U.S. patent application number 14/490420 was filed with the patent office on 2017-02-16 for l-arabinose fermenting yeast.
The applicant listed for this patent is ALLIANCE FOR SUSTAINABLE ENERGY, LLC. Invention is credited to Mary Ann FRANDEN, Eric JARVIS, Eric KNOSHAUG, Arjun SINGH, Pirkko SUOMINEN, Min ZHANG.
Application Number | 20170044554 14/490420 |
Document ID | / |
Family ID | 38802154 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170044554 |
Kind Code |
A1 |
ZHANG; Min ; et al. |
February 16, 2017 |
L-ARABINOSE FERMENTING YEAST
Abstract
An L-arabinose utilizing yeast strain is provided for the
production of ethanol by introducing and expressing bacterial araA,
araB and araD genes, L-arabinose transporters are also introduced
into the yeast to enhance the uptake of arabinose. The yeast
carries additional genomic mutations enabling it to consume
L-arabinose, even as the only carbon source, and to produce
ethanol. A yeast strain engineered to metabolize arabinose through
a novel pathway is also disclosed. Methods of producing ethanol
include utilizing these modified yeast strains.
Inventors: |
ZHANG; Min; (Lakewood,
CO) ; SINGH; Arjun; (Lakewood, CO) ; SUOMINEN;
Pirkko; (Minnetonka, MN) ; KNOSHAUG; Eric;
(Golden, CO) ; FRANDEN; Mary Ann; (Centennial,
CO) ; JARVIS; Eric; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALLIANCE FOR SUSTAINABLE ENERGY, LLC |
GOLDEN |
CO |
US |
|
|
Family ID: |
38802154 |
Appl. No.: |
14/490420 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13744023 |
Jan 17, 2013 |
8841090 |
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14490420 |
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12909523 |
Oct 21, 2010 |
8372626 |
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13744023 |
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11912493 |
Oct 24, 2007 |
7846712 |
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PCT/US07/64330 |
Mar 19, 2007 |
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12909523 |
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60810562 |
Jun 1, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/81 20130101;
C12P 7/08 20130101; C12P 7/06 20130101; Y02E 50/17 20130101; Y02E
50/10 20130101 |
International
Class: |
C12N 15/81 20060101
C12N015/81; C12P 7/06 20060101 C12P007/06 |
Goverment Interests
CONTRACTUAL ORIGIN
[0002] The United States Government has rights in this invention
under Contract No. DB-AC36-99GO010337 between the United States
Department of Energy and the National Renewable Energy Laboratory,
a Division of the Midwest Research Institute.
Claims
1-37. (canceled)
38. A transgenic yeast strain for fermenting arabinose to ethanol,
the transgenic yeast strain comprising genes for the expression of
an araA peptide, an araB peptide and an araD peptide and wherein
the transgenic strain is aldose reductase deficient (AR.sup.-).
39. The transgenic yeast strain of claim 38, wherein the araA gene
is derived from E. coli or B. subtilis.
40. The transgenic yeast strain of claim 38, wherein the transgenic
yeast strain has a deletion or disruption of the aldose reductase
gene.
41. The transgenic yeast strain of claim 40, wherein the transgenic
yeast strain has a deletion or disruption of the open reading frame
(ORF) yhr104w.
42. The transgenic yeast strain of claim 38, wherein the transgenic
yeast strain is capable of producing at least 4.7 g/liter of
ethanol from 19 g/L of arabinose.
43. The transgenic yeast strain of claim 38, wherein the transgenic
yeast strain has a mutation of the aldose reductase gene.
44. The transgenic yeast strain of claim 38, wherein the yeast
strain includes at least one arabinose transporter gene selected
from the group consisting of GAL2, KmLAT1 and PgLAT2.
45. An ethanol producing yeast strain, comprising at least one
heterologous arabinose transporter wherein the ethanol producing
yeast strain is aldose reductase (AR) deficient (AR-).
46. The ethanol producing yeast strain of claim 45, further
comprising at least two heterologous arabinose transporters.
47. The ethanol producing yeast strain of claim 45, wherein the
heterologous arabinose transporter is from a non-conventional yeast
species.
48. The ethanol producing yeast strain of claim 47, wherein the
heterologous arabinose transporter is KmLat1 or PgLat2.
49. The ethanol producing yeast strain of claim 45, wherein the
yeast strain overexpresses a GAL2-encoded galactose permease.
50. The ethanol producing yeast strain of claim 49, wherein the
yeast derived GAL2-encoded galactose permease is under control of a
TDH3 promoter.
51. The ethanol producing yeast strain of claim 45, further
comprising one or more bacterial genes that facilitate arabinose
utilization and fermentation in the ethanol producing yeast
strain.
52. The ethanol producing yeast strain of claim 51, wherein the one
or more bacterial genes for facilitated arabinose utilization and
fermentation are selected from the group consisting of araA, araB
and araD.
53. The ethanol producing yeast strain of claim 51, comprising
araA, araB and araD.
54. The ethanol producing yeast strain of claim 52, wherein at
least one of araA, araB and araD is derived from E. coli.
55. A method for producing ethanol utilizing a transgenic yeast
strain, comprising the steps of: providing a material comprising
arabinose, performing a fermentation of the material using the
transgenic yeast strain, wherein the transgenic yeast strain
comprises genes for the expression of an araA peptide, an araB
peptide and an araD peptide and wherein the transgenic yeast strain
is aldose reductase deficient (AR.sup.-); and producing at least 8
g/liter ethanol.
56. The method of claim 55, wherein the material comprises
arabinose and glucose, and the method produces at least 17 g/liter
ethanol.
57. The method of claim 55, wherein the transgenic yeast strain has
a deletion or disruption of the aldose reductase gene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/912,493, filed Oct. 24, 2007, which is a
national stage entry of International Application No.
PCT/US07/64330, filed Mar. 19, 2007, which claims priority to U.S.
Provisional Application No. 60/810,562, filed Jun. 1, 2006. The
contents of each application listed above are incorporated by
reference in their entirety.
BACKGROUND
[0003] Fuel ethanol is a suitable alternative to fossil fuels.
Ethanol may be produced from plant biomass, which is an economical
and renewable resource that is available in large amounts. Examples
of biomass include agricultural feedstocks, paper wastes, wood
chips and so on. The sources of biomass vary from region to region
based on the abundance of natural or agricultural biomass that is
available in a particular region. For example, while sugar cane is
the primary source of biomass used to produce ethanol in Brazil,
corn-derived biomass, corn starch is a large source of biomass to
produce ethanol in the United States. Other agricultural feedstocks
include, by way of example: straw; grasses such as switchgrass;
grains; and any other lignocellulosic or starch-bearing
material.
[0004] A typical biomass substrate contains from 35-45% cellulose,
25-40% hemicellulose, and 15-30% lignin, although sources may be
found that deviate from these general ranges. As is known in the
art, cellulose is polymer of glucose subunits, and hemicellulose
contains mostly xylose. Arabinose is also a significant fermentable
substrate that is found in biomass, such as corn fiber and many
herbaceous crops in varying amounts. Other researchers have
investigated the utilization of arabinose and hemicellulose, as
reported by Hespell, R. B. 1998. Extraction and characterization of
hemicellulose from the corn fiber produced by corn wet-milling
processes. J. Agric Food Chem. 46:2615-2619, and McMillan, J. D.,
and B. L. Boynton. 1994. Arabinose utilization by xylose-fermenting
yeasts and fungi. Appl. Biochem. Biotechnol. 45-46:569-584. The two
most abundant types of pentose that exist naturally are D-xylose
and L-arabinose.
[0005] It is problematic that most of the currently available
ethanol-producing microorganisms are only capable of utilizing
hexose sugar, such as glucose. This is confirmed by a review of the
art, such as is reported by Barnett, J. A. 1976. The utilization of
sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32:125-234. Many
types of yeast, especially Saccharomyces cerevisiae and related
species, are very effective in fermenting glucose-based feedstocks
into ethanol through anaerobic fermentation. However, these
glucose-fermenting yeasts are unable to ferment xylose or
L-arabinose, and are unable to grow solely on these pentose sugars.
Although other yeast species, such as Pichia stipitis and Candida
shehatae, can ferment xylose to ethanol, they are not as effective
as Saccharomyces for fermentation of glucose and have a relatively
low level of ethanol tolerance. Thus, the present range of
available yeast are not entirely suitable for large scale
industrial production of ethanol from biomass.
[0006] Most bacterial, including E. coli and Bacillus subtilis,
utilize L-arabinose for aerobic growth, but they do not ferment
L-arabinose to ethanol. These and other microorganisms, such as
Zymononas mobilis, have also been genetically modified to produce
ethanol from hexose or pentose. This has been reported, for
example, in Deanda, K., M. Zhang, C. Eddy, and S. Picatagglo, 1906,
Development of an arabinose-fermenting Zymomonas mobilis strain by
metabolic pathway engineering. Appl. Environ. Microbiol.
62:4465-4470; and Zhang, M., C. Eddy, K. Deanda, M. Finkelstein,
and S. Picataggio, 1995 Metabolic engineering of a pentose
metabolism pathway in ethanologenic Zymomonas mobilis. Science
267:240-243. However, it remains the case that the low alcohol
tolerance of these non-yeast microorganisms limits their utility in
the ethanol industry.
[0007] Much effort has been made over the last decade or so,
without truly overcoming the problem of developing new strains that
ferment xylose to generate ethanol. Such efforts are reported, for
example, in Kotter, P., R. Amore, C. P. Hollenberg, and M. Ciriacy.
1990. Isolation and characterization of the Pichia stipitis xylitol
dehydrogenase gene, XYL2, and construction of a xylose-utilizing
Saccharomyces cerevisiae transformant. Curr. Genet. 18:493-500; and
Wahlbom, C. F., and B. Hahn-Hagardal. 2002 Recent studies have been
conducted on yeast strains that potentially ferment arabinose.
Sedlak, M., and N. W. Ho. 2001. Expression of E. coli araBAD operon
encoding enzymes for metabolizing L-arabinose in Saccharomyces
cerevisiae, Enzyme Microb. Technol. 28:16-24 discloses the
expression of an E. coli araBAD operon encoding enzymes for
metabolizing L-arabinose in Saccharomyces cerevisiae. Although this
strain expresses araA, araB and araD proteins, it is incapable of
producing ethanol.
[0008] U.S. patent application Ser. No. 10/983,951 by Boles and
Becker discloses the creation of a yeast strain that may ferment
L-arabinose. However, the overall yield is relatively low, at about
60% of theoretical value. The rate of arabinose transport into S.
cerevisiae may be a limiting factor for complete utilization of the
pentose substrate. Boles and Becker attempted to enhance arabinose
uptake by overexpressing the GAL2-encoded galactose permease in S.
cerevisiae. However, the rate of arabinose transport using
galactose permease was still much lower when compared to that
exhibited by non-conventional yeast such as Kluyveromyces
marxianus. Another limitation that may have contributed to the low
yield of ethanol in the modified strain of Becker and Boles is the
poor activity of the L-arabinose isomerase encoded by the bacterial
araA gene. Although Becker and Boles used an araA gene from B.
subtilis instead of one from E. coli, the specific activity of the
enzyme was still low. Other workers in the field have reported that
low isomerase activity is a bottleneck in L-arabinose utilization
by yeast.
[0009] There remains a need for new arabinose-fermenting strains
that are capable of producing ethanol at high yield. There is
further a need to identify novel arabinose transporters for
introduction into Saccharomyces cerevisiae to boost the production
of ethanol from arabinose.
[0010] The foregoing examples of the related art and limitations
related therewith are intended to be illustrative and not
exclusive. Other limitations of the related art will become
apparent to those of skill in the art upon a reading of the
specification and a study of the drawings.
SUMMARY
[0011] The following embodiments and aspects thereof are described
and illustrated in conjunction with systems, tools and methods
which are meant to be exemplary and illustrative, not limiting in
scope. In various embodiments, one or more of the above-described
problems have been reduced or eliminated, while other embodiments
are directed to other improvements.
[0012] The presently disclosed instrumentalities overcome some of
the problems outlined above and advance the art by providing new
yeast strains that are capable of using L-arabinose to produce
ethanol at a relatively high yield. Since the yeast galactose
permease may facilitate uptake of arabinose, any Gal.sup.+ strain
possessing endogenous galactose permease activity may be used as
described below. Although S. cerevisiae is used by way of example,
the scope of coverage extends to any organisms possessing
endogenous pathways to generate ethanol from arabinose and to
organisms into which components of such arabinose metabolic
pathways or arabinose transporters may be introduced. The use of S.
cerevisiae is preferred.
[0013] In a brief overview of the recombinant technique, the
endogenous yeast aldose reductase (AR) gene is disrupted by
replacing the AR coding sequence with the yeast LEU2 gene. Because
the yeast aldose reductase is the first enzyme to metabolize
arabinose in yeast, an AR.sup.31 strain is used to reduce diversion
of arabinose to unwanted byproducts and to prevent possible
inhibition of the isomerase by arabitol. The bacterial araA, araB,
and araD genes are cloned into appropriate yeast expression
vectors. The expression constructs containing all three ara genes
are introduced in the AR.sup.- strain and the transformants were
capable of making ethanol from L-arabinose.
[0014] In another aspect of this disclosure, two novel arabinose
transporter genes, termed KmLAT1 and PgLAT2, have been cloned and
characterized from two non-conventional yeast species,
Kluyveromyces marxianus and Pichia guilliermondii (also known as
Candida guilliermondii), respectively. Both Kluyveromyces marxianus
and Pichia guilliermondii are efficient utilizers of L-arabinose,
which renders them ideal sources for cloning L-arabinose
transporter genes.
[0015] The KmLAT1 gene may be isolated using functional
complementation of an adapted S. cerevisiae strain that could not
grow on L-arabinose because it lacked sufficient L-arabinose
transport activity. KmLat1 protein has a predicted length of 556
amino acids encoded by a single ORF of 1668 bp. It is a
transmembrane protein having high homology to sugar transporters of
many different yeast species. When KmLat1 is expressed in S.
cerevisiae, transport assays using labeled L-arabinose show that
tills transporter has the kinetic characteristics of a low affinity
arabinose transporter, with K.sub.m=230 mM and V.sub.max=55
nmol/mgmin. Transport of L-arabinose by KmLat1 is not significantly
inhibited by common uncoupling agents but is out-competed by
glucose, galactose, xylose, and maltose.
[0016] The PgLAT2 gene may be isolated using the technique of
differential display from Pichia guilliermondii. The PgLAT2 gene
has an ORF of 1617 nucleotides encoding a protein with a predicted
length of 539 amino acids. When PgLAT2 is expressed in S.
cerevisiae, transport assays show that this transporter has almost
identical L-arabinose transport kinetics as that of wildtype Pichia
guilliermondii. The PgLat2 transporter when expressed in S.
cerevisiae has a K, of 0.07 mM and V.sub.max of 18 nmol/mg-min for
L-arabinose transport. Inhibition experiments show significant
inhibition of the PgLat2 transporter by protonophores (e.g.,
NaN.sub.3, DNP, and CCP) and H+-adenosine triphosphatase (ATPase)
inhibitors (e.g., DESB and DCCD) similar to inhibition in wildtype
P. guilliermondii. Competition experiments show that L-arabinose
uptake by the PgLat2 transporter is inhibited by glucose,
galactose, xylose and to a lesser extent by maltose.
[0017] The transport kinetics of S. cerevisiae Gal2 have been
measured and compared to those of KmLat1. The S. cerevisiae GAL2
gene (SEQ ID NO 5) under control of a TDH3 promoter exhibits 28
times greater (8.9 nmol/mgmm) L-arabinose transport rate as
compared to GAL2 gene under control of a ADH1 promoter. The
GAL2-encoded permease (SEQ ID NO 6) shows a K, of 550 mM and a
V.sub.max of 425 nmol/mgmin for L-arabinose transport and a K.sub.m
of 25 mM and a V.sub.max of 76 nmol/mgmin for galactose transport.
Although L-arabinose transport by both KmLAT1 and GAL2 encoded
permeases is out-competed by glucose or galactose, the inhibitory
effects of glucose or galactose are greater on the GAL2 encoded
permease than on the KmLAT1 encoded transporter.
[0018] It is farther disclosed here that a S. cerevisiae strain may
be transformed with different combinations of the KmLAT1 and PgLAT2
transporter genes and a plasmid carrying the GAL2 gene native to S.
cerevisiae. The doubling time for the PgLat2p and Gal2p
co-expressing cells grown on L-arabinose is markedly shorter than
that of the cells expressing only Gal2p, suggesting that
L-arabinose uptake may have been enhanced in these cells. In
addition, the PgLat2p and Gal2p co-expressing cells appear to grow
to a higher optical density at saturation, suggesting that this
strain may be able to utilize the L-arabinose in the medium more
completely. This conclusion is supported by HPLC analysis which
shows significantly less residual L-arabinose in the culture of
cells expressing PgLat2p and Gal2p.
[0019] In one embodiment, the transformed strains that carry the
new transporter genes may be further transformed with plasmids
carrying three bacterial genes, araA, araB and araC, which encode
proteins that may be utilized for arabinose utilization and
fermentation. In another embodiment, the bacterial genes, araA,
araB and araC, may be transformed into a yeast strain that does not
carry any of the new transporter genes.
[0020] In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become
apparent by reference to the drawings and by study of the following
descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Exemplary embodiments are illustrated in referenced figures
of the drawings. It is intended that the embodiments and figures
disclosed herein are to be considered illustrative rather than
limiting.
[0022] FIG. 1 shows the relationship between KmLAT1 and other
transporters based on the neighbor-joining method (Saitou and Nei
1987).
[0023] FIG. 2 shows the DNA (SEQ ID NO. 1) sequence of
Kluyveromyces marxianus KmLAT1, and the predicted protein sequence
(SEQ ID NO. 2).
[0024] FIG. 3 is a schematic presentation of the arabinose
metabolic pathway in recombinant yeast containing proteins encoded
by three bacterial genes araB, araA, and araD.
[0025] FIG. 4 shows the library insert from genomic K. marxianus
DNA complements adapted S. cerevisiae for growth on L-arabinose.
Cloning into the library expression vector is at the indicated
BamHI restriction sites. The black block arrow is the L-arabinose
transporter ORF responsible for complementation (KmLAT1). The block
arrow with vertical stripes is the interrupted transporter ORF. The
block arrow with the horizontal stripes is an un-related ORF
ligated in place gratuitously during library construction. The
Sau3AI restriction site where the transporter ORF was interrupted
is shown. The primer used for PCR based genomic walking in K.
marxianus is shown.
[0026] FIG. 5 shows the growth curve of S. cerevisiae expressing
KmLAT1 (.DELTA.), GAL2 (.box-solid.) or a control vector
(.diamond-solid.) on 2% L-arabinose.
[0027] FIG. 6A shows Eadie-Hofstee plot of L-arabinose uptake by
KmLat1 (.diamond-solid.) or Gal2 (.box-solid.) expressed in S.
cerevisiae grown on 2% L-arabinose. FIG. 6B shows comparison of
Eadie-Hofstee plots of KmLat1 expressed in S. cerevisiae
(.diamond-solid.) and wild type transport activity of K. marxianus
(.DELTA.) both grown on 2% L-arabinose.
[0028] FIG. 7 shows the DNA (SEQ ID NO. 3) sequence of Pichia
guilliermondii PgLAT2, and the predicted protein sequence (SEQ ID
NO. 4).
[0029] FIG. 8 shows the induction of L-arabinose transport in P.
guilliermondii. Uptake of 13 mM labeled sugar was assayed for cells
grown in minimal media containing 2% L-arabinose, D-galactose or
D-xylose. White bars indicated labeled L-arabinose transport. Black
bars indicate labeled galactose transport. Bars with vertical
stripes indicate labeled xylose transport.
[0030] FIG. 9 shows the sugar transport competition analysis in P.
guilliermondii grown in minimal L-arabinose medium.
[0031] FIG. 10 shows the transport kinetics of L-arabinose by the
PgLAT2 transporter expressed in S. cerevisiae. Open triangles
indicate transport for wild type P. guilliermondii grown on
L-arabinose. Black diamonds indicate transport for PgLAT2 expressed
in S. cerevisiae grown on L-arabinose.
[0032] FIG. 11 comparison of the growth curves in 0.2% L-arabinose
for S. cerevisiae cells expressing either Gal2p alone or both Gal2p
and PgLAT2. The maximum growth density and growth rate are
significantly enhanced in the strain expressing both Gal2p and
PgLAT2.
[0033] FIGS. 12A and 12B show the growth curves of BFY001 (parent)
(black square) and BFY002 (.DELTA.AR) (black triangle) on glucose
and xylulose.
[0034] FIGS. 13A, 13B and 13C show the diagrams of the expression
plasmids with araB, araA, and araD genes carrying the His3
selectable marker.
[0035] FIGS. 14A, 14B and 14C show the diagrams of the expression
plasmids with araB, araA, and araD genes carrying the Ura3
selectable marker.
[0036] FIG. 15 shows histogram with the result of ethanol
production in whole-cell fermentation using yeast cells expressing
the three bacterial genes araB, araA, and araD.
[0037] FIGS. 16A, 16B and 16C show histogram with the result of
ethanol production in cell-free fermentation using yeast cells
expressing the three bacterial genes araB, araA, and araD.
[0038] FIGS. 17A, 17B and 17C plot ethanol production using yeast
cells expressing the three bacterial genes araB, araA, and araD, as
a function of the incubation time in a cell-free fermentation
system.
[0039] FIG. 18 shows histogram with the result of ethanol
production in cell-free fermentation using yeast cells expressing
the three bacterial genes araB, araA, and araD.
[0040] FIG. 19 plots ethanol production using yeast cells
expressing the three bacterial genes araB, araA, and araD, under
single- and mixed-sugar fermentations.
DETAILED DESCRIPTION
[0041] There will now be shown and described methods for producing
transgenic yeast that we capable of metabolizing arabinose and
producing ethanol. In the discussion below, parenthetical mention
is made to publications from the references section for a
discussion of related procedures that may be found useful from a
perspective of one skilled in the art. This is done to demonstrate
what is disclosed by way of nonlimiting example.
[0042] The following definitions arc provided to facilitate,
understanding of certain terms used frequently herein and are not
meant to limit the scope of the present disclosure:
[0043] "Amino acid" refers to any of the twenty naturally occurring
amino acids as well as any modified amino acid sequences.
Modifications may include natural processes such as
postranslational processing, or may include chemical modifications
which are known in the art. Modifications include but are not
limited to: phosphorylation, ubiquitination, acetylation,
amidation, glycosylation, covalent attachment of flavin,
ADP-ribosylation, cross linking, iodination, methylation, and the
like.
[0044] "Antibody" refers to a generally Y-shaped molecule having a
pair of antigen binding sites, a hinge region and a constant
region. Fragments of antibodies, for example an antigen binding
fragment (Fab), chimeric antibodies, antibodies haying a human
constant region coupled to a murine antigen binding region, and
fragments thereof, as well as other well known recombinant
antibodies are included in this definition.
[0045] "Antisense" refers to polynucleotide sequences that are
complementary to target "sense" polynucleotide sequence.
[0046] "Biomass" refers collectively to organic non-fossil
material. "Biomass" in the present disclosure refers particularly
to plant material that is used to generate fuel, such as ethanol.
Examples of biomass includes but are not limited, to corn fiber,
dried distiller's grain, jatropha, manure, meal and bone meal,
miscanthus, peat, plate waste, landscaping waste, maize, rich
hulls, silage, stover, maiden grass, switchgrass, whey, and bagasse
from sugarcane.
[0047] "Complementary" or "complementarity" refers to the ability
of a polynucleotide in a polynucleotide molecule to form a base
pair with another polynucleotide in a second polynucleotide
molecule. For example, the sequence A-G-T is complementary to the
sequence T-C-A. Complementarity may be partial, in which only some
of the polynucleotides match according to base pairing, or
complete, where all the polynucleotide match according to base
pairing.
[0048] The term "derivative" refers to compounds that are derived
from a predecessor compound by way of chemical or physical
modification. For example, a compound is a sugar derivatives if it
is formed by oxidization of one or more terminal groups to
carboxylic acids, by reduction of a carbonyl group, by substitution
of hydrogen(s), amino group(s), thiol group(s), etc, for one or
more hydroxyl groups on a sugar, or if it is formed by
phosphorylation on a sugar molecule.
[0049] "Expression" refers to transcription and translation
occurring within a host cell. The level of expression of a DNA
molecule in a host cell may be determined on the basis of either
the amount of corresponding mRNA that is present within the cell or
the amount of DNA molecule encoded protein produced by the host
cell (Sambrook et al., 1989, Molecular cloning: A Laboratory
Manual, 18.1-18.88).
[0050] "Fusion protein" refers to a first protein attached to a
second, heterologous protein. Preferably, the heterologous protein
is fused via recombinant DNA techniques, such that the first and
second proteins arc expressed in frame. The heterologous protein
may confer a desired characteristic to the fusion protein, for
example, a detection signal, enhanced stability or stabilization of
the protein, facilitated oligomerization of the protein, or
facilitated purification of the fusion protein. Examples of
heterologous proteins useful as fusion proteins include molecules
having full-length or partial protein in sequence of KmLat1 or
PgLat2. Further examples include peptide tags such as histidine tag
(6-His), leucine zipper, substrate targeting moieties, signal
peptides, and the like. Fusion proteins are also meant to encompass
variants and derivatives of KmLat1 or PgLat2 polypeptides that are
generated by conventional site-directed mutagenesis and more modern
techniques such as directed evolution, discussed infra.
[0051] "Genetically engineered" refers to any recombinant DNA or
RNA method used to create a prokaryotic or eukaryotic host ceil
that expresses a protein at elevated levels, at lowered levels, or
in a mutated form. In other words, the host cell has been
transacted, transformed, or transduced with a recombinant
polynucleotide molecule, and thereby been altered so as to cause
the cell to alter expression of the desired protein. Methods and
vectors for genetically engineering host cells are well known in
the art; for example various techniques are illustrated in Current
Protocols In Molecular Biology, Ausubel el al., eds. (Wiley &
Sons, New York, 1988, and quarterly updated). Genetic engineering
techniques include but are not limited to expression vectors,
targeted homologous recombination and gene activation (see, for
example, U.S. Pat. No. 5,272,071 to Chappel) and trans activation
by engineered transcription factors (see, for Example, Segal, et
al., 1999, Proc Natl Acad Sci USA 96(6):2758-63). Genetic
engineering also encompasses any mutagenesis techniques wherein a
cell is exposed to chemicals to induce errors in DNA replication or
to accelerate gene recombination. The term "spontaneous mutation"
refers to mutations that occurs at a much lower rate as a result of
genetic recombination or DNA replication errors that occur
naturally from generation to generation.
[0052] "Heterologous" refers to DNA, RNA and/or polypeptides
derived from different organisms or species, for example a bacteria
polypeptide is heterologous to yeast.
[0053] "Homology" refers to a degree of similarity between
polynucleotides, haying significant effect on the efficiency and
strength of hybridization between polynucleotide molecules. The
term also refers, to a degree of similarity between polypeptides.
Two polypeptides having greater than or equal to about 60%
similarity are presumptively homologous.
[0054] "Host," "Host cell" or "host cells" refers to cells
expressing a heterologous polynucleotide molecule. The term
"heterologous" means non-native. For instance, when a gene that is
not normally expressed in an organism is introduced and expressed
in that host organism, such an expression is heterologous. Host
cells of the present disclosure express polynucleotides encoding
KmLAT1 or PgLAT2 or a fragment thereof. Examples of suitable host
cells useful in the present disclosure include, but are not limited
to, prokaryotic and eukaryotic cells. Specific examples of such
cells include bacteria of the genera Escherichia, Bacillus and
Salmonella, as well as members of the genera Pseudomonas,
Streptomyces, and Staphylococcus; fungi, particularly filamentous
fungi such as Trichoderma and Aspergillus, Phanerochaete
chrysosporium and other white not fungi; also other fungi including
Fusaria, molds, and yeast including Saccharomyces sp., Pichia sp.,
and Candida sp. and the like; plants e.g. Arabidopsis, cotton,
barley, tobacco, potato, and aquatic plants and the like; SF9
insect cells (Summers and Smith, 1987, Texas Agriculture Experiment
Station Bulletin, 1555), and the like. Other specific examples
include mammalian cells such as human embryonic kidney cells (293
cells), Chinese hamster ovary (CHO) cells (Puck et al., 1958, Proc.
Natl. Acad. Sci. USA 60, 1275-1281), human cervical carcinoma cells
(HELA) (ATCC CCL-2), human liver cells (Hep G2) (ATCC HB8065),
human breast cancer cells (MCF-7) (ATCC HTB22), human colon
carcinoma cells (DLD-1) (ATCC CCL 221), Daudi cells (ATCC CRL-213),
murine myeloma cells such as P3/NSI/1-Ag4-1 (ATCC TIB-18), P3X63Ag8
(ATCC TIB-9), SP2/0-Ag14 (ATCC CRL-1581) and the like. The most
preferred host is Saccharomyces cerevisiae.
[0055] "Hybridization" refers to the pairing of complementary
polynucleotides during an annealing period. The strength of
hybridization between two polynucleotide molecules is impacted by
the homology between the two molecules, stringency of the
conditions involved, the melting temperature of the formed hybrid
and the G:C ratio within the polynucleotides.
[0056] "Identity" refers to a comparison of two different DNA or
protein sequences by comparing pairs of nucleic acid or amino acids
within the two sequences. Methods for determining sequence identity
are known. See, for example, computer programs commonly employed
for this purpose, such as the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), that uses the algorithm of
Smith and Waterman, 1981, Adv. Appl. Math., 2:482-489.
[0057] "Isolated" refers to a polynucleotide or polypeptide that
has been separated from at least one contaminant (polynucleotide or
polypeptide) with which it is normally associated. For example, an
isolated polynucleotide or polypeptide is in a context or in a form
that is different from that in which it is found in nature.
[0058] "Nucleic acid sequence" refers to the order or sequence of
deoxyribonuclrotides along a strand of deoxyribonucleic acid. The
order of these deoxyribonucleotides determines the order of amino
acids along a polypeptide chain. The deoxyribonucleotide sequence
thus codes for the amino acid sequence.
[0059] "Polynucleotide" refers to a linear sequence of nucleotides.
The nucleotides may be ribonucleotides, or deoxyribonucleotides, or
a mixture of both. Examples of polynucleotides in this context
include single and double stranded DNA, single and double stranded
RNA, and hybrid molecules having mixtures of single and double
stranded DNA and RNA. The polynucleotides may contain one or more
modified nucleotides.
[0060] "Protein," "peptide," and "polypeptide" are used
interchangeably to denote an amino acid polymer or a set of two or
more interacting or bound amino acid polymers.
[0061] "Purify," or "purified" refers to a target protein makes up
for at last about 90% of a composition. In other words, it refers
to a target protein that is free from at least 5-10% of
contaminating proteins. Purification of a protein from
contaminating proteins may be accomplished using known techniques,
including ammonium sulfate or ethanol precipitation, acid
precipitation, heat precipitation, anion or cation exchange
chromatography, phosphocelluse chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, size-exclusion chromatography, and
lectin chromatography. Various protein purification techniques are
illustrated in Current Protocols in Molecular Biology, Ausubel et
al., eds. (Wiley & Sons, New York, 1988, and quarterly
updates).
[0062] "Selectable marker" refers to a marker that identifies a
cell as having undergone a recombinant DNA or RNA event. Selectable
markers include, for example, genes that encode antimetabolite
resistance such as the DHFR protein that confers resistance to
methotrexate (Wigler et al, 1980, Proc Natl Acad Sci USA 77:3567;
O'Hare et al., 1981, Proc Natl Acad Sci USA, 78:1527), the GPT
protein that confers resistance to mycophenolic acid (Mulligan
& Berg, 1981, PNAS USA, 78:2072), the neomycin resistance
marker that confers resistance to the aminoglycoside G-418
(Calberre-Garapin et al., 1981, J Mol Biol, 150:1), the Hygro
protein that confers resistance to hygromycin B (Santerre et. al.,
1984, Gene 30:147), and the Zeocin.TM. resistance marker
(Invitrogen). In addition, the herpes simplex virus thymidine
kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine
phosphoribosyltransferase genes may be employed in tk.sup.-
hgprt.sup.- and aprt.sup.- cells, respectively.
[0063] "Transform" means the process of introducing a gene into a
host cell. The gene may be foreign in origin, but the gene may also
derive from the host. A transformed host cell is termed a
"transformant." The introduced gene may be integrated onto the
chromosome of the host, or the gene may remain on a stand-alone
vector independent of the host chromosomes.
[0064] "Variant", as used herein, means a polynucleotide or
polypeptide molecule that differs from a reference molecule.
Variants may include nucleotide changes that result in amino acid
substitutions, deletions, fusions, or truncations in the resulting
variant polypeptide when compared to the reference polypeptide.
[0065] "Vector," "extra-chromosomal vector" or "expression vector"
refers to a first polynucleotide molecule, usually double-stranded,
which may have inserted into it a second polynucleotide molecule,
for example a foreign or heterologous polynucleotide. The
heterologous polynucleotide molecule may of may not be naturally
found in the host cell, and may be, for example, one or more
additional copy of the heterologous polynucleotide naturally
present in the host genome. The vector is adapted for transporting
the foreign polynucleotide molecule into a suitable host cell. Once
in the host cell, the vector may be capable of integrating-into the
host cell chromosomes. The vector may optionally contain additional
elements for selecting cells containing the integrated
polynucleotide molecule as well as elements to promote
transcription of mRNA from transacted DNA. Examples of vectors
useful in the methods disclosed herein include, but are not limited
to, plasmids, bacteriophages, cosmids, retroviruses, and artificial
chromosomes.
[0066] For purpose of this disclosure, unless otherwise stated, the
techniques used may be found in any of several well-known
references, such as: Molecular Cloning: A Laboratory Manual
(Sambrook et al. (1989) Molecular cloning: A Laboratory Manual),
Gene Expression Technology (Methods in Enzymology, Vol. 185, edited
by D. Goeddel, 1991 Academic Press, San Diego, Calif.), "Guide to
Protein Purification" in Methods in Enzymology (M. P. Deutshcer,
3d., (1990) Academic Press, Inc.), PCR Protocols; A Guide to
Methods and Applications (Innis et al. (1990) Academic Press, San
Diego, Calif.), Culture of Animal Cells: A Manual of Basic
Technique, 2.sup.nd ed. (R. I. Freshney (1987) Liss, Inc., New
York, N.Y.), and Gene Transfer and Expression Protocols, pp
109-128, ed. E. J. Murray, The Humana Press Inc., Clifton,
N.J.).
[0067] Unless otherwise indicated, the term "yeast," "yeast strain"
or "yeast cell" refers to baker's yeast, Saccharomyces cerevisiae.
Other yeast species, such as Kluyveromyces marxianus or Pichia
guilliermondii, are referred to as non-conventional yeast in this
disclosure. Strains of S. cerevisiae, depository information, and
plasmids used for this disclosure are listed in Table 1, 2 and
Table 3, respectively, The yeast Kluyveromyces marxianus CBS-1089
is obtained from the Centraalbureau voor Schimmelcultures (CBS)
collection. Pichia guilliermondii NRRL Y-2075 is obtained from the
Agricultural Research Service Culture Collection (NRRL).
TABLE-US-00001 TABLE 1 S. cerevisiae Strains Used in this
Disclosure Strain Genotype Plasmids BFY001 MATa ura3-52
trp1-.DELTA.63 his3-.DELTA.200 leu2-.DELTA.1 BFY002 MATa ura3-52
trp1-.DELTA.63 his3-.DELTA.200 leu2-.DELTA.1 yhr104w::LEU2 BFY507
MATa ura3-52 trp1-.DELTA.63 his3-.DELTA.200 leu2-.DELTA.1 p138, p42
yhr104w::LEU2 adapted for growth on L-arabinose BFY518 same as
BFY507 p138 BFY566 same as BFY518 p138, p171 BFY590 same as BFY518
gal2dcHIS3 p138 BFY597 same as BFY590 p138, p42 BFY598 same as
BFY590 p138, p187 BFY012 same as BFY002 pBFY004, pBFY013, pBFY012
BFY013 same as BFY002 pBFY007, pBFY016, pBFY014 BFY014 same as
BFY002 pBFY007, pBFY015, pBFY017 BFY015 same as BFY002 pBFY005,
pBFY016, pBFY019 BFY016 same as BFY002 pBFY005, pBFY018, pBFY017
BFY017 same as BFY002 pBFY009, pBFY018, pBFY014 BFY018 same as
BFY002 pBFY009, pBFY015, pBFY019 BFY057 MATa his3D1 leu2D0 ura3D0
met15D0 gal80D::G418 yhr104w::LEU2 BFY534 same as BFY057 p144, p165
BFY535 same as BFY057 p144, pBFY13 BFY605 same as BFY590 p244
BFY625 MATa his3.DELTA.1 leu2.DELTA.0 ura3.DELTA.0 trp1.DELTA.
pBFY12, pBFY13, p138 met15.DELTA.0 gal80.DELTA.::G418 adapted for
growth on L-arabinose BFY626 Same as BFY625 pBFY12, p138, p204
[0068] Yeast strains may be grown on liquid or solid media with 2%
agar for solid media. Where appropriate, some amino acids or
nucleic acids are purposely left out from the media for plasmid
maintenance. Growth conditions are typically 30.degree. C. unless
otherwise indicated, with shaking in liquid cultures. Anaerobic
conditions are generally more favorable to metabolize the various
sugars to ethanol.
[0069] A number of the yeast strains listed in Table 1 have been
deposited at the American Type Culture Collection (ATCC) in
accordance with the provisions of the Budapest Treatey on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedures. In each instance, the yeast strain
was deposited by the inventors listed herein on Mar. 16, 2007 at
American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110 U.S.A.
TABLE-US-00002 TABLE 2 Depository Information Yeast
Strain/Accession Number BFY013/PTA-8258 BFY534/PTA-8257
BFY598/PTA-8256 BFY626/PTA-8255
TABLE-US-00003 TABLE 3 Plasmids Used in this Disclosure Plasmid
Marker and expressed genes p42 URA3, GAL2 over-expression p138
TRP1, B. subtilis araA, E. coli araB, E. coli araD p144 E. coli
araB, D; B. subtilis araA in pBFY012 p165 HIS3, GAL2
over-expression p171 HIS3, 8.8 kb K. marxianus genomic DNA fragment
p187 URA3, KmLAT1 over-expression plasmid p204 HIS3, PgLAT2
over-expression plasmid p244 URA3, PgLAT2 over-expression plasmid
pBFY004 control 2.mu. vector with PGK promoter, GAL10 terminator
and Trp1 marker pBFY005 E. coli araB in pBFY004 pBFY007 E. coli
araA in pBFY004 pBFY009 E. coli araD in pBFY004 pBFY012 control
2.mu. vector with PGK promoter, GAL10 terminator and Ura3 marker
pBFY013 control 2.mu. vector with PGK promoter, GAL10 terminator
and His3 marker pBFY014 E. coli araB in pBFY012 pBFY015 E. coli
araB in pBFY013 pBFY016 E. coli araD in pBFY013 pBFY017 E. coli
araD in pBFY012 pBFY018 E. coli araA in pBFY013 pBFY019 E. coli
araA in pBFY012
[0070] Yeast cells may be grown in rich media YPD or minimum media
conventionally used in the field. YPD medium contains about 1%
yeast extract, 2% peptone and 2% dextrose. Yeast minimum media
typically contains 0.67% of yeast nitrogen base ("YNB") without
amino acids supplemented with appropriate amino acids or purine or
pyrimidine bases. An amount of sugar, typically 2% unless otherwise
indicated, may be used as carbon source, including glucose
(dextrose), galactose, maltose or L-arabinose among others.
Adaptation for growth on L-arabinose is performed as described in,
for example, Becker and Boles (2003) with modifications as detailed
in Example 3.
[0071] Over-expression plasmids are constructed by cloning the gene
for over-expression downstream of the S. cerevisiae PGK1 or TDH3
promoter in a 2.mu.-based vector. Construction of a DNA library is
detailed in the Examples. Note that other like S. cerevisiae
prompters can also be used for overexpression, including ADH2,
PDC1, PGI1, etc.
[0072] E. coli cells may be grown in LB liquid media or on LB agar
plates supplemented with ampicillin at 100 .mu.g/ml as needed.
Transformation of E. coli DH5.alpha. is by electrotransformation
according to a protocol by Invitrogen (Invitrogen 11319-019). After
transformation, the baterial cells are plated on LB plates
containing 100 .mu.g/ml ampicillin for selection. Transformation of
S. cerevisiae was performed using a DMSO-enhanced lithium-acetate
procedure as described with the following modifications (Hill et
al., 1991). Cells are harvested and initially washed in water. 600
.mu.l of PBG4000 solution is added and 70 .mu.l DMSO is added just
prior to heat shocking. Cells are heat-shocked for 15 min at
42.degree.0 C. and the last wash stop is skipped. Cells are
resuspended in 10 mM TE solution and plated.
[0073] Yeast DNA is isolated using the Easy DNA kit according to
manufacturer's protocol (Invitrogen, K1800-01). DNA manipulations
and library construction are performed as described in Molecular
Cloning: A Laboratory Manual (1989), except otherwise specifically
indicated in this disclosure. Plasmids are cured from yeast by
growing the strain in rich non-selective media overnight followed
by plating on non-selective media. Isolated colonies are replica
plated to screen for loss of selective markers. Plasmid rescue is
performed by transforming isolated yeast DNA into E. coli followed
by isolation and characterization, E. coli plasmid isolation is
accomplished using plasmid spin mini-prep kit according to the
manufacturer's manual (Siegen, 27106). PCR-based chromosomal
walking is performed using the Universal GenomeWalker Kit as
described (BD Biosciences, K1807-1).
[0074] For the transport assays, cells may be grown in minimal
media supplemented with 20 g/L of L-arabinose. Cells are collected
in mid-growth and washed twice before suspension in water at 30
mg/ml. Uptake of L-(1-.sup.14C) arabinose (54 mCi/mmol, Moravek
Biochemicals Inc.) or D-(1-.sup.14C) galactose (57 mCi/mmol,
Amersham Biosciences) is measured as previously described by
Stambuk et al. (2003). Assays are performed in 30 seconds to
maintain initial rates after appropriate experiments to ensure
uptake is linear for at least 1 minute. Transport activity is
described as nano-moles of labeled sugar transported per mg cell
dry weight per minute. Inhibition and competition assays are
performed as previously described by Stambuk et al. (2003).
[0075] Sequencing results showed that the KmLAT1 gene contains on
ORF of 1668 bp in length. The predicted amino acid sequence of
KmLAT1 shares homology With high-affinity glucose transporters, in
particular, with HGT1 with high-affinity glucose transporters from
non-conventional yeast than With transporter proteins encoded by
the bacterial araE gene or hexose transporters from S. cerevisiae
(FIG. 1).
TABLE-US-00004 TABLE 4 Properties and similarities of KmLat1 to
other sugar transporters. Predicted protein Predicted Degree of
(no. of aa/ pl of transmembrane identity (%)/ Putative function
gene no. of kDa) protein regions similarity (%) Organism of gene
product KmLat1 556/61.3 8.22 12 -- K. marxianus.sup.1 L-arabinose
transporter KlHgt1 551/60.8 5.76 12 77/89 K. lactis.sup.2 high
affinity glucose transporter AEL042Cp 547/59.8 8.82 12 65/82 A.
gossypii.sup.3 putative hexose transporter DEHA0E01738g 545/61.1
5.55 12 52/70 D. hansenii.sup.4 hexose transporter CaHgt1 545/60.7
8.05 12-13 50/71 C. albicans.sup.5 putative hexose transporter
CaHgt2 545/60.4 8.48 12-14 51/71 C. albicans.sup.6 putative hexose
transporter Accession numbers: .sup.1Not yet assigned,
.sup.21346290, .sup.3AEL042C, .sup.4DEHA0E01738g, .sup.5CAA76406,
.sup.6orf19.3668
[0076] Transmembrane regions predicted for KmLat1 and PgLat2 by the
software Tmpred shows 12 transmembrane regions with a larger
intercellular loop between regions 6 and 7 (FIG. 2) (See Hofmann et
al, 1993), typical of GAL2 and other yeast sugar transporters
having 10-12 transmembrane regions (See e.g., Alves-Araujo et al.,
2004; Day et al., 2002; Kruckeberg et al., 1996; Pina et al., 2004;
and Welerstall et al. 1999).
[0077] Like other members of the transporter family, and in
particular sugar transporters, KmLat1 and PgLat2 polypeptides are
useful in facilitating the uptake of various sugar molecules into
the cells. It is envisioned that KmLat1 or PgLat2 polypeptides
could be used for other purposes, for example, in analytical
instruments or other processes where uptake of sugar is required.
KmLat1 or PgLat2 polypeptides may be used alone or in combination
with one or more other transporters to facilitate the movement of
molecules across a membrane structure, which function may be
modified by one skilled in the relevant art, all of which are
within the scope of the present disclosure.
[0078] The KmLAT1 polypeptides include isolated polypeptides having
an amino acid sequence as shown below in Example 2; and in SEQ ID
NO:2, as well as variants and derivatives, including fragments,
having substantial sequence similarity of the amino acid sequence
of SEQ ID NO:2 and that retain any of the functional activities of
KmLAT1. PgLAT2 polypeptides include isolated polypeptides having an
amino acid sequence as shpwn below in Example 5; and in SEQ ID
NO:4, as well as variants and derivatives, including fragments,
having substantial sequence similarity to the amino acid sequence
of SEQ ID NO:4 and that retain any of the functional activities of
PgLAT2. The functional activities of the KmLAT1 or PgLAT2
polypeptides include but are not limited to transport of
L-arabinose across cell membrane. Such activities may be
determined, for example, by subjecting the variant, derivative, or
fragment to a arabinose transport assay as detailed, for example in
Example 4.
[0079] Variants and derivatives of KmLAT1 or PgLAT2 include, for
example, KmLAT1 or PgLAT2 polypeptides modified by convalent or
aggregative conjugation with other chemical moieties, such as
glycosyl groups, polyethylene glycol (PEG) groups, lipids,
phosphate, acetyl groups, and the like.
[0080] The amino acid sequence of KmLAT1 or PgLAT2 polypeptides is
preferably at least about 60% identical, more preferably at least
about 70% identical, more preferably still at least about 80%
identical, and in some embodiments at least about 90%, 95%, 96%,
97%, 98%, and 99% identical, to the KmLAT1 and PgLAT2 amino acid
sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The
percentage sequence identity, also termed homology (see definition
above) may be readily determined, for example, by comparing the two
polypeptide sequences using any of the computer programs commonly
employed for this purpose; such as the Gap program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, Madison Wis.), which uses the
algorithm of Smith and Waterman, 1981, Adv. Appl. Math.
2:482-489.
[0081] Variants and derivatives of the KmLAT1 or PgLAT2
polypeptides may further include, for example, fusion proteins
formed of a KmLAT1 or PgLAT2 polypeptide and another polypeptide.
Fusion protein may be formed between a fragment of the KmLAT1
polypeptide and another polypeptide, such that the fusion protein
may retain none or only part of the activities normally performed
by the full-length KmLAT1 or PgLAT2 polypeptide. Preferred
polypeptides, for constructing the fusion protein include those
that facilitate purification or oligomerization, or those that
enhance KmLAT1 or PgLAT2 stability and/or transport capacity or
transport rate for sugars, especially for arabinose. Preferred
polypeptides may also include those that gain enhanced transport
capability when fused with KmLAT1, PgLAT2 or fragments thereof.
[0082] KmLAT1 or PgLAT2 variants and derivatives may contain
conservatively substituted amino acids, meaning that one or more
amino acid may be replaced by an amino acid that does not alter the
secondary and/or tertiary structure of the polypeptide. Such
substitutions may include the replacement of an amino acid, by a
residue having similar physicochemical properties, such as
substituting one aliphatic residue (Ile, Val, Leu, or Ala) for
another, or substitutions between basic residues Lys and Arg,
acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl
residues Ser and Tyr, or aromatic residues Phe and Tyr.
Phenotypically silent amino acid exchanges are described more fully
in Bowie et al., 1990. In addition, functional KmLAT1 or PgLAT2
polypeptide variants include those having amino acid substitutions,
deletions, or additions to the amino acid sequence outside
functional regions of the protein. Techniques for making these
substitutions and deletions are well known in the art and include,
for example, site-directed mutagenesis.
[0083] The KmLAT1 or PgLAT2 polypeptides may be provided in an
isolated form, or in a substantially purified form. The
polypeptides may be recovered and purified from recombinant cell
cultures by known methods, including, for example, ammonium sulfate
or ethanol precipitation, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, and lectin chromatography. Preferably, protein
chromatography is employed for purification.
[0084] A preferred form of KmLAT1 or PgLAT2 polypeptides is that of
recombinant polypeptides expressed by suitable hosts. In one
preferred embodiment, when heterologous expression of KmLAT1 or
PgLAT2 is desired, the coding sequences of KmLAT1 or PgLAT2 may be
modified in accordance with the codon usage of the host. Such
modification may result in increase protein expression of a foreign
in the host. Furthermore, the hosts may simultaneously produce
other transporters such that multiple transporters are expressed in
the same cell, wherein the different transporters may form
oligomers to transport the same sugar. Alternatively, the different
transporters may function independently to transport different
sugars. Such recombinant cells may ne useful in crude fermentation
processing or in other industrial processing.
[0085] KmLAT1 or PgLAT2 polypeptides may be fused to heterologous
polypeptides to facilitate purification. Many available
heterologous peptides (peptide tags) allow selective binding of the
fusion protein to a blinding partner. Non-limiting examples of
peptide tags include 6-His, thioredoxin, hemaglutinin, GST, and the
OmpA signal sequence tag. A binding partner that recognizes and
binds to the heterologous peptide may be any molecule or compound,
including metal ions (for example, metal affinity columns),
antibodies, antibody fragments, or any protein or peptide that
preferentially binds the heterologous peptide to permit
purification of the fusion protein.
[0086] KmLAT1 or PgLAT2 polypeptides may be modified to facilitate
formation of KmLAT1 or PgLAT2 oligomers. For example, KmLAT1
polypeptides may be fused to peptide moieties that promote
oligomerization, such as leucine zippers and certain antibody
fragment polypeptides, for example, Fc polypeptides. Techniques for
preparing these fusion proteins are known, and are described, for
example, in WO 99/31241 and in Cosman et. al., 2001. Fusion to an
Fc polypeptide offers the additional advantage of facilitating
purification by affinity chromatography over Protein A or Protein G
columns. Fusion to a leucine-zipper (LZ), for example, a repetitive
heptad repeat, often with four or five leucine residues
interspersed with other amino acids, is described in Landschultz et
al., 1988.
[0087] It is also envisioned that an expanded set of variants and
derivatives of KmLAT1 or PgLAT2 polynucleotides and/or polypeptides
may be generated to select for useful molecules, where such
expansion is achieved not only by conventional methods such as
site-directed mutagenesis but also by more modern techniques,
either independently or in combination.
[0088] Site-directed-mutagenesis is considered an informational
approach to protein engineering and may rely on high-resolution
crystallographic structures of target proteins for specific amino
acid changes (van den Burg et al. 1998). For example, modification
of the amino acid sequence of KmLAT1 or PgLAT2 polypeptides may be
accomplished as is known in the art, such as by introducing
mutations at particular locations by oligonucleotide-directed
mutagenesis Site-directed-mutagenesis may also take advantage of
the recent advent of computational methods for identifying
site-specific changes for a variety of protein engineering
objectives (Hellinga, 1998).
[0089] The more modern techniques include, but are not limited to,
non-informational mutagenesis techniques (referred to generically
as "directed evolution"). Directed evolution, in conjunction with
high-throughput screening, allows testing of statistically
meaningful variations in protein conformation (Arnold, 1998).
Directed evolution technology may include diversification methods
similar to that described by Crameri et al. (1998), site-saturation
mutagenesis, staggered extension process (StEP) (Zhao et al.,
1998), and DNA synthesis/reassembly (U.S. Pat. No. 5,965,408).
[0090] Fragments of the KmLAT1 or PgLAT2 polypeptide maybe used,
for example, to generate specific anti-KmLAT1 antibodies. Using
known selection techniques, specific epitopes may be selected and
used to generate monoclonal or polyclonal antibodies. Such
antibodies have utility in the assay of KmLAT1 or PgLAT2 activity
as well as in purifying recombinant KmLAT1 or PgLAT2 polypeptides
from genetically engineered host cells.
[0091] The disclosure also provides polynucleotide molecules
encoding the KmLAT1 or PgLAT2 polypeptides discussed above. KmLAT1
or PgLAT2 polynucleotide molecules include polynucleotide molecules
having the nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID
NO:3, respectively; polynucleotide molecules that hybridize to the
nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively,
under high stringency hybridization conditions (for example,
42.degree., 2.5 hr., 6.times.SCC, 0.1% SDS); and polynucleotide
molecules having substantial nucleic acid sequence identity with
the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3,
respectively. It will be appreciated that such polynucleotide
molecules also broadly encompass equivalent substitutions of codons
that may be translated to produce the same amino acid sequences,
truncated fragments of the polynucelotide molecues, and
polynucleotide molecules with a high incidence of homology, such as
90%, 95%, 96%, 97%, 98%, or 99% or more homology with respect to
what is disclosed.
[0092] The KmLAT1 or PgLAT2 polynucleotide molecules of the
disclosure are preferably isolated molecules encoding the KmLAT1 or
PgLAT2 polypeptide having an amino acid sequence as shown in SEQ ID
NO:2 and SEQ ID NO:4, respectively, as well as derivatives,
variants, and useful fragments of the KmLAT1 or PgLAT2
polynucleotide. The KmLAT1 or PgLAT2 polynucleotide sequence may
include deletions, substitutions, or additions to the nucleic acid
sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively.
[0093] The KmLAT1 or PgLAT2 polynucleotide molecule may be cDNA,
chemically synthesized DNA, DNA amplified by PCR, RNA, or
combinations thereof. Due to the degeneracy of the genetic code,
two DNA sequences may differ and yet encode identical amino acid
sequences. The present disclosure thus provides an isolated
polynucleotide molecule having a KmLAT1 or PgLAT2 nucleic acid
sequence encoding KmLAT1 or PgLAT2 polypeptide, wherein the nucleic
acid sequence encodes a polypeptide haying the complete amino acid
sequences as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, or
variants, derivatives, and fragments thereof.
[0094] The KmLAT1 or PgLAT2 polynucleotides of the disclosure have
a nucleic acid sequence that is at least about 60% identical to the
nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,
respectively, in some embodiments at least about 70% identical to
the nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,
respectively, at least, about 80% identical to the nucleic acid
sequence shown in SEQ ID NO:1 and SEQ ID NO:3, respectively, and in
other embodiments al least about 90%-95%, 96%, 97%, 98%, 99%%
identical to the nucleic acid sequence shown in SEQ ID NO:1 and SEQ
ID NO:3, respectively. Nucleic acid sequence identity is determined
by known methods, for example by aligning two sequences in a
software program such as the BLAST program (Altschul, S. F et al.
(1990) J. Mol. Biol. 215:403-410, from the National Center for
Biotechnology Information
(http://www.ncbi.nlm.nih.gov//BLAST/).
[0095] The KmLAT1 or PgLAT2 polynucleotide molecules of the
disclosure also include isolated polynucleotide molecules having a
nucleic acid sequence that hybridizes under high stringency
conditions (as defined above) to a the nuclei acid sequence shown
in SEQ ID NO:1 and SEQ ID NO:3, respectively. Hybridization of the
polynucleotide is to at least about 15 contiguous nucleotides, oral
least about 20 contiguous nucleotides, and in other embodiments at
least about 30 contiguous nucleotides, and in still other
embodiments at least about 100 contiguous nucleotides of the
nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,
respectively.
[0096] Useful fragments of the KmLAT1 or PgLAT2 polynucleotide
molecules described herein, include probes and primers. Such probes
and primers may be used, for example, in PCR methods to amplify and
detect the presence of KmLAT1 or PgLAT2 polynucleotides in vitro,
as well as in Southern and Northern blots for analysis of KmLAT1 or
PgLAT2. Cells expressing the KmLAT1 or PgLAT2 polynucleotide
molecules may also be identified by the use of such probes. Methods
for the production and use of such primers and probes are known.
For PCR, 5' and 3' primers corresponding to a region at the termini
of the KmLAT1 or PgLAT2 polynucleotide molecule may be employed to
isolate and amplify the KmLAT1 PgLAT2 polynucleotide using
conventional techniques,
[0097] Other useful fragments of the KmLAT1 or PgLAT2
polynucleotides include antisense or sense oligonucleotides
comprising a single-stranded nucleic acid sequence capable of
binding to a target KmLAT1 or PgLAT2 mRNA (using a sense strand),
or DNA (using an antisense strand) sequence.
[0098] The present disclosure also provides vectors containing the
polynucleotide molecules, as well as host cells transformed with
such vectors. Any of the polynucleotide molecules of the disclosure
may be contained in a vector, which generally includes a selectable
marker and an origin of replication, for propagation in a host. The
vectors may further include suitable transcriptional or
translational regulatory sequences, such as those derived from a
mammalian, fungal, bacterial, viral, or insect genes, operably
linked to the KmLAT1 or PgLAT2 polynucleotide molecule. Examples of
such regulatory sequences include transcriptional promoters,
operators, or enhancers, mRNA ribosomal binding sites, and
appropriate sequences which control transcription and translation.
Nucleotide sequences are operably linked when the regulatory
sequence functionally relates to the DNA encoding the target
protein. Thus, a promoter nucleotide sequence is operably linked to
a KmLAT1 or PgLAT2 DNA sequence if the promoter nucleotide sequence
directs the transcription of the KmLAT1 or PgLAT2 sequence.
[0099] Selection of suitable vectors for the cloning of KmLAT1 or
PgLAT2 polynucleotide molecules encoding the KmLAT1 or PgLAT2
polypeptides of this disclosure depends upon the host cell in which
the vector will be transformed, and, where applicable, the host
cell from which the target polypeptide is to be expressed. Suitable
host cells for expression of KmLAT1 or PgLAT2 polypeptides include
prokaryotes, yeast, and higher eukaryotic cells, each of which is
discussed below. Selection of suitable combinations of vectors and
host organisms is a routine matter from a perspective of skill.
[0100] The KmLAT1 or PgLAT2 polypeptides to be expressed in such
host cells may also be fusion proteins that include sequences from
other proteins. As discussed above, such regions may be included to
allow, for example, enhanced functionality, improved stability, or
facilitated purification of the KmLAT1 or PgLAT2 polypeptide. For
example, a nucleic acid sequence encoding a peptide that binds
strongly to arabinose may be fused in-frame to the transmembrane
sequence of the KmLAT1 or PgLAT2 polypeptides so that the resulting
fusion protein binds arabinose and transports the sugar across the
cell membrane at a higher rate than the KmLAT1 or PgLAT2
transporter.
[0101] Suitable host cells for expression of target polypeptides
include prokaryotes, yeast, and higher eukaryotic ceils. Suitable
prokaryotic hosts to be used for the expression of these
polypeptides include bacteria of the genera Escherichia, Bacillus,
and Salmonella, as well as members of the genera Pseudomonas,
Streptomyces, and Staphylococcus.
[0102] Expression vectors for use in prokaryotic hosts generally
comprise one or more phenotypic selectable marker genes. Such genes
encode, for example, a protein that confers antibiotic resistance
or that supplies auxotrophic requirement. A wide variety of such
vectors are readily available from commercial sources. Examples
include pSPORT vectors, pGEM vectors (Promega, Madison, Wis.),
pPROEX vectors (LTI, Bethesda, Md.), Bluescript vectors
(Stratagene), and pQE vectors (Qiagen).
[0103] KmLAT1 or PgLAT2 may also be expressed in yeast host cells
from genera including Saccharomyces, Pichia, and Kluveromyces.
Preferred yeast host is S. cerevisiae. Yeast vectors will often
contain an origin of replication sequence from a 2.mu. yeast
plasmid for high copy vectors and a CEN sequence for a low copy
number vector. Other sequences on a yeast vector may include an
autonomously replicating sequence (ARS), a promoter region,
sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Vectors replicable in
both yeast and E. coli (termed shuttle vectors) are preferred. In
addition to the above-mentioned features of yeast vectors, a
shuttle vector will also include sequences for replication and
selection in E. coli.
[0104] Insect host cell culture systems may also be used for the
expression of KmLAT1 or PgLAT2 polypeptides. The target
polypeptides are preferably expressed using a baculovirus
expression system, as described, for example, in the review by
Luckow and Summers, 1988.
[0105] The choice of a suitable expression vector for expression of
KmLAT1 or PgLAT2 polypeptides will depend upon the host cell to be
used. Examples of suitable expression vectors for E. coli include
pET, pUC, and similar vectors as is known in the art. Preferred
vectors for expression of the KmLAT1 or PgLAT2 polypeptides include
the shuttle plasmid plJ702 for Streptomyces lividans, pGAPZalpha-A,
B, G and pPICZalpha-A, B, C (Invitrogen) for Pichia pastoris, and
pFE-1 and pFE-2 for filamentous fungi and similar vectors as is
known in the art. The vectors preferred for expression in S.
cerevisiae are listed in Table 2.
[0106] Modification of a KmLAT1 or PgLAT2 polynucleotide molecule
to facilitate insertion into a particular vector (foe example, by
modifying restriction sites), ease of use in a particular
expression system or host (for example, using preferred host
codons), and the like, are known and are contemplated for use.
Genetic engineering methods for the production of KmLAT1 or PgLAT2
polypeptides include the expression of the polynucleotide molecules
in cell free expression systems, in host cells, in tissues, and in
animal models, according to known methods.
[0107] This disclosure also provides reagents, compositions, and
methods that are useful for analysis of KmLAT1 or PgLAT2 activity
and for assessing the amount and rate of arabinose transport.
[0108] The KmLAT1 or PgLAT2 polypeptides of the present disclosure,
in whole or in part, may be used to raise polyclonal and monoclonal
antibodies that are useful in purifying KmLAT1 or PgLAT2, or
detecting KmLAT1 or PgLAT2 polypeptide expression, as well as a
reagent tool for characterizing the molecular actions of the KmLAT1
or PgLAT2 polypeptide. Preferably, a peptide containing a unique
epitope of the KmLAT1 or PgLAT2 polypeptide is used in preparation
of antibodies, using conventional techniques. Methods for the
selection of peptide epitopes and production of antibodies are
known. See, for example, Antibodies: A Laboratory Manual, Harlow
and Land (eds.), 1988 Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.; Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.), 1980 Plenum
Press, New York.
[0109] Agents that modify, for example, to increase or decrease,
KmLAT1 or PgLAT2 transport of arabinose or other sugars may be
identified by the transport assay described in Example 4, for
example. Performing the transport assay in the presence or absence
of a test agent permits screening of such agents.
[0110] The KmLAT1 or PgLAT2 transport activity is determined in the
presence or absence of a test agent and then compared. For
instance, a lower KmLAT1 transport activity in the presence of the
test agent, than in the absence of the test agent, indicates that
the test agent has decreased the activity of the KmLAT1.
Stipulators and inhibitors of KmLAT1 or PgLAT2 may be used to
augment, inhibit, or modify KmLAT1 or PgLAT2 transport activity,
and therefore may have potential industrial uses as veil as
potential use in further elucidation of the molecular actions of
KmLAT1 or PgLAT2.
[0111] The KmLAT1 or PgLAT2 polypeptide of the disclosure is an
effective arabinose transporter. In the methods of the disclosure,
the sugar transporting effects of KmLAT1 or PgLAT2 are achieved by
mixing cells expressing KmLAT1 or PgLAT2 with pure sugar or
sugar-containing biomass. KmLAT1 or PgLAT2 may also be used in a
cell-free system. KmLAT1 or PgLAT2 may be used under other
conditions, for example, at elevated temperatures or under acidic
pH. Other methods of using KmLAT1 or PgLAT2 to transport sugar,
especially arabinose, for fermentation, are envisioned to be within
the scope of what is disclosed, KmLAT1 or PgLAT2 polypeptides may
be used in any known application currently utilizing a sugar
transporter, all of which are within the scope of this
disclosure.
[0112] It is shown in this disclosure that Gal2p is an effective
L-arabinose transporter at high concentrations of arabinose,
whereas KmLAT1 or PgLAT2 may be more effective at different
concentrations of L-arabinose. Combination of the Gal2p and the two
new transporters from non-conventional yeast may be employed to
provide complementary transport into S. ceretfsiae of L-arabinose
down to very low residual concentration of arabinose.
[0113] It is shown here that combinatorial expression of Gal2p,
KmLAT1 and PgLAT2 may enhance the overall rate and extent of
arabinose utilization by recombinant S. cereviciae cells expressing
these transporters. As shown in Example 8, the doubling time for S.
cereviciae strain expressing both PgLAT2 and Gal2p is shorter than
S. cereviciae cells expressing Gal2p alone (15 hours vs. 19 hours),
suggesting that L-arabinose uptake may be enhanced by the
synergistic effect of PgLAT2 and Gal2p in these cells. Moreover the
PgLAT2 expressing strain appears to grow to a higher overall
optical density at saturation, suggesting that this strain was able
to utilize the carbon source (L-arabinose) in the medium more
completely. This hypothesis is supported by HPLC analysis of the
final culture media (Table 5) which indicates that there is
significantly less residual L-arabinose in the culture of cells
expressing Gal2p and PgLAT2 than in the culture of those expressing
Gal2p alone. Thus, heterologous expression of either or both KmLAT1
and PgLAT2 in S. cereviciae may enhance arabinose utilization by
facilitating arabinose transport when the concentration of
arabinose is relatively low.
TABLE-US-00005 TABLE 5 Doubling times and HPLC Measurement of
Residual Arabinose Concentration in Cultures Described in FIG. 11.
Transporters Doubling L-arabinose Flask Expressed Time (hours)
Final OD.sub.600 (g/L) by HPLC 1 Gal2p only 19.2 0.72 0.68 2 18.6
0.72 0.67 3 Gal2p + PgLat2 15.0 0.85 0.49 4 14.8 0.85 0.48
*starting L-arabinose concentration 1.89 g/L and media without
L-arabinose had an undetectable level (<0.1 g/L). ND = not
determined.
[0114] L-arabinose metabolism in bacteria involves three enzymes:
L-arabinose isomerase (araA), L-ribulokinase (araB), and
L-ribulose-5-p 4-epimerase (araD), which may be collectively
referred to as the "araBAD" proteins in this disclosure. The genes
encoding these three enzymes may be referred to as the "araBAD"
genes in this disclosure. The combined action of these three
bacterial proteins, convert L-arabinose to Xylulose-5-phosphate
(See FIG. 3). S. cerevisiae contains the pathway to utilize and
ferment the final product xylulose-5-phosphate and produce ethanol
under certain conditions (see FIG. 3).
[0115] S. cerevisiae strain to be used to construct an arabinose
fermenting yeast strain preferably possesses Gal.sup.+ phenotype. A
Gal.sup.+ strain is likely to express galactose permease which may
facilitate the uptake of arabinose by S. cerevisiae.
[0116] S. cerevisiae typically possesses endogenous aldose
reductase ("AR") activity, which may divert arabinose to a pathway
different from the one that may lead to the production of ethnaol
through the action of the bacterial araBAD proteins. Moreover, the
arabitol generated by the AR protein may inhibit the isomerase
encoded by araA. In order to increase the overall yield of ethanol
from arabinose, it is preferable to use an AR-deficient strain to
construct the arabinose fermenting yeast of the present disclosure.
The AR-deficient strain may be obtained by screening for
spontaneous mutations, or preferably by targeted gene disruption or
mutation. An example of such gene disruption is detailed in Example
10.
[0117] As shown in FIG. 3, the engineered pathway utilizing
bacterial araBAD converts L-arabinose to xylulose-5-P that S.
cerevisiae can convert to ethanol using endogenous enzymatic
activities. It is thus desirable to ensure that the arabinose
metabolic pathway starting from xylulose to ethanol remains intact
in the AR-deficient strain. This may be tested by comparing the
growth of the AR-deficient strain with its parental strain on
glucose or xylulose. If both strains proliferate at about the same
rate on glucose or xylulose, it is likely that the AR gene
disruption event has not negatively impacted the catabolism of
glucose of xylulose.
[0118] The present disclosure also provides a new method to measure
arabinose uptake by yeast cells. Traditionally, L-arabinose
transport is measured by using radio-labeled substrate. Since
aldose reductase, which converts L-arabinose to arabitol, is
cytosolic, it is possible to use the formation of new arabitol as
an indicator of arabinose uptake. Higher levels of arabitol
indicates higher uptake of L-arabinose. To confirm the validity of
this method, L-arabinose transport was measured using the
traditional .sup.14C-labelled L-arabinose in various yeast strains
with different levels of arabitol formation. These experiments show
that the level of arabitol formation corresponds well with the
level of L-arabinose uptake.
[0119] Using this method, several high arabitol producing strains
have been isolated, including two gal80 mutants and two otherwise
wildtype strains, which have 3 to 4 folds higher L-arabonise and
D-galactose transport activity than the BFY001 originally used to
construct the arabinose fermenting strains. Bacterial genes
encoding the araBAD proteins may be introduced into these strains
to achieve higher rate of arabinose uptake and thus higher overall
yield. This result also validates the indirect screening method for
strains with higher arabinose transport activity.
[0120] Although the present disclosure teaches the introduction of
foreign genes such as E. coil araBAD genes, into yeast cells, genes
from other species encoding proteins that perform the same or
similar function as the E. coli araBAD proteins, i.e., converting
L-arabinose into various intermediates and eventually into ethanol,
may be used in place of the E. coli araBAD genes (See e.g., Becker
and Boles, 2003, using araA from Bacillus subtilis). The DNA of the
foreign genes may be present in a host cell at one copy, or
preferably, in more than one copy. The foreign genes maybe under
control of a constitutive promoter or an inducible promoter.
[0121] The foreign genes may be present as plasmids or
minichromosomes in the host yeast cells, or alternatively, the
plasmids carrying the foreign genes may be engineered so that the
foreign genes are integrated into the chromosomes of the host
through genetic recombination. In the latter case, the foreign
tends to be maintained after generations, even when the host cells
are grown in rich media where no selective pressure is present. By
contrast, in the former case where the genes remain on a vector,
the genes may be lost after a few generations. Under those
circumstances, the host yeast cells are preferably grown in a
minimum media supplemented with appropriate amino acids or purine
or pyrimidine bases so that a selective pressure helps maintain the
plasmids.
[0122] Cell-free or whole-cell fermentation may be used to convert
arabinose to ethanol. In the whole cell fermentation process, the
transformants may be grown on minimum media with appropriate
supplementation to maintain the plasmids. The transformant cells
are preferably grown on galactose to induce the expression of
galactose permease. More preferably, the cells are grown on both
arabinose and galactose before the fermentation assays. In
addition, transformant cells can be grown on both arabinose and
glucose before the fermentation assays (see FIG. 19).
[0123] In the cell-free fermentation system, cells are harvested
and the cells are lysed to release enzymes for the conversion of
the sugar to ethanol. One bottleneck for a whole-cell fermentation
system is the uptake of arabinose by the cells, which may explain
its lower overall yield of ethanol than the cell-free system.
However, whole-cell fermentation is preferred because it is easier
to perform. In a whole-cell-fermentation system, the cells may be
mixed directly with the biomass or other substrates, requiring no
extra steps of cell-handling.
[0124] In a preferred embodiment, the various arabinose metabolic
pathways disclosed here may be introduced into a S. cerevisiae
strain that have been modified to facilitate its arabinose uptake.
Such strain may include but are not limited to strains that express
both the Gal2p and one or two of the novel arabinose transporters
similar to the ones disclosed here. The expression levels of the
array of arabinose transporters may be fine-tuned such that they
are commensurate with the rate of arabinose metabolism inside the
engineered yeast cells. Most preferably, the expression levels of
the transporters may be linked to the arabinose metabolic rate in
each cell, such that the arabinose is taken in more rapidly by
those cells that convert arabinose to ethanol more efficiently.
[0125] The examples herein illustrate the present instrumentalities
by way of illustration, and not by limitation. The chemicals,
biological agents and other ingredients are presented as typical
components or reactants, and the procedures described herein may
represent but one of the typical ways to accomplish the goal of the
particular experiment. It is understood that various modification
may be derived in view of the foregoing disclosure without
departing from the spirit of the present disclosure.
Example 1
Cloning of the New Transporter Gene KmLAT1
[0126] A K. marxianus genomic library was constructed in our yeast
-vector puffy13 which contains the yeast 2.mu. origin of
replication, a URI3 selection cassette, and a BamHI site located
between the PGK1 promoter and GAL10 terminator. After partial
digestion of 200 .mu.g of genomic DNA with Sau3AI restriction
enzyme, fragments of 2-8 kb in length were gel-isolated and ligated
into the BamHI site of pBFY013. This ligation reaction was then
transformed into E. coli and plated for recovery. Plate counts
produced .about.3000 cfu's/10 .mu.l of transformed cells and the
plasmid DNA from 24 colonics was screened for presence of insert
revealing 22 of 24 transformants had an insert ranging from 1 kb to
.about.8 kb giving an average insert size of 3.2 kb. The
transformed cells were scraped from the plates, DNA recovered, and
5 .mu.l was transformed into competent BFY518 cells. The strain,
BFY518, was cured of the GAL2 over-expression plasmid negating its
ability to form colonies on agar plates containing L-arabinose as
the sole carbon source enabling restoration of colony formation by
complementation with a heterologous L-arabinose transporter. To
count the number of transformed yeast cells, 10 .mu.l of the yeast
library transformation were plated onto minimal glucose media yet
the colonies were so dense that only an estimate of .about.5000
colonies was possible. The rest of the transformation mix
(.about.140.mu.l) was plated onto minimal media containing 2%
L-arabinose for selection from which a small amount of background
growth was noticed. The plates were then replica plated to fresh
L-arabinose minimal media. The total number of cells plated for
selection represented .about.280,000 transformants representing
.about.8 fold coverage of the 10.7 mb K. marxianus genome (See
Dujon et al., 2004). Two colonies grew on the replica plates and
the plasmid DNA was rescued and re-transformed into BFY518 allowing
growth once again on L-arabinose confirming that the K. marxianus
genomic insert carried on these plasmids was responsible for
growth. Restriction analysis suggested both plasmids harbored the
same insert of approximately 8.8 kb in size.
Example 2
Sequence Analysis of the KmLAT1Gene
[0127] Sequencing results showed that both plasmids had identical
inserts of 8838 kb containing two ORFs on the 5' end of the insert.
Both of these ORFs showed strong homology to yeast sugar
transporters. One transporter ORF was interrupted by a fragment of
an unrelated ORF suggesting that recombination of fragments during
ligation into the vector occurred in library construction (FIG. 4).
Recombination of library fragments during ligation into the vector
was shown by PCR walking experiments performed on K. marxianus
genomic DNA. Walking was performed out of the transporter in a 5'
direction and additional transporter sequences including the start
codon was recovered rather than the additional sequence from the
undated ORF. The uninterrupted transporter ORF, termed KmLAT1, was
recovered twice more in another subsequent library screening. This
ORF was 1668 bp in length and shared homology with high-affinity
glucose transporters in particular, HGT1 from K. lactis (Table 4)
and showed a much closer association with high-affinity glucose
transporters from non-conventional yeasts than the bacterial araE
genes or S. cerevisiae hexose transporters (FIG. 1).
[0128] Transmembrane region prediction by the software Tmpred shows
12 transmembrane regions with a larger intercellular loop between
regions 6 and 7 (FIG. 2) (See Hoffmann et al, 1993), typical of
GAL2 and other yeast sugar transporters having 10-12 transmembrane
regions (See e.g., Alves-Araujo et al., 2004; Day et al., 2002;
Kruckeberg et al., 1996; Pina et al., 2004; and Welerstall et al.
1999).
Example 3
KmLAT1 Expressed in S. cerevisiae Enables Growth on Arabinose
[0129] The coding sequence of KmLAT1 was isolated by PCR from
genomic DNA of K. marxianus and cloned into a yeast 2.mu. plasmid
under control of the PGK1 promoter of S. cerevisiae. This construct
was transformed into a GAL2 deleted strain of S. cerevisiae adapted
to L-arabinose. Briefly, cells are grown in appropriate selective
glucose minimal media until saturation then washed and diluted to a
starting OD.sub.600 of 0.2 in minimal media supplemented with 2%
L-arabinose. Cultures are incubated until exponential growth Is
observed then the cultures are diluted twice into the same media
for continued growth to establish the final L-arabinose utilizing
adapted strain which is purified on streak plates. Control plasmids
carrying the yeast GAL2 gene and an empty vector were also used to
transform yeast cells.
[0130] Yeast cells with a 2.mu. plasmid carrying the KmLAT1 or GAL2
gene or cells with an empty 2.mu. plasmid were grown with shaking
in liquid minimum media containing 2% L-arabinose as the sole
carbon source. The OD.sub.600 of each culture was measured and
monitored by 140 hours. Growth curve results show that KmLAT1 is
sufficient to support growth on L-arabinose when compared to cells
harboring the empty vector which does not show any signs of growth
(FIG. 5). This result confirms that the KmLAT1 gene encodes an
arabinose transporter that enables yeast cells to grow on
L-arabinose.
Example 4
Comparison of the Arabinose Transport Kinetics between Gal2 and
KmLAT1 Expressed in S. cerevisiae
[0131] The transport characteristics of the KmLAT1 and tire Gal2
transporters expressed in S. cerevisiae were compared. Both
transporters were expressed in a host background adapted for growth
on L-arabinose in which the endogenous copy of GAL2 had been
entirely replaced with a HIS3 selection marker. The KmLat1
transporter showed a low-affinity transporter having a K.sub.m=230
mM and a V.sub.max=55 nmol/mgmin (FIG. 6A). This is in contrast to
the high-affinity active transport activity induced in the wild
type K. marxianus when grown on 2% L-arabinose (FIG. 6B). These
results suggest there are at least 2 transporters in K. marxianus
that may transport L-arabinose but just the high-affinity activity
is induced in the wild type when grown 2% L-arabinose. Inhibition
experiments showed that when KmLAT1 is expressed in S. cerevisiae
it is not significantly inhibited by protonophores such as
NaN.sub.3, DNP, and CCP. Neither is KmLAT1 inhibited by
H+-adenosine triphosphatase (ATPase) inhibitors such as DESB and
DCCD (Table 6). This is in contrast to the transport activity in
wild type K. marxianus, suggesting that KmLAT1 is a facilitated
diffusion permease similar to the Gal2 permease. Competition
experiments showed that KmLAT1 is out-competed by glucose,
galactose, xylose, and maltose when expressed in S. cerevisiae
(Table 6).
TABLE-US-00006 TABLE 6 Effect of Inhibitors or Competing Sugars on
the Rate of L-Arabinose Transport in L-Arabinose-Grown S.
cerevisiae Expressing GAL2 or KmLAT1 Relative L-arabinose Inhibitor
or Concentration transport (%) Competing Sugar (mM) Gal2 KmLat1
None NA 100.sup.a 100.sup.c NaN.sub.3 10 66 11 CCCP 5 46 61 DCCD 5
69 55 DNP 5 72 75 DESB 5 81 100 None NA 100.sup.b 100.sup.d Glucose
900 10 17 Galactose 900 3 23 Xylose 900 25 25 Maltose 450 ND 38
.sup.aUptake rate was 66.0 nmol mg.sup.-1 min.sup.-1 determined
with 118 mM labeled L-arabinose. .sup.bUptake rate was 18.9 nmol
mg.sup.-1 min.sup.-1 determined with 30 mM labeled L-arabinose.
.sup.cUptake rate was 7.7 nmol mg.sup.-1 min.sup.-1 determined with
118 mM labeled L-arabinose. .sup.dUptake rate was 3.6 nmol
mg.sup.-1 min.sup.-1 determined with 30 mM labeled L-arabinose. ND,
Not Done.
[0132] Transport kinetics of S. cerevisiae BFY597 over-expressing
the Gal2 permease grown on 2% L-arabinose showed a of K.sub.m of
550 and a V.sub.max of 425 nmol/mgmin for L-arabinose transport
(FIG. 6A). Inhibition assays showed a reduction but not a complete
inhibition of transport suggestive of facilitated diffusion
transport (Table 6). Competition studies showed that glucose,
galactose, and xylose significantly reduced L-arabinose transport
indicating that those sugars are preferentially transported over
L-arabinose (Table 6). The kinetics of galactose transport were
also measured in this strain and indicate that Gal2p has a K.sub.m
of 25 mM and a V.sub.max of 76 nmol/mgm for galactose transport
(data not shown) demonstrating a higher affinity for galactose that
would out-compete L-arabinose for transport.
Example 5
Cloning of the New Transporter Gene PgLAT2
[0133] Wildtype Pichia guilliermondii NRRL Y-2075 was obtained from
the Agricultural Research Service Culture Collection (NRRL). Pichia
guilliermondii cells were grown in minimal media supplemented with
2% L-arabinose, galactose, or xylose. Cells were collected in
mid-growth and washed twice in water before suspension in water at
about 30 mg/ml. RNA was extracted from the cells using the acid
phenol method (Ausubel, et al., Short Protocols in Molecular
Biology, John Wiley and Sons, 1999). Briefly, approximately 15 mL
of fresh culture was added to about 25 mL of crushed ice and
centrifuged at 4.degree. C. for 5 min at 3840.times.g. Cells were
washed twice with cold DEPC-treated water, and the pellets were
frozen at -80.degree. C. After the pellets were resuspended in 400
ul TES (10 mM Tris HCl, pH 7.5, 5 mM EDTA, 0.5% SDS), 400 ul of
acid phenol was added. The samples were vortexed vigorously for 10
sec, followed by incubation for 30-60 min at 65.degree. C. with
occasional vortexing. The tubes with the samples were then chilled
on ice and spun for 5 min at 4.degree. C. The aqueous phase was
removed and re-extracted with chloroform. The aqueous phase was
then ethanol precipitated using 0.1 volume of 3 M sodium acetate
(pH 5.3) and two volumes of 100% ethanol. The pellet was washed
using 80% ethanol, dried, and resuspended in 50 ul DEPC H.sub.2O.
Total RNA concentration-was quantitated by measuring the OD.sub.200
and visualized on agarose gels.
[0134] RNA purification, synthesis of cDNA, and differential
display were performed at GenHunter Corporation according to
standard techniques. DNA Bands showing higher levels of expression
from arabinose-grown cells relative to xylose- or galactose-grown
cells were reamplified using the differential display amplification
primers. Direct sequencing was performed on the PCR products using
the GenHunter arbitrary primers. In cases that did not yield clean
sequence, the amplification products were cloned in the TOPO-TA
vector pCR2.1 (Invitrogen) and individual clones were sequenced.
Sequences were then compared to the databases using BLASTX analysis
and those that showed similarity to known transporters on
transporter-like proteins were examined further. One of these
sequences led to the identification of a novel transporter gene,
PgLAT2 from Pichia guilliermondii, PgLAT2 gene has an ORF of 1617
nucleotides encoding a protein with a predicted length of 539 amino
acids (FIG. 7).
Example 6
Characteristics of Sugar Transport by Pichia guilliermondii
[0135] The induction of L-arabinose transport in wild type P.
guilllermondii was examined. Wildtype Pichia guilliermondii cells
were grown in minimal media supplemented with 2% L-arabinose,
galactose, or xylose while BFY605 cells were grown in the same
media supplemented with 0.2% L-arabinose. Cells were collected in
mid-growth and washed twice in water before suspension in water at
about 30 mg/ml. Uptake of L-(1-.sup.14C)arabinose (54 mCi/mmol,
Moravek Biochemicals Inc.), D-(1-.sup.14C)galactose (57 mCi/mmol,
Amersham Biosciences), or D-(1-.sup.14C)xylose (53 mCi/mmol,
Moravek Biochemicals Inc.) was measured as previously described
(Stambuk, Franden et al. 2003). Assays were performed in 5, 10, or
30 second periods to maintain initial rates. Appropriate
experiments ensured uptake was linear for at least 1 minute.
Transport activity was described as nmoles of labeled sugar
transported per mg cell dry weight per minute. Inhibition and
competition assays were performed as previously described (Stambuk,
Franden et al. 2003).
[0136] Cells grown on L-arabinose were able to transport
L-arabinose whereas cells grown on galactose or xylose were not
able to transport L-arabinose. Additionally, xylose transport was
about double in cells grown in L-arabinose media compared to cells
grown in xylose media. Galactose was transported at the same rate
independent of growth substrate (FIG. 8). Transport competition
between L-arabinose and xylose was also examined. Uptake of labeled
L-arabinose was reduced by 96% when 100 .times. un-labeled xylose
was included in the transport assay whereas uptake of labeled
xylose was only reduced by 16% when 100 .times. un-labeled
L-arabinose was included in the assay (FIG. 9). This data suggests
that in P. guilliermondii, growth on L-arabinose induces expression
of a specific transport system capable of transporting L-arabinose
and xylose. Additionally, this system preferentially transports
xylose at the expense of L-arabinose if both sugars are present and
has a higher transport velocity for xylose than the transport
system induced when grown on xylose. By contrast, transport
activity for L-arabinose is not induced when grown on xylose.
Example 7
Arabinose Transport Kinetics of PgLAT2 Expressed in S.
cerevisiae
[0137] The L-arabinose transport characteristics of the PgLAT2
transporter expressed in S. cerevisiae grown on 0.2% L-arabinose
medium showed the same L-arabinose transport characteristics as
wildtype P. guilliermondii (FIG. 10). The PgLAT2 transporter when
expressed in S. cerevisiae has a K.sub.m=0.07 mM and V.sub.max=18
nmol/mgmin. Inhibition experiments showed significant inhibition of
transport by protonophores (NaN.sub.3, DNP, and CCP) and
H+-adenosine triphosphatase (ATPase) inhibitors (DESB and DCCD)
similar to the inhibition Observed in wildtype P. guilliermondii
(Table 7). Compaction experiments showed that L-arabinose uptake by
the PgLAT2 transporter was inhibited by glucose, galactose, xylose
and to a lesser extent by maltose (Table 7).
TABLE-US-00007 TABLE 7 Effect of Inhibitors or Competing Sugars on
the Rate of L-Arabinose Transport in L-Arabinose-Grown P.
guilliermondii Y-2075 and S. cerevisiae BFY605 Inhibitor or
Relative L-arabinose transport Competing Concentration S.
cerevisiae (PgLAT2 Sugar (mM) P. guilliermondii transporter)
None.sup.a -- 100 100 NaN.sub.3 10 1 16 DNP 5 0 4 CCCP 5 0 2 DCCD 5
22 36 DESB 5 8 1 None.sup.b -- 100 100 Glucose 120 ND 17 Galactose
120 ND 20 Xylose 120 4 0 Maltose 120 ND 30 .sup.aRate of
L-arabinose transport was 11.2 nmol mg.sup.-1 min.sup.-1 for P.
guilliermondii and 10.4 nmol mg.sup.-1 min.sup.-1 for S. cerevisiae
(PgLAT2 transporter) determined with 0.33 mM labeled L-arabinose.
.sup.bRate of L-arabinose transport was 14.2 nmol mg.sup.-1
min.sup.-1 for P. guilliermondii and 14.4 nmol mg.sup.-1 min.sup.-1
for S. cerevisiae (PgLAT2 transporter) determined with 1.2 mM
labeled L-arabinose.
[0138] The transport activities, inhibition profiles, and
competition rates with respect to xylose of wildtype P.
guilliermondii and of the PgLAT2 transporter expressed in S.
cerevisiae are identical suggesting that P. guilliermondii has a
single, high affinity, active transporter charged with uptake of
L-arabinose. There are no L-arabinose transport activities that are
unaccounted which suggests the presence of a single L-arabinose
transporter in P. guilliermondii.
Example 8
Synergistic Effect on Growth Rate and Sugar Utilization by S.
cerevisiae Expressing Gal2p and the New Transporter Proteins-PgLat2
and KmLat1
[0139] To determine the complementary effects on arabinose
transport by the three transporters, namely, Gal2p, PgLat2 and
KmLAT1, yeast strains were constructed with appropriate selection
markers to allow different pathway and transporter combinations to
be expressed. All possible transporter combinations were generated
by introducing transporter expression plasmids for PgLat2 and
KmLat1 (or empty vectors) into S. cerevisiae strains expressing the
bacterial genes araA; araB and araD (See e.g., Becker and Boles).
All strains expressing Gal2p (due to the gal80A genotype), plus or
minus other transporters, were able to grow on 2% or 0.2%
L-arabinose after extensive lag times (a process termed
"adaptation."). A relatively low concentration of L-arabinose
(0.2%) was used in this experiment as strain differences are more
pronounced at this concentration. Once "adapted" to growth on 0.2%
L-arabinose, the strains were able to grow more quickly and growth
curves for each transporter combination were generated.
[0140] FIG. 11 shows a comparison of shake flask-growth curves for
four strains on 0.2% L-arabinose, all of which express Gal2p (via
the GAL80 deletion) in the absence or presence of the novel
transporter PgLat2 (also see Table 5). A significant lag time was
observed due to their inoculation from stationary cultures.
However, once growth initiated, the growth rate was relatively
rapid. The doubling time for each culture in the exponential phase
of the curve is shown in Table 5. The doubling time for the PgLat2
and Gal2p co-expressing cells was markedly shorter than in the
cells expressing only Gal2p (15 hours vs. 19 hours). A second
observation relates to the overall extent of growth. The PgLat2
expressing strain appeared to grow to a higher overall optical
density at saturation, suggesting that this strain was able to
utilize the carbon source (L-arabinose) in the medium more
completely (FIG. 11).
Example 9
Co-expression of Gal2p with PgLAT2 or KmLAT1 Enables more Complete
Utilization of Arabinose by Recombinant S. cerevisiae
[0141] Doubling limes for the cultures described above in Example 8
were measured in early exponential phase for each culture. Doubling
time was measured by the period of time taken for the number of
cells to double in a given cell culture (See generally, Guthirie
and Fink, 1991). The concentration of remaining L-arabinose at the
276 hour time point was determined by HPLC (for saturated cultures
only). The concentration of L-arabinose in the starting media was
about 1.89 g/L and the concentration of L-arabinose in media
without L-arabinose had an undetectable level (<0.1 g/L). As
shown in Table 5, significantly less residual L-arabinose remained
in the culture of cells expressing both Gal2p and PgLAT2 than in
the culture of cells expressing Gal2p alone.
Example 10
Construction of S. cerevisiae Strain Deficient in Aldose Reductase
(AR)
[0142] Based on the sequence of a presumptive AR gene, two
oligonucleotide primers were designed and the AR gene along with
600 bp of flanking DNA were cloned by PCR using genomic DNA
isolated from yeast as template. Using another set of primers, an
AR deletion construct was made in which all the coding sequences of
the AR gene were replaced with a restriction enzyme site (SalI).
The yeast LEU2 gene was isolated as a SalI-XhoI fragment and cloned
into the SalI site of the AR deletion construct. The DNA fragment
containing the LEU2 gene and AR flanking sequences was used to
transform the leu2.sup.- yeast strain BFY001. LEU.sup.+
transformants were isolated, grown and analyzed by Southern and PCR
analysis to confirm that the AR gene in the genome had been deleted
and replaced with the LEU2 gene by homologous recombination.
[0143] One such transformant, designated BFY002, was chosen as a
host for further construction of arabinose fermenting yeast
strains. Shake-flask experiments were conducted and the results
showed that arabitol formation in BFY002 had been reduced to about
50% in comparison with the parental strain BFY001.
[0144] Growth of BFY002 on glucose and xylulose was compared with
that of BFY001. Briefly, yeast strains BFY001 and BFY002 were grown
in rich medium YPD. Cells were collected by centrifugation and
washed with sterile water. The washed cells were suspended in water
at the original density. 50 .mu.l of this cell suspension was used
to inoculate 5 ml of medium containing yeast nitrogen base ("YNB")
supplemented with leucine ("Leu"), tryptophan ("Trp"), histidine
("His") and uracil ("Ura"), plus with 1% glucose or 1% xylulose.
The growth of each strain on both media at 30.degree. C. with
shaking was monitored by measuring the OD.sub.600 for about 6
generation times. As expected, both strains had much shorter
doubling time on glucose than on xylulose. No significant
difference in the growth curves was observed between BFY001 and
BFY002 regardless of whether glucose or xylulose was used (FIG. 12A
and FIG. 12B).
Example 11
Isolation of E. coli araBAD Genes and B. subtillis araA and
Introduction into S. cerevisiae
[0145] Primers were designed to isolate E. coli araA gene as a
BgIII fragment, and the araB and araD genes were isolated as BamHI
fragments by PCR from plasmid pZB206. The fragments containing the
three genes were cloned into a yeast expression vector pBFY004,
which contains PGK promoter, GAL10 terminator, and TRP1 selection
marker. Each plasmid carrying individual gene was then transformed
into the yeast strain BFY002 in separate experiments. Transformants
carrying individual plasmid were analyzed for the expression levels
of each ara protein. L-ribulokinase (araB) and
L-ribulose-5-P-4-epimerase (araD) were expressed at a higher level
while L-arabinose isomerase (araA) was expressed at a lower
level.
[0146] In order to introduce all three ara genes into the same
cell, URA3 and HIS3 expression vectors for each ara gene were
constructed by re-engineering the TRP1 plasmid, pBFY004. Briefly,
the TRP1 coding sequence was removed and replaced with a SalI
restriction site to generate a plasmid designated as pBFY011. Other
selection markers, HIS3 or URA3, were then cloned into this
plasmid. Another strategy for introducing all three ara genes into
the same cell was to construct a plasmid carrying all three genes.
Briefly this was done by combining each ara gene with a different
promoter/terminator combination to prevent homologus recombination
and thus loop-out of the genes with corresponding loss of function.
Primers were designed to clone the E. coli araD gene between the
TDH3 promoter and GAL2 terminator. This expression cassette was
then moved to pBFY007 (which already has the E. coli araA gene
cloned between the PGK1 promoter and GAL10 terminator) and
designated pBFY051. Similarly primers were designed to clone the E.
coli araB gene between the PGII promoter and the PDC1 terminator.
This expression cassette was then moved to pBFY051 to create
pBFY090 which now has all three E. coli ara genes. The B. subtilis
araA gene was then isolated using PCR and cloned between the PGK1
promoter and GAL10 terminator to replace the E. coli araA. The URA3
gene was isolated as a SalI fragment and cloned into the SalI site
to construct the plasmid pBFY012. Similarly, a HIS3 expression
vector, pBFY013, was contructed by engineering and cloning the HIS3
gene into pBFY011.
[0147] The engineered ara genes were cloned into each of these
expression vectors to generate a series of expression vectors
carrying each of the araBAD genes with either Trp1, Ura3 or His3
markers (Table 3; also see FIG. 13A, FIG. 13B, FIG. 13C, FIG. 14A,
FIG. 14B and FIG. 14C). Appropriate combinations of these
expression vectors were introduced into the strain BFY002.
Similarly, the plasmid containing all three ara genes was
introduced into Hip strain BFY057. The transformants were
characterized and assayed for growth and fermentation of
arabinose.
Example 12
Determination of the Copy Numbers of Plasmids Carrying araBAD in S.
cerevisiae
[0148] The copy numbers of the three plasmids present m the strain
BFY013 (Table 8) transformed as described in Example 11 were
determined. On colony of BFY013 was isolated and used to inoculate
a flask with yeast minimum medium. The cells were allowed to grown
to exponential phase and the cells were harvested by
centrifugation. Spheroplasts of the cell were prepared and DNA was
extracted from these spheroplasts (See Guthrie and Fink, 1991). E.
coli strain DH5.alpha. cells were then transformed with the
extracted yeast DNA. Bacterial transformants were plated out and
plasmid DNA in individual colonies was isolated and characterized
by restriction digest followed by agarose, gel electrophoresis.
Assuming that the individual plasmids carrying each of the araBAD
genes possessed the same capability to transform bacterial cells,
the ratio between the copy numbers of each plasmids present in the
original yeast cells was estimated based on the number of E. coli
transformants harboring each plasmid (Table 8).
TABLE-US-00008 TABLE 8 Ratio of the 3 plasmids in BFY013 ara gene
Number araB 11 araA 4 araD 15
Example 13
Assays of Enzymntic Activities of araBAD Proteins Expressed in S.
cerevisiae
[0149] The activities of the three E. coli enzymes heterologously
expressed in S. cerevisiae were measured in the crude extracts of
the yeast transformants according to protocol described in Becker
and Boles, 2003. The results of these assays are summarized in
Table 9. Table 10 compares the enzymatic activities of the two
strains used for subsequent fermentation. The enzymatic assays were
performed in the presence of absence of 20 mM MnCl.sub.2, but it
appears that MnCl.sub.2 does not have significant effect on the
overall results (Table 10).
TABLE-US-00009 TABLE 9 Enzyme activities in transformants carrying
all 3 ara genes L-arabinose isomerase (araA) L-ribulokinase (araB)
L-ribulose 5-P4-epimerase (araD) Sp. act umol/min/mg Sp. act
umol/min/mg Sp. act umol/min/mg Strain Oct 20-21 (1) Oct 20-21 (2)
Dec 20-22 (2) Oct 20-21 Dec 20-22 Oct 20-21 Dec 20-22 BFY012 nd nd
nd nd nd nd BFY013 0.05 0.10 0.11 trp 1.2 1.3 ura 0.5 1.0 his
BFY014 0.04 0.11 0.11 trp 1.2 1.9 his nd nd ura BFY015 nd nd ura
2.4 2.8 trp 0.39 0.9 his BFY016 0.03 0.06 his 2.4 2.4 trp nd nd ura
BFY017 0.02 nd his 0.9 1.1 ura 1.56 >3.5 trp BFY018 0.01 nd ura
1.2 1.0 his 1.12 >2.2 trp Zymomonas 0.85 0.9 1.9 nd = not
detected (1) = cysteine-carbozole (2) = new method, NADH
disappearance All cultures were grown in selective medium (YNB + 2%
glo-trp-his-ura)
TABLE-US-00010 TABLE 10 Enzyme Activities in Strains Used for
Fermentation Isomerase Kinase Epimerase (araA) (araB) (araD) Sp.
act Sp. act Sp. act MnCl2 Protein umol/ umol/ umol/ Strain (20 mM)
mg/ml min/mg min/mg min/mg BFY012 No 6.8 nd nd nd BFY012 Yes 6.1 nd
nd nd BFY013 No 7.0 0.09 2.2 0.6 BFY013 Yes 6.7 0.09 1.9 0.5
Example 14
Whole-cell Fermentation in S. cerevisiae
[0150] The transformed yeast cells carrying bacterial genes araBAD
were first grown in galactose or arabinose alone or in the presence
of both galactose and arabinose in YNB. Cells were collected by
centrifugation and washed in water. The washed cells were
resuspended in liquid media-containing 1% yeast extract and 2%
peptone (YP). The cell suspensions were aliquoted into various
tubes before appropriate sugars were added. The tubes were
incubated at 30.degree. C. and cell samples were taken at the time
indicated. The samples were filtered and analyzed for ethanol
concentration by gas chromatography (GC) according to Tietz, 1976
(Table 11 and FIG. 15) or by high performance liquid chromatography
(HPLC). As shown in Table 11, cells that were grown in both
galactose and arabinose immediately before the fermentation assay
had a slightly higher overall yield of ethanol than cells grown in
galactose alone during the same period. The strains BFY534 and
BFY535 were grown in arabinose alone prior to fermentation. From a
starting concentration of 19 g/L of L-arabinose, BFY534 and BFY535
used 12.7 and 11.8 g/L of L-arabinose to yield 4.7 and 4.9 g/L of
ethanol in 48 hours respectively. The percentage of maximum
theoretical conversion would thus be 75% and 78% respectively and a
productivity of 0.012 g EtOH/g cells hr for both strains. In an
additional fermentation with strain BFY534 performed in shake
flasks, 19.7 g/L of L-arabinose was converted to 8.5 g/L ethanol in
96 hrs giving 85% of the theoretical maximum conversion and a
productivity of 0.017 g EtOH/g cells hr.
TABLE-US-00011 TABLE 11 Ethanol Concentration (g/l) from Whole Cell
Fermentation Glucose Arabinose (66.7 g/l) (66.7 g/l) No sugar Time
(hrs) 0 24 0 24 120 0 24 120 BFY012 (Gal) 1.9 33.6 1.0 1.1 1.8 1.0
0.9 1.8 BFY013 (Gal) 1.6 33.3 0.9 1.3 2.6 0.0 0.9 0 BFY015 (Gal)
1.5 33.8 0.9 1.3 2.5 0.0 0.9 1.3 BFY012 (G + A) 2.1 34.3 1.0 1.2
1.9 1.0 1.0 1 BFY013 (G + A) 1.7 34.2 1.0 1.8 4.2 0.9 1.0 0 BFY015
(G + A) 1.5 33.8 0.9 1.5 3.2 0.0 1.0 0
Example 15
Cell-free Fermentation in S. cerevisiae
[0151] For fermentation of arabinose in a cell-free system, yeast
transformants were grown in the presence of both galactose and
arabinose in YNB. Cells were collected by centrifugation and washed
in water. Cell walls were removed by enzymatic digestion and the
cells were then lysed in a lysis buffer containing 20 mM potassium
phosphate buffer, pH 7 and 10 mM MgCl.sub.2 and 1 mM DTT. Cells
debris were removed by centrifugation, and the supernatant was
transferred to a tube where various chemicals were added to the
supernatant such that the fermentation mix contained 7 mM Mg
acetate, 5 mM ATP, 0.1 mM diphosphoglyceric acid, 4 mM Na arsenate
and 2 mM NAD.sup.+. Fermentation was started by adding appropriate
sugar to the fermentation mix in the tube. The tube was incubated
at 30.degree. C. and samples were taken at the time indicated. The
samples were boiled, centrifuged, filtered and analyzed for ethanol
concentration by gas chromatography (GC) as previously described.
Results of the cell-free fermentation are shown in Table 12, FIG.
16A, FIG. 16B, FIG. 16C, FIG. 17A, FIG. 17B, FIG. 17C and FIG.
18.
TABLE-US-00012 TABLE 12 Ethanol Concentration (g/l) from Cell-Free
Fermentation Glucose Arabinose No sugar Time (hrs) 0 2 24 48 72 0 2
24 48 72 0 2 24 48 72 BFY012 0.0 2.7 8.1 8.3 8.2 0.0 1.4 1.9 1.9
1.9 1.2 1.5 1.9 1.9 2.0 BFY013 0.0 3.1 12.6 13.2 12.8 0.0 1.7 4.5
6.0 6.8 0.0 1.5 2.1 2.1 2.0 BFY012 (20 mM MnCl.sub.2) 1.3 2.6 9.2
9.0 8.5 1.2 1.4 1.9 1.9 2.1 0.0 1.4 2.0 2.0 1.8 BFY013 (20 mM
MnCl.sub.2) 1.3 3.0 12.8 13.1 -- 1.3 1.8 4.6 6.1 6.8 1.3 1.6 2.0
2.0 --
Example 16
Mixed-Sugar Fermentation in S. cerevisiae
[0152] Yeast attains adapted to contain the araB and araD genes
from E. coli, the araA isomerase gene from B. subtilis, and the
GAL2 overexpression plasmid have been described previously (see for
example, Example 11); Adapted strains, BFY534 for example, were
tested for fermentation of glucose, arabinose, and a mixture of
arabinose and glucose. In particular, 125 ml non-baffled flasks
containing 50 ml of yeast-extract peptone media (including adapted
yeast) and either no sugar, glucose, L-arabinose, or both glucose
and L-arabinose were prepared. The flasks were closed with
Saranwrap held in place with rubber bands. The fermentations were
performed a 30.degree. C. with gentle shaking (80 rpm). In all
cases, each sugar was present at a concentration of 20 g/L.
[0153] The results are illustrated in FIG. 19, where a greater than
50% increase in ethanol production was obtained in co-fermentation
of glucose and L-arabinose, compared to the ethanol yield of
glucose alone.
[0154] While a number of exemplary aspects and embodiments have
been discussed above, those of skill in the art will recognize
certain modifications, permulations, additions and sub-combinations
thereof. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such modifications, permulations, additions and
sub-combinations as are within their true spirit and scope.
[0155] This specification contains numerous citations to references
such as patents, patent applications, and scientific publications.
Each is hereby incorporated by reference for all purposes.
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Biotechnol. Bioeng. 78:172-178. [0183] Welerstall, T., C.
Hollenberg, and E. Boles, 1999,Cloning and characterization of
three genes (SUT1-3) encoding glucose transporters of the yeast
Pichia stipitis, Mol Microbiol 31:871-883. [0184] Zhang, M., C.
Eddy, K. Deanda, M. Finkolstein, and S. Picatagglo, 1995, Metabolic
engineering of a pentose metabolism pathway in ethanologenic
Zymomonas mobilis. Science 267:240-243. [0185] Zhao, H.; Giver, L.;
Shao, Z.; Affholter, J. A.; Arnold, F. H. Nature Biotechnol. 1998,
16, 258-62.
Sequence CWU 1
1
611671DNAKluyveromyces marxianusCDS(1)..(1668) 1atg act tta aaa gat
aaa cta ttg ctc cgc aat atc gaa ttc aag gga 48Met Thr Leu Lys Asp
Lys Leu Leu Leu Arg Asn Ile Glu Phe Lys Gly 1 5 10 15 act ttc tat
gcg aag ttc ccg caa att cac aac att tac gca atc ggt 96Thr Phe Tyr
Ala Lys Phe Pro Gln Ile His Asn Ile Tyr Ala Ile Gly 20 25 30 gtg
att tcg tgt ata tct ggt ctc atg ttt ggt ttc gat atc tct tca 144Val
Ile Ser Cys Ile Ser Gly Leu Met Phe Gly Phe Asp Ile Ser Ser 35 40
45 atg tct tcc atg atc ggt act gaa act tac aaa aaa tat ttt gac cat
192Met Ser Ser Met Ile Gly Thr Glu Thr Tyr Lys Lys Tyr Phe Asp His
50 55 60 cca aaa tcc att acc caa ggt ggt atc acc gcg tca atg tcc
ggt ggt 240Pro Lys Ser Ile Thr Gln Gly Gly Ile Thr Ala Ser Met Ser
Gly Gly 65 70 75 80 tcc ttc tta ggc tct tta ctc tct cct gct att tcc
gat acc ttt ggc 288Ser Phe Leu Gly Ser Leu Leu Ser Pro Ala Ile Ser
Asp Thr Phe Gly 85 90 95 aga aaa gtg tcg ttg cac att tgt gcc gtc
ttg tgg atc gtc gga tgc 336Arg Lys Val Ser Leu His Ile Cys Ala Val
Leu Trp Ile Val Gly Cys 100 105 110 att ttg caa agt gct gcc caa gac
caa cca atg cta atc gct ggc cgt 384Ile Leu Gln Ser Ala Ala Gln Asp
Gln Pro Met Leu Ile Ala Gly Arg 115 120 125 gtt atc gca ggg ttg ggt
atc ggg ttc ggc tct ggt tct gct cca att 432Val Ile Ala Gly Leu Gly
Ile Gly Phe Gly Ser Gly Ser Ala Pro Ile 130 135 140 tac tgt tct gaa
atc tcc cca cca aag gtt aga ggc ttg atc acc ggt 480Tyr Cys Ser Glu
Ile Ser Pro Pro Lys Val Arg Gly Leu Ile Thr Gly 145 150 155 160 ctt
ttc cag ttc tct atc act gtt ggt att atg att ctc ttc tac gtt 528Leu
Phe Gln Phe Ser Ile Thr Val Gly Ile Met Ile Leu Phe Tyr Val 165 170
175 ggt tac ggg tgc cac ttc ctc agt ggt aat ctt tca ttc aga ttg act
576Gly Tyr Gly Cys His Phe Leu Ser Gly Asn Leu Ser Phe Arg Leu Thr
180 185 190 tgg ggt ttg caa gtt atc cca gga ttt gtg ttg ctg gtc ggt
gtc cta 624Trp Gly Leu Gln Val Ile Pro Gly Phe Val Leu Leu Val Gly
Val Leu 195 200 205 ttc ttg cca gaa tcc cca cgt tgg ttg gct aac cac
gac cgt tgg gaa 672Phe Leu Pro Glu Ser Pro Arg Trp Leu Ala Asn His
Asp Arg Trp Glu 210 215 220 gaa act gag tca atc gtc gcc aag gtc gtc
gcc aag ggt aac gta gac 720Glu Thr Glu Ser Ile Val Ala Lys Val Val
Ala Lys Gly Asn Val Asp 225 230 235 240 gat gaa gaa gtc aag ttc caa
ttg gaa gaa att aaa gag cag gtg att 768Asp Glu Glu Val Lys Phe Gln
Leu Glu Glu Ile Lys Glu Gln Val Ile 245 250 255 ctt gat gct gcc gcc
aag aac ttc tcc ttc aag gat ttg cta aga cca 816Leu Asp Ala Ala Ala
Lys Asn Phe Ser Phe Lys Asp Leu Leu Arg Pro 260 265 270 aag acc aga
aag aag ctc ttt gtt ggt gtg tgt gct caa atg tgg caa 864Lys Thr Arg
Lys Lys Leu Phe Val Gly Val Cys Ala Gln Met Trp Gln 275 280 285 caa
ttg tgt ggt atg aac gtt atg atg tac tac att gtg tac gtc ttt 912Gln
Leu Cys Gly Met Asn Val Met Met Tyr Tyr Ile Val Tyr Val Phe 290 295
300 aac atg gct ggt tac act ggt aac acc aac ttg gtt gca tct tcc att
960Asn Met Ala Gly Tyr Thr Gly Asn Thr Asn Leu Val Ala Ser Ser Ile
305 310 315 320 caa tac gtc ttg aac gtg cta atg act ttc cct gca cta
ttc tta atc 1008Gln Tyr Val Leu Asn Val Leu Met Thr Phe Pro Ala Leu
Phe Leu Ile 325 330 335 gat aaa gtc ggt aga aga cct gtc ttg atc gtt
ggt ggt att ttc atg 1056Asp Lys Val Gly Arg Arg Pro Val Leu Ile Val
Gly Gly Ile Phe Met 340 345 350 ttc aca tgg ttg ttc gct gtc gct ggt
ttg ttg gca tca tat tcc gtc 1104Phe Thr Trp Leu Phe Ala Val Ala Gly
Leu Leu Ala Ser Tyr Ser Val 355 360 365 cca gct cca aat ggt gtt aac
ggt gat gat act gtc aca atc aga atc 1152Pro Ala Pro Asn Gly Val Asn
Gly Asp Asp Thr Val Thr Ile Arg Ile 370 375 380 cca gac aag cac aag
tcc gcc gct aag ggt gtc att gca tgt tca tac 1200Pro Asp Lys His Lys
Ser Ala Ala Lys Gly Val Ile Ala Cys Ser Tyr 385 390 395 400 ttg ttc
gtc tgc tct ttc gct cca acc tgg ggt att ggt atc tgg att 1248Leu Phe
Val Cys Ser Phe Ala Pro Thr Trp Gly Ile Gly Ile Trp Ile 405 410 415
tac tgt tcc gaa att ttc aac aac atg gaa aga gcc aag ggt tcc tct
1296Tyr Cys Ser Glu Ile Phe Asn Asn Met Glu Arg Ala Lys Gly Ser Ser
420 425 430 gtg gct gct gct acc aac tgg gca ttc aac ttc gct ttg gcg
atg ttc 1344Val Ala Ala Ala Thr Asn Trp Ala Phe Asn Phe Ala Leu Ala
Met Phe 435 440 445 gtc cca tct gca ttc aag aac atc tca tgg aaa aca
tac atc gtc ttt 1392Val Pro Ser Ala Phe Lys Asn Ile Ser Trp Lys Thr
Tyr Ile Val Phe 450 455 460 ggt gtc ttt tca gtt gca ttg act gtc caa
acc tac ttc atg ttc cca 1440Gly Val Phe Ser Val Ala Leu Thr Val Gln
Thr Tyr Phe Met Phe Pro 465 470 475 480 gaa act aga ggt aag acc ttg
gaa gaa atc gac caa atg tgg gtc gac 1488Glu Thr Arg Gly Lys Thr Leu
Glu Glu Ile Asp Gln Met Trp Val Asp 485 490 495 aac atc cca gcc tgg
aag act agc agc tac atc cca caa ttg cct atc 1536Asn Ile Pro Ala Trp
Lys Thr Ser Ser Tyr Ile Pro Gln Leu Pro Ile 500 505 510 atc gaa gat
gaa ttt ggt aac aag ttg ggt ttg ttg ggt aac cca caa 1584Ile Glu Asp
Glu Phe Gly Asn Lys Leu Gly Leu Leu Gly Asn Pro Gln 515 520 525 cat
ctc gag cat gtt aaa tcc gtc gaa aag gat act gta gtg gaa aaa 1632His
Leu Glu His Val Lys Ser Val Glu Lys Asp Thr Val Val Glu Lys 530 535
540 tta gaa tcg tca gag gct aat agc agc agc tcg gtc tag 1671Leu Glu
Ser Ser Glu Ala Asn Ser Ser Ser Ser Val 545 550 555
2556PRTKluyveromyces marxianus 2Met Thr Leu Lys Asp Lys Leu Leu Leu
Arg Asn Ile Glu Phe Lys Gly 1 5 10 15 Thr Phe Tyr Ala Lys Phe Pro
Gln Ile His Asn Ile Tyr Ala Ile Gly 20 25 30 Val Ile Ser Cys Ile
Ser Gly Leu Met Phe Gly Phe Asp Ile Ser Ser 35 40 45 Met Ser Ser
Met Ile Gly Thr Glu Thr Tyr Lys Lys Tyr Phe Asp His 50 55 60 Pro
Lys Ser Ile Thr Gln Gly Gly Ile Thr Ala Ser Met Ser Gly Gly 65 70
75 80 Ser Phe Leu Gly Ser Leu Leu Ser Pro Ala Ile Ser Asp Thr Phe
Gly 85 90 95 Arg Lys Val Ser Leu His Ile Cys Ala Val Leu Trp Ile
Val Gly Cys 100 105 110 Ile Leu Gln Ser Ala Ala Gln Asp Gln Pro Met
Leu Ile Ala Gly Arg 115 120 125 Val Ile Ala Gly Leu Gly Ile Gly Phe
Gly Ser Gly Ser Ala Pro Ile 130 135 140 Tyr Cys Ser Glu Ile Ser Pro
Pro Lys Val Arg Gly Leu Ile Thr Gly 145 150 155 160 Leu Phe Gln Phe
Ser Ile Thr Val Gly Ile Met Ile Leu Phe Tyr Val 165 170 175 Gly Tyr
Gly Cys His Phe Leu Ser Gly Asn Leu Ser Phe Arg Leu Thr 180 185 190
Trp Gly Leu Gln Val Ile Pro Gly Phe Val Leu Leu Val Gly Val Leu 195
200 205 Phe Leu Pro Glu Ser Pro Arg Trp Leu Ala Asn His Asp Arg Trp
Glu 210 215 220 Glu Thr Glu Ser Ile Val Ala Lys Val Val Ala Lys Gly
Asn Val Asp 225 230 235 240 Asp Glu Glu Val Lys Phe Gln Leu Glu Glu
Ile Lys Glu Gln Val Ile 245 250 255 Leu Asp Ala Ala Ala Lys Asn Phe
Ser Phe Lys Asp Leu Leu Arg Pro 260 265 270 Lys Thr Arg Lys Lys Leu
Phe Val Gly Val Cys Ala Gln Met Trp Gln 275 280 285 Gln Leu Cys Gly
Met Asn Val Met Met Tyr Tyr Ile Val Tyr Val Phe 290 295 300 Asn Met
Ala Gly Tyr Thr Gly Asn Thr Asn Leu Val Ala Ser Ser Ile 305 310 315
320 Gln Tyr Val Leu Asn Val Leu Met Thr Phe Pro Ala Leu Phe Leu Ile
325 330 335 Asp Lys Val Gly Arg Arg Pro Val Leu Ile Val Gly Gly Ile
Phe Met 340 345 350 Phe Thr Trp Leu Phe Ala Val Ala Gly Leu Leu Ala
Ser Tyr Ser Val 355 360 365 Pro Ala Pro Asn Gly Val Asn Gly Asp Asp
Thr Val Thr Ile Arg Ile 370 375 380 Pro Asp Lys His Lys Ser Ala Ala
Lys Gly Val Ile Ala Cys Ser Tyr 385 390 395 400 Leu Phe Val Cys Ser
Phe Ala Pro Thr Trp Gly Ile Gly Ile Trp Ile 405 410 415 Tyr Cys Ser
Glu Ile Phe Asn Asn Met Glu Arg Ala Lys Gly Ser Ser 420 425 430 Val
Ala Ala Ala Thr Asn Trp Ala Phe Asn Phe Ala Leu Ala Met Phe 435 440
445 Val Pro Ser Ala Phe Lys Asn Ile Ser Trp Lys Thr Tyr Ile Val Phe
450 455 460 Gly Val Phe Ser Val Ala Leu Thr Val Gln Thr Tyr Phe Met
Phe Pro 465 470 475 480 Glu Thr Arg Gly Lys Thr Leu Glu Glu Ile Asp
Gln Met Trp Val Asp 485 490 495 Asn Ile Pro Ala Trp Lys Thr Ser Ser
Tyr Ile Pro Gln Leu Pro Ile 500 505 510 Ile Glu Asp Glu Phe Gly Asn
Lys Leu Gly Leu Leu Gly Asn Pro Gln 515 520 525 His Leu Glu His Val
Lys Ser Val Glu Lys Asp Thr Val Val Glu Lys 530 535 540 Leu Glu Ser
Ser Glu Ala Asn Ser Ser Ser Ser Val 545 550 555 31620DNAPichia
guilliermondiiCDS(1)..(1617) 3atg gct tac gag gac aaa cta gtg gct
ccg gcc ttg aag ttt aga aac 48Met Ala Tyr Glu Asp Lys Leu Val Ala
Pro Ala Leu Lys Phe Arg Asn 1 5 10 15 ttt ctt gac aaa act ccc aat
atc tac aat cca tat atc att tct ata 96Phe Leu Asp Lys Thr Pro Asn
Ile Tyr Asn Pro Tyr Ile Ile Ser Ile 20 25 30 atc tcg tgc att gcg
ggt atg atg ttc ggt ttt gat att tct tca atg 144Ile Ser Cys Ile Ala
Gly Met Met Phe Gly Phe Asp Ile Ser Ser Met 35 40 45 tca gcg ttt
gtc agt tta cca gca tac gtg aat tat ttc gat aca cct 192Ser Ala Phe
Val Ser Leu Pro Ala Tyr Val Asn Tyr Phe Asp Thr Pro 50 55 60 tca
gca gtg att caa gga ttt atc aca tct gcc atg gct ttg ggt tca 240Ser
Ala Val Ile Gln Gly Phe Ile Thr Ser Ala Met Ala Leu Gly Ser 65 70
75 80 ttt ttc ggg tca att gct tct gcg ttt gtg tct gag cca ttt gga
aga 288Phe Phe Gly Ser Ile Ala Ser Ala Phe Val Ser Glu Pro Phe Gly
Arg 85 90 95 cga gct tcc tta cta act tgt tcg tgg ttt tgg atg ata
gga gca gcc 336Arg Ala Ser Leu Leu Thr Cys Ser Trp Phe Trp Met Ile
Gly Ala Ala 100 105 110 atc caa gcg tct tcg cag aac cga gct caa ttg
att att ggt cgg att 384Ile Gln Ala Ser Ser Gln Asn Arg Ala Gln Leu
Ile Ile Gly Arg Ile 115 120 125 ata tct gga ttt ggg gtt ggt ttc ggg
tcg tct gtg gct ccc gta tat 432Ile Ser Gly Phe Gly Val Gly Phe Gly
Ser Ser Val Ala Pro Val Tyr 130 135 140 ggc tcc gag atg gca cct aga
aaa att aga gga aga att ggt gga att 480Gly Ser Glu Met Ala Pro Arg
Lys Ile Arg Gly Arg Ile Gly Gly Ile 145 150 155 160 ttt caa tta tct
gtc acc ctc ggt atc atg att atg ttc ttc ata agt 528Phe Gln Leu Ser
Val Thr Leu Gly Ile Met Ile Met Phe Phe Ile Ser 165 170 175 tac gga
act tct cat att aag act gcg gca gct ttc agg tta gcc tgg 576Tyr Gly
Thr Ser His Ile Lys Thr Ala Ala Ala Phe Arg Leu Ala Trp 180 185 190
gca ctc cag atc att cct gga ctc ctc atg tgt att ggt gtc ttc ttt
624Ala Leu Gln Ile Ile Pro Gly Leu Leu Met Cys Ile Gly Val Phe Phe
195 200 205 att cca gaa tct cct aga tgg ttg gcc aaa caa ggt cac tgg
gac gaa 672Ile Pro Glu Ser Pro Arg Trp Leu Ala Lys Gln Gly His Trp
Asp Glu 210 215 220 gcc gaa atc att gta gcc aaa att caa gcc aaa gga
gat cga gaa aat 720Ala Glu Ile Ile Val Ala Lys Ile Gln Ala Lys Gly
Asp Arg Glu Asn 225 230 235 240 ccc gat gtt ttg att gaa att tcg gaa
ata aaa gac caa ttg atg gtt 768Pro Asp Val Leu Ile Glu Ile Ser Glu
Ile Lys Asp Gln Leu Met Val 245 250 255 gac gag aat gcc aaa gcc ttt
acc tat gct gac ttg ttt tcg aaa aaa 816Asp Glu Asn Ala Lys Ala Phe
Thr Tyr Ala Asp Leu Phe Ser Lys Lys 260 265 270 tat ctt ccc aga acc
atc aca gcc atg ttc gct caa atc tgg caa caa 864Tyr Leu Pro Arg Thr
Ile Thr Ala Met Phe Ala Gln Ile Trp Gln Gln 275 280 285 ttg aca gga
atg aat gtc atg atg tac tat atc gtt tac att ttc gaa 912Leu Thr Gly
Met Asn Val Met Met Tyr Tyr Ile Val Tyr Ile Phe Glu 290 295 300 atg
gct ggc tac ggt gga aat gga gtg ttg gta tca tcg aca att cag 960Met
Ala Gly Tyr Gly Gly Asn Gly Val Leu Val Ser Ser Thr Ile Gln 305 310
315 320 tac gtt atc ttt gtc gtt gtt aca ttt gtc tca tta ttc ttt ttg
gac 1008Tyr Val Ile Phe Val Val Val Thr Phe Val Ser Leu Phe Phe Leu
Asp 325 330 335 aaa ttt gga aga aga aaa att tta ctt gtc gga gca gct
tcc atg atg 1056Lys Phe Gly Arg Arg Lys Ile Leu Leu Val Gly Ala Ala
Ser Met Met 340 345 350 acc tgg cag ttt gca gtg gca ggg atc ttg gcc
agg tac tcg gtc ccg 1104Thr Trp Gln Phe Ala Val Ala Gly Ile Leu Ala
Arg Tyr Ser Val Pro 355 360 365 tac gat ctc agc gat act gtc aaa att
aaa att cct gac aat cac aaa 1152Tyr Asp Leu Ser Asp Thr Val Lys Ile
Lys Ile Pro Asp Asn His Lys 370 375 380 tcg gct gca aaa ggt gtc att
gca tgc tgc tat ctt ttc gta gca tcg 1200Ser Ala Ala Lys Gly Val Ile
Ala Cys Cys Tyr Leu Phe Val Ala Ser 385 390 395 400 ttc gga ttt tcc
tgg gga gtt ggt atc tgg tta tac tgc tct gaa gtc 1248Phe Gly Phe Ser
Trp Gly Val Gly Ile Trp Leu Tyr Cys Ser Glu Val 405 410 415 tgg gga
gac tca caa tcg aga cag aga gga gcc gct gtg tca act gct 1296Trp Gly
Asp Ser Gln Ser Arg Gln Arg Gly Ala Ala Val Ser Thr Ala 420 425 430
tca aat tgg att ttc aat ttt gcg ctc gcc atg ttc aca cca tct tcg
1344Ser Asn Trp Ile Phe Asn Phe Ala Leu Ala Met Phe Thr Pro Ser Ser
435 440 445 ttt aaa aat atc acc tgg aag aca tac tgt att tat gcc act
ttc tgc
1392Phe Lys Asn Ile Thr Trp Lys Thr Tyr Cys Ile Tyr Ala Thr Phe Cys
450 455 460 gca tgt atg ttc atc cat gtg ttc ttc ttc ttc cca gaa acc
aag ggg 1440Ala Cys Met Phe Ile His Val Phe Phe Phe Phe Pro Glu Thr
Lys Gly 465 470 475 480 aag cgc ttg gaa gaa att gct caa att tgg gaa
gaa aaa att cca gct 1488Lys Arg Leu Glu Glu Ile Ala Gln Ile Trp Glu
Glu Lys Ile Pro Ala 485 490 495 tgg aaa acc acc aac tgg caa cct cat
gtt cct ttg ttg tcg gac cac 1536Trp Lys Thr Thr Asn Trp Gln Pro His
Val Pro Leu Leu Ser Asp His 500 505 510 gaa ctt gcg gaa aag atc aat
gcc gaa cat gtg gag aac gtg aat tct 1584Glu Leu Ala Glu Lys Ile Asn
Ala Glu His Val Glu Asn Val Asn Ser 515 520 525 agg gaa caa tcg gat
gac gag aag tcg cag gta taa 1620Arg Glu Gln Ser Asp Asp Glu Lys Ser
Gln Val 530 535 4539PRTPichia guilliermondii 4Met Ala Tyr Glu Asp
Lys Leu Val Ala Pro Ala Leu Lys Phe Arg Asn 1 5 10 15 Phe Leu Asp
Lys Thr Pro Asn Ile Tyr Asn Pro Tyr Ile Ile Ser Ile 20 25 30 Ile
Ser Cys Ile Ala Gly Met Met Phe Gly Phe Asp Ile Ser Ser Met 35 40
45 Ser Ala Phe Val Ser Leu Pro Ala Tyr Val Asn Tyr Phe Asp Thr Pro
50 55 60 Ser Ala Val Ile Gln Gly Phe Ile Thr Ser Ala Met Ala Leu
Gly Ser 65 70 75 80 Phe Phe Gly Ser Ile Ala Ser Ala Phe Val Ser Glu
Pro Phe Gly Arg 85 90 95 Arg Ala Ser Leu Leu Thr Cys Ser Trp Phe
Trp Met Ile Gly Ala Ala 100 105 110 Ile Gln Ala Ser Ser Gln Asn Arg
Ala Gln Leu Ile Ile Gly Arg Ile 115 120 125 Ile Ser Gly Phe Gly Val
Gly Phe Gly Ser Ser Val Ala Pro Val Tyr 130 135 140 Gly Ser Glu Met
Ala Pro Arg Lys Ile Arg Gly Arg Ile Gly Gly Ile 145 150 155 160 Phe
Gln Leu Ser Val Thr Leu Gly Ile Met Ile Met Phe Phe Ile Ser 165 170
175 Tyr Gly Thr Ser His Ile Lys Thr Ala Ala Ala Phe Arg Leu Ala Trp
180 185 190 Ala Leu Gln Ile Ile Pro Gly Leu Leu Met Cys Ile Gly Val
Phe Phe 195 200 205 Ile Pro Glu Ser Pro Arg Trp Leu Ala Lys Gln Gly
His Trp Asp Glu 210 215 220 Ala Glu Ile Ile Val Ala Lys Ile Gln Ala
Lys Gly Asp Arg Glu Asn 225 230 235 240 Pro Asp Val Leu Ile Glu Ile
Ser Glu Ile Lys Asp Gln Leu Met Val 245 250 255 Asp Glu Asn Ala Lys
Ala Phe Thr Tyr Ala Asp Leu Phe Ser Lys Lys 260 265 270 Tyr Leu Pro
Arg Thr Ile Thr Ala Met Phe Ala Gln Ile Trp Gln Gln 275 280 285 Leu
Thr Gly Met Asn Val Met Met Tyr Tyr Ile Val Tyr Ile Phe Glu 290 295
300 Met Ala Gly Tyr Gly Gly Asn Gly Val Leu Val Ser Ser Thr Ile Gln
305 310 315 320 Tyr Val Ile Phe Val Val Val Thr Phe Val Ser Leu Phe
Phe Leu Asp 325 330 335 Lys Phe Gly Arg Arg Lys Ile Leu Leu Val Gly
Ala Ala Ser Met Met 340 345 350 Thr Trp Gln Phe Ala Val Ala Gly Ile
Leu Ala Arg Tyr Ser Val Pro 355 360 365 Tyr Asp Leu Ser Asp Thr Val
Lys Ile Lys Ile Pro Asp Asn His Lys 370 375 380 Ser Ala Ala Lys Gly
Val Ile Ala Cys Cys Tyr Leu Phe Val Ala Ser 385 390 395 400 Phe Gly
Phe Ser Trp Gly Val Gly Ile Trp Leu Tyr Cys Ser Glu Val 405 410 415
Trp Gly Asp Ser Gln Ser Arg Gln Arg Gly Ala Ala Val Ser Thr Ala 420
425 430 Ser Asn Trp Ile Phe Asn Phe Ala Leu Ala Met Phe Thr Pro Ser
Ser 435 440 445 Phe Lys Asn Ile Thr Trp Lys Thr Tyr Cys Ile Tyr Ala
Thr Phe Cys 450 455 460 Ala Cys Met Phe Ile His Val Phe Phe Phe Phe
Pro Glu Thr Lys Gly 465 470 475 480 Lys Arg Leu Glu Glu Ile Ala Gln
Ile Trp Glu Glu Lys Ile Pro Ala 485 490 495 Trp Lys Thr Thr Asn Trp
Gln Pro His Val Pro Leu Leu Ser Asp His 500 505 510 Glu Leu Ala Glu
Lys Ile Asn Ala Glu His Val Glu Asn Val Asn Ser 515 520 525 Arg Glu
Gln Ser Asp Asp Glu Lys Ser Gln Val 530 535 51725DNASaccharomyces
cerevisiaeCDS(1)..(1722) 5atg gca gtt gag gag aac aat gtg cct gtt
gtt tca cag caa ccc caa 48Met Ala Val Glu Glu Asn Asn Val Pro Val
Val Ser Gln Gln Pro Gln 1 5 10 15 gct ggt gaa gac gtg atc tct tca
ctc agt aaa gat tcc cat tta agc 96Ala Gly Glu Asp Val Ile Ser Ser
Leu Ser Lys Asp Ser His Leu Ser 20 25 30 gca caa tct caa aag tat
tcc aat gat gaa ttg aaa gcc ggt gag tca 144Ala Gln Ser Gln Lys Tyr
Ser Asn Asp Glu Leu Lys Ala Gly Glu Ser 35 40 45 ggg cct gaa ggc
tcc caa agt gtt cct ata gag ata ccc aag aag ccc 192Gly Pro Glu Gly
Ser Gln Ser Val Pro Ile Glu Ile Pro Lys Lys Pro 50 55 60 atg tct
gaa tat gtt acc gtt tcc ttg ctt tgt ttg tgt gtt gcc ttc 240Met Ser
Glu Tyr Val Thr Val Ser Leu Leu Cys Leu Cys Val Ala Phe 65 70 75 80
ggc ggc ttc atg ttt ggc tgg gat acc agt act att tct ggg ttt gtt
288Gly Gly Phe Met Phe Gly Trp Asp Thr Ser Thr Ile Ser Gly Phe Val
85 90 95 gtc caa aca gac ttt ttg aga agg ttt ggt atg aaa cat aag
gat ggt 336Val Gln Thr Asp Phe Leu Arg Arg Phe Gly Met Lys His Lys
Asp Gly 100 105 110 acc cac tat ttg tca aac gtc aga aca ggt tta atc
gtc gcc att ttc 384Thr His Tyr Leu Ser Asn Val Arg Thr Gly Leu Ile
Val Ala Ile Phe 115 120 125 aat att ggc tgt gcc ttt ggt ggt att ata
ctt tcc aaa ggt gga gat 432Asn Ile Gly Cys Ala Phe Gly Gly Ile Ile
Leu Ser Lys Gly Gly Asp 130 135 140 atg tat ggc cgt aaa aag ggt ctt
tcg att gtc gtc tcg gtt tat ata 480Met Tyr Gly Arg Lys Lys Gly Leu
Ser Ile Val Val Ser Val Tyr Ile 145 150 155 160 gtt ggt att atc att
caa att gcc tct atc aac aag tgg tac caa tat 528Val Gly Ile Ile Ile
Gln Ile Ala Ser Ile Asn Lys Trp Tyr Gln Tyr 165 170 175 ttc att ggt
aga atc ata tct ggt ttg ggt gtc ggc ggc atc gct gtc 576Phe Ile Gly
Arg Ile Ile Ser Gly Leu Gly Val Gly Gly Ile Ala Val 180 185 190 tta
tgt cct atg ttg atc tct gaa att gct cca aag cac ttg aga ggc 624Leu
Cys Pro Met Leu Ile Ser Glu Ile Ala Pro Lys His Leu Arg Gly 195 200
205 aca cta gtt tct tgt tat cag ctg atg att act gca ggt atc ttt ttg
672Thr Leu Val Ser Cys Tyr Gln Leu Met Ile Thr Ala Gly Ile Phe Leu
210 215 220 ggc tac tgt act aat tac ggt aca aag agc tat tcg aac tca
gtt caa 720Gly Tyr Cys Thr Asn Tyr Gly Thr Lys Ser Tyr Ser Asn Ser
Val Gln 225 230 235 240 tgg aga gtt cca tta ggg cta tgt ttc gct tgg
tca tta ttt atg att 768Trp Arg Val Pro Leu Gly Leu Cys Phe Ala Trp
Ser Leu Phe Met Ile 245 250 255 ggc gct ttg acg tta gtt cct gaa tcc
cca cgt tat tta tgt gag gtg 816Gly Ala Leu Thr Leu Val Pro Glu Ser
Pro Arg Tyr Leu Cys Glu Val 260 265 270 aat aag gta gaa gac gcc aag
cgt tcc att gct aag tct aac aag gtg 864Asn Lys Val Glu Asp Ala Lys
Arg Ser Ile Ala Lys Ser Asn Lys Val 275 280 285 tca cca gag gat cct
gcc gtc cag gcc gag tta gat ctg atc atg gcc 912Ser Pro Glu Asp Pro
Ala Val Gln Ala Glu Leu Asp Leu Ile Met Ala 290 295 300 ggt ata gaa
gct gaa aaa ctg gct ggc aat gcg tcc tgg ggg gaa tta 960Gly Ile Glu
Ala Glu Lys Leu Ala Gly Asn Ala Ser Trp Gly Glu Leu 305 310 315 320
ttt tcc acc aag acc aaa gta ttt caa cgt ttg ttg atg ggt gtg ttt
1008Phe Ser Thr Lys Thr Lys Val Phe Gln Arg Leu Leu Met Gly Val Phe
325 330 335 gtt caa atg ttc caa caa tta acc ggt aac aat tat ttt ttc
tac tac 1056Val Gln Met Phe Gln Gln Leu Thr Gly Asn Asn Tyr Phe Phe
Tyr Tyr 340 345 350 ggt acc gtt att ttc aag tca gtt ggc ctg gat gat
tcc ttt gaa aca 1104Gly Thr Val Ile Phe Lys Ser Val Gly Leu Asp Asp
Ser Phe Glu Thr 355 360 365 tcc att gtc att ggt gta gtc aac ttt gcc
tcc act ttc ttt agt ttg 1152Ser Ile Val Ile Gly Val Val Asn Phe Ala
Ser Thr Phe Phe Ser Leu 370 375 380 tgg act gtc gaa aac ttg ggg cgt
cgt aaa tgt tta ctt ttg ggc gct 1200Trp Thr Val Glu Asn Leu Gly Arg
Arg Lys Cys Leu Leu Leu Gly Ala 385 390 395 400 gcc act atg atg gct
tgt atg gtc atc tac gcc tct gtt ggt gtt act 1248Ala Thr Met Met Ala
Cys Met Val Ile Tyr Ala Ser Val Gly Val Thr 405 410 415 aga tta tat
cct cac ggt aaa agc cag cca tct tct aaa ggt gcc ggt 1296Arg Leu Tyr
Pro His Gly Lys Ser Gln Pro Ser Ser Lys Gly Ala Gly 420 425 430 aac
tgt atg att gtc ttt acc tgt ttt tat att ttc tgt tat gcc aca 1344Asn
Cys Met Ile Val Phe Thr Cys Phe Tyr Ile Phe Cys Tyr Ala Thr 435 440
445 acc tgg gcg cca gtt gcc tgg gtc atc aca gca gaa tca ttc cca ctg
1392Thr Trp Ala Pro Val Ala Trp Val Ile Thr Ala Glu Ser Phe Pro Leu
450 455 460 aga gtc aag tcg aaa tgt atg gcg ttg gcc tct gct tcc aat
tgg gta 1440Arg Val Lys Ser Lys Cys Met Ala Leu Ala Ser Ala Ser Asn
Trp Val 465 470 475 480 tgg ggg ttc ttg att gca ttt ttc acc cca ttc
atc aca tct gcc att 1488Trp Gly Phe Leu Ile Ala Phe Phe Thr Pro Phe
Ile Thr Ser Ala Ile 485 490 495 aac ttc tac tac ggt tat gtc ttc atg
ggc tgt ttg gtt gcc atg ttt 1536Asn Phe Tyr Tyr Gly Tyr Val Phe Met
Gly Cys Leu Val Ala Met Phe 500 505 510 ttt tat gtc ttt ttc ttt gtt
cca gaa act aaa ggc cta tcg tta gaa 1584Phe Tyr Val Phe Phe Phe Val
Pro Glu Thr Lys Gly Leu Ser Leu Glu 515 520 525 gaa att caa gaa tta
tgg gaa gaa ggt gtt tta cct tgg aaa tct gaa 1632Glu Ile Gln Glu Leu
Trp Glu Glu Gly Val Leu Pro Trp Lys Ser Glu 530 535 540 ggc tgg att
cct tca tcc aga aga ggt aat aat tac gat tta gag gat 1680Gly Trp Ile
Pro Ser Ser Arg Arg Gly Asn Asn Tyr Asp Leu Glu Asp 545 550 555 560
tta caa cat gac gac aaa ccg tgg tac aag gcc atg cta gaa taa 1725Leu
Gln His Asp Asp Lys Pro Trp Tyr Lys Ala Met Leu Glu 565 570
6574PRTSaccharomyces cerevisiae 6Met Ala Val Glu Glu Asn Asn Val
Pro Val Val Ser Gln Gln Pro Gln 1 5 10 15 Ala Gly Glu Asp Val Ile
Ser Ser Leu Ser Lys Asp Ser His Leu Ser 20 25 30 Ala Gln Ser Gln
Lys Tyr Ser Asn Asp Glu Leu Lys Ala Gly Glu Ser 35 40 45 Gly Pro
Glu Gly Ser Gln Ser Val Pro Ile Glu Ile Pro Lys Lys Pro 50 55 60
Met Ser Glu Tyr Val Thr Val Ser Leu Leu Cys Leu Cys Val Ala Phe 65
70 75 80 Gly Gly Phe Met Phe Gly Trp Asp Thr Ser Thr Ile Ser Gly
Phe Val 85 90 95 Val Gln Thr Asp Phe Leu Arg Arg Phe Gly Met Lys
His Lys Asp Gly 100 105 110 Thr His Tyr Leu Ser Asn Val Arg Thr Gly
Leu Ile Val Ala Ile Phe 115 120 125 Asn Ile Gly Cys Ala Phe Gly Gly
Ile Ile Leu Ser Lys Gly Gly Asp 130 135 140 Met Tyr Gly Arg Lys Lys
Gly Leu Ser Ile Val Val Ser Val Tyr Ile 145 150 155 160 Val Gly Ile
Ile Ile Gln Ile Ala Ser Ile Asn Lys Trp Tyr Gln Tyr 165 170 175 Phe
Ile Gly Arg Ile Ile Ser Gly Leu Gly Val Gly Gly Ile Ala Val 180 185
190 Leu Cys Pro Met Leu Ile Ser Glu Ile Ala Pro Lys His Leu Arg Gly
195 200 205 Thr Leu Val Ser Cys Tyr Gln Leu Met Ile Thr Ala Gly Ile
Phe Leu 210 215 220 Gly Tyr Cys Thr Asn Tyr Gly Thr Lys Ser Tyr Ser
Asn Ser Val Gln 225 230 235 240 Trp Arg Val Pro Leu Gly Leu Cys Phe
Ala Trp Ser Leu Phe Met Ile 245 250 255 Gly Ala Leu Thr Leu Val Pro
Glu Ser Pro Arg Tyr Leu Cys Glu Val 260 265 270 Asn Lys Val Glu Asp
Ala Lys Arg Ser Ile Ala Lys Ser Asn Lys Val 275 280 285 Ser Pro Glu
Asp Pro Ala Val Gln Ala Glu Leu Asp Leu Ile Met Ala 290 295 300 Gly
Ile Glu Ala Glu Lys Leu Ala Gly Asn Ala Ser Trp Gly Glu Leu 305 310
315 320 Phe Ser Thr Lys Thr Lys Val Phe Gln Arg Leu Leu Met Gly Val
Phe 325 330 335 Val Gln Met Phe Gln Gln Leu Thr Gly Asn Asn Tyr Phe
Phe Tyr Tyr 340 345 350 Gly Thr Val Ile Phe Lys Ser Val Gly Leu Asp
Asp Ser Phe Glu Thr 355 360 365 Ser Ile Val Ile Gly Val Val Asn Phe
Ala Ser Thr Phe Phe Ser Leu 370 375 380 Trp Thr Val Glu Asn Leu Gly
Arg Arg Lys Cys Leu Leu Leu Gly Ala 385 390 395 400 Ala Thr Met Met
Ala Cys Met Val Ile Tyr Ala Ser Val Gly Val Thr 405 410 415 Arg Leu
Tyr Pro His Gly Lys Ser Gln Pro Ser Ser Lys Gly Ala Gly 420 425 430
Asn Cys Met Ile Val Phe Thr Cys Phe Tyr Ile Phe Cys Tyr Ala Thr 435
440 445 Thr Trp Ala Pro Val Ala Trp Val Ile Thr Ala Glu Ser Phe Pro
Leu 450 455 460 Arg Val Lys Ser Lys Cys Met Ala Leu Ala Ser Ala Ser
Asn Trp Val 465 470 475 480 Trp Gly Phe Leu Ile Ala Phe Phe Thr Pro
Phe Ile Thr Ser Ala Ile 485 490 495 Asn Phe Tyr Tyr Gly Tyr Val Phe
Met Gly Cys Leu Val Ala Met Phe 500 505 510 Phe Tyr Val Phe Phe Phe
Val Pro Glu Thr Lys Gly Leu Ser Leu Glu 515 520 525 Glu Ile Gln Glu
Leu Trp Glu Glu Gly Val Leu Pro Trp Lys Ser Glu 530 535 540 Gly Trp
Ile Pro Ser Ser Arg Arg Gly Asn Asn Tyr Asp Leu Glu Asp 545 550 555
560 Leu Gln His Asp Asp Lys Pro Trp Tyr Lys Ala Met Leu Glu 565
570
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References