glucose or per dry weight in the yeast cell and/or in the
culture medium with low C/N ratio at different stages of the
cultivation.
Example 41. Aerobic Shake Flask Characterization of Strains
Y23/101-57 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-13 (Ex.
37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-1 (Ex. 37B),
Y23/81/95/101-20 (Ex. 37B) and Y23/85/95/101-8 (Ex. 37B) in Xylose
Medium with C/N Ratio of 103
[0262] Transformants were separately cultivated in 50 ml of Yeast
culture medium IV (pH 5.5, 20 g xylose, 0.15 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus xylose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction from the yeast cells and
triacylglycerol measurements were carried out as described in
Example 21 and lipid extraction from the culture medium as
described in Example 39.
[0263] After 47 hours cultivation (Table 21) Y23/101-57,
Y23/81/101-4, Y23/85/101-13 and Y23/95/101-1 transformants produced
12, 18, 4 and 5% more triacylglycerol with higher rate,
respectively, than the control strain in xylose medium. Also
triacylglycerol yields on biomass and per used xylose were up to
28% and 16% higher with the transformants Y23/101-57, Y23/81/101-4,
Y23/85/101-13, Y23/86/101-23 and Y23/95/101-1 than the control
strain, respectively. Also total lipid concentration, rate and
yields on biomass and per used xylose were 8 to 29%, 8 to 29%, 19
to 44% and 11 to 33% higher with the transformants than the control
strain, respectively, after 47 hours cultivation in xylose
medium.
TABLE-US-00025 TABLE 21 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW) and used xylose in the yeast cells after 47 hours cultivation
in xylose medium with C/N ratio of 103 Yield TAG TAG Yield TAG (%
used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 1.90 38.5 16.0
41 Y23/101-57 (PDC) 2.12 44.7 18.0 45 Y23/81/101-4 2.24 49.2 18.6
48 (PDC + ALD Y23/85/101-13 1.98 44.3 18.1 42 (PDC + ALD + ACS)
Y23/86/101-23 1.85 47.7 18.3 39 (PDC + ACS) Y23/95/101-1 2.00 38.5
16.5 43 (PDC + PDAT) Yield lipid Lipid Yield lipid (% used Lipid
Strain (g/l) (% CDW) xylose) mg/l/h Control 2.40 48.5 20.2 51
Y23/101-57 (PDC) 2.75 57.9 23.3 59 Y23/81/101-4 2.70 59.3 22.4 57
(PDC + ALD Y23/85/101-13 2.60 58.1 23.7 55 (PDC + ALD + ACS)
Y23/86/101-23 2.70 69.7 26.8 57 (PDC + ACS) Y23/95/101-1 3.10 59.6
25.6 66 (PDC + PDAT)
[0264] After 94 hours cultivation (Table 22A) Y23/101-57,
Y23/81/101-4, Y23/85/101-13, Y23/86/101-23, Y23/95/101-1,
Y23/81/95/101-20 and Y23/85/95/101-8 transformants produced 7, 9,
8, 7, 6, 6 and 4% more triacylglycerol with higher rate,
respectively, than the control strain in xylose medium. Also
triacylglycerol yields on biomass and per used xylose were 4 to 25%
and 5 to 17% higher with the transformants than the control strain,
respectively. Triacylglycerol concentration in the culture medium
with the transformants Y23/81/101-4 and Y23/86/101-23 was 222 and
156% higher, respectively, than in the cultivations with the
control strain (Table 22B). Additionally, the total triacylglycerol
yields per used xylose calculated from the intracellular
triacylglycerol concentration and the triacylglycerol concentration
detected from the culture medium were 7 to 17% higher with the
transformants than with the control strain.
TABLE-US-00026 TABLE 22A Triacylglycerol (TAG) concentration (g/l),
rate (mg/l/h) and yield (%) per biomass (CDW) and used xylose in
the yeast cells after 94 hours cultivation in xylose medium with
C/N ratio of 103 Yield TAG TAG Yield TAG (% used TAG Strain (g/l)
(% CDW) xylose) mg/l/h Control 4.50 72.0 23.9 48 Y23/101-57 (PDC)
4.82 79.4 25.6 51 Y23/81/101-4 4.92 84.2 26.1 52 (PDC + ALD
Y23/85/101-13 4.85 75.1 25.7 52 (PDC + ALD + ACS) Y23/86/101-23
4.82 88.5 25.6 51 (PDC + ACS) Y23/95/101-1 4.79 79.8 25.7 51 (PDC +
PDAT) Y23/81/95/101-20 4.78 89.7 27.9 51 (PDC + ALD + PDAT)
Y23/85/95/101-8 4.66 80.4 25.2 50 (PDC + ALD + ACS + PDAT)
TABLE-US-00027 TABLE 22B Triacylglycerol (TAG) concentration (g/l)
in the culture medium and calculated total TAG concentration (g/l),
rate (mg/l/h) and yield (%) per used xylose in cultivation after 94
hours cultivation in xylose medium with C/N ratio of 103 Yield
total TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l)
xylose) mg/l/h Control 0.09 4.59 24.3 49 Y23/101-57 (PDC) 0.07 4.89
26.0 52 Y23/81/101-4 0.29 5.22 27.7 55 (PDC + ALD Y23/85/101-13
0.10 4.94 26.2 53 (PDC + ALD + ACS) Y23/86/101-23 0.23 5.05 26.8 54
(PDC + ACS) Y23/95/101-1 0.07 4.86 26.1 52 (PDC + PDAT)
Y23/81/95/101-20 0.09 4.86 28.4 52 (PDC + ALD + PDAT)
Y23/85/95/101-8 0.10 4.76 25.7 51 (PDC + ALD + ACS + PDAT)
[0265] This example shows that expression of PDC1, ALD6, ACS2 and
PDAT genes in different combinations enhanced triacylglycerol and
total lipid concentrations and rates of production and
triacylglycerol and total lipid yields from used xylose or per dry
weight in the yeast cell and/or in the culture medium with high C/N
ratio in different stages of the cultivation.
Example 42. Aerobic Shake Flask Characterization of Strains
Y23/101-57 (Ex. 37B), Y23/85/101-13 (Ex. 37B) and Y23/81/95/101-20
(Ex. 37B) in Xylose Medium with C/N Ratio of 20
[0266] Transformants were separately cultivated in 50 ml of
Xylose-CN20 medium (pH 5.5, 20 g xylose, 0.3 g
(NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g
Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g
Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1
mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was
inoculated to an OD.sub.600 of 0.3 with cells grown on yeast
peptone plus xylose plates. The cultivations were maintained at a
temperature of 30.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as a control. Cell dry
weight was determined and HPLC analysis was carried out as
described in Example 22. Lipid extraction from the yeast cells and
triacylglycerol measurements were carried out as described in
Example 21 and lipid extraction from the culture medium as
described in Example 39.
[0267] After 94 hours cultivation (Table 23A) transformants
Y23/101-57 and Y23/85/101-13 produced 9 and 5% more triacylglycerol
with higher rate, respectively, than the control strain in xylose
medium. Also triacylglycerol yields on biomass and per used xylose
were 2 to 10% and 4 to 8% higher with the transformants than the
control strain, respectively. Triacylglycerol concentration in the
culture medium with the transformants Y23/101-57, Y23/85/101-13 and
Y23/81/95/101-20 was 163, 163 and 188% higher, respectively, than
in the cultivations with the control strain (Table 21B).
Additionally, the total triacylglycerol yields per used xylose
calculated from the intracellular triacylglycerol concentration and
the triacylglycerol concentration detected from the culture medium
were 4 to 12% higher with the transformants than with the control
strain.
TABLE-US-00028 TABLE 23A Triacylglycerol (TAG) concentration (g/l)
and yield (%) per biomass (CDW) and used xylose in the yeast cells
after 94 hours cultivation in xylose medium with C/N ratio of 20
Yield TAG TAG Yield TAG (% used Strain (g/l) (% CDW) xylose)
Control 4.21 68.7 21.1 Y23/101-57 (PDC) 4.57 75.8 22.8
Y23/85/101-13 4.41 69.9 22.0 (PDC + ALD + ACS)
TABLE-US-00029 TABLE 23B Triacylglycerol (TAG) concentration (g/l)
in the culture medium and calculated total TAG concentration (g/l),
rate (mg/l/h) and yield (%) per used xylose in cultivation after 94
hours cultivation in xylose medium with C/N ratio of 20 Yield total
TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l) xylose)
mg/l/h Control 0.08 4.28 21.4 46 Y23/101-57 (PDC) 0.21 4.78 23.9 51
Y23/85/101-13 0.21 4.61 23.1 49 (PDC + ALD + ACS) Y23/81/95/101-20
0.23 4.45 22.3 47 (PDC + ALD + PDAT)
[0268] This example shows that expression of PDC1, ALD6, ACS2 and
PDAT genes in different combinations enhanced triacylglycerol
concentrations and rates of production and triacylglycerol yields
from used xylose or per dry weight in the yeast cell and/or in the
culture medium with low C/N ratio.
Example 43. Aerobic Shake Flask Characterization of Strains
M22/94-16 (Ex. 16B) and M22/94/102-31 (Ex. 38B), in Xylose Medium
with C/N Ratio of 21
[0269] Transformants were separately cultivated in 50 ml of mould
C/N 21 medium (pH 5.5, 10 g glucose, 1.4 g yeast extract, 2.5 g
KH.sub.2PO.sub.4, 0.3 g (NH.sub.4).sub.2SO.sub.4, 10 mg
ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4,
0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg
FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated
with 1*10.sup.7 spores. The cultivations were maintained at a
temperature of 28.degree. C. with shaking at 250 rpm. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Mucor
circinelloides wild type strain M22 was used as a control. Cell dry
weight was determined as described in Example 25 and HPLC analysis
was carried out as described in Example 22. Lipid extraction and
total lipid and triacylglycerol measurements as described in
Example 21.
[0270] After 96 hours cultivation (Table 24) transformants
M22/94-16 and M22/94/102-31 produced 18 and 30% more
triacylglycerol with higher rate, respectively, than the control
strain in xylose medium. Also triacylglycerol yields on biomass and
per used xylose were 10 to 34% and 1 to 24% higher with the
transformants than the control strain, respectively. Also total
lipid concentration and yield on biomass were 15 to 16% and 6 to
33% higher in the transformants than in the control.
TABLE-US-00030 TABLE 24 Triacylglycerol (TAG) and total lipid
concentrations (g/l), rates (mg/l/h) and yields (%) per biomass
(CDW; cell dry weight) and used xylose after 96 hours cultivation
in xylose medium with C/N ratio of 21 Yield TAG TAG Yield TAG (%
used TAG Strain g/l (% CDW) xylose mg/l/h Control 0.60 34.9 13.9 6
M22/94-16 0.71 38.4 14.0 7 (ALD + ACS) M22/94/102-31 0.78 53.2 17.2
8 (PDC + ALD + ACS) Yield lipid Lipid Yield lipid (% used Lipid
Strain g/l (% CDW) xylose mg/l/h Control 0.81 47.6 18.9 8 M22/94-16
0.94 50.5 18.5 10 (ALD + ACS) M22/94/102-31 0.93 63.5 20.5 10 (PDC
+ ALD + ACS)
[0271] This example shows that expression of PDC1, ALD6 and ACS2
genes in different combinations enhanced triacylglycerol and total
lipid concentrations and rates of production and triacylglycerol
and total lipid yields from used xylose or per dry weight in the
yeast cell culture me with low C/N ratio.
Example 44. Production of Triacylglycerol or Lipid by Strains of C.
curvatus Modified by Addition of Genes Encoding PDC and ALD and
PDAT and ACS (Y23/85/95/101-8, Ex. 37B) in High Cell Density
Cultures Grown on Glucose with C/N Ratio of 28
[0272] Transformant (Y23/85/95/101-8) was cultivated in Multifors
bioreactors (max. working volume 500 ml, Infors HT, Switzerland) at
pH 4.0, 30.degree. C., in 500 ml medium containing 90 to 96 g
glucose, 6.74 g (NH.sub.4).sub.2SO.sub.4, 1.2 g KH.sub.2PO.sub.4,
0.3 g Na.sub.2HPO.sub.4.2H.sub.2O, 1.5 g MgSO.sub.4.7H.sub.2O, 0.1
g CaCl.sub.2.6H.sub.2O, 5.26 mg citric acid.H.sub.2O, 5.26 mg
ZnSO.sub.4.7H.sub.2O, 0.1 mg MnSO.sub.4.4H.sub.2O, 0.5 mg
CoCl.sub.2.6H.sub.2O, 0.26 mg CuSO.sub.4.5H.sub.2O, 0.1 mg
Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg FeSO.sub.4.7H.sub.2O, 0.1 mg
H.sub.3BO.sub.4, 0.05 mg D-biotin, 1.0 mg CaPantothenate, 5.0 mg
nicotinic acid, 25 mg myoinositol, 1.0 mg thiamine.HCl, 1.0 mg
pyridoxine.HCl and 0.2 mg p-aminobenzoic acid per litre. The pH was
maintained constant by addition of 1 M KOH or 1 M H.sub.2PO.sub.4.
Cultures were agitated at 1000 rpm (2 Rushton turbine impellors)
and aerated at 2 volumes air per volume culture per minute (vvm).
Clerol FBA 3107 antifoaming agent (Cognis, SaintFargeau-Ponthierry
Cedex France, 1 ml l.sup.-1) was added to prevent foam
accumulation. Bioreactors were inoculated to initial OD.sub.600 of
0.5 to 4.0 with cells grown in the same medium (substituting 1.5 g
urea per litre for (NH.sub.4).sub.2SO.sub.4 and omitting the
CaCl.sub.2.6H.sub.2O) in 50 ml volumes in 250 ml flasks at
30.degree. C. with shaking at 200 rpm for 24 to 42 h. Samples for
cell dry weight measurement, lipid extraction and HPLC analysis
were withdrawn periodically during cultivation. Cryptococcus
curvatus wild type strain Y23 was used as the control.
[0273] Lipid extraction and triacylglycerol concentration
measurements were carried out as described in Example 21. Lipid
extraction from the culture medium was carried out as described in
Example 39. Cell dry weight was determined as described in Example
30. HPLC analyses were carried out as described in Example 22.
[0274] Table 25A shows that a transformant containing the genes
PDC, ALD, ACS and PDAT produced 58% more triacylglycerol than Y23,
with a 67% and 185% increase in the yields on glucose consumed and
on biomass, respectively. Additionally, the transformant containing
the genes PDC, ALD, ACS and PDAT produced 20% more triacylglycerol
than Y23, with a 111% increase in the yield on glucose consumed in
the culture medium (Table 25B).
TABLE-US-00031 TABLE 25A Triacylglycerol produced in pH controlled
bioreactor culture of Y23 and transformant of Y23 expressing PDC +
ALD + ACS + PDAT, with glucose as carbon source and C/N 28. Data is
the average of 2 cultures .+-. standard error of the mean.
Percentage increase is shown in parenthesis. Yield TAG TAG (%
glucose Yield TAG Strain (g/l) consumed) (% per CDW) Y23 1.30 .+-.
0.06 1.22 .+-. 0.01 2.67 .+-. 0.14 Y23/85/95/101-8 2.05 .+-. 0.34
2.04 .+-. 0.22 4.95 .+-. 0.33 (PDC + ALD + ACS + (+58%) (+67%)
(+185%) PDAT)
TABLE-US-00032 TABLE 25B Triacylglycerol produced in the culture
medium in pH controlled bioreactor culture of Y23 and transformant
of Y23 expressing PDC + ALD + ACS + PDAT, with glucose as carbon
source and C/N 28. Data is the average of 2 cultures .+-. standard
error of the mean. Percentage increase is shown in parenthesis.
Yield TAG TAG (% glucose Strain (g/l) consumed) Y23 0.15 .+-. 0.04
0.18 .+-. 0.03 Y23/85/95/101-8 0.18 .+-. 0.00 0.38 .+-. 0.07 (PDC +
ALD + ACS + (+20%) (+111%) PDAT)
[0275] This example shows that expression of PDC1, ALD6, ACS1 and
PDAT enhanced triacylglycerol concentrations and triacylglycerol
yields from used glucose or per dry weight in high cell density
cultures in the yeast cell and/or in the culture medium.
Sequences used:
[0276] SEQ ID NOs: 1 and 2 correspond to primers YeastTEF1 and
YeastTEF4, respectively, used to isolate genomic fragment of the C.
curvatus TEF gene.
[0277] SEQ ID NOs: 3 and 4 correspond to primers PCR linker I and
PCR linker II, respectively, used in chromosome walk
experiments.
[0278] SEQ ID NOs: 5, 6, 7 and 8 correspond to primers CC_TEF2,
CC_TEF1, CC_TEF6 and CC_TEF5 respectively, used to isolate genomic
fragments of the C. curvatus TEF promoter region in chromosome walk
experiments.
[0279] SEQ ID NOs: 9 and 10 correspond to primers CC_TEF10 and
CC_TEF11, respectively, used to isolate promoter of the C. curvatus
TEF gene.
[0280] SEQ ID NOs: 11 and 12 correspond to primers CC_TEF3 and
CC_TEF4, respectively, used to isolate genomic fragment of the C.
curvatus TEF terminator region in chromosome walk experiments.
[0281] SEQ ID NOs: 13 and 14 correspond to primers CC_TEF7 and
CC_TEF8, respectively, used to isolate terminator of the C.
curvatus TEF gene.
[0282] SEQ ID NOs: 15 and 16 correspond to primers Yeast TPI5 and
Yeast TPI8, respectively, used to isolate genomic fragment of the
C. curvatus TPI gene.
[0283] SEQ ID NOs: 17 and 18 correspond to primers CC_TPI2 and
CC_TPI1, respectively, used to isolate genomic fragment of the C.
curvatus TPI promoter region in chromosome walk experiments.
[0284] SEQ ID NOs: 19 and 20 correspond to primers CC_TPI7 and
CC_TPI9, respectively, used to isolate promoter of the C. curvatus
TPI gene.
[0285] SEQ ID NOs: 21 and 22 correspond to primers CC_TPI4 and
CC_TPI3, respectively, used to isolate genomic fragment of the C.
curvatus TPI terminator region in chromosome walk experiments.
[0286] SEQ ID NOs: 23 and 24 correspond to primers CC_TPI5 and
CC_TPI6, respectively, used to isolate terminator of the C.
curvatus TPI gene.
[0287] SEQ ID NOs: 25 and 26 correspond to primers YeastENO5 and
YeastENO10, respectively, used to isolate genomic fragment of the
C. curvatus ENO gene.
[0288] SEQ ID NOs: 27, 28, 29 and 30 correspond to primers CC_ENO2,
CC_ENO1, CC_ENO5 and CC_ENO6, respectively, used to isolate genomic
fragments of the C. curvatus ENO promoter region in chromosome walk
experiments.
[0289] SEQ ID NOs: 31 and 32 correspond to primers CC_ENO9 and
CC_ENO10, respectively, used to isolate promoter of the C. curvatus
ENO gene.
[0290] SEQ ID NOs: 33 and 34 correspond to primers CC_ENO4 and
CC_ENO3, respectively, used to isolate genomic fragment of the C.
curvatus ENO terminator region in chromosome walk experiments.
[0291] SEQ ID NOs: 35 and 36 correspond to primers CC_ENO7 and
CC_ENO8, respectively, used to isolate terminator of the C.
curvatus ENO gene.
[0292] SEQ ID NOs: 37 and 38 correspond to primers CC_GPD3 and
CC_GPD4, respectively, used to isolate genomic fragment of the C.
curvatus GPD terminator region in chromosome walk experiments.
[0293] SEQ ID NOs: 39 and 40 correspond to primers CC_GPD6 and
CC_GPD7, respectively, used to isolate terminator of the C.
curvatus GPD gene.
[0294] SEQ ID NOs: 41 and 42 correspond to primers Hph 5 and Hph 3,
respectively, used to isolate E. coli hygromycin gene.
[0295] SEQ ID NOs: 43 and 44 correspond to primers Kan 5 and Kan 3,
respectively, used to isolate E. coli G418 resistance gene.
[0296] SEQ ID NOs: 45 and 46 correspond to primers CERR 5 and CERR
3, respectively, used to isolate S. cerevisiae cerulenin resistance
gene.
[0297] SEQ ID NO: 47 corresponds to the amino acid sequence of the
S. cerevisiae ALD6 gene, with GenBank accession number AAB68304
(version number AAB68304.1).
[0298] SEQ ID NO: 48 corresponds to S. cerevisiae ALD6 protein
encoding DNA codon optimized according to Ustilago maydis-fungus
codon usage.
[0299] SEQ ID NO: 49 corresponds to S. cerevisiae ALD6 protein
encoding DNA codon optimized according to Rhizopus
oryzae-filamentous fungus codon usage.
[0300] SEQ ID NO: 50 corresponds to the amino acid sequence of the
S. cerevisiae ACS2 gene, with GenBank accession number CAA97725
(version number CAA97725.1.
[0301] SEQ ID NO: 51 corresponds to S. cerevisiae ACS2 protein
encoding DNA codon optimized according to Ustilago maydis-fungus
codon usage.
[0302] SEQ ID NO: 52 corresponds to the amino acid sequence of the
Rhizopus oryzae PDAT gene, encoded by gene with locus number
RO3G_07851.3 in Broad Institute Rhizopus oryzae database.
[0303] SEQ ID NO: 53 corresponds to Rhizopus oryzae PDAT protein
encoding DNA codon optimized according to Ustilago maydis-fungus
codon usage.
[0304] SEQ ID NOs: 54 and 55 correspond to primers Mould TPI1 and
mould TPI3, respectively, used to isolate genomic fragment of the
Mucor circinelloides TPI gene.
[0305] SEQ ID NOs: 56 and 57 correspond to primers MC_TPI2 and
MC_TPI1, respectively, used to isolate genomic fragment of the
Mucor circinelloides TPI promoter region in chromosome walk
experiments.
[0306] SEQ ID NOs: 58 and 59 correspond to primers MC_TPI7 and
MC_TPI8, respectively, used to clone promoter of the Mucor
circinelloides TPI gene.
[0307] SEQ ID NOs: 60 and 61 correspond to primers MC_TPI4 and
MC_TPI3, respectively, used to isolate genomic fragment of the
Mucor circinelloides TPI terminator region in chromosome walk
experiments.
[0308] SEQ ID NOs: 62 and 63 correspond to primers MC_TPI5 and
MC_TPI6, respectively, used to clone terminator of the Mucor
circinelloides TPI gene.
[0309] SEQ ID NOs: 64 and 65 correspond to primers Mould TEF1 and
Mould TEF4, respectively, used to isolate genomic fragment of the
Mucor circinelloides TEF gene.
[0310] SEQ ID NOs: 66, 67, 68 and 69 correspond to primers MC_TEF2,
MC_TEF1, MC_TEF6 and MC_TEF5, respectively, used to isolate genomic
fragments of the Mucor circinelloides TEF promoter region in
chromosome walk experiments.
[0311] SEQ ID NOs: 70 and 71 correspond to primers MC_TEF9 and
MC_TEF10, respectively, used to clone promoter of the Mucor
circinelloides TEF gene.
[0312] SEQ ID NOs: 72, 73, 74 and 75 correspond to primers MC_TEF4,
MC_TEF3, MC_TEF8 and MC_TEF7, respectively, used to isolate genomic
fragments of the Mucor circinelloides TEF terminator region in
chromosome walk experiments.
[0313] SEQ ID NOs: 76 and 77 correspond to primers MC_TEF11 and
MC_TEF12, respectively, used to clone terminator of the Mucor
circinelloides TEF gene.
[0314] SEQ ID NOs: 78 and 79 correspond to primers Mould PGK4 and
Mould PGK2, respectively, used to isolate genomic fragment of the
Mucor circinelloides PGK gene.
[0315] SEQ ID NOs: 80, 81, 82 and 83 correspond to primers MC_PGK2,
MC_PGK1, MC_PGK4 and MC_PGK3, respectively, used to isolate genomic
fragments of the Mucor circinelloides PGK promoter region in
chromosome walk experiments.
[0316] SEQ ID NOs: 84 and 85 correspond to primers MC_PGK5 and
MC_PGK6, respectively, used to clone promoter of the Mucor
circinelloides PGK gene.
[0317] SEQ ID NOs: 86, 87, 88 and 89 correspond to primers MC_GPD2,
MC_GPD1, MC_GPD10 and MC_GPD9, respectively, used to isolate
genomic fragment of the Mucor circinelloides GPD promoter region in
chromosome walk experiments.
[0318] SEQ ID NOs: 90 and 91 correspond to primers MC_GPD11 and
MC_GPD12, respectively, used to clone promoter of the Mucor
circinelloides GPD gene.
[0319] SEQ ID NO: 92 corresponds to S. cerevisiae ACS2 protein
encoding DNA codon optimized according to Rhizopus oryzae-fungus
codon usage.
[0320] SEQ ID NO: 93 corresponds to Rhizopus oryzae PDAT protein
encoding DNA codon optimised according to Rhizopus oryzae-fungus
codon usage.
[0321] SEQ ID NO: 94 corresponds to S. cerevisiae PDC1 protein
encoding DNA, with GenBank accession number X77316 (version number
X77316.1).
[0322] SEQ ID NO: 95 corresponds to the amino acid sequence of the
S. cerevisiae PDC1 gene, with GenBank accession number CAA54522
(version number CAA54522.1.
[0323] SEQ ID NO: 96 corresponds to S. cerevisiae PDC1 protein
encoding DNA codon optimized according to Ustilago maydis-fungus
codon usage.
[0324] SEQ ID NO: 97 corresponds to S. cerevisiae PDC1 protein
encoding DNA codon optimized according to Rhizopus oryzae-fungus
codon usage.
REFERENCES
[0325] Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. 1997. Nucleic Acids Res.
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Microbiol. 127:169-176. [0327] Folch J., Lees M. and Stqanley, G.
H. S. J. Biol. Chem 226:497-509 (1957) [0328] van den Berg, M. A.,
de Jong-Gubbels, P., Steensma, H. Y., van Dijken, J. P. and Pronk,
J. T. 1996. J. Biol. Chem. 271:28953-28959. [0329] Connerton, I.
F., Fincham, J. R. S., Sandeman, R. A. and Hynes, M. J. 1990. Mol.
Microbiol. 4:451-460. [0330] Dahlqvist, A., Stahl, U., Lenman, M.,
Banas, A., Lee, M., Sandager, L., Ronne, H. and Stymne, S. 2000.
PNAS 97:6487-6492 [0331] Flikweert, M. T., Van der Zanden, L.,
Janssen, W. M. TH. M., Steensma, H. Y., Van Dijken, J. P. and
Pronk, J. T. 1996. Yeast 12:247-257. [0332] Flipphi, M., Mathieu,
M., Cirpus, I., Panozzo, C. and Felenbok, B. 2001. J. Biol. Chem.
276:6950-6958. [0333] Hiesinger, M., Wagner, C. and Schuller, H.-J.
1997. FEBS Lett. 415:16-20. Hynes, M. J. and Murray, S. L. 2010.
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Kubicek, C. P. 1994. Curr Genet. 25, 567-570 Meesters, P. A. E. P.,
Springer, J. and Eggink, G. 1997. Appl. Microbiol. Biotechnol.
47:663-667. [0335] Mueller, P. R. and Wold, B. 1989, "In vivo
footprinting of a muscle specific enhancer by ligation mediated
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Harashima, S. and Oshima, Y. 1993. J. Ferm. Bioeng. 76:60-63.
[0337] Postma, E., Verduyn, C., Scheffers, W. A. and van Dijken, J.
P. 1989. Appl. Environ. Microbiol. 55:468-477. [0338] Pronk, J. T.,
Steensma, H. Y. and Van Dijken, J. P. 1996. Yeast 12:1607-1633.
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[0347] Wolff, A. M., Appel, K. F., Petersen, J. B., Poulsen, U. and
Arnau, J. 2002 FEMS Yeast Res. 2:203-213
Sequence CWU 1
1
97124DNAArtificial Sequenceprimer 1tacaagtgyg gtggtatyga caag
24223DNAArtificial Sequenceprimer 2tcwacggayt tgacttcagt ggt
23325DNAArtificial Sequenceprimer 3gcggtgaccc gggagatctg aattc
25412DNAArtificial Sequenceprimer 4gaattcagat ct 12517DNAArtificial
Sequenceprimer 5cacgctcacg ctcggcc 17615DNAArtificial
Sequenceprimer 6ccgaggtcgg cggcc 15719DNAArtificial Sequenceprimer
7ggcaggcgca aagctggac 19826DNAArtificial Sequenceprimer 8gcagtcactg
tcattgtcgc actacc 26932DNAArtificial Sequenceprimer 9tccccgcggg
gatccatcac gcctgcccgt cc 321047DNAArtificial Sequenceprimer
10gctctagagc ctgcaggttt ttataggttc tgcgaatggt tagtacg
471125DNAArtificial Sequenceprimer 11cctccaggac gtctacaaga tcggc
251216DNAArtificial Sequenceprimer 12cccgtcggcc gtgtcg
161338DNAArtificial Sequenceprimer 13tccccccggg cctgcaggtt
gtagagccct cggttctg 381431DNAArtificial Sequenceprimer 14ggaattccgg
aggcttgtca tcatacgaga c 311523DNAArtificial Sequenceprimer
15ggtaactkka agatgaacgg ctc 231623DNAArtificial Sequenceprimer
16gckccrccga craggaawcc rtc 231720DNAArtificial Sequenceprimer
17gggaagttgg cgctgtggac 201817DNAArtificial Sequenceprimer
18cgctgagctt ggcgtcg 171929DNAArtificial Sequenceprimer
19cgggatcccg gaattcctga ccacccgcg 292039DNAArtificial
Sequenceprimer 20tgtgtgcctg caggcttgga tatgctgttt taggtttgg
392119DNAArtificial Sequenceprimer 21aagcgcgtct cgcagaagg
192217DNAArtificial Sequenceprimer 22acggcggctc cgtcaac
172335DNAArtificial Sequenceprimer 23gctctagagc ctgcagggat
gaggcgtggc atagg 352426DNAArtificial Sequenceprimer 24cgggatcccg
cagctgacga caggct 262525DNAArtificial Sequenceprimer 25cccgtcacct
cycagaagga gattg 252619DNAArtificial Sequenceprimer 26cggtctcacc
ggatcggtg 192716DNAArtificial Sequenceprimer 27ttggcagcgg cctcgg
162818DNAArtificial Sequenceprimer 28ccaaggatgg cgttggcg
182919DNAArtificial Sequenceprimer 29cgtgctcctg cccaggagg
193020DNAArtificial Sequenceprimer 30cgatgctctc ggcagttgcg
203129DNAArtificial Sequenceprimer 31cggaattctg tctgtacgag
tctgtacac 293238DNAArtificial Sequenceprimer 32ggaattcctg
caggtttgag gtgaggttgt tgttttgg 383317DNAArtificial Sequenceprimer
33ggcgtgcaac gccctcc 173421DNAArtificial Sequenceprimer
34ccatccaggc gtgggtactg a 213532DNAArtificial Sequenceprimer
35cccaagcttc ctgcagggtg cgcgtagtgc gc 323629DNAArtificial
Sequenceprimer 36cccaagcttg ggacgccgag gagcatctc
293721DNAArtificial Sequenceprimer 37cgtcggtctt tgacgccaag g
213823DNAArtificial Sequenceprimer 38cgtggtacga caacgagtac ggc
233943DNAArtificial Sequenceprimer 39gctctagagc ctgcaggatc
ccttcgagga tgtagttagg ttg 434031DNAArtificial Sequenceprimer
40cgggatcccg tggaggtgtc tgtgatgacg a 314138DNAArtificial
Sequenceprimer 41ggactagtcc tgcaggatga aaaagcctga actcaccg
384237DNAArtificial Sequenceprimer 42ggactagtcc tgcaggctat
tcctttgccc tcggacg 374335DNAArtificial Sequenceprimer 43ggactagtcc
tgcaggatga gccatattca acggg 354439DNAArtificial Sequenceprimer
44ggactagtcc tgcaggttag aaaaactcat cgagcatca 394538DNAArtificial
Sequenceprimer 45acacaccctg caggatgagt gtgtctaccg ccaagagg
384634DNAArtificial Sequenceprimer 46gtgtgtcctg caggttaatt
tgcggccggt accg 3447500PRTSaccharomyces cerevisiae 47Met Thr Lys
Leu His Phe Asp Thr Ala Glu Pro Val Lys Ile Thr Leu 1 5 10 15 Pro
Asn Gly Leu Thr Tyr Glu Gln Pro Thr Gly Leu Phe Ile Asn Asn 20 25
30 Lys Phe Met Lys Ala Gln Asp Gly Lys Thr Tyr Pro Val Glu Asp Pro
35 40 45 Ser Thr Glu Asn Thr Val Cys Glu Val Ser Ser Ala Thr Thr
Glu Asp 50 55 60 Val Glu Tyr Ala Ile Glu Cys Ala Asp Arg Ala Phe
His Asp Thr Glu 65 70 75 80 Trp Ala Thr Gln Asp Pro Arg Glu Arg Gly
Arg Leu Leu Ser Lys Leu 85 90 95 Ala Asp Glu Leu Glu Ser Gln Ile
Asp Leu Val Ser Ser Ile Glu Ala 100 105 110 Leu Asp Asn Gly Lys Thr
Leu Ala Leu Ala Arg Gly Asp Val Thr Ile 115 120 125 Ala Ile Asn Cys
Leu Arg Asp Ala Ala Ala Tyr Ala Asp Lys Val Asn 130 135 140 Gly Arg
Thr Ile Asn Thr Gly Asp Gly Tyr Met Asn Phe Thr Thr Leu 145 150 155
160 Glu Pro Ile Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Ile
165 170 175 Met Met Leu Ala Trp Lys Ile Ala Pro Ala Leu Ala Met Gly
Asn Val 180 185 190 Cys Ile Leu Lys Pro Ala Ala Val Thr Pro Leu Asn
Ala Leu Tyr Phe 195 200 205 Ala Ser Leu Cys Lys Lys Val Gly Ile Pro
Ala Gly Val Val Asn Ile 210 215 220 Val Pro Gly Pro Gly Arg Thr Val
Gly Ala Ala Leu Thr Asn Asp Pro 225 230 235 240 Arg Ile Arg Lys Leu
Ala Phe Thr Gly Ser Thr Glu Val Gly Lys Ser 245 250 255 Val Ala Val
Asp Ser Ser Glu Ser Asn Leu Lys Lys Ile Thr Leu Glu 260 265 270 Leu
Gly Gly Lys Ser Ala His Leu Val Phe Asp Asp Ala Asn Ile Lys 275 280
285 Lys Thr Leu Pro Asn Leu Val Asn Gly Ile Phe Lys Asn Ala Gly Gln
290 295 300 Ile Cys Ser Ser Gly Ser Arg Ile Tyr Val Gln Glu Gly Ile
Tyr Asp 305 310 315 320 Glu Leu Leu Ala Ala Phe Lys Ala Tyr Leu Glu
Thr Glu Ile Lys Val 325 330 335 Gly Asn Pro Phe Asp Lys Ala Asn Phe
Gln Gly Ala Ile Thr Asn Arg 340 345 350 Gln Gln Phe Asp Thr Ile Met
Asn Tyr Ile Asp Ile Gly Lys Lys Glu 355 360 365 Gly Ala Lys Ile Leu
Thr Gly Gly Glu Lys Val Gly Asp Lys Gly Tyr 370 375 380 Phe Ile Arg
Pro Thr Val Phe Tyr Asp Val Asn Glu Asp Met Arg Ile 385 390 395 400
Val Lys Glu Glu Ile Phe Gly Pro Val Val Thr Val Ala Lys Phe Lys 405
410 415 Thr Leu Glu Glu Gly Val Glu Met Ala Asn Ser Ser Glu Phe Gly
Leu 420 425 430 Gly Ser Gly Ile Glu Thr Glu Ser Leu Ser Thr Gly Leu
Lys Val Ala 435 440 445 Lys Met Leu Lys Ala Gly Thr Val Trp Ile Asn
Thr Tyr Asn Asp Phe 450 455 460 Asp Ser Arg Val Pro Phe Gly Gly Val
Lys Gln Ser Gly Tyr Gly Arg 465 470 475 480 Glu Met Gly Glu Glu Val
Tyr His Ala Tyr Thr Glu Val Lys Ala Val 485 490 495 Arg Ile Lys Leu
500 481506DNAArtificial SequenceS. cerevisiae codon optimized to
Ustilago maydis 48atgaccaagc tccacttcga caccgccgag cccgtcaaga
tcaccctccc caacggcctc 60acctacgagc agcccaccgg cctcttcatc aacaacaagt
tcatgaaggc ccaggacggc 120aagacctacc ccgtcgagga cccctcgacc
gagaacaccg tctgcgaggt cagctcggcc 180accaccgagg acgtggagta
cgccatcgag tgcgccgacc gcgccttcca cgacaccgag 240tgggccaccc
aggaccctcg cgagcgcggt cgcctcctct ccaaactcgc cgacgaactc
300gagtcgcaga tcgacctcgt cagctcgatc gaggccctcg acaacggcaa
gaccctcgcc 360ctcgctcgcg gcgacgtgac gatcgccatc aactgcctcc
gcgacgccgc tgcctacgcc 420gacaaggtca acggccgcac catcaacacc
ggcgacggct acatgaactt caccaccctc 480gagcccatcg gcgtctgcgg
ccagatcatc ccctggaact tccccatcat gatgctcgcc 540tggaagatcg
cccctgccct cgccatgggc aacgtctgca tcctcaagcc tgccgccgtc
600acccccctca acgccctcta cttcgcctcg ctctgcaaga aggtcggcat
ccctgccggc 660gtcgtcaaca tcgtccctgg ccctggccgc accgtcggcg
ctgccctcac caacgacccc 720cgcatccgca agctcgcctt caccggctcg
accgaggtcg gcaagtcggt cgccgtcgac 780tcgtcggagt cgaacctcaa
gaagatcacg ctcgaactcg gcggcaagtc ggcccacctc 840gtgtttgacg
acgccaacat caagaaaacc ctccctaacc tcgtcaacgg catcttcaag
900aacgccggcc agatctgctc gtcgggctcg cgcatctacg tccaggaagg
catctacgac 960gaactcctcg ccgccttcaa ggcctacctc gaaaccgaga
tcaaggtcgg caaccccttc 1020gacaaggcca acttccaggg cgccatcacc
aaccgccagc agttcgacac catcatgaac 1080tacatcgaca tcggcaagaa
ggaaggcgct aagatcctca cgggcggtga aaaggtcggc 1140gacaagggct
acttcatccg ccccaccgtc ttttacgacg tgaacgagga catgcgcatc
1200gtcaaggaag agatcttcgg ccccgtcgtc accgtcgcca agttcaagac
cctcgaagag 1260ggcgtcgaga tggctaactc gagcgaattt ggcctcggct
cgggcatcga aaccgagtcg 1320ctctcgaccg gcctcaaggt cgccaagatg
ctcaaggccg gcaccgtctg gatcaacacc 1380tacaacgact tcgactcgcg
cgtccccttc ggcggcgtca agcagtcggg ctacggccgc 1440gagatgggcg
aggaagtcta ccacgcctac accgaggtca aggccgtccg catcaagctc 1500tgatga
1506491506DNAArtificial SequenceS. cerevisiae codon optimized to
Rhizopus oryzae 49atgacaaaac ttcattttga tacagctgaa ccagttaaaa
tcacacttcc aaatggtctt 60acttatgaac aaccaacagg tctttttatc aacaacaaat
ttatgaaagc tcaggatggt 120aaaacatatc cagttgaaga tccatcaaca
gaaaatacag tttgtgaagt ttcatcagct 180acaacagagg atgttgaata
tgctattgaa tgtgctgatc gagcttttca tgatacagaa 240tgggctacac
aagatccacg agaacgaggt cgacttcttt caaaacttgc tgatgaactt
300gaatcacaaa ttgatcttgt ttcatcaatc gaagctcttg ataatggtaa
aacacttgct 360cttgctcgag gtgatgttac aattgctatc aattgtttgc
gagatgctgc tgcttatgct 420gataaagtta atggtcgaac aattaataca
ggtgatggtt atatgaattt tacaacgctt 480gaaccaattg gtgtttgtgg
tcaaattatt ccatggaact ttccaattat gatgcttgct 540tggaaaattg
ctccagctct tgctatgggt aatgtttgta ttcttaaacc agctgctgtt
600acaccactta atgcacttta ttttgcttca ctttgtaaaa aagttggtat
tccagctggt 660gttgttaata ttgttccagg tccaggtcga acagttggtg
ctgctcttac aaatgatcca 720cgtattcgaa aacttgcttt tacaggttca
acagaagttg gtaaatcagt tgctgttgat 780tcatcagagt caaacttgaa
aaaaatcact cttgaacttg gtggtaaatc agctcatctt 840gtctttgatg
atgctaacat caaaaaaaca cttccaaacc ttgtcaatgg tatctttaaa
900aacgctggtc aaatttgttc atcaggttca cgaatttatg tccaagaggg
tatctatgat 960gaacttcttg ctgcttttaa agcatatctt gagacagaaa
ttaaagtcgg taacccattt 1020gataaagcta attttcaagg tgctatcaca
aatcgacaac aatttgatac gatcatgaac 1080tatattgata tcggtaaaaa
agaaggtgct aaaattctta caggtggtga aaaagttggt 1140gataaaggtt
attttatccg accaacagtc ttttatgatg tcaatgaaga tatgcgaatt
1200gtcaaagaag aaatttttgg tccagttgtt acagttgcta aattcaagac
acttgaagag 1260ggtgttgaaa tggctaattc atcagaattt ggtcttggtt
caggtattga aacagaatca 1320ctttcaacag gtcttaaagt cgctaaaatg
cttaaagctg gtacagtttg gattaacacg 1380tataacgatt ttgattcacg
agttccattt ggtggtgtta aacaatcagg ttatggtcga 1440gaaatgggtg
aagaagttta tcacgcttat acagaagtta aagctgtccg aatcaaactt 1500taataa
150650683PRTSaccharomyces cerevisiae 50Met Thr Ile Lys Glu His Lys
Val Val Tyr Glu Ala His Asn Val Lys 1 5 10 15 Ala Leu Lys Ala Pro
Gln His Phe Tyr Asn Ser Gln Pro Gly Lys Gly 20 25 30 Tyr Val Thr
Asp Met Gln His Tyr Gln Glu Met Tyr Gln Gln Ser Ile 35 40 45 Asn
Glu Pro Glu Lys Phe Phe Asp Lys Met Ala Lys Glu Tyr Leu His 50 55
60 Trp Asp Ala Pro Tyr Thr Lys Val Gln Ser Gly Ser Leu Asn Asn Gly
65 70 75 80 Asp Val Ala Trp Phe Leu Asn Gly Lys Leu Asn Ala Ser Tyr
Asn Cys 85 90 95 Val Asp Arg His Ala Phe Ala Asn Pro Asp Lys Pro
Ala Leu Ile Tyr 100 105 110 Glu Ala Asp Asp Glu Ser Asp Asn Lys Ile
Ile Thr Phe Gly Glu Leu 115 120 125 Leu Arg Lys Val Ser Gln Ile Ala
Gly Val Leu Lys Ser Trp Gly Val 130 135 140 Lys Lys Gly Asp Thr Val
Ala Ile Tyr Leu Pro Met Ile Pro Glu Ala 145 150 155 160 Val Ile Ala
Met Leu Ala Val Ala Arg Ile Gly Ala Ile His Ser Val 165 170 175 Val
Phe Ala Gly Phe Ser Ala Gly Ser Leu Lys Asp Arg Val Val Asp 180 185
190 Ala Asn Ser Lys Val Val Ile Thr Cys Asp Glu Gly Lys Arg Gly Gly
195 200 205 Lys Thr Ile Asn Thr Lys Lys Ile Val Asp Glu Gly Leu Asn
Gly Val 210 215 220 Asp Leu Val Ser Arg Ile Leu Val Phe Gln Arg Thr
Gly Thr Glu Gly 225 230 235 240 Ile Pro Met Lys Ala Gly Arg Asp Tyr
Trp Trp His Glu Glu Ala Ala 245 250 255 Lys Gln Arg Thr Tyr Leu Pro
Pro Val Ser Cys Asp Ala Glu Asp Pro 260 265 270 Leu Phe Leu Leu Tyr
Thr Ser Gly Ser Thr Gly Ser Pro Lys Gly Val 275 280 285 Val His Thr
Thr Gly Gly Tyr Leu Leu Gly Ala Ala Leu Thr Thr Arg 290 295 300 Tyr
Val Phe Asp Ile His Pro Glu Asp Val Leu Phe Thr Ala Gly Asp 305 310
315 320 Val Gly Trp Ile Thr Gly His Thr Tyr Ala Leu Tyr Gly Pro Leu
Thr 325 330 335 Leu Gly Thr Ala Ser Ile Ile Phe Glu Ser Thr Pro Ala
Tyr Pro Asp 340 345 350 Tyr Gly Arg Tyr Trp Arg Ile Ile Gln Arg His
Lys Ala Thr His Phe 355 360 365 Tyr Val Ala Pro Thr Ala Leu Arg Leu
Ile Lys Arg Val Gly Glu Ala 370 375 380 Glu Ile Ala Lys Tyr Asp Thr
Ser Ser Leu Arg Val Leu Gly Ser Val 385 390 395 400 Gly Glu Pro Ile
Ser Pro Asp Leu Trp Glu Trp Tyr His Glu Lys Val 405 410 415 Gly Asn
Lys Asn Cys Val Ile Cys Asp Thr Met Trp Gln Thr Glu Ser 420 425 430
Gly Ser His Leu Ile Ala Pro Leu Ala Gly Ala Val Pro Thr Lys Pro 435
440 445 Gly Ser Ala Thr Val Pro Phe Phe Gly Ile Asn Ala Cys Ile Ile
Asp 450 455 460 Pro Val Thr Gly Val Glu Leu Glu Gly Asn Asp Val Glu
Gly Val Leu 465 470 475 480 Ala Val Lys Ser Pro Trp Pro Ser Met Ala
Arg Ser Val Trp Asn His 485 490 495 His Asp Arg Tyr Met Asp Thr Tyr
Leu Lys Pro Tyr Pro Gly His Tyr 500 505 510 Phe Thr Gly Asp Gly Ala
Gly Arg Asp His Asp Gly Tyr Tyr Trp Ile 515 520 525 Arg Gly Arg Val
Asp Asp Val Val Asn Val Ser Gly His Arg Leu Ser 530 535 540 Thr Ser
Glu Ile Glu Ala Ser Ile Ser Asn His Glu Asn Val Ser Glu 545 550 555
560 Ala Ala Val Val Gly Ile Pro Asp Glu Leu Thr Gly Gln Thr Val Val
565 570 575 Ala Tyr Val Ser Leu Lys Asp Gly Tyr Leu Gln Asn Asn Ala
Thr Glu 580 585 590 Gly Asp Ala Glu His Ile Thr Pro Asp Asn Leu Arg
Arg Glu Leu Ile 595 600 605 Leu Gln Val Arg Gly Glu Ile Gly Pro Phe
Ala Ser Pro Lys Thr Ile 610 615 620 Ile Leu Val Arg Asp Leu Pro Arg
Thr Arg Ser Gly Lys Ile Met Arg 625 630 635 640 Arg Val Leu Arg Lys
Val Ala Ser Asn Glu Ala Glu Gln Leu Gly Asp 645 650 655 Leu Thr Thr
Leu Ala Asn Pro Glu Val Val Pro Ala Ile Ile Ser
Ala 660 665 670 Val Glu Asn Gln Phe Phe Ser Gln Lys Lys Lys 675 680
512052DNAArtificial SequenceS.cerevisiae codon optimized to
Ustilago maydis 51atgaccatca aggagcacaa ggtcgtctac gaggcccaca
acgtcaaggc cctcaaggct 60ccccagcact tctacaactc gcagcccggc aagggctacg
tcaccgacat gcagcactac 120caggagatgt accagcagtc gatcaacgag
cccgagaagt tcttcgacaa gatggccaag 180gagtacctcc actgggacgc
cccctacacc aaggtccagt cgggctcgct caacaacggc 240gatgtcgcct
ggttcctcaa cggcaagctc aacgcctcgt acaactgcgt cgaccgccac
300gccttcgcca accccgacaa gcccgccctc atctacgagg ccgacgacga
gtcggacaac 360aagatcatca ccttcggcga actcctccgc aaggtctcgc
agatcgccgg cgtcctcaag 420tcgtggggcg tcaagaaggg cgacaccgtc
gccatctacc tccccatgat ccccgaggcc 480gtcatcgcca tgctcgccgt
cgcccgcatc ggcgccatcc actcggtcgt ctttgccggc 540ttctcggccg
gctcgctcaa ggaccgcgtc gtcgatgcca actcgaaggt cgtcatcacc
600tgcgacgagg gcaagcgcgg tggcaagacc atcaacacca agaagatcgt
cgacgaaggc 660ctcaacggcg tcgacctcgt ctcgcgcatc ctcgtctttc
agcgcaccgg caccgagggc 720atccccatga aggctggccg cgactactgg
tggcacgagg aggccgccaa gcagcgcacc 780tacctccccc ctgtctcgtg
cgacgccgag gaccccctct tcctcctcta cacctcgggc 840tcgaccggca
gccctaaggg cgtcgtccat acgaccggcg gctacctcct cggcgctgcc
900ctcaccaccc gctacgtctt tgacatccac cccgaggacg tgctcttcac
cgctggcgac 960gtgggctgga tcaccggcca cacctacgcc ctctacggcc
ccctcaccct cggcaccgcc 1020tcgatcatct tcgagtcgac ccccgcctac
cccgactacg gccgctactg gcgcatcatc 1080cagcgccaca aggccaccca
cttctacgtc gcccccaccg ccctccgcct catcaagcgc 1140gtcggcgagg
ccgagatcgc caagtacgac acctcgtcgc tccgcgtcct cggctcggtc
1200ggcgagccca tctcgcccga cctctgggag tggtatcacg agaaggtcgg
caacaagaac 1260tgcgtcatct gcgacaccat gtggcagacc gagtcgggtt
cgcacctcat cgcccccctc 1320gctggcgccg tccctaccaa gcctggctcg
gccaccgtcc ctttcttcgg catcaacgcc 1380tgcatcatcg accccgtcac
cggcgtcgaa ctcgagggca acgacgtgga gggcgtcctc 1440gccgtcaagt
cgccctggcc ctcgatggcc cgcagcgtct ggaaccacca cgaccgctac
1500atggacacct acctcaagcc ctaccccggc cactacttca ccggcgacgg
cgctggtcgc 1560gatcacgatg gctactactg gattcgcggt cgcgtcgacg
atgtcgtcaa cgtctcgggc 1620caccgcctct cgacctcgga gatcgaggcc
agcatcagca accacgagaa cgtctcggag 1680gccgccgtcg tcggcatccc
cgacgaactc accggccaga ccgtcgtcgc ctacgtctcg 1740ctcaaggacg
gctacctcca gaacaacgcc accgagggcg acgccgagca catcaccccc
1800gacaacctcc gccgcgaact catcctccag gtccgcggcg agatcggccc
cttcgcctcg 1860cccaagacca tcatcctcgt ccgcgacctc cctcgcaccc
gctcgggcaa gatcatgcgc 1920cgcgtcctcc gcaaggtcgc ctcgaacgag
gccgagcagc ttggtgacct caccaccctc 1980gccaaccctg aggtcgtccc
cgccatcatc tcggccgtcg agaaccagtt cttctcgcaa 2040aagaagaagt ga
205252611PRTRhizopus oryzae 52Met Ser Lys Leu Arg Arg Arg Lys Gln
Glu Lys Thr Thr Lys Thr Asn 1 5 10 15 Asp Gln Glu Leu Pro Glu Asp
Ile Met Ala Asp Ser Ala Ala Asn Val 20 25 30 Phe Lys Glu Lys Arg
Pro Phe Trp Gly Arg Lys Arg Phe Asn Phe Ile 35 40 45 Val Gly Leu
Ser Val Gly Leu Leu Ala Met Tyr Ala Ala Ser Thr Thr 50 55 60 Pro
Val Ala Gln Ser His Ile Asn Ser Leu Gln His Tyr Leu Leu Leu 65 70
75 80 Gln Leu Ala Asp Ile Asp Leu Ala Ser Ile Leu Pro Ala Thr Glu
Met 85 90 95 Val Asp Glu Phe Leu Gly Asn Phe Thr Asn Leu Ile Thr
Pro Thr Pro 100 105 110 Ala Thr Glu Met Ser Phe Met Pro Ala Leu Glu
Tyr Lys Glu Ser Leu 115 120 125 Asp Leu Lys Pro Gln Phe Pro Val Val
Met Ile Pro Ala Met Val Arg 130 135 140 Ser Val Leu Leu Asp Lys Glu
Ser Trp Thr Glu His Ile Met Leu Asp 145 150 155 160 Pro Glu Thr Gly
Leu Asp Pro Pro Gly Tyr Lys Val Arg Ala Val His 165 170 175 Glu Lys
Lys Gly Val Glu Ala Ala Asp Tyr Phe Ile Thr Gly Tyr Trp 180 185 190
Val Trp Ala Lys Val Ile Glu Asn Leu Ala Thr Ile Gly Tyr Asp Thr 195
200 205 Asn Asn Met Tyr Phe Ala Ser Tyr Asp Trp Arg Leu Ser Phe Ser
Asn 210 215 220 Leu Glu Val Arg Asp Gly Tyr Phe Ser Lys Leu Lys His
Thr Ile Glu 225 230 235 240 Leu Ser Lys Lys Gln Ser Gly Gln Lys Ser
Val Ile Ile Thr His Ser 245 250 255 Met Gly Gly Thr Met Phe Pro Tyr
Phe Leu Lys Trp Val Glu Ser Lys 260 265 270 Gly His Gly Gln Gly Gly
Gln Lys Trp Val Asp Glu His Ile Glu Ser 275 280 285 Phe Val Asn Ile
Ala Ala Pro Leu Val Gly Val Pro Lys Ala Val Thr 290 295 300 Ser Leu
Leu Ser Gly Glu Thr Arg Asp Thr Met Ala Leu Gly Ser Phe 305 310 315
320 Gly Ala Tyr Val Leu Glu Lys Phe Phe Ser Arg Arg Glu Arg Ala Lys
325 330 335 Leu Met Arg Ser Trp Met Gly Gly Ala Ser Met Leu Pro Lys
Gly Gly 340 345 350 Glu Ala Ile Trp Gly Arg Gly Gly Asn Ala Pro Asp
Asp Glu Glu Asp 355 360 365 Glu Lys Tyr Gln Ser Phe Gly Asn Met Ile
Ser Phe Val Pro Arg Pro 370 375 380 Glu Gly Phe Asn Glu Asn Ser Thr
Asp Ile Pro Ser Asn Ser Gly Asp 385 390 395 400 Pro Leu Val Arg Asn
Tyr Thr Val Gln Gly Ser Ile Gln Leu Leu Thr 405 410 415 Lys Asn Ala
Asp Ile Lys Phe Gly Lys Gln Leu Tyr Ala Asn Tyr Ser 420 425 430 Phe
Gly Leu Thr Thr Ser Ser Lys Gln Leu Lys Arg Asn Glu Asn Asp 435 440
445 Pro Thr Lys Trp Ser Asn Pro Leu Glu Ser Arg Leu Pro Asn Ala Pro
450 455 460 Asn Met Lys Ile Tyr Cys Phe Tyr Gly Ile Glu Val Pro Thr
Glu Arg 465 470 475 480 Ser Tyr Tyr Tyr Ala Ile Leu Asn Glu Asn Met
Asp Gln Glu Cys Gly 485 490 495 His Ser Asn Ser Thr Ala Glu Cys Thr
Thr Glu Gln Asn Ala Glu Pro 500 505 510 Asn Ser Ser Pro Ala Val Ala
Lys Thr Ser Ser Ala Ala Phe Pro Asp 515 520 525 Lys Thr Pro Ser Leu
His Ile Asp Ala Ser Ile Asn Asp Pro Val Gln 530 535 540 Arg Ile Glu
Thr Gly Ile Arg Phe Ser Asn Gly Asp Gly Thr Val Pro 545 550 555 560
Leu Leu Ser Leu Gly Tyr Met Cys Ala Pro Ser Gly Gly Trp Arg Lys 565
570 575 His Ala Asp Leu Tyr Asn Pro Gly His Ser Pro Val Val Leu Arg
Glu 580 585 590 Tyr Lys His Glu Val Ser Thr Ser Lys Leu Asp Val Arg
Gly Gly Trp 595 600 605 Ile Ser Tyr 610 531836DNAArtificial
SequenceRhizopus oryzae codon optimized to Ustilago maydis
53atgtcgaagc tccgccgtcg caagcaggaa aagaccacca agaccaacga ccaggaactc
60cccgaggaca tcatggctga ctcggccgcc aacgtcttta aggagaagcg ccccttctgg
120ggccgcaagc gcttcaactt catcgtcggc ctcagcgtcg gcctcctcgc
catgtacgcc 180gccagcacca cccctgtcgc ccagtcgcac atcaactcgc
tccagcacta cctcctcctc 240caactcgccg acatcgacct cgcctcgatc
ctccccgcca ccgagatggt cgacgagttc 300ctcggcaact tcaccaacct
catcaccccc acccctgcta cggagatgtc gttcatgccc 360gccctcgagt
acaaggagtc gctcgacctc aagccccagt tccccgtcgt catgatcccc
420gctatggtcc gctcggtcct cctcgacaag gaaagctgga ccgagcacat
catgctcgac 480cccgaaacgg gcctcgaccc acccggctac aaggtccgcg
ccgtccacga gaagaagggc 540gtcgaggccg ccgactactt catcaccggc
tactgggtct gggccaaggt catcgagaac 600ctcgccacca tcggctacga
caccaacaac atgtacttcg cctcgtacga ctggcgcctc 660tcgttctcga
acctcgaagt ccgcgacggc tacttctcga agctcaagca caccatcgaa
720ctctcgaaga agcagtcggg ccagaagtcg gtcatcatca cccactcgat
gggcggcacc 780atgttccctt actttctcaa gtgggtcgag tcgaagggcc
acggccaggg cggtcagaag 840tgggtcgacg agcacatcga gtcgttcgtc
aacatcgctg cccccctcgt cggcgtcccc 900aaggccgtca cctcgctcct
ctcgggcgag acccgcgaca ccatggccct cggctcgttc 960ggcgcctacg
tcctcgagaa gttcttctcg cgtcgagagc gcgccaagct catgcgctcg
1020tggatgggcg gtgcctcgat gctccccaag ggcggcgagg ctatctgggg
tcgcggcggt 1080aacgcccccg acgacgagga ggacgagaag taccaatcgt
tcggtaacat gatctcgttc 1140gtccctcgcc ccgagggctt caacgagaac
tcgaccgaca tcccctcgaa ctcgggcgac 1200cccctcgtcc gcaactacac
cgtccagggc tcgatccagc tcctcaccaa gaacgccgac 1260atcaagttcg
gcaagcagct ctacgccaac tactcgttcg gcctcaccac ctcgtcgaag
1320cagctcaagc gcaacgagaa cgaccccacc aagtggtcga accccctcga
gtcgcgcctc 1380cccaacgccc ccaacatgaa gatctactgc ttctacggca
tcgaggtccc caccgaacgc 1440tcgtactact acgccatcct caacgagaac
atggaccagg agtgcggcca ctcgaactcg 1500accgccgagt gcaccaccga
gcagaacgcc gagcccaact cgtcgcctgc tgtcgccaag 1560acctcgtcgg
ccgccttccc cgacaagacc ccttcgctcc acatcgacgc ctcgatcaac
1620gaccctgtcc agcgcatcga gaccggcatc cgcttctcga acggcgacgg
caccgtcccg 1680ctcctctcgc tcggctacat gtgcgccccc tcgggcggtt
ggcgcaagca cgccgacctc 1740tacaaccccg gccactcgcc cgtcgtcctc
cgcgagtaca agcacgaggt ctcgacctcg 1800aagctcgatg tccgcggtgg
ctggatctcg tactga 18365422DNAArtificial Sequenceprimer 54gaggtygtcg
tykcycctcc yg 225525DNAArtificial Sequenceprimer 55gcgaccttrc
crgtrccgat rgccc 255625DNAArtificial Sequenceprimer 56aacaacccaa
tcaacaccca tatcc 255724DNAArtificial Sequenceprimer 57ttttgagcag
caaccttgat ttcc 245842DNAArtificial Sequenceprimer 58cgggatcccg
gaattccagc ctctctaaac gagagttatc cc 425950DNAArtificial
Sequenceprimer 59tccccccggg cctgcagggt tgaatatata aagttttttt
ttagaaaaaa 506025DNAArtificial Sequenceprimer 60ggtgtctccg
tcattgcctg tattg 256125DNAArtificial Sequenceprimer 61gtcgctcgtc
aaatgaaggc tattg 256236DNAArtificial Sequenceprimer 62gctctagagc
ctgcagggtt gcttcgctcc cctccc 366333DNAArtificial Sequenceprimer
63cgggatcccg cactgccaga ctaaacgcca gag 336424DNAArtificial
Sequenceprimer 64cggtaagggt tcyttcaagt acgc 246519DNAArtificial
Sequenceprimer 65ggaagacgga grggcttgt 196625DNAArtificial
Sequenceprimer 66ttgtacttgg gggtctcgaa cttcc 256722DNAArtificial
Sequenceprimer 67gataccacgc tcagcttcag cc 226826DNAArtificial
Sequenceprimer 68ggatggatgg atggatgcat agtatg 266927DNAArtificial
Sequenceprimer 69cacaacactt ctccaaatct gagaagc 277033DNAArtificial
Sequenceprimer 70cccaagcttg ggagcaatgc taaaaaagcc tgg
337153DNAArtificial Sequenceprimer 71tgtgtgcctg caggtttgaa
taactatagt atagattttt agtacatcga tgg 537223DNAArtificial
Sequenceprimer 72gggatggaac aaggagacca agg 237323DNAArtificial
Sequenceprimer 73gactctcctc gaagccatcg atg 237414DNAArtificial
Sequenceprimer 74ccaaggccgc cgcc 147525DNAArtificial Sequenceprimer
75gcccttgtat aaagtgtgct tttgg 257642DNAArtificial Sequenceprimer
76tccccccggg cctgcaggat tgctacctgc tagttttttc tt
427732DNAArtificial Sequenceprimer 77ggaattccaa aagtcttttt
gggtgtcttt ag 327822DNAArtificial Sequenceprimer 78tcaccaacaa
cmancgtaty gt 227923DNAArtificial Sequenceprimer 79tggaaacgma
ggttytcnar aag 238019DNAArtificial Sequenceprimer 80caacagcctc
accgttggg 198120DNAArtificial Sequenceprimer 81gcagcacctt
gttcaagggc 208229DNAArtificial Sequenceprimer 82gacactgttt
tgaaatgcct aacccatac 298324DNAArtificial Sequenceprimer
83gtacggaaaa cagagtcatg gtgc 248435DNAArtificial Sequenceprimer
84tccccgcggg gatcccaagg cactaccact acttc 358548DNAArtificial
Sequenceprimer 85gctctagagc ctgcagggat taattatatg attcaatgat
gaagataa 488621DNAArtificial Sequenceprimer 86ctcgacggaa ccatcgaaac
g 218726DNAArtificial Sequenceprimer 87gaaggacaat acgaccaata cgaccg
268832DNAArtificial Sequenceprimer 88ggcttctttc cttggtgaat
aaagtgagtt ac 328928DNAArtificial Sequenceprimer 89cggaaattat
ccgttcaaac tatcgccc 289020DNAArtificial Sequenceprimer 90agccaaaagt
tgaattcgac 209159DNAArtificial Sequenceprimer 91cccaagcttc
ctgcaggttt tagaatttat gaaatatata tatataaaga tataatatg
59922052DNAArtificial SequenceS.cerevisiae codon optimized to
Rhizopus oryzae 92atgacaatta aagaacataa agtcgtttat gaagctcata
atgttaaagc tcttaaagca 60ccacaacatt tctataattc acaaccaggt aaaggttatg
tcacagatat gcagcattat 120caagaaatgt atcaacaatc aatcaacgaa
ccagagaaat ttttcgataa gatggctaaa 180gaatatcttc attgggatgc
tccatataca aaagttcaat caggttcact taacaatggt 240gatgttgctt
ggtttcttaa tggtaaactt aacgcttcat ataattgtgt tgatcgacat
300gcttttgcta atccagataa accagctctt atctatgaag ctgatgatga
atcagataac 360aaaatcatca catttggtga acttttgcga aaagtttcac
aaattgctgg tgttcttaaa 420tcatggggtg ttaaaaaagg tgatacagtt
gctatctatc ttccaatgat tcctgaagct 480gttattgcta tgcttgctgt
tgctcgaatt ggtgctattc attcagttgt ttttgctggt 540ttttcagctg
gttcacttaa agatcgagtt gttgatgcta attcaaaagt tgttatcaca
600tgtgatgaag gtaaacgagg tggtaaaaca attaacacga aaaaaatcgt
tgatgaaggt 660cttaatggtg ttgatcttgt ttcacgtatt cttgtttttc
aacgaacagg tactgaaggt 720attccaatga aagctggtcg agattattgg
tggcatgaag aagctgctaa acaacgaaca 780tatcttccac cagtttcatg
tgatgctgaa gatccacttt ttcttttgta tacatctggt 840tcaacaggtt
ctccaaaagg tgttgttcat acaacaggtg gttatcttct tggtgctgct
900cttacaacac gatatgtctt tgatattcat ccagaagatg ttctttttac
agcaggtgat 960gttggttgga ttacaggtca tacttatgca ctttatggtc
cacttacact tggtacagct 1020tcaattatct ttgaatcaac gccagcttat
ccagattatg gtcgatattg gcgaattatt 1080caacgacata aagctacaca
tttttatgtc gctccaacag cacttcgact tattaaacga 1140gttggtgaag
ctgaaattgc aaaatatgat acatcatcac ttcgagttct tggttcagtt
1200ggtgaaccaa tttcaccaga tctttgggaa tggtatcatg aaaaagttgg
taacaaaaat 1260tgtgttatct gtgatacaat gtggcaaaca gaatcaggtt
ctcatcttat tgctccactt 1320gctggtgctg ttccaacaaa accaggttca
gctacagttc cattttttgg tatcaatgct 1380tgtattattg atccagttac
aggtgttgaa cttgaaggta atgatgttga aggtgttctt 1440gctgttaaat
caccatggcc atcaatggct cgatcagttt ggaatcatca tgatcgttat
1500atggatacgt atcttaaacc atatccaggt cactatttta caggtgatgg
tgcaggtcga 1560gatcatgatg gttattattg gattcgaggt cgagttgatg
atgttgttaa tgtttcaggt 1620catcgacttt caacatcaga aattgaagca
tcaatttcaa accatgaaaa tgtttcagaa 1680gctgctgttg ttggtattcc
agatgaactt acaggtcaaa cagttgttgc ttatgtctct 1740cttaaagatg
gttatcttca aaacaatgct actgaaggtg atgctgaaca tattacacca
1800gataatcttc gacgagaact tattcttcaa gttcgaggtg aaattggtcc
atttgcttca 1860ccaaaaacaa ttattcttgt tcgagatctt ccacgaacac
gatcaggtaa aatcatgcga 1920cgagttcttc gaaaagttgc ttcaaatgaa
gctgaacaac ttggtgatct tacaacactt 1980gcaaatccag aagttgttcc
agctattatt tcagctgtcg aaaaccagtt tttctcacag 2040aaaaagaagt aa
2052931836DNAArtificial SequenceRhizopus oryzae codon optimized to
Rhizopus oryzae 93atgtcaaaac ttcgacgacg aaaacaagaa aaaacaacga
aaacaaacga tcaagaactt 60cctgaagata ttatggctga ttcagctgct aatgtcttta
aagagaaacg accattttgg 120ggtcgaaaac gatttaactt tatcgttggt
ctttcagttg gtcttttggc aatgtatgct 180gcttcaacaa caccagttgc
tcaatcacat attaactcac ttcagcacta tcttttgctt 240caacttgctg
atattgatct tgcttcaatt cttccagcta cagaaatggt tgatgaattt
300cttggtaact ttacaaatct tatcacacca acaccagcaa cagaaatgtc
atttatgcca 360gctcttgaat ataaagagtc acttgatctt aaaccacaat
ttccagttgt tatgattcca 420gctatggttc gatcagttct tcttgataaa
gaatcatgga cagaacatat tatgcttgat 480ccagaaacag gtcttgatcc
accaggttat aaagttcgag ctgtccatga aaaaaaaggt 540gttgaagctg
ctgattattt cattacaggt tattgggttt gggctaaagt tattgaaaat
600cttgctacga ttggttatga tacgaacaac atgtattttg cttcatatga
ttggcgactt 660tcattttcaa atcttgaagt ccgagatggt tatttttcaa
aacttaaaca cacgatcgag 720ctttcaaaaa aacaatcagg tcagaaatca
gtcattatca cacattcaat gggtggtaca 780atgtttccat attttcttaa
atgggtcgaa tcaaaaggtc atggtcaagg tggtcaaaaa 840tgggttgatg
aacatattga atcatttgtc aatattgctg ctccacttgt tggtgttcca
900aaagctgtta catcacttct ttcaggtgaa acacgagata caatggctct
tggttcattt 960ggtgcttatg tccttgagaa attcttttca cgacgagaac
gagctaaact tatgcgatca 1020tggatgggtg gtgcatcaat gcttccaaaa
ggtggtgaag caatttgggg tcgaggtggt 1080aatgctccag atgatgaaga
agatgagaaa tatcagtcat
ttggtaacat gatttcattt 1140gttccacgac cagaaggttt taacgaaaat
tcaacggata ttccatcaaa ttcaggtgat 1200ccacttgttc gaaattatac
agtccaaggt tcaattcaac ttcttacgaa aaacgctgat 1260atcaaatttg
gtaaacagct ttatgctaac tattcatttg gtcttacaac atcatcaaaa
1320cagcttaaac gaaatgaaaa cgatccaaca aaatggtcaa atccacttga
atcacgactt 1380ccaaatgctc caaacatgaa aatctattgt ttttatggta
ttgaagttcc aacagagcga 1440tcatattatt atgctatcct taacgaaaat
atggatcaag aatgtggtca ttcaaattca 1500acagctgaat gtacaacaga
acaaaatgct gaaccaaatt catcaccagc tgttgctaaa 1560acatcatcag
ctgcttttcc agataaaaca ccatcacttc atattgatgc ttcaattaat
1620gatccagtcc aacgaattga aacaggtatt cgattttcaa atggtgatgg
tacagttcca 1680cttctttcac ttggttatat gtgtgctcca tcaggtggtt
ggcgaaaaca tgctgatttg 1740tataatccag gtcattcacc agttgttctt
cgagaatata aacatgaagt ctcaacgtca 1800aaacttgatg ttcgaggtgg
ttggatttca tattaa 1836941701DNASaccharomyces cerevisiae
94atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac
60accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt
120gaaggtatga gatgggctgg taacgccaac gaattgaacg ctcgttacgc
cgctgatggt 180tacgctcgta tcaagggtat gtcttgtatc atcaccacct
tcggtgtcgg tgaattgtct 240gctttgaacg gtattgccgg ttcttacgct
gaacacgtcg gtgttttgca cgttgttggt 300gtcccatcca tctcttctca
agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg
ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact
420gacatctgta ccgccccagc tgaaattgac agatgtatca gaaccactta
cgtcacccaa 480agaccagtct acttaggttt gccagctaac ttggtcgact
tgaacgtccc agctaagttg 540ttgcaaactc caattgacat gtctttgaag
ccaaacgatg ctgaatccga aaaggaagtc 600attgacacca tcttggtctt
ggctaaggat gctaagaacc cagttatctt ggctgatgct 660tgttgttcca
gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc
720ccagctttcg tcaccccaat gggtaagggt tccattagcg aacaacaccc
aagatacggt 780ggtgtttacg tcggtacctt gtccaagcca gaagttaagg
aagccgttga atctgctgac 840ttgattttgt ctgtcggtgc tttgttgtct
gatttcaaca ccggttcttt ctcttactct 900tacaagacca agaacattgt
cgaattccac tccgaccaca tgaagatcag aaacgccact 960ttcccaggtg
tccaaatgaa attcgttttg caaaagttgt tgaccaatat tgctgacgcc
1020gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc
tgctgtccca 1080gcttctaccc cattgaagca agaatggatg tggaaccaat
tgggtaactt cttgcaagaa 1140ggtgatgttg tcattgctga aaccggtacc
tccgctttcg gtatcaacca aaccactttc 1200ccaaacaaca cctacggtat
ctctcaagtc ttatggggtt ccattggttt caccactggt 1260gctaccttgg
gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta
1320ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat
gatcagatgg 1380ggcttgaagc catacttgtt cgtcttgaac aacgatggtt
acaccattga aaagttgatt 1440cacggtccaa aggctcaata caacgaaatt
caaggttggg accacctatc cttgttgcca 1500actttcggtg ctaaggacta
cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560ttgacccaag
acaagtcttt caacgacaac tctaagatca gaatgattga ggttatgttg
1620ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc
tgctaccaac 1680gctaagcaat aagcgattta a 170195563PRTSaccharomyces
cerevisiae 95Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg
Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly
Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu
Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Arg
Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys
Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn
Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His
Val Val Gly Val Pro Ser Ile Ser Ser Gln Ala Lys Gln Leu Leu 100 105
110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met
115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile
Cys Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr
Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala
Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr
Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu
Lys Glu Val Ile Asp Thr Ile Leu Val Leu Ala 195 200 205 Lys Asp Ala
Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His
Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230
235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Ser Glu Gln
His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys
Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu
Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe
Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser
Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val
Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Asn 325 330 335 Ile Ala
Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350
Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355
360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val
Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln
Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val
Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly
Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val
Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln
Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu
Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475
480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu
485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His
Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp
Lys Ser Phe Asn 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Val
Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Glu Gln
Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln
961692DNASaccharomyces cerevisiae 96atgtcggaga tcaccctcgg
caagtacctc ttcgagcgcc tcaagcaggt caacgtcaac 60accgtctttg gcctccccgg
cgacttcaac ctctcgctcc tcgacaagat ctacgaggtc 120gagggcatgc
gctgggccgg caacgccaac gaactcaacg ccgcctacgc cgccgacggc
180tacgcccgca tcaagggcat gtcgtgcatc atcaccacct tcggtgtcgg
cgaactctcg 240gccctcaacg gtatcgccgg ctcgtacgct gagcacgtcg
gcgtcctcca cgtcgtcggc 300gtcccttcga tctcggccca ggccaagcag
ctcctcctcc accacaccct cggtaacggc 360gacttcaccg tctttcaccg
catgtcggcc aacatctcgg aaaccaccgc catgatcacc 420gatatcgcca
ccgcccctgc cgagatcgac cgctgcatcc gcaccaccta cgtcacccag
480cgccccgtct acctcggcct ccccgccaac ctcgtcgacc tcaacgtccc
cgccaagctc 540ctccagaccc ccatcgacat gtcgctcaag cccaacgacg
ccgagtcgga gaaggaagtc 600atcgacacca tcctcgccct cgtcaaggac
gccaagaacc ccgtcatcct cgccgacgcc 660tgctgctcgc gccacgacgt
caaggccgaa acgaagaagc tcatcgatct cacccagttc 720cccgccttcg
tcacccccat gggcaagggc tcgatcgacg agcagcaccc ccgctacggc
780ggcgtctacg tcggcaccct ctcgaagccc gaggtcaagg aagccgtcga
gtcggccgac 840ctcatcctct cggtcggcgc actcctctcg gacttcaaca
ccggctcgtt ctcgtactcg 900tacaagacca agaacatcgt cgagttccac
tcggaccaca tgaagatccg caacgccacc 960ttccccggcg tccagatgaa
gttcgtcctc cagaagctcc tcaccacgat cgccgacgcc 1020gccaagggct
acaagcccgt cgccgtccct gcccgcacac cagccaacgc agccgtccct
1080gcctcgaccc ccctcaagca agagtggatg tggaaccagc tcggcaactt
cctccaagag 1140ggcgacgtcg tgatcgccga aaccggcacc tcggccttcg
gcatcaacca gaccaccttc 1200cccaacaaca cctacggcat ctcgcaggtc
ctctggggct cgatcggctt caccaccggc 1260gccaccctcg gcgctgcctt
cgcagctgag gaaatcgacc ccaagaagcg tgtcatcctc 1320ttcatcggcg
acggctcgct ccagctcacc gtccaagaga tctcgaccat gatccgctgg
1380ggcctcaagc cctacctctt cgtcctcaac aacgacggct acaccatcga
gaagctcatc 1440cacggcccca aggcccagta caacgagatc cagggctggg
accacctctc gctcctccct 1500accttcggcg ccaaggacta cgaaacccac
cgcgtcgcca caaccggcga gtgggacaag 1560ctcacccagg acaagtcgtt
caacgacaac tcgaagatcc gcatgatcga gatcatgctc 1620cccgtctttg
acgcccccca gaacctcgtc gagcaggcca agctcaccgc cgccaccaac
1680gccaagcagt ga 1692971692DNASaccharomyces cerevisiae
97atgtcagaaa tcacgcttgg taaatatctt ttcgagcgac ttaaacaggt caatgtcaat
60actgtctttg gtttgccagg tgatttcaac ctttcacttc ttgacaagat ctatgaagtc
120gaaggtatgc gttgggcagg taatgcaaat gaacttaatg cagcatatgc
agcagatggt 180tatgcacgaa tcaaaggtat gtcatgtatc atcacaacgt
ttggtgtcgg tgaactttca 240gcacttaatg gtattgcagg ttcatatgca
gaacatgttg gtgttcttca tgttgttggt 300gttccatcaa tttcagctca
agcaaaacag cttcttcttc atcatacact tggtaacggt 360gattttacgg
tctttcatcg aatgtcagca aacatctcag aaacaacagc aatgatcaca
420gatattgcaa cagcaccagc agaaattgat cgatgtatcc gaacaacata
tgtcacacaa 480cgaccagttt atttgggttt gccagcaaat cttgtcgatc
ttaatgtccc agcaaaactt 540cttcagacac caattgacat gtcacttaaa
cctaacgatg ccgaatcaga aaaagaagtc 600atcgatacaa tccttgcact
tgtcaaagat gcaaaaaacc cagttatcct tgcagatgca 660tgttgttcac
gacatgatgt caaagcagaa acgaagaaac ttatcgatct tacacagttt
720ccagcatttg ttacgccaat gggtaaaggt tcaatcgatg aacaacatcc
acgatatggt 780ggtgtttatg ttggtacact ttcaaaacca gaggtcaaag
aagcagttga atcagcagat 840cttatccttt cagttggtgc acttctttca
gattttaaca cgggttcatt ctcatattct 900tataagacga agaacatcgt
cgagtttcac tcagatcata tgaagatccg aaacgcaaca 960tttccaggtg
tccaaatgaa gtttgtcctt cagaaacttc ttacgacaat tgcagatgca
1020gccaaaggtt ataagccagt tgcagttcca gcacgaacac cagcaaatgc
agcagttcca 1080gcttcaacac cacttaaaca agaatggatg tggaatcagc
ttggtaactt tcttcaagag 1140ggtgatgttg ttatcgcaga aacaggtaca
tcagcatttg gtatcaacca gacaacgttt 1200ccaaacaaca cgtatggtat
ctcacaagtt ctttggggtt caatcggttt tacaacaggt 1260gcaacacttg
gtgcagcatt tgctgcagaa gaaatcgatc caaaaaagcg agtcatcctt
1320tttatcggtg atggttcact tcagcttaca gttcaagaaa tctctacaat
gatccgatgg 1380ggtcttaagc catatctttt tgtccttaac aacgacggtt
atacgatcga aaaacttatc 1440catggtccaa aggcacagta taacgaaatt
caaggttggg atcacctttc acttttgcca 1500acatttggtg caaaggatta
tgagacacat cgagttgcaa caacaggtga atgggacaaa 1560cttacacagg
ataagtcatt caacgacaac tcaaagatcc gaatgatcga aattatgctt
1620ccagtctttg atgcaccaca aaatcttgtt gagcaggcaa aacttacagc
agcaacaaat 1680gccaaacagt aa 1692
* * * * *