U.S. patent application number 16/746062 was filed with the patent office on 2020-05-07 for cells with improved pentose conversion.
The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Rene Marcel DE JONG, Maria Bernedina Elizabeth JONKERS, Paul KLAASSEN, Aloysius Wilhelmus Rudolphus Hubertus TEUNISSEN.
Application Number | 20200140900 16/746062 |
Document ID | / |
Family ID | 67058054 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140900 |
Kind Code |
A1 |
JONKERS; Maria Bernedina Elizabeth
; et al. |
May 7, 2020 |
CELLS WITH IMPROVED PENTOSE CONVERSION
Abstract
The invention relates to a cell capable of converting one or
more pentose sugar and one or more hexose sugar into fermentation
product constitutively expressing one or more heterologous or
homologous polypeptide having the amino acid sequence set out in
SEQ ID NO: 20, or a variant polypeptide thereof having at least 45%
identity to SEQ ID NO 20. In an embodiment the heterologous
polypeptide has glyoxalase activity.
Inventors: |
JONKERS; Maria Bernedina
Elizabeth; (Echt, NL) ; KLAASSEN; Paul; (Echt,
NL) ; TEUNISSEN; Aloysius Wilhelmus Rudolphus Hubertus;
(Echt, NL) ; DE JONG; Rene Marcel; (Echt,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Family ID: |
67058054 |
Appl. No.: |
16/746062 |
Filed: |
January 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16352415 |
Mar 13, 2019 |
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16746062 |
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14434116 |
Apr 8, 2015 |
10273507 |
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PCT/EP2013/071462 |
Oct 15, 2013 |
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16352415 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/52 20130101;
C12Y 404/01005 20130101; C12Y 503/01006 20130101; C12Y 501/03004
20130101; C12P 7/06 20130101; C12Y 503/01005 20130101; C12N 1/16
20130101; C12N 1/22 20130101; C12P 2203/00 20130101; C12N 2330/50
20130101; C12N 9/88 20130101; C12P 7/10 20130101 |
International
Class: |
C12P 7/10 20060101
C12P007/10; C12N 1/22 20060101 C12N001/22; C12N 1/16 20060101
C12N001/16; C12N 9/88 20060101 C12N009/88; C12P 7/06 20060101
C12P007/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 16, 2012 |
EP |
12188715.2 |
May 8, 2013 |
EP |
13166959.0 |
Claims
1. A cell capable of converting one or more pentose sugar and one
or more hexose sugar into fermentation product constitutively
expressing one or more heterologous or homologous polypeptide
having the amino acid sequence set out in SEQ ID NO: 20 or a
variant polypeptide thereof, having at least 45% identity to SEQ ID
NO 20.
2. A cell according to claim 1, wherein the heterologous
polypeptide has glyoxalase activity, optionally comprising
glyoxalase I activity.
3. An cell capable of converting one or more pentose sugar and or
one or more hexose sugar into fermentation product comprising a
constitutively expressed heterologous or homologous polynucleotide
which comprises: (a) the nucleotide sequence as set out in SEQ ID
NO: 27; (b) a nucleotide sequence having at least about 50%
sequence identity with the nucleotide sequence of SEQ ID NO: 27;
(c) a fragment of a nucleotide sequence as defined in (a), (b) or
(c) having at least 100 nucleotides; (d) a sequence which is
degenerate as a result of the genetic code to a sequence as defined
in any one of (a), (b), or (c); (e) a nucleotide sequence which is
the reverse complement of a nucleotide sequence as defined in (a),
(b), (c), or (d).
4. A cell according to claim 1, comprising a nucleotide sequence
encoding a xylose isomerase.
5. A cell according to claim 1, wherein the cell comprises one or
more genetic modifications resulting in: (a) an increase in
transport of xylose in the cell; (b) an increase in xylulose kinase
activity; (c) an increase in flux through the pentose phosphate
pathway; (d) a decrease in aldose reductase activity; (e) a
decrease in sensitivity to catabolite repression; (f) an increase
in tolerance to ethanol, osmolarity or organic acid; or (g) a
reduced production of by-product.
6. A cell according to claim 5, wherein the one or more genetic
modifications result in overexpression of at least one gene
encoding an enzyme of the non-oxidative part of the pentose
phosphate pathway.
7. A cell according to claim 6, wherein the gene is a gene encoding
a ribulose-5-phosphate isomerase, a ribulose-5-phosphate epimerase,
a transketolase or a transaldolase.
8. A cell according to claim 5, wherein the one or more genetic
modifications result in overexpression of a gene encoding a
xylulose kinase.
9. A cell according to claim 5, wherein the one or more genetic
modifications result in a decrease in aldose reductase activity in
the cell.
10. A cell according to claim 5 which has the ability to use
L-arabinose, wherein the genes TAL1, TKL1, RPE1 and RKI1 are
overexpressed.
11. A cell according to claim 1, wherein the coding region of the
GRE3-gene is inactivated by replacement of the coding region with a
nucleotide sequence comprising the genes TAL1, TKL1, RPE1 and
RKI1.
12. A cell according to claim 1, wherein the genes araA, araB and
araD are expressed.
13. A cell according to claim 1, wherein one or more constitutively
expressed or constitutively overexpressed genes are stably
integrated into the genome of the cell.
14. A process for producing a fermentation product which process
comprises fermenting a medium comprising a source of xylose and/or
arabinose with a cell according to claim 1 such that the cell
ferments xylose and/or to the fermentation product.
15. A process according to claim 14, wherein the fermentation
product is chosen from the list including ethanol, butanol, lactic
acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic
acid, citric acid, malic acid, fumaric acid, itaconic acid, an
amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a
.beta.-lactam antibiotic and a cephalosporin.
16. A process according to claim 15 wherein the fermentation
product is ethanol.
17. A process according to claim 14, wherein the process is
anaerobic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/352,415, filed 13 Mar. 2019, which is a
continuation of U.S. patent application Ser. No. 14/434,116, filed
8 Apr. 2015, which is a .sctn. 371 National Stage of International
Application No. PCT/EP2013/071462, filed 15 Oct. 2013, which claims
priority to European Patent Application Nos. 12188715.2, filed 16
Oct. 2012 and 13166959.0, filed 8 May 2013. The disclosures of the
priority applications are incorporated in their entirety herein by
reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.txt)
[0002] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_2919208-316002_ST25.txt" created on 8 Jan. 2020,
and 36,155 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
BACKGROUND
Field of the Invention
[0003] The invention is directed to cells with improved pentose
conversion. More specifically the invention relates to cells that
have improved conversion of xylose and/or L-arabinose. The cells
are useful for the production of fermentation products, for
instance for the production of ethanol from sugars that are derived
from lignocellulosic material.
Description of Related Art
[0004] Large-scale consumption of traditional, fossil fuels
(petroleum-based fuels) in recent decades has contributed to high
levels of pollution. This, along with the realisation that the
world stock of fossil fuels is not unlimited and a growing
environmental awareness, has stimulated new initiatives to
investigate the feasibility of alternative fuels such as ethanol,
which is a particulate-free burning fuel source that releases less
CO.sub.2 than unleaded gasoline on a per litre basis.
[0005] Although biomass-derived ethanol may be produced by the
fermentation of hexose sugars obtained from many different sources,
the substrates typically used for commercial scale production of
fuel alcohol, such as cane sugar and corn starch, are expensive.
Increases in the production of fuel ethanol will therefore require
the use of lower-cost feedstocks.
[0006] Currently, only lignocellulosic feedstock derived from plant
biomass is available in sufficient quantities to substitute the
crops currently used for ethanol production. In most
lignocellulosic material, the second-most-common sugar, after
glucose, is xylose. Also L-arabinose is a sugar derived from some
lignocellulosic material. Thus, for an economically feasible fuel
production process, both hexose and pentose sugars must be
fermented to form ethanol. The yeast Saccharomyces cerevisiae is
robust and well adapted for ethanol production, but it is unable to
produce ethanol using xylose as a carbon source. There is therefore
a need for an organism possessing these properties so as to enable
the commercially-viable production of ethanol from lignocellulosic
feedstocks. Xylose isomerase from the anaerobic fungus Piromyces
Sp.E2 was introduced in S. cerevisiae and high levels of enzyme
activities were observed enabling this strain to grow anaerobically
and produce ethanol from xylose (WO2003/062430 and WO06/009434).
Such yeast strains for the first time provided specific rates of
xylose consumption and ethanol formation that are compatible with
ethanol production at a commercial scale.
[0007] However, it is still desirable to improve pentose conversion
and to reduce fermentation time.
SUMMARY
[0008] An object of the invention is to provide cells with improved
pentose conversion. Another object of the invention is to provide
recombinant strains that have improved conversion of xylose and/or
L-arabinose. Another object is to provide strains that have
improved conversion of xylose and/or L-arabinose in the presence of
glucose. Another object is to reduce the fermentation time in
fermentation of pentose and hexose comprising sugar mixtures.
[0009] One or more of these objects are attained according to the
invention. According to the present invention, there is provided a
cell which constitutively expresses a heterologous or homologous
polypeptide, having the amino acid sequence of SEQ ID NO: 20 or an
amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 27, or a variant thereof having an amino acid sequence having
at least 45% sequence identity to SEQ ID NO: 20.
[0010] The cells according to the examples have improved pentose
conversion and lead to reduced fermentation times in fermentation
of pentose and hexose comprising sugar mixtures. In an embodiment
the polypeptide has glyoxalase activity.
[0011] The invention also provides: [0012] a process for producing
a fermentation product which process comprises fermenting a medium
containing a source of xylose and or L-arabinose with a cell of the
invention such that the cell ferments xylose and/or L-arabinose to
the fermentation product; [0013] the use of a cell of the invention
in a process for the production of a fermentation product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: Graph of GLO1 expression cassette with the PGK1
promoter and PGI1 terminator;
[0015] FIG. 2: Map of plasmid pRN228 (see examples);
[0016] FIG. 3: Map of plasmid pRN935 (see examples);
[0017] FIG. 4: Map of plasmid pRN685 (see examples);
[0018] FIG. 5: Map of plasmid pRN1048 (see examples);
[0019] FIG. 6: Map of plasmid pRN1129 (see examples);
[0020] FIG. 7: Map of yeast shuttle vector pRN599 (see
examples);
[0021] FIG. 8: Map of plasmid pRN1049;
[0022] FIG. 9: Gene expression data. Normalized Fold Expression for
Strains RN1001Epl and RN1001Gpl;
[0023] FIG. 10: CO.sub.2 production curve for growth of strains
RN1001Gpl (top) and RN1001Epl (bottom);
[0024] FIG. 11: Growth curve of strains RN1001Epl and RN1001Gpl
showing sugar consumption.
[0025] FIG. 12: Map of plasmid pRN324;
[0026] FIG. 13: Map of plasmid pRN1142;
[0027] FIG. 14: Map showing integration site in S. cervisiae
genome: Integration site 1 (Chr XV, coordinates 458319 bp to 459320
bp). The specified map is indicated by the region between the two
vertical dashed lines;
[0028] FIG. 15: Scheme that shows mechanism of integration of the
PCR fragments (His or His-Glo) into the S. cervisiae genome;
[0029] FIG. 16: Gene expression data. Normalized Fold Expression
for GLO1 of strains RN1041 (top) and RN1216 (bottom);
[0030] FIG. 17: CO.sub.2 production curve for growth of strains
RN1041H, RN1041HG-1 and RN1041 HG-2;
[0031] FIG. 18: Sugar consumption curve of strains RN1041H (top),
RN1041 HG-1 (mid) and RN1041 HG-2 (bottom);
[0032] FIG. 19: CO.sub.2 production curve for growth of strains
RN1216H, RN1216HG-1 and RN1216HG-2;
[0033] FIG. 20: Sugar consumption curve of strains RN1216H (top),
RN1216HG-1 (mid) and RN1216HG-2 (bottom) showing sugar
consumption.
[0034] FIG. 21: Map of plasmid pDB1175 (see examples);
[0035] FIG. 22: Map of plasmid pDB1176 (see examples);
[0036] FIG. 23: Map of plasmid pDB1177 (see examples);
[0037] FIG. 24: Map of plasmid pDB1178 (see examples);
[0038] FIG. 25: Map of plasmid pRN1179 (see examples);
[0039] FIG. 26: Scheme that shows mechanism of integration of the
PCR fragments into the S. cervisiae genome;
[0040] FIG. 27: CO.sub.2 production curve for growth of strains
RN1216 ScG_H, RN1216 CglaG_H, RN1216 ZrouG_H, RN1216 KIG_H, RN1216
CmagG_H and RN1216 H;
[0041] FIG. 28: Sugar consumption curve of strain RN1216 ScG_H
[0042] FIG. 29: Sugar consumption curve of strain RN1216
CglaG_H
[0043] FIG. 30: Sugar consumption curve of strain RN1216
ZrouG_H
[0044] FIG. 31: Sugar consumption curve of strain RN1216 KIG_H
[0045] FIG. 32: Sugar consumption curve of strain RN1216
CmagG_H
[0046] FIG. 33: Sugar consumption curve of strain RN1216 H
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0047] SEQ ID NO 1: Forward primer PGK1 promoter;
[0048] SEQ ID NO 2: Reverse primer PGI1 terminator;
[0049] SEQ ID NO 3: Forward primer ACT1 gene;
[0050] SEQ ID NO 4: Reverse primer ACT1 gene;
[0051] SEQ ID NO 5: Forward primer GLO1 gene, Q-PCR;
[0052] SEQ ID NO 6: Reverse primer GLO1 gene, Q-PCR;
[0053] SEQ ID NO 7: Forward primer ALG9 gene, Q-PCR;
[0054] SEQ ID NO 8: Reverse primer ALG9 gene, Q-PCR;
[0055] SEQ ID NO 9: Forward primer UBC6 gene, Q-PCR;
[0056] SEQ ID NO 10: Reverse primer UBC6 gene, Q-PCR;
[0057] SEQ ID NO 11: Forward primer HIS3 cassette, 5' flank
INT1;
[0058] SEQ ID NO 12: Reverse primer GLO1 cassette, 3' flank
INT1;
[0059] SEQ ID NO 13: Reverse primer HIS3 cassette, 3' flank
INT1;
[0060] SEQ ID NO 14: Forward primer 5' flank INT1;
[0061] SEQ ID NO 15: Reverse primer 5' flank INT1;
[0062] SEQ ID NO 16: Forward primer 3' flank INT1;
[0063] SEQ ID NO 17: Reverse primer 3' flank INT1;
[0064] SEQ ID NO 18: Nucleotide sequence 5' flank of integration
site INT1;
[0065] SEQ ID NO 19: Nucleotide sequence 3' flank of integration
site INT1;
[0066] SEQ ID NO 20: Protein sequence of GLO1 from S.
cerevisiae;
[0067] SEQ ID NO 21: Protein sequence of GLO1 from Candida
glabrata;
[0068] SEQ ID NO 22: Protein sequence of GLO1 from
Zygosaccharomyces rouxii;
[0069] SEQ ID NO 23: Protein sequence of GLO1 from Kluyveromyces
lactis;
[0070] SEQ ID NO 24: Protein sequence of GLO1 from Candida
magnolia;
[0071] SEQ ID NO 25: Forward primer of the GLO1 ORF;
[0072] SEQ ID NO 26: Reverse primer of the GLO1 ORF;
[0073] SEQ ID NO 27: Nucleotide sequence of GLO1 from S.
cerevisiae;
[0074] SEQ ID NO 28: Forward primer of the homologous GLO1
expression cassettes (including promoter and terminator);
[0075] SEQ ID NO 29: Reverse primer of the homologous GLO1
expression cassettes (including promoter and terminator);
[0076] SEQ ID NO 30: Forward primer of the HIS3 cassette,
consisting of 20 nucleotides and a tail of 50 nucleotides on the
5'-end, identical to the 50 nucleotides of the 3'-end of the
homologous GLO1 expression cassettes;
[0077] SEQ ID NO 31: Reverse primer of the HIS3 cassette,
consisting of 21 nucleotides and a tail of 50 nucleotides on the
5'-end, identical to the 50 nucleotides of the 5'-end of the 3' 500
bp INT1 flank;
[0078] SEQ ID NO 32: Reverse primer of the 5' 500 bp INT1 flank,
consisting of 23 nucleotides and a tail of 50 nucleotides on the
5'-end, identical to the 50 nucleotides of the 5'-end of the
homologous GLO1 expression cassettes;
[0079] SEQ ID NO 33: Forward primer of the 3' 500 bp INT1 flank,
consisting of 24 nucleotides and a tail of 50 nucleotides on the
5'-end, identical to the 50 nucleotides of the 3'-end of the HIS3
expression cassette;
[0080] SEQ ID NO 34: Codon pair optimized nucleotide sequence of
GLO1 from S. cerevisiae;
[0081] SEQ ID NO 35: Codon pair optimized nucleotide sequence of
GLO1 from Candida glabrata;
[0082] SEQ ID NO 36: Codon pair optimized nucleotide sequence of
GLO1 from Candida magnolia;
[0083] SEQ ID NO 37: Codon pair optimized nucleotide sequence of
GLO1 from Kluyveromyces lactis;
[0084] SEQ ID NO 38: Codon pair optimized nucleotide sequence of
GLO1 from Zygosaccharomyces rouxii.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0085] Throughout the present specification and the accompanying
claims the words "comprise" and "include" and variations such as
"comprises", "comprising", "includes" and "including" are to be
interpreted inclusively. That is, these words are intended to
convey the possible inclusion of other elements or integers not
specifically recited, where the context allows.
[0086] According to the invention, the cell may comprises a
glyoxalase activity, preferably a glyoxalase I activity.
Glyoxalase1 (GLO1) is a gene that encodes that encodes glyoxalase I
(EC 4.4.1.5), which is involved in methylglyoxal catabolism (2).
Methylglyoxal is a toxic compound formed as a by-product of
glycolysis.
[0087] Alternative names for glyoxalase are for instance
lactoylglutathione lyase, aldoketomutase, ketone-aldehyde mutase,
methylglyoxalase, S-D-lactoylglutathione methylglyoxal lyase.
[0088] One method of methylglyoxal catabolism comprises a
glyoxalase system in which methylglyoxal is condensed with
glutathione by Glo1p to produce S-D-lactoylglutathione. This
glutathione thiolester is then hydrolyzed to lactic acid and
glutathione by glyoxalase II (Glo2p and Glo4p). GLO1 expression is
induced by methylglyoxal and is specifically induced by osmotic
stress in a high osmolarity glycerol (Hog1p)-mitogen-activated
protein (MAP) kinase-dependent manner (1).
[0089] Deletion of GLO1 results in hypersensitivity to
methylglyoxal. In S. cerevisiae, glyoxalase I (Glo1p) is native and
occurs is a monomer. This system shows many of the typical features
of the enzymes that dispose of endogenous toxins. Firstly, in
contrast to the amazing substrate range of many of the enzymes
involved in xenobiotic metabolism, it shows narrow substrate
specificity. Secondly, intracellular thiols are required as part of
its enzymatic mechanism and thirdly, the system acts to recycle
reactive metabolites back to a form which may be useful to cellular
metabolism.
[0090] In an embodiment, the invention relates to a cell which
expresses a glyoxalase, wherein the amino acid sequence of the
glyoxalase has at least 45% identity to the amino acid sequence set
out in SEQ ID NO: 20 and wherein the nucleotide sequence is
constitutively integrated homologous or heterologous to the
cell.
Amino Acid/Polynucleotide Sequence
[0091] The cell of the invention is defined with reference to a
protein having the amino acid sequence of SEQ ID NO: 20 or a
sequence having at least 45% sequence identity thereto. In an
embodiment, the protein has at least about 50%, at least about 55%,
at least about 60%, at least about 65%, at least about 70%, at
least about 80%, at least about 90%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98% or at least about 99% sequence identity with the amino acid
sequence of SEQ ID NO: 20.
[0092] In an embodiment, the cell comprises and/or expresses a
polypeptide having amino acids H, E, H and E corresponding to the
positions H25, E89, H269 and E318 and/or H185, E242, H117 and E163
in SEQ ID NO: 20. In an embodiment, the cell comprises and/or
expresses a polypeptide having a fragment (E,s,d)-L-X-(H,Y)-(N,s)
corresponding to the fragment E242-L243-X-H245-N246 in SEQ ID NO:
20. In an embodiment the cell comprises and/or expresses a
polypeptide having a fragment G-(F,Y)-G-H corresponding to the
fragment G266-Y267-G268-H269 in SEQ ID NO: 20. In an embodiment the
cell comprises and/or expresses a polypeptide having a fragment
G-X(6)-(F,i)-X(2,3)-D-X(3)-Y corresponding to the fragment
G301-X(6)-F308-X(2)-D311-X(3)-Y315 in SEQ ID NO: 20.
[0093] In the above, amino acids are indicated with one letter
code. X is any amino acid; (X,Y) aminoacid X or Y; X(y) an y number
aminoacids X and X(y,z) an y or z number of aminoacids X. Small
letter code indicates an amino acid that has minor occurrence.
[0094] In an embodiment the cell comprises and/or expresses a
polypeptide having glyoxalase activity.
[0095] A cell according to the present invention may comprise a
nucleotide sequence encoding a glyoxalase having the nucleotide
sequence of SEQ ID NO: 27 or a sequence which has at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, preferably at least about 80%,
at least about 85%, at least about 90%, at least about 92%, at
least about 93%, at least about 94%, at least about 95%, at least
about 96%, at least about 97%, at least about 98% or at least about
99% or at least 75%, at least 80%, at least 85%, at least 90%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98% or at least 99% sequence identity with
the nucleic acid sequence set out in SEQ ID NO: 27.
[0096] The invention therefore provides cells with polynucleotide
sequences comprising the gene encoding the glyoxalase polypeptide,
as well as its coding sequence.
[0097] The polynucleotides of the invention may be isolated or
synthesized. The glyoxalase polypeptides and glyoxalase
polynucleotides herein may be synthetic polypeptides, respectively
polynucleotides. The synthetic polynucleotides may be optimized in
codon use, preferably according to the methods described in
WO2006/077258 and/or PCT/EP2007/055943, which are herein
incorporated by reference. PCT/EP2007/055943 addresses codon-pair
optimization.
[0098] The term refers to a polynucleotide molecule, which is a
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule,
either single stranded or double stranded. A polynucleotide may
either be present in isolated form, or be comprised in recombinant
nucleic acid molecules or vectors, or be comprised in a host
cell.
[0099] The word "polypeptide" is used herein for chains containing
more than seven amino acid residues. All oligopeptide and
polypeptide formulas or sequences herein are written from left to
right and in the direction from amino terminus to carboxy terminus.
The one-letter code of amino acids used herein is commonly known in
the art.
[0100] By "isolated" polypeptide or protein is intended a
polypeptide or protein removed from its native environment. For
example, recombinantly produced polypeptides and proteins expressed
in host cells are considered isolated for the purpose of the
invention as are native or recombinant polypeptides which have been
substantially purified by any suitable technique such as, for
example, the single-step purification method disclosed in Smith and
Johnson, Gene 67:31-40 (1988).
[0101] The polynucleotides of the present invention, such as a
polynucleotide encoding the glyoxalase polypeptide can be isolated
or synthesized using standard molecular biology techniques and the
sequence information provided herein.
[0102] The polynucleotide encoding the glyoxalase polypeptide of
the invention can be amplified using cDNA, mRNA or alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers
according to standard PCR amplification techniques. The nucleic
acid so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis.
Enzyme Kinetics
[0103] Enzymes are protein catalysts that, like all catalysts,
speed up the rate of a chemical reaction without being used up in
the process.
[0104] They achieve their effect by temporarily binding to the
substrate and, in doing so, lowering the activation energy needed
to convert it into a product.
[0105] The rate at which an enzyme works is influenced by several
factors, e.g., the concentration of substrate molecules (designated
[S], expressed in units of molarity), the temperature, the presence
of inhibitors (competitive inhibitors binding to the same site as
the substrate or noncompetitive inhibitors binding to another site
on the enzyme reducing its catalytic power), pH, and the like.
[0106] Enzyme kinetics studies and describes the rate at which
enzymes work. There are many methods of measurement. The enzyme
converts substrate into product at an initial rate that is
approximately linear for a short period after the start of the
reaction. As the reaction proceeds and substrate is consumed, the
rate continuously slows, as substrate is still present at
saturating levels. To measure the initial (and maximal) rate,
enzyme assays are typically carried out while the reaction has
progressed only a few percent towards total completion. The length
of the initial rate period depends amongst others on the assay
conditions.
[0107] The study of enzyme kinetics is important for two basic
reasons. Firstly, it helps to explain how enzymes work, and
secondly, it helps to predict how enzymes behave in living
organisms. The kinetic constants, Km and Vmax, are critical to
attempts to understand how enzymes work together to control
metabolism.
[0108] The Michaelis constant Km is experimentally defined as the
concentration at which the rate of the enzyme reaction is half
Vmax. Vmax is the maximal velocity at which the enzyme catalyzes
the reaction: as [S] gets higher, the enzyme becomes saturated with
substrate and the rate reaches Vmax, the enzyme's maximum rate.
[0109] Km is (roughly) an inverse measure of the affinity or
strength of binding between the enzyme and its substrate. The lower
the Km value (in mM), the greater the affinity, hence the lower the
concentration of substrate needed to achieve a given rate.
The Host Cell
[0110] The host cell may be any host cell suitable for production
of a useful product. A host cell may be any suitable cell, such as
a prokaryotic cell, such as a bacterium, or a eukaryotic cell.
Typically, the cell will be a eukaryotic cell, for example a yeast
or a filamentous fungus. The host cell is capable of converting one
or more pentose sugar, such as L-arabinose or xylose into
fermentation product.
[0111] The host cell may be a bacterium. In an embodiment the
bacterium is genetically engineered for pentose conversion. In an
embodiment the bacterium is not Escherichia coli (E-coli). An
example of a bacterium that is a suitable host for the application
of the invention is genetically engineered Zymomonas mobilis. Such
strain is for example described in Yanna et al, Appl Microbiol
Biotechnol. 2012 June; 94(6):1667-78.
[0112] The host for the invention may be (genetically engineered)
yeast. Genetic engineering is hereinafter described in detail.
Yeasts are herein defined as eukaryotic microorganisms and include
all species of the subdivision Eumycotina (Alexopoulos, C. J.,
1962, In: Introductory Mycology, John Wiley & Sons, Inc., New
York) that predominantly grow in unicellular form.
[0113] Yeasts may either grow by budding of a unicellular thallus
or may grow by fission of the organism. A preferred yeast as a
transformed host cell may belong to the genera Saccharomyces,
Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula,
Kloeckera, Schwanniomyces or Yarrowia. Preferably the yeast is one
capable of anaerobic fermentation, more preferably one capable of
anaerobic alcoholic fermentation.
[0114] In one embodiment the host cell is yeast.
[0115] Preferably the host is an industrial host, more preferably
an industrial yeast. An industrial host and industrial yeast cell
may be defined as follows. The living environments of yeast cells
in industrial processes are significantly different from that in
the laboratory. Industrial yeast cells must be able to perform well
under multiple environmental conditions which may vary during the
process. Such variations include change in nutrient sources, pH,
ethanol concentration, temperature, oxygen concentration, etc.,
which together have potential impact on the cellular growth and
ethanol production of Saccharomyces cerevisiae. Under adverse
industrial conditions, the environmental tolerant strains should
allow robust growth and production. Industrial yeast strains are
generally more robust towards these changes in environmental
conditions which may occur in the applications they are used, such
as in the baking industry, brewing industry, wine making and the
ethanol industry. Examples of industrial yeast (S. cerevisiae) are
Ethanol Red.RTM. (Fermentis) Fermiol.RTM. (DSM) and Thermosacc.RTM.
(Lallemand).
[0116] In an embodiment the host is inhibitor tolerant. Inhibitor
tolerant host cells may be selected by screening strains for growth
on inhibitors containing materials, such as illustrated in Kadar et
al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858,
wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was
selected.
Transformation
[0117] The polynucleotides according to the invention may be
expressed in a suitable host. Therefore standard transformation
techniques may be used.
[0118] The invention further relates to a nucleic acid construct
comprising the polynucleotide as described before, e.g. a
vector.
[0119] Another aspect of the invention thus pertains to vectors,
including cloning and expression vectors, comprising a
polynucleotide of the invention encoding a glyoxalase polypeptide
protein or a functional equivalent thereof and methods of growing,
transforming or transfecting such vectors in a suitable host cell,
for example under conditions in which expression of a glyoxalase of
the invention occurs. As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked.
[0120] Polynucleotides of the invention can be incorporated into a
recombinant replicable vector, for example a cloning or expression
vector. The vector may be used to replicate the nucleic acid in a
compatible host cell. Thus in a further embodiment, the invention
provides a method of making polynucleotides of the invention by
introducing a polynucleotide of the invention into a replicable
vector, introducing the vector into a compatible host cell, and
growing the host cell under conditions which bring about
replication of the vector. The vector may be recovered from the
host cell. Suitable host cells are described below.
[0121] It will be appreciated by those skilled in the art that the
design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression
of protein desired, etc. The vectors, such as expression vectors,
of the invention can be introduced into host cells to thereby
produce proteins or peptides, encoded by nucleic acids as described
herein. The vectors, such as recombinant expression vectors, of the
invention can be designed for expression of glyoxalase polypeptide
proteins in prokaryotic or eukaryotic cells.
[0122] For example, glyoxalase polypeptides can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors), filamentous fungi, yeast cells or mammalian
cells. Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Representative examples of appropriate
hosts are described hereafter.
[0123] Appropriate culture mediums and conditions for the
above-described host cells are known in the art.
[0124] For most filamentous fungi and yeast, the vector or
expression construct is preferably integrated in the genome of the
host cell in order to obtain stable transformants. However, for
certain yeasts also suitable episomal vectors are available into
which the expression construct can be incorporated for stable and
high level expression, examples thereof include vectors derived
from the 2.mu. and pKD1 plasmids of Saccharomyces and
Kluyveromyces, respectively, or vectors containing an AMA sequence
(e.g. AMA1 from Aspergillus). In case the expression constructs are
integrated in the host cells genome, the constructs are either
integrated at random loci in the genome, or at predetermined target
loci using homologous recombination, in which case the target loci
preferably comprise a highly expressed gene.
[0125] Accordingly, expression vectors useful in the present
invention include chromosomal-, episomal- and virus-derived vectors
e.g., vectors derived from bacterial plasmids, bacteriophage, yeast
episome, yeast chromosomal elements, viruses such as baculoviruses,
papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagemids.
[0126] The vector may further include sequences flanking the
polynucleotide giving rise to RNA which comprise sequences
homologous to eukaryotic genomic sequences or viral genomic
sequences. This will allow the introduction of the polynucleotides
of the invention into the genome of a host cell.
[0127] An integrative cloning vector may integrate at random or at
a predetermined target locus in the chromosome(s) of the host cell
into which it is to be integrated.
[0128] The vector system may be a single vector, such as a single
plasmid, or two or more vectors, such as two or more plasmids,
which together contain the total DNA to be introduced into the
genome of the host cell.
[0129] The vector may contain a polynucleotide of the invention
oriented in an antisense direction to provide for the production of
antisense RNA.
[0130] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, transduction, infection,
lipofection, cationic lipid-mediated transfection or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual, 2.sup.nd, ed. Cold Spring Harbor Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989), Davis et al., Basic Methods in Molecular Biology (1986) and
other laboratory manuals.
[0131] The polynucleotide according to the invention is
constitutively expressed. Herein, the term "constitutive", usually
with respect to the host cell, means that the polynucleotide is
present not (or not only) as it is present naturally in the host.
In that sense it can also be explained as that the polynucleotide
is brought into the cell by human intervention. A characteristic of
constitutive expressed gene is that the gene is transcribed
continuously, irrespective of the growth conditions, such as the
carbon source or the growth phase of the cells. The polynucleotide
may be heterologous to the genome of the host cell. The term
"heterologous", usually with respect to the host cell, means that
the polynucleotide does not naturally occur in the genome of the
host cell or that the polypeptide is not naturally produced by that
cell.
[0132] The polynucleotide may be homologous to the genome of the
host cell. The term "homologous", usually with respect to the host
cell, means that the polynucleotide naturally occurs in the genome
of the host cell or that the polypeptide is naturally produced by
that cell.
[0133] In another embodiment, the invention features cells, e.g.,
transformed host cells or recombinant host cells that contain a
nucleic acid encompassed by the invention. A "transformed cell" or
"recombinant cell" is a cell into which (or into an ancestor of
which) has been introduced, by means of recombinant DNA techniques,
a nucleic acid according to the invention. Both prokaryotic and
eukaryotic cells are included, e.g., bacteria, fungi, yeast, and
the like, especially preferred are yeast cells including e.g.
Saccharomyces, for example Saccharomyces cerevisiae.
[0134] A host cell can be chosen that modulates the expression of
the inserted sequences, or modifies and processes the gene product
in a specific, desired fashion. Such modifications (e.g.,
glycosylation) and processing (e.g. cleavage) of protein products
may facilitate optimal functioning of the protein.
[0135] Various host cells have characteristic and specific
mechanisms for post-translational processing and modification of
proteins and gene products. Appropriate cell lines or host systems
familiar to those of skill in the art of molecular biology and/or
microbiology can be chosen to ensure the desired and correct
modification and processing of the foreign protein expressed. To
this end, eukaryotic host cells that possess the cellular machinery
for proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product can be used. Such host cells
are well known in the art.
[0136] If desired, a cell as described above may be used to in the
preparation of a polypeptide according to the invention. Such a
method typically comprises cultivating a host cell (e.g.
transformed or transfected with an expression vector as described
above) under conditions to provide for expression (by the vector)
of a coding sequence encoding the polypeptide, and optionally
recovering the expressed polypeptide. Polynucleotides of the
invention can be incorporated into a recombinant replicable vector,
e.g. an expression vector. The vector may be used to replicate the
nucleic acid in a compatible host cell. Thus in a further
embodiment, the invention provides a method of making a
polynucleotide of the invention by introducing a polynucleotide of
the invention into a replicable vector, introducing the vector into
a compatible host cell, and growing the host cell under conditions
which bring about the replication of the vector. The vector may be
recovered from the host cell.
[0137] The vectors may be transformed or transfected into a
suitable host cell as described above to provide for expression of
a polypeptide of the invention. This process may comprise culturing
a host cell transformed with an expression vector as described
above under conditions to provide for expression by the vector of a
coding sequence encoding the polypeptide.
[0138] Herein standard isolation, hybridization, transformation and
cloning techniques are used (e.g., as described in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Homology & Identity
[0139] Amino acid or nucleotide sequences are said to be homologous
when exhibiting a certain level of similarity. Two sequences being
homologous indicate a common evolutionary origin. Whether two
homologous sequences are closely related or more distantly related
is indicated by "percent identity" or "percent similarity", which
is high or low respectively. Although disputed, to indicate
"percent identity" or "percent similarity", "level of homology" or
"percent homology" is frequently used interchangeably.
[0140] A comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. The skilled person will be aware of the
fact that several different computer programs are available to
align two sequences and determine the homology between two
sequences (Kruskal, J. B. (1983) An overview of sequence comparison
In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits
and macromolecules: the theory and practice of sequence comparison,
pp. 1-44 Addison Wesley). The percent identity between two amino
acid sequences can be determined using the Needleman and Wunsch
algorithm for the alignment of two sequences. (Needleman, S. B. and
Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm
aligns amino acid sequences as well as nucleotide sequences. The
Needleman-Wunsch algorithm has been implemented in the computer
program NEEDLE. For the purpose of this invention the NEEDLE
program from the EMBOSS package was used (version 2.8.0 or higher,
EMBOSS: The European Molecular Biology Open Software Suite (2000)
Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp
276-277, http://emboss.bioinformatics.nl/). For protein sequences,
EBLOSUM62 is used for the substitution matrix. For nucleotide
sequences, EDNAFULL is used. Other matrices can be specified. The
optional parameters used for alignment of amino acid sequences are
a gap-open penalty of 10 and a gap extension penalty of 0.5. The
skilled person will appreciate that all these different parameters
will yield slightly different results but that the overall
percentage identity of two sequences is not significantly altered
when using different algorithms.
Global Homology Definition
[0141] The homology or identity is the percentage of identical
matches between the two full sequences over the total aligned
region including any gaps or extensions. The homology or identity
between the two aligned sequences is calculated as follows: Number
of corresponding positions in the alignment showing an identical
amino acid in both sequences divided by the total length of the
alignment including the gaps. The identity defined as herein can be
obtained from NEEDLE and is labelled in the output of the program
as "IDENTITY".
Longest Identity Definition
[0142] The homology or identity between the two aligned sequences
is calculated as follows: Number of corresponding positions in the
alignment showing an identical amino acid in both sequences divided
by the total length of the alignment after subtraction of the total
number of gaps in the alignment. The identity defined as herein can
be obtained from NEEDLE by using the NOBRIEF option and is labelled
in the output of the program as "longest-identity".
[0143] With the identity to SEQ ID NO: 20 as defined herein above
an overview of GLO1 aminoacids that are active in cells according
to the invention is given in table 1.
TABLE-US-00001 TABLE 1 Overview of GLO1 aminoacids active in cell
according to invention (see examples) and identity to GLO1 of
Saccharomyces cerevisiae (SEQ ID NO: 20). % identity to SEQ Source
of GLO1 ID NO: 20 SEQ ID NO Saccharomyces cerevisiae 100 20 Candida
glabrata 69 21 Kluyveromyces lactis 61 23 Zygosaccharomyces rouxii
61 22 Candida magnoliae 45 24
[0144] Therefore, in an embodiment the GLO1 nucleotide or amino
acid is derived from a microorganism chosen from the group
consisting of Saccharomyces cerevisiae, Candida glabrata,
Kluyveromyces lactis, Zygosaccharomyces rouxii and Candida
magnolia.
[0145] In an embodiment the GLO1 nucleotide or amino acid is
derived from a microorganism chosen from the group consisting of
Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis
and Candida magnolia.
[0146] In an embodiment, the GLO nucleotide is a codon pair
optimized nucleotide sequence, wherein the sequence is SED ID NO:
34, SED ID NO: 35, SED ID NO: 36, SED ID NO: 37, SED ID NO: 38.
[0147] In an embodiment, the GLO nucleotide is a codon pair
optimized nucleotide sequence, wherein the sequence is SED ID NO:
34, SED ID NO: 35, SED ID NO: 36, or SED ID NO: 37.
[0148] The various embodiments of the invention described herein
may be cross-combined. The invention relates to a cell according to
claim 1. Such cell is herein also designated as transformed host
cell. Embodiments thereof are now described.
[0149] The cell is capable of using L-arabinose and xylose. In an
embodiment, the cell is capable of converting L-arabinose into
L-ribulose and/or xylulose 5-phosphate and/or into a desired
fermentation product, for example one of those mentioned
herein.
[0150] Organisms, for example S. cerevisiae strains, able to
produce ethanol from L-arabinose may be produced by modifying a
host cell introducing the araA (L-arabinose isomerase), araB
(L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes from a
suitable source. Such genes may be introduced into a host cell in
order that it is capable of using arabinose. Such an approach is
given is described in WO2003/095627. araA, araB and araD genes from
Lactobacillus plantarum may be used and are disclosed in
WO2008/041840. The araA gene from Bacillus subtilis and the araB
and araD genes from Escherichia coli may be used and are disclosed
in EP1499708. In another embodiment, araA, araB and araD genes may
derived from of at least one of the genus Clavibacter, Arthrobacter
and/or Gramella, in particular one of Clavibacter michiganensis,
Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in
WO 2009011591.
[0151] In an embodiment, the transformed host cell may also
comprise one or more copies of xylose isomerase gene and/or one or
more copies of xylose reductase and/or xylitol dehydrogenase.
[0152] The number of copies may be determined by the skilled person
by any known method. In an embodiment, the transformed host cell is
able to ferment glucose, arabinose, xylose and galactose.
[0153] In an embodiment, the cell is capable of converting 90% or
more glucose, xylose arabinose, galactose and mannose available,
into a fermentation product. In an embodiment, cell is capable of
converting 91% or more, 92% or more, 94% or more, 95% or more, 96%
or more, 97% or more, 98% or more or 100% of all glucose, xylose
arabinose, galactose and mannose available, into a fermentation
product.
[0154] In one embodiment of the invention the transformed host cell
is able to ferment one or more additional sugar, preferably C5
and/or C6 sugar e.g. mannose. In an embodiment of the invention the
transformed host cell comprises one or more of: a xylA-gene, XYL1
gene and XYL2 gene and/or XKS1-gene, to allow the transformed host
cell to ferment xylose; deletion of the aldose reductase (GRE3)
gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to
allow the increase of the flux through the pentose phosphate
pathway in the cell.
[0155] In an embodiment, the transformed host cell is an industrial
cell, more preferably an industrial yeast (as defined herein above
in the section host).
[0156] In an embodiment the transformed host cell is inhibitor
tolerant. Inhibitor tolerance is resistance to inhibiting
compounds. The presence and level of inhibitory compounds in
lignocellulose may vary widely with variation of feedstock,
pretreatment method hydrolysis process. Examples of categories of
inhibitors are carboxylic acids, furans and/or phenolic compounds.
Examples of carboxylic acids are lactic acid, acetic acid or formic
acid. Examples of furans are furfural and hydroxy-methylfurfural.
Examples or phenolic compounds are vanillin, syringic acid, ferulic
acid and coumaric acid. The typical amounts of inhibitors are for
carboxylic acids: several grams per liter, up to 20 grams per liter
or more, depending on the feedstock, the pretreatment and the
hydrolysis conditions. For furans: several hundreds of milligrams
per liter up to several grams per liter, depending on the
feedstock, the pretreatment and the hydrolysis conditions.
[0157] For phenolics: several tens of milligrams per liter, up to a
gram per liter, depending on the feedstock, the pretreatment and
the hydrolysis conditions.
[0158] The transformed host cells according to the invention may be
inhibitor tolerant, i.e. they can withstand common inhibitors at
the level that they typically have with common pretreatment and
hydrolysis conditions, so that the transformed host cells can find
broad application, i.e. it has high applicability for different
feedstock, different pretreatment methods and different hydrolysis
conditions.
[0159] In one embodiment, the industrial transformed host cell is
constructed on the basis of an inhibitor tolerant host cell,
wherein the construction is conducted as described hereinafter.
Inhibitor tolerant host cells may be selected by screening strains
for growth on inhibitors containing materials, such as illustrated
in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140,
847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC
26602 was selected.
[0160] In an embodiment, the transformed host cell is marker-free.
As used herein, the term "marker" refers to a gene encoding a trait
or a phenotype which permits the selection of, or the screening
for, a host cell containing the marker. Marker-free means that
markers are essentially absent in the transformed host cell. Being
marker-free is particularly advantageous when antibiotic markers
have been used in construction of the transformed host cell and are
removed thereafter. Removal of markers may be done using any
suitable prior art technique, e.g intramolecular recombination. A
suitable method of marker removal is illustrated in the
examples.
[0161] A transformed host cell may be able to convert plant
biomass, celluloses, hemicelluloses, pectins, starch, starch
derivatives, for example into fermentable sugars. Accordingly, a
transformed host cell may express one or more enzymes such as a
cellulase (an endocellulase or an exocellulase), a hemicellulase
(an endo- or exo-xylanase or arabinase) necessary for the
conversion of cellulose into glucose monomers and hemicellulose
into xylose and arabinose monomers, a pectinase able to convert
pectins into glucuronic acid and galacturonic acid or an amylase to
convert starch into glucose monomers.
[0162] The transformed host cell further may comprise those
enzymatic activities required for conversion of sugar to a desired
fermentation product, such as ethanol, butanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, fumaric acid, malic acid, itaconic acid, an amino
acid, 1,3-propane-diol, ethylene, glycerol, a .beta.-lactam
antibiotic or a cephalosporin.
[0163] In an embodiment, the transformed host cell is a cell that
is naturally capable of alcoholic fermentation, preferably,
anaerobic alcoholic fermentation. A transformed host cell
preferably has a high tolerance to ethanol, a high tolerance to low
pH (i.e. capable of growth at a pH lower than about 5, about 4,
about 3, or about 2.5) and towards organic and/or a high tolerance
to elevated temperatures.
[0164] Any of the above characteristics or activities of a
transformed host cell may be naturally present in the cell or may
be introduced or modified by genetic modification.
Construction of the Transformed Host Cell
[0165] According to an embodiment, the genes may be introduced in
the host cell by introduction into a host cell one or more of:
[0166] a) the genes araA, araB and araD under control of strong
constitutive promoter(s); [0167] b) PPP-genes TAL1, TKL1, RPE1 and
RKI1, optionally under control of one or more strong constitutive
promoter; [0168] c) deletion of an aldose reductase gene; [0169] d)
a xylA-gene and a XKS1-gene under control of strong constitutive
promoter(s); [0170] e) a xylA gene under control of a strong
constitutive promoter, which has the ability to integrate into the
genome on multiple loci; and adaptive evolution to produce the
transformed host cell. The above cell may be constructed using
recombinant expression techniques.
[0171] Recombinant Expression
[0172] The transformed host cell is a recombinant cell. That is to
say, a transformed host cell comprises, or is transformed with or
is genetically modified with a nucleotide sequence that does not
naturally occur in the cell in question.
[0173] Techniques for the recombinant expression of enzymes in a
cell, as well as for the additional genetic modifications of a
transformed host cell are well known to those skilled in the art.
Typically such techniques involve transformation of a cell with
nucleic acid construct comprising the relevant sequence. Such
methods are, for example, known from standard handbooks, such as
Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual
(3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, or F. Ausubel et al., eds., "Current protocols in
molecular biology", Green Publishing and Wiley Interscience, New
York (1987). Methods for transformation and genetic modification of
host cells are known from e.g. EP-A-0635 574, WO 98/46772, WO
99/60102, WO 00/37671, WO90/14423, EP-A-0481008, EP-A-0635574 and
U.S. Pat. No. 6,265,186.
[0174] Typically, the nucleic acid construct may be a plasmid, for
instance a low copy plasmid or a high copy plasmid. The cell
according to the present invention may comprise a single or
multiple copies of the nucleotide sequence encoding a enzyme, for
instance by multiple copies of a nucleotide construct or by use of
construct which has multiple copies of the enzyme sequence.
[0175] The nucleic acid construct may be maintained episomally and
thus comprise a sequence for autonomous replication, such as an
autosomal replication sequence. A suitable episomal nucleic acid
construct may e.g. be based on the yeast 2.mu. or pKD1 plasmids
(Gleer et al., 1991, Biotechnology 9: 968-975), or the AMA plasmids
(Fierro et al., 1995, Curr Genet. 29:482-489). Alternatively, each
nucleic acid construct may be integrated in one or more copies into
the genome of the cell. Integration into the cell's genome may
occur at random by non-homologous recombination but preferably, the
nucleic acid construct may be integrated into the cell's genome by
homologous recombination as is well known in the art (see e.g.
WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No.
6,265,186).
[0176] Most episomal or 2.mu. plasmids are relatively unstable in
yeast, being lost in approximately 10.sup.-2 or more cells after
each generation. Even under conditions of selective growth, only
60% to 95% of the cells retain the episomal plasmid. The copy
number of most episomal plasmids ranges from 20-100 per cell of
cir.sup.+ hosts. However, the plasmids are not equally distributed
among the cells, and there is a high variance in the copy number
per cell in populations. Strains transformed with integrative
plasmids are extremely stable, even in the absence of selective
pressure. However, plasmid loss can occur at approximately
10.sup.-3 to 10.sup.-4 frequencies by homologous recombination
between tandemly repeated DNA, leading to looping out of the vector
sequence. Preferably, the vector design in the case of stable
integration is thus, that upon loss of the selection marker genes
(which also occurs by intramolecular, homologous recombination)
that looping out of the integrated construct is no longer possible.
Preferably the genes are thus stably integrated. Stable integration
is herein defined as integration into the genome, wherein looping
out of the integrated construct is no longer possible. Preferably
selection markers are absent. Typically, the enzyme encoding
sequence will be operably linked to one or more nucleic acid
sequences, capable of providing for or aiding the transcription
and/or translation of the enzyme sequence.
[0177] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. For instance, a promoter or
enhancer is operably linked to a coding sequence the said promoter
or enhancer affects the transcription of the coding sequence.
[0178] As used herein, the term "promoter" refers to a nucleic acid
fragment that functions to control the transcription of one or more
genes, located upstream with respect to the direction of
transcription of the transcription initiation site of the gene, and
is structurally identified by the presence of a binding site for
DNA-dependent RNA polymerase, transcription initiation sites and
any other DNA sequences known to one of skilled in the art. A
"constitutive" promoter is a promoter that is active under most
environmental and developmental conditions. An "inducible" promoter
is a promoter that is active under environmental or developmental
regulation.
[0179] The promoter that could be used to achieve the expression of
a nucleotide sequence coding for an enzyme according to the present
invention, may be not native to the nucleotide sequence coding for
the enzyme to be expressed, i.e. a promoter that is heterologous to
the nucleotide sequence (coding sequence) to which it is operably
linked. The promoter may, however, be homologous, i.e. endogenous,
to the host cell.
[0180] Promotors are widely available and known to the skilled
person. Suitable examples of such promoters include e.g. promoters
from glycolytic genes, such as the phosphofructokinase (PFK),
triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate
dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK),
phosphoglycerate kinase (PGK) promoters from yeasts or filamentous
fungi; more details about such promoters from yeast may be found in
(WO 93/03159). Other useful promoters are ribosomal protein
encoding gene promoters, the lactase gene promoter (LAC4), alcohol
dehydrogenase promoters (ADH1, ADH4, and the like), and the enolase
promoter (ENO). Other promoters, both constitutive and inducible,
and enhancers or upstream activating sequences will be known to
those of skill in the art. The promoters used in the host cells of
the invention may be modified, if desired, to affect their control
characteristics. Suitable promoters in this context include both
constitutive and inducible natural promoters as well as engineered
promoters, which are well known to the person skilled in the art.
Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or
GAD, CYC1, HIS3, ADH1, PGL, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2,
ENO1, TPI1, and AOX1. Other suitable promoters include PDC1, GPD1,
PGK1, TEF1, and TDH3.
[0181] In a transformed host cell, the 3'-end of the nucleotide
acid sequence encoding enzyme preferably is operably linked to a
transcription terminator sequence. Preferably the terminator
sequence is operable in a host cell of choice, such as e.g. the
yeast species of choice. In any case the choice of the terminator
is not critical; it may e.g. be from any yeast gene, although
terminators may sometimes work if from a non-yeast, eukaryotic,
gene. Usually a nucleotide sequence encoding the enzyme comprises a
terminator. Preferably, such terminators are combined with
mutations that prevent nonsense mediated mRNA decay in the host
transformed host cell (see for example: Shirley et al., 2002,
Genetics 161:1465-1482).
[0182] The transcription termination sequence further preferably
comprises a polyadenylation signal.
[0183] Optionally, a selectable marker may be present in a nucleic
acid construct suitable for use in the invention. As used herein,
the term "marker" refers to a gene encoding a trait or a phenotype
which permits the selection of, or the screening for, a host cell
containing the marker. The marker gene may be an antibiotic
resistance gene whereby the appropriate antibiotic can be used to
select for transformed cells from among cells that are not
transformed. Examples of suitable antibiotic resistance markers
include e.g. dihydrofolate reductase,
hygromycin-B-phosphotransferase, 3'-O-phosphotransferase II
(kanamycin, neomycin and G418 resistance). Antibiotic resistance
markers may be most convenient for the transformation of polyploid
host cells, Also non-antibiotic resistance markers may be used,
such as auxotrophic markers (URA3, TRP1, LEU2) or the S. pombe TPI
gene (described by Russell P R, 1985, Gene 40: 125-130). In a
preferred embodiment the host cells transformed with the nucleic
acid constructs are marker gene free. Methods for constructing
recombinant marker gene free microbial host cells are disclosed in
EP-A-0635 574 and are based on the use of bidirectional markers
such as the A. nidulans amdS (acetamidase) gene or the yeast URA3
and LYS2 genes. Alternatively, a screenable marker such as Green
Fluorescent Protein, lacL, luciferase, chloramphenicol
acetyltransferase, beta-glucuronidase may be incorporated into the
nucleic acid constructs of the invention allowing to screen for
transformed cells.
[0184] Optional further elements that may be present in the nucleic
acid constructs suitable for use in the invention include, but are
not limited to, one or more leader sequences, enhancers,
integration factors, and/or reporter genes, intron sequences,
centromers, telomers and/or matrix attachment (MAR) sequences. The
nucleic acid constructs of the invention may further comprise a
sequence for autonomous replication, such as an ARS sequence.
[0185] The recombination process may thus be executed with known
recombination techniques. Various means are known to those skilled
in the art for expression and overexpression of enzymes in a
transformed host cell. In particular, an enzyme may be
overexpressed by increasing the copy number of the gene coding for
the enzyme in the host cell, e.g. by integrating additional copies
of the gene in the host cell's genome, by expressing the gene from
an episomal multicopy expression vector or by introducing a
episomal expression vector that comprises multiple copies of the
gene.
[0186] Alternatively, overexpression of enzymes in the host cells
of the invention may be achieved by using a promoter that is not
native to the sequence coding for the enzyme to be overexpressed,
i.e. a promoter that is heterologous to the coding sequence to
which it is operably linked. Although the promoter preferably is
heterologous to the coding sequence to which it is operably linked,
it is also preferred that the promoter is homologous, i.e.
endogenous to the host cell. Preferably the heterologous promoter
is capable of producing a higher steady state level of the
transcript comprising the coding sequence (or is capable of
producing more transcript molecules, i.e. mRNA molecules, per unit
of time) than is the promoter that is native to the coding
sequence. Suitable promoters in this context include both
constitutive and inducible natural promoters as well as engineered
promoters.
[0187] In an embodiment, the transformed host cell is markerfree,
which means that no auxotrophic or dominant markers, in particular
antibiotic resistance markers, are present in the genome or
extra-chromosomally.
[0188] The coding sequence used for overexpression of the enzymes
mentioned above may preferably be homologous to the host cell.
However, coding sequences that are heterologous to the host may be
used.
[0189] Overexpression of an enzyme, when referring to the
production of the enzyme in a genetically modified cell, means that
the enzyme is produced at a higher level of specific enzymatic
activity as compared to the unmodified host cell under identical
conditions. Usually this means that the enzymatically active
protein (or proteins in case of multi-subunit enzymes) is produced
in greater amounts, or rather at a higher steady state level as
compared to the unmodified host cell under identical conditions.
Similarly this usually means that the mRNA coding for the
enzymatically active protein is produced in greater amounts, or
again rather at a higher steady state level as compared to the
unmodified host cell under identical conditions. Preferably in a
host, an enzyme to be overexpressed is overexpressed by at least a
factor of about 1.1, about 1.2, about 1.5, about 2, about 5, about
10 or about 20 as compared to a strain which is genetically
identical except for the genetic modification causing the
overexpression. It is to be understood that these levels of
overexpression may apply to the steady state level of the enzyme's
activity, the steady state level of the enzyme's protein as well as
to the steady state level of the transcript coding for the
enzyme.
[0190] Preferably, the glyoxalase is expressed in the cytosol.
[0191] Adaptation
[0192] Adaptation is the evolutionary process whereby a population
becomes better suited (adapted) to its habitat or habitats. This
process takes place over several to many generations, and is one of
the basic phenomena of biology.
[0193] The term adaptation may also refer to a feature which is
especially important for an organism's survival. Such adaptations
are produced in a variable population by the better suited forms
reproducing more successfully, by natural selection.
[0194] Changes in environmental conditions alter the outcome of
natural selection, affecting the selective benefits of subsequent
adaptations that improve an organism's fitness under the new
conditions. In the case of an extreme environmental change, the
appearance and fixation of beneficial adaptations can be essential
for survival. A large number of different factors, such as e.g.
nutrient availability, temperature, the availability of oxygen,
etcetera, can drive adaptive evolution.
[0195] Fitness
[0196] There is a clear relationship between adaptedness (the
degree to which an organism is able to live and reproduce in a
given set of habitats) and fitness. Fitness is an estimate and a
predictor of the rate of natural selection. By the application of
natural selection, the relative frequencies of alternative
phenotypes will vary in time, if they are heritable.
[0197] Genetic Changes
[0198] When natural selection acts on the genetic variability of
the population, genetic changes are the underlying mechanism. By
this means, the population adapts genetically to its circumstances.
Genetic changes may result in visible structures, or may adjust the
physiological activity of the organism in a way that suits the
changed habitat.
[0199] It may occur that habitats frequently change. Therefore, it
follows that the process of adaptation is never finally complete.
In time, it may happen that the environment changes gradually, and
the species comes to fit its surroundings better and better. On the
other hand, it may happen that changes in the environment occur
relatively rapidly, and then the species becomes less and less well
adapted. Adaptation is a genetic process, which goes on all the
time to some extent, also when the population does not change the
habitat or environment.
[0200] The Adaptive Evolution
[0201] The transformed host cells may in their preparation be
subjected to adaptive evolution. A transformed host cell may be
adapted to sugar utilisation by selection of mutants, either
spontaneous or induced (e.g. by radiation or chemicals), for growth
on the desired sugar, preferably as sole carbon source, and more
preferably under anaerobic conditions. Selection of mutants may be
performed by techniques including serial transfer of cultures as
e.g. described by Kuyper et al. (2004, FEMS Yeast Res. 4: 655-664)
or by cultivation under selective pressure in a chemostat culture.
E.g. in a preferred host cell at least one of the genetic
modifications described above, including modifications obtained by
selection of mutants, confer to the host cell the ability to grow
on the xylose as carbon source, preferably as sole carbon source,
and preferably under anaerobic conditions. When XI is used as gene
to convert xylose, preferably the cell produce essentially no
xylitol, e.g. the xylitol produced is below the detection limit or
e.g. less than about 5, about 2, about 1, about 0.5, or about 0.3%
of the carbon consumed on a molar basis.
[0202] Adaptive evolution is also described e.g. in Wisselink H. W.
et al, Applied and Environmental Microbiology August 2007, p.
4881-4891
[0203] In one embodiment of adaptive evolution a regimen consisting
of repeated batch cultivation with repeated cycles of consecutive
growth in different media is applied, e.g. three media with
different compositions (glucose, xylose, and arabinose; xylose and
arabinose. See Wisselink et al. (2009) Applied and Environmental
Microbiology, February 2009, p. 907-914.
[0204] Yeast Transformation and Genetic Stability
[0205] Genetic engineering, i.e. transformation of yeast cells with
recombinant DNA, became feasible for the first time in 1978 [Beggs,
1978; Hinnen et al., 1978]. Recombinant DNA technology in yeast has
established itself since then. A multitude of different vector
constructs are available. Generally, these plasmid vectors, called
shuttle vectors, contain genetic material derived from E. coli
vectors consisting of an origin of replication and a selectable
marker (often the .beta.-lactamase gene, ampR), which enable them
to be propagated in E. coli prior to transformation into yeast
cells. Additionally, the shuttle vectors contain a selectable
marker for selection in yeast. Markers can be genes encoding
enzymes for the synthesis of a particular amino acid or nucleotide,
so that cells carrying the corresponding genomic deletion (or
mutation) are complemented for auxotrophy or autotrophy.
Alternatively, these vectors contain heterologous dominant
resistance markers, which provides recombinant yeast cells (i.e.
the cells that have taken up the DNA and express the marker gene)
resistance towards certain antibiotics, like G418 (geneticin),
hygromycin B or phleomycin. In addition, these vectors may contain
a sequence of (combined) restriction sites (multiple cloning site
or MCS) which will allow cloning foreign DNA into these sites,
although alternative methods exist as well.
[0206] Traditionally, four types of shuttle vectors can be
distinguished by the absence or presence of additional genetic
elements: [0207] Integrative plasmids (YIp) which by homologous
recombination are integrated into the host genome at the locus of
the marker or another gene, when this is opened by restriction and
the linearized DNA is used for transformation of the yeast cells.
This generally results in the presence of one copy of the foreign
DNA inserted at this particular site in the genome. [0208] Episomal
plasmids (YEp) which carry part of the 2.mu. plasmid DNA sequence
necessary for autonomous replication in yeast cells. Multiple
copies of the transformed plasmid are propagated in the yeast cell
and maintained as episomes. [0209] Autonomously replicating
plasmids (YRp) which carry a yeast origin of replication (ARS,
autonomously replicated sequence) that allows the transformed
plasmids to be propagated several hundred-fold. [0210] CEN plasmids
(YCp) which carry in addition to an ARS sequence a centromeric
sequence (derived from one of the nuclear chromosomes) which
normally guarantees stable mitotic segregation and usually reduces
the copy number of self-replicated plasmid to just one.
[0211] These plasmids are being introduced into the yeast cells by
transformation. Transformation of yeast cells may be achieved by
several different techniques, such as permeabilization of cells
with lithium acetate (Ito et al, 1983) and electroporation
methods.
[0212] In commercial application of recombinant microorganisms,
plasmid instability is the most important problem. Instability is
the tendency of the transformed cells to lose their engineered
properties because of changes to, or loss of, plasmids. This issue
is discussed in detail by Zhang et al (Plasmid stability in
recombinant Saccharomyces cerevisiae. Biotechnology Advances, Vol.
14, No. 4, pp. 401-435, 1996). Strains transformed with integrative
plasmids are extremely stable, even in the absence of selective
pressure (Sherman, F.
http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/9.html and
references therein).
[0213] The heterologous DNA is usually introduced into the organism
in the form of extra-chromosomal plasmids (YEp, YCp and YRp).
Unfortunately, it has been found with both bacteria and yeasts that
the new characteristics may not be retained, especially if the
selection pressure is not applied continuously. This is due to the
segregational instability of the hybrid plasmid when recombinant
cells grow for a long period of time. This leads to population
heterogeneity and clonal variability, and eventually to a cell
population in which the majority of the cells has lost the
properties that were introduced by transformation. If vectors with
auxotrophic markers are being used, cultivation in rich media often
leads to rapid loss of the vector, since the vector is only
retained in minimal media. The alternative, the use of dominant
antibiotic resistance markers, is often not compatible with
production processes. The use of antibiotics may not be desired
from a registration point of view (the possibility that trace
amounts of the antibiotic end up in the end product) or for
economic reasons (costs of the use of antibiotics at industrial
scale).
[0214] Loss of vectors leads to problems in large scale production
situations. Alternative methods for introduction of DNA do exist
for yeasts, such as the use of integrating plasmids (YIp). The DNA
is integrated into the host genome by recombination, resulting in
high stability. (Caunt, P. Stability of recombinant plasmids in
yeast. Journal of Biotechnology 9 (1988) 173-192). We have found
that an integration method using the host transposons are a good
alternative. In an embodiment genes may be integrated into the
transformed host cell genome. Initial introduction (i.e. before
adaptive evolution) of multiple copies be executed in any way known
in the art that leads to introduction of the genes. In an
embodiment, this may be accomplished using a vector with parts
homologous to repeated sequences (transposons), of the host cell.
When the host cell is a yeast cell, suitable repeated sequences are
the long terminal repeats (LTR) of the Ty element, known as delta
sequence. Ty elements fall into two rather similar subfamilies
called Ty1 and Ty2. These elements are about 6 kilobases (kb) in
length and are bounded by long terminal repeats (LTR), sequences of
about 335 base pairs (Boeke J D et al, The Saccharomyces cerevisiae
Genome Contains Functional and Nonfunctional Copies of Transposon
Ty1. Molecular and Cellular Biology, April 1988, p. 1432-1442 Vol.
8, No. 4). In the fully sequenced S. cerevisiae strain, S288c, the
most abundant transposons are Ty1 (31 copies) and Ty2 (13 copies)
(Gabriel A, Dapprich J, Kunkel M, Gresham D, Pratt S C, et al.
(2006) Global mapping of transposon location. PLoS Genet 2(12):
e212.doi:10.1371/journal.pgen.0020212). These transposons consist
of two overlapping open reading frames (ORFs), each of which encode
several proteins. The coding regions are flanked by the
aforementioned, nearly identical LTRs. Other, but less abundant and
more distinct Ty elements in S. cerevisiae comprise Ty3, Ty4 and
Ty5. For each family of full-length Ty elements there are an order
of magnitude more solo LTR elements dispersed through the genome.
These are thought to arise by LTR-LTR recombination of full-length
elements, with looping out of the internal protein encoding
regions.
[0215] The retrotransposition mechanism of the Ty retrotransposon
has been exploited to integrate multiple copies throughout the
genome (Boeke et al., 1988; Jacobs et al., 1988). The long terminal
repeats (LTR) of the Ty element, known as delta sequences, are also
good targets for integration by homologous recombination as they
exist in about 150-200 copies that are either Ty associated or solo
sites (Boeke, 1989; Kingsman and Kingsman, 1988). (Parekh R. N.
(1996). An Integrating Vector for Tunable, High Copy, Stable
Integration into the Dispersed Ty DELTA Sites of Saccharomyces
cerevisiae. Biotechnol. Prog. 1996, 12, 16-21). By adaptive
evolution, the number of copies may change.
[0216] araA, araB and araD Genes
[0217] A transformed host cell is capable of using arabinose. A
transformed host cell is therefore, be capable of converting
L-arabinose into L-ribulose and/or xylulose 5-phosphate and/or into
a desired fermentation product, for example one of those mentioned
herein.
[0218] Organisms, for example S. cerevisiae strains, able to
produce ethanol from L-arabinose may be produced by modifying a
cell introducing the araA (L-arabinose isomerase), araB
(L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes from a
suitable source. Such genes may be introduced into a transformed
host cell is order that it is capable of using arabinose. Such an
approach is given is described in WO2003/095627. araA, araB and
araD genes from Lactobacillus plantarum may be used and are
disclosed in WO2008/041840. The araA gene from Bacillus subtilis
and the araB and araD genes from Escherichia coli may be used and
are disclosed in EP1499708. In another embodiment, araA, araB and
araD genes may derived from of at least one of the genus
Clavibacter, Arthrobacter and/or Gramella, in particular one of
Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella
forsetii, as disclosed in WO 2009011591.
[0219] PPP-Genes
[0220] A transformed host cell may comprise one or more genetic
modifications that increases the flux of the pentose phosphate
pathway (PPP). In particular, the genetic modification(s) may lead
to an increased flux through the non-oxidative part of the pentose
phosphate pathway. A genetic modification that causes an increased
flux of the non-oxidative part of the pentose phosphate pathway is
herein understood to mean a modification that increases the flux by
at least a factor of about 1.1, about 1.2, about 1.5, about 2,
about 5, about 10 or about 20 as compared to the flux in a strain
which is genetically identical except for the genetic modification
causing the increased flux. The flux of the non-oxidative part of
the pentose phosphate pathway may be measured by growing the
modified host on xylose as sole carbon source, determining the
specific xylose consumption rate and subtracting the specific
xylitol production rate from the specific xylose consumption rate,
if any xylitol is produced. However, the flux of the non-oxidative
part of the pentose phosphate pathway is proportional with the
growth rate on xylose as sole carbon source, preferably with the
anaerobic growth rate on xylose as sole carbon source. There is a
linear relation between the growth rate on xylose as sole carbon
source (.mu..sub.max) and the flux of the non-oxidative part of the
pentose phosphate pathway. The specific xylose consumption rate
(Q.sub.s) is related to the specific growth rate (.mu.) and to the
yield of biomass on sugar (Y.sub.xs) according to:
Q.sub.s=m.sub.s+.rho./y.sub.sx.sup.max
or
1/y.sub.sx=m.sub.s/.mu.+1/y.sub.sx.sup.max
[0221] Therefore if .mu. is constant, the increased flux of the
non-oxidative part of the pentose phosphate pathway may be deduced
from the increase in maximum growth rate under these conditions
unless transport (uptake is limiting).
[0222] One or more genetic modifications that increase the flux of
the pentose phosphate pathway may be introduced in the host cell in
various ways. These including e.g. achieving higher steady state
activity levels of xylulose kinase and/or one or more of the
enzymes of the non-oxidative part pentose phosphate pathway and/or
a reduced steady state level of unspecific aldose reductase
activity. These changes in steady state activity levels may be
effected by selection of mutants (spontaneous or induced by
chemicals or radiation) and/or by recombinant DNA technology e.g.
by overexpression or inactivation, respectively, of genes encoding
the enzymes or factors regulating these genes.
[0223] In a preferred host cell, the genetic modification comprises
overexpression of at least one enzyme of the (non-oxidative part)
pentose phosphate pathway. Preferably the enzyme is selected from
the group consisting of the enzymes encoding for
ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase,
transketolase and transaldolase. Various combinations of enzymes of
the (non-oxidative part) pentose phosphate pathway may be
overexpressed. E.g. the enzymes that are overexpressed may be at
least the enzymes ribulose-5-phosphate isomerase and
ribulose-5-phosphate epimerase; or at least the enzymes
ribulose-5-phosphate isomerase and transketolase; or at least the
enzymes ribulose-5-phosphate isomerase and transaldolase; or at
least the enzymes ribulose-5-phosphate epimerase and transketolase;
or at least the enzymes ribulose-5-phosphate epimerase and
transaldolase; or at least the enzymes transketolase and
transaldolase; or at least the enzymes ribulose-5-phosphate
epimerase, transketolase and transaldolase; or at least the enzymes
ribulose-5-phosphate isomerase, transketolase and transaldolase; or
at least the enzymes ribulose-5-phosphate isomerase,
ribulose-5-phosphate epimerase, and transaldolase; or at least the
enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate
epimerase, and transketolase. In one embodiment of the invention
each of the enzymes ribulose-5-phosphate isomerase,
ribulose-5-phosphate epimerase, transketolase and transaldolase are
overexpressed in the host cell. More preferred is a host cell in
which the genetic modification comprises at least overexpression of
both the enzymes transketolase and transaldolase as such a host
cell is already capable of anaerobic growth on xylose. In fact,
under some conditions xylose converting host cells overexpressing
only the transketolase and the transaldolase already have the same
anaerobic growth rate on xylose as do host cells that overexpress
all four of the enzymes, i.e. the ribulose-5-phosphate isomerase,
ribulose-5-phosphate epimerase, transketolase and transaldolase.
Moreover, host cells overexpressing both of the enzymes
ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase
are preferred over host cells overexpressing only the isomerase or
only the epimerase as overexpression of only one of these enzymes
may produce metabolic imbalances.
[0224] The enzyme "ribulose 5-phosphate epimerase" (EC 5.1.3.1) is
herein defined as an enzyme that catalyses the epimerisation of
D-xylulose 5-phosphate into D-ribulose 5-phosphate and vice versa.
The enzyme is also known as phosphoribulose epimerase;
erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase;
xylulose phosphate 3-epimerase; phosphoketopentose epimerase;
ribulose 5-phosphate 3-epimerase; D-ribulose phosphate-3-epimerase;
D-ribulose 5-phosphate epimerase; D-ribulose-5-P 3-epimerase;
D-xylulose-5-phosphate 3-epimerase; pentose-5-phosphate
3-epimerase; or D-ribulose-5-phosphate 3-epimerase. A ribulose
5-phosphate epimerase may be further defined by its amino acid
sequence. Likewise a ribulose 5-phosphate epimerase may be defined
by a nucleotide sequence encoding the enzyme as well as by a
nucleotide sequence hybridising to a reference nucleotide sequence
encoding a ribulose 5-phosphate epimerase. The nucleotide sequence
encoding for ribulose 5-phosphate epimerase is herein designated
RPE1.
[0225] The enzyme "ribulose 5-phosphate isomerase" (EC 5.3.1.6) is
herein defined as an enzyme that catalyses direct isomerisation of
D-ribose 5-phosphate into D-ribulose 5-phosphate and vice versa.
The enzyme is also known as phosphopentosisomerase;
phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose
isomerase; D-ribose 5-phosphate isomerase; D-ribose-5-phosphate
ketol-isomerase; or D-ribose-5-phosphate aldose-ketose-isomerase. A
ribulose 5-phosphate isomerase may be further defined by its amino
acid sequence. Likewise a ribulose 5-phosphate isomerase may be
defined by a nucleotide sequence encoding the enzyme as well as by
a nucleotide sequence hybridising to a reference nucleotide
sequence encoding a ribulose 5-phosphate isomerase. The nucleotide
sequence encoding for ribulose 5-phosphate isomerase is herein
designated RKI1.
[0226] The enzyme "transketolase" (EC 2.2.1.1) is herein defined as
an enzyme that catalyses the reaction: D-ribose
5-phosphate+D-xylulose 5-phosphate <->sedoheptulose
7-phosphate+D-glyceraldehyde 3-phosphate and vice versa. The enzyme
is also known as glycolaldehydetransferase or
sedoheptulose-7-phosphate; D-glyceraldehyde-3-phosphate
glycolaldehydetransferase. A transketolase may be further defined
by its amino acid. Likewise a transketolase may be defined by a
nucleotide sequence encoding the enzyme as well as by a nucleotide
sequence hybridising to a reference nucleotide sequence encoding a
transketolase. The nucleotide sequence encoding for transketolase
is herein designated TKL1.
[0227] The enzyme "transaldolase" (EC 2.2.1.2) is herein defined as
an enzyme that catalyses the reaction: sedoheptulose
7-phosphate+D-glyceraldehyde 3-phosphate <->D-erythrose
4-phosphate+D-fructose 6-phosphate and vice versa. The enzyme is
also known as dihydroxyacetonetransferase; dihydroxyacetone
synthase; formaldehyde transketolase; or
sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate
glyceronetransferase. A transaldolase may be further defined by its
amino acid sequence. Likewise a transaldolase may be defined by a
nucleotide sequence encoding the enzyme as well as by a nucleotide
sequence hybridising to a reference nucleotide sequence encoding a
transaldolase. The nucleotide sequence encoding for transketolase
from is herein designated TAL1.
[0228] Xylose Isomerase or Xylose Reductase Genes
[0229] According to the invention, one or more copies of one or
more xylose isomerase gene and/or one or more xylose reductase and
xylitol dehydrogenase are introduced into the genome of the host
cell. The presence of these genetic elements confers on the cell
the ability to convert xylose by isomerisation or reduction.
[0230] In one embodiment, the one or more copies of one or more
xylose isomerase gene are introduced into the genome of the host
cell.
[0231] A "xylose isomerase" (EC 5.3.1.5) is herein defined as an
enzyme that catalyses the direct isomerisation of D-xylose into
D-xylulose and/or vice versa. The enzyme is also known as a
D-xylose ketoisomerase. A xylose isomerase herein may also be
capable of catalysing the conversion between D-glucose and
D-fructose (and accordingly may therefore be referred to as a
glucose isomerase). A xylose isomerase herein may require a
bivalent cation, such as magnesium, manganese or cobalt as a
cofactor.
[0232] Accordingly, such a transformed host cell is capable of
isomerising xylose to xylulose. The ability of isomerising xylose
to xylulose is conferred on the host cell by transformation of the
host cell with a nucleic acid construct comprising a nucleotide
sequence encoding a defined xylose isomerase. A transformed host
cell isomerises xylose into xylulose by the direct isomerisation of
xylose to xylulose.
[0233] The Xylose isomerase gene may have various origin, such as
for example Piromyces sp. as disclosed in WO2006/009434. Other
suitable origins are Bacteroides, in particular Bacteroides
uniformis as described in PCT/EP2009/526231n another embodiment,
one or more copies of one or more xylose reductase and xylitol
dehydrogenase genes are introduced into the genome of the host
cell. In this embodiment the conversion of xylose is conducted in a
two-step conversion of xylose into xylulose via a xylitol
intermediate as catalyzed by xylose reductase and xylitol
dehydrogenase, respectively. In an embodiment thereof xylose
reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK)
may be overexpressed, and optionally one or more of genes encoding
NADPH producing enzymes are up-regulated and one or more of the
genes encoding NADH consuming enzymes are up-regulated, as
disclosed in WO 2004085627.
[0234] XKS1 Gene
[0235] A transformed host cell may comprise one or more genetic
modifications that increase the specific xylulose kinase activity.
Preferably the genetic modification or modifications causes
overexpression of a xylulokinase, e.g. by overexpression of a
nucleotide sequence encoding a xylulokinase. The gene encoding the
xylulokinase may be endogenous to the host cell or may be a
xylulokinase that is heterologous to the host cell. A nucleotide
sequence used for overexpression of xylulokinase in the host cell
is a nucleotide sequence encoding a polypeptide with xylulokinase
activity.
[0236] The enzyme "xylulokinase" (EC 2.7.1.17) is herein defined as
an enzyme that catalyses the reaction ATP+D-xylulose=ADP+D-xylulose
5-phosphate. The enzyme is also known as a phosphorylating
xylulokinase, D-xylulokinaseor ATP:D-xylulose 5-phosphotransferase.
A xylulokinase used in the invention may be further defined by its
amino acid sequence. Likewise a xylulokinase may be defined by a
nucleotide sequence encoding the enzyme as well as by a nucleotide
sequence hybridising to a reference nucleotide sequence encoding a
xylulokinase.
[0237] In a transformed host cell, a genetic modification or
modifications that increase(s) the specific xylulokinase activity
may be combined with any of the modifications increasing the flux
of the pentose phosphate pathway as described above. This is not,
however, essential.
[0238] In the host cells of the invention, a xylulokinase to be
overexpressed is overexpressed by at least a factor of about 1.1,
about 1.2, about 1.5, about 2, about 5, about 10 or about 20 as
compared to a strain which is genetically identical except for the
genetic modification(s) causing the overexpression. It is to be
understood that these levels of overexpression may apply to the
steady state level of the enzyme's activity, the steady state level
of the enzyme's protein as well as to the steady state level of the
transcript coding for the enzyme.
[0239] Aldose Reductase (GRE3) Gene Deletion
[0240] In the embodiment, where XI is used as gene to convert
xylose, it may be advantageous to reduce aldose reductase activity.
A transformed host cell may therefore comprise one or more genetic
modifications that reduce aldose reductase activity in the host
cell. Preferably, unspecific aldose reductase activity is reduced
in the host cell by one or more genetic modifications that reduce
the expression of or inactivates a gene encoding an unspecific
aldose reductase. Preferably, the genetic modification(s) reduce or
inactivate the expression of each endogenous copy of a gene
encoding an aldose reductase in the host cell, in an embodiment
GRE3 aldose reductase deletion (herein called GRE3 deletion).
Transformed host cells may comprise multiple copies of genes
encoding unspecific aldose reductases as a result of di-, poly- or
aneuploidy, and/or the host cell may contain several different
(iso)enzymes with aldose reductase activity that differ in amino
acid sequence and that are each encoded by a different gene. Also
in such instances preferably the expression of each gene that
encodes an unspecific aldose reductase is reduced or inactivated.
Preferably, the gene is inactivated by deletion of at least part of
the gene or by disruption of the gene, whereby in this context the
term gene also includes any non-coding sequence up- or down-stream
of the coding sequence, the (partial) deletion or inactivation of
which results in a reduction of expression of unspecific aldose
reductase activity in the host cell.
[0241] A nucleotide sequence encoding an aldose reductase whose
activity is to be reduced in the host cell is a nucleotide sequence
encoding a polypeptide with aldose reductase activity.
[0242] Thus, a host cell comprising only a genetic modification or
modifications that reduce(s) unspecific aldose reductase activity
in the host cell is specifically included in the invention.
[0243] The enzyme "aldose reductase" (EC 1.1.1.21) is herein
defined as any enzyme that is capable of reducing xylose or
xylulose to xylitol. In the context of the present invention an
aldose reductase may be any unspecific aldose reductase that is
native (endogenous) to a host cell of the invention and that is
capable of reducing xylose or xylulose to xylitol. Unspecific
aldose reductases catalyse the reaction:
aldose+NAD(P)H+H.sup.+alditol+NAD(P).sup.+
[0244] The enzyme has a wide specificity and is also known as
aldose reductase; polyol dehydrogenase (NADP.sup.+); alditol:NADP
oxidoreductase; alditol:NADP.sup.+1-oxidoreductase;
NADPH-aldopentose reductase; or NADPH-aldose reductase.
[0245] A particular example of such an unspecific aldose reductase
that is endogenous to S. cerevisiae and that is encoded by the GRE3
gene (Traff et al., 2001, Appl. Environ. Microbiol. 67: 5668-74).
Thus, an aldose reductase of the invention may be further defined
by its amino acid sequence. Likewise an aldose reductase may be
defined by the nucleotide sequences encoding the enzyme as well as
by a nucleotide sequence hybridising to a reference nucleotide
sequence encoding an aldose reductase.
[0246] Bioproducts Production
[0247] Over the years suggestions have been made for the
introduction of various organisms for the production of bio-ethanol
from crop sugars. In practice, however, all major bio-ethanol
production processes have continued to use the yeasts of the genus
Saccharomyces as ethanol producer. This is due to the many
attractive features of Saccharomyces species for industrial
processes, i.e., a high acid-, ethanol- and osmo-tolerance,
capability of anaerobic growth, and of course its high alcoholic
fermentative capacity. Preferred yeast species as host cells
include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S.
uvarum, S. diastaticus, K. lactis, K. marxianus or K fragilis.
[0248] A transformed host cell may be a cell suitable for the
production of ethanol. A transformed host cell may, however, be
suitable for the production of fermentation products other than
ethanol
[0249] Such non-ethanolic fermentation products include in
principle any bulk or fine chemical that is producible by a
eukaryotic microorganism such as a yeast or a filamentous
fungus.
[0250] A transformed host cell that may be used for production of
non-ethanolic fermentation products is a host cell that contains a
genetic modification that results in decreased alcohol
dehydrogenase activity.
[0251] In an embodiment the transformed host cell may be used in a
process wherein sugars originating from lignocellulose are
converted into ethanol.
[0252] Lignocellulose
[0253] Lignocellulose, which may be considered as a potential
renewable feedstock, generally comprises the polysaccharides
cellulose (glucans) and hemicelluloses (xylans, heteroxylans and
xyloglucans). In addition, some hemicellulose may be present as
glucomannans, for example in wood-derived feedstocks. The enzymatic
hydrolysis of these polysaccharides to soluble sugars, including
both monomers and multimers, for example glucose, cellobiose,
xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose,
galacturonic acid, glucoronic acid and other hexoses and pentoses
occurs under the action of different enzymes acting in concert.
[0254] In addition, pectins and other pectic substances such as
arabinans may make up considerably proportion of the dry mass of
typically cell walls from non-woody plant tissues (about a quarter
to half of dry mass may be pectins).
[0255] Pretreatment
[0256] Before enzymatic treatment, the lignocellulosic material may
be pretreated. The pretreatment may comprise exposing the
lignocellulosic material to an acid, a base, a solvent, heat, a
peroxide, ozone, mechanical shredding, grinding, milling or rapid
depressurization, or a combination of any two or more thereof. This
chemical pretreatment is often combined with heat-pretreatment,
e.g. between 150-220.degree. C. for 1 to 30 minutes.
[0257] Enzymatic Hydrolysis
[0258] The pretreated material is commonly subjected to enzymatic
hydrolysis to release sugars that may be fermented according to the
invention. This may be executed with conventional methods, e.g.
contacting with cellulases, for instance cellobiohydrolase(s),
endoglucanase(s), beta-glucosidase(s) and optionally other enzymes.
The conversion with the cellulases may be executed at ambient
temperatures or at higher temperatures, at a reaction time to
release sufficient amounts of sugar(s). The result of the enzymatic
hydrolysis is hydrolysis product comprising C5/C6 sugars, herein
designated as the sugar composition.
The Sugar Composition
[0259] The sugar composition according to the invention comprises
glucose, arabinose and xylose. Any sugar composition may be used in
the invention that suffices those criteria. Optional sugars in the
sugar composition are galactose and mannose. In a preferred
embodiment, the sugar composition is a hydrolysate of one or more
lignocellulosic material. Lignocellulose herein includes
hemicellulose and hemicellulose parts of biomass. Also
lignocellulose includes lignocellulosic fractions of biomass.
Suitable lignocellulosic materials may be found in the following
list: orchard primings, chaparral, mill waste, urban wood waste,
municipal waste, logging waste, forest thinnings, short-rotation
woody crops, industrial waste, wheat straw, oat straw, rice straw,
barley straw, rye straw, flax straw, soy hulls, rice hulls, rice
straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn
stalks, corn cobs, corn husks, switch grass, miscanthus, sweet
sorghum, canola stems, soybean stems, prairie grass, gamagrass,
foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic
animal wastes, lawn clippings, cotton, seaweed, trees, softwood,
hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar
cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from
kernels, products and by-products from wet or dry milling of
grains, municipal solid waste, waste paper, yard waste, herbaceous
material, agricultural residues, forestry residues, municipal solid
waste, waste paper, pulp, paper mill residues, branches, bushes,
canes, corn, corn husks, an energy crop, forest, a fruit, a flower,
a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log,
a root, a sapling, a shrub, switch grass, a tree, a vegetable,
fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls,
hard or soft wood, organic waste material generated from an
agricultural process, forestry wood waste, or a combination of any
two or more thereof.
[0260] An overview of some suitable sugar compositions derived from
lignocellulose and the sugar composition of their hydrolysates is
given in table 2. The listed lignocelluloses include: corn cobs,
corn fiber, rice hulls, melon shells, sugar beet pulp, wheat straw,
sugar cane bagasse, wood, grass and olive pressings.
TABLE-US-00002 TABLE 2 Overview of sugar compositions from
lignocellulosic materials. Lignocellulosic material Gal Xyl Ara Man
Glu Rham Sum %. Gal. Corn cob a 10 286 36 227 11 570 1.7 Corn cob b
131 228 160 144 663 19.8 Rice hulls a 9 122 24 18 234 10 417 2.2
Rice hulls b 8 120 28 209 12 378 2.2 Melon Shells 6 120 11 208 16
361 1.7 Sugar beet pulp 51 17 209 11 211 24 523 9.8 Wheat straw
Idaho 15 249 36 396 696 2.2 Corn fiber 36 176 113 372 697 5.2 Cane
Bagasse 14 180 24 5 391 614 2.3 Corn stover 19 209 29 370 626 Athel
(wood) 5 118 7 3 493 625 0.7 Eucalyptus (wood) 22 105 8 3 445 583
3.8 CWR (grass) 8 165 33 340 546 1.4 JTW (grass) 7 169 28 311 515
1.3 MSW 4 24 5 20 440 493 0.9 Reed Canary Grass Veg 16 117 30 6 209
1 379 4.2 Reed Canary Grass Seed 13 163 28 6 265 1 476 2.7 Olive
pressing residu 15 111 24 8 329 487 3.1 Gal = galactose, Xyl =
xylose, Ara = arabinose, Man = mannose, Glu = glucose, Rham =
rhamnose. The percentage galactose (% Gal) is given.
[0261] It is clear from table 2 that in these lignocelluloses a
high amount of sugar is presence in de form of glucose, xylose,
arabinose and galactose. The conversion of glucose, xylose,
arabinose and galactose to fermentation product is thus of great
economic importance. Also mannose is present in some lignocellulose
materials be it usually in lower amounts than the previously
mentioned sugars. Advantageously therefore also mannose is
converted by the transformed host cell.
[0262] Fermentation
[0263] The fermentation process may be an aerobic or an anaerobic
fermentation process. An anaerobic fermentation process is herein
defined as a fermentation process run in the absence of oxygen or
in which substantially no oxygen is consumed, preferably less than
about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h
is consumed (i.e. oxygen consumption is not detectable), and
wherein organic molecules serve as both electron donor and electron
acceptors. In the absence of oxygen, NADH produced in glycolysis
and biomass formation, cannot be oxidised by oxidative
phosphorylation. To solve this problem many microorganisms use
pyruvate or one of its derivatives as an electron and hydrogen
acceptor thereby regenerating NAD.sup.+.
[0264] Thus, in a preferred anaerobic fermentation pentose is
converted into fermentation products such as ethanol, butanol,
lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid,
succinic acid, citric acid, malic acid, fumaric acid, an amino
acid, 1,3-propane-diol, ethylene, glycerol, a .beta.-lactam
antibiotic and a cephalosporin.
[0265] The fermentation process is preferably run at a temperature
that is optimal for the cell. Thus, for most yeasts or fungal host
cells, the fermentation process is performed at a temperature which
is less than about 42.degree. C., preferably less than about
38.degree. C. For yeast or filamentous fungal host cells, the
fermentation process is preferably performed at a temperature which
is lower than about 35, about 33, about 30 or about 28.degree. C.
and at a temperature which is higher than about 20, about 22, or
about 25.degree. C.
[0266] The ethanol yield on xylose and/or glucose in the process
preferably is at least about 50, about 60, about 70, about 80,
about 90, about 95 or about 98%. The ethanol yield is herein
defined as a percentage of the theoretical maximum yield.
[0267] The invention also relates to a process for producing a
fermentation product.
[0268] The fermentation process according to the present invention
may be run under aerobic and anaerobic conditions. In an
embodiment, the process is carried out under micro-aerophilic or
oxygen limited conditions.
[0269] An anaerobic fermentation process is herein defined as a
fermentation process run in the absence of oxygen or in which
substantially no oxygen is consumed, preferably less than about 5,
about 2.5 or about 1 mmol/L/h, and wherein organic molecules serve
as both electron donor and electron acceptors.
[0270] In a preferred process the cell ferments both the xylose and
glucose, preferably simultaneously in which case preferably a cell
is used which is insensitive to glucose repression. In addition to
a source of xylose (and glucose) as carbon source, the fermentation
medium will further comprise the appropriate ingredient required
for growth of the cell. Compositions of fermentation media for
growth of microorganisms such as yeasts are well known in the
art
[0271] The fermentation processes may be carried out in batch,
fed-batch or continuous mode. A separate hydrolysis and
fermentation (SHF) process or a simultaneous saccharification and
fermentation (SSF) process may also be applied. A combination of
these fermentation process modes may also be possible for optimal
productivity. These processes are described hereafter in more
detail.
[0272] SSF Mode
[0273] For Simultaneous Saccharification and Fermentation (SSF)
mode, the reaction time for liquefaction/hydrolysis or
presaccharification step is dependent on the time to realize a
desired yield, i.e. cellulose to glucose conversion yield. Such
yield is preferably as high as possible, preferably 60% or more,
65% or more, 70% or more, 75% or more 80% or more, 85% or more, 90%
or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or
more, even 99.5% or more or 99.9% or more.
[0274] According to the invention very high sugar concentrations in
SHF mode and very high product concentrations (e.g. ethanol) in SSF
mode are realized. In SHF operation the glucose concentration is 25
g/L or more, 30 g/L or more, 35 g/L or more, 40 g/L or more, 45 g/L
or more, 50 g/L or more, 55 g/L or more, 60 g/L or more, 65 g/L or
more, 70 g/L or more, 75 g/L or more, 80 g/L or more, 85 g/L or
more, 90 g/L or more, 95 g/L or more, 100 g/L or more, 110 g/L or
more, 120 g/L or more or may e.g. be 25 g/L-250 g/L, 30 gl/L-200
g/L, 40 g/L-200 g/L, 50 g/L-200 g/L, 60 g/L-200 g/L, 70 g/L-200
g/L, 80 g/L-200 g/L, 90 g/L, 80 g/L-200 g/L.
[0275] Product Concentration in SSF Mode
[0276] In SSF operation, the product concentration (g/L) is
dependent on the amount of glucose produced, but this is not
visible since sugars are converted to product in the SSF, and
product concentrations can be related to underlying glucose
concentration by multiplication with the theoretical maximum yield
(Yps max in gr product per gram glucose)
[0277] The theoretical maximum yield (Yps max in gr product per
gram glucose) of a fermentation product can be derived from
textbook biochemistry. For ethanol, 1 mole of glucose (180 gr)
yields according to normal glycolysis fermentation pathway in yeast
2 moles of ethanol (=2.times.46=92 gr ethanol. The theoretical
maximum yield of ethanol on glucose is therefore 92/180=0.51 gr
ethanol/gr glucose.
[0278] For Butanol (MW 74 gr/mole) or iso butanol, the theoretical
maximum yield is 1 mole of butanol per mole of glucose. So Yps max
for (iso-)butanol=74/180=0.41 gr (iso-) butanol/gr glucose.
[0279] For lactic acid the fermentation yield for homolactic
fermentation is 2 moles of lactic acid (MW=90 gr/mole) per mole of
glucose. According to this stoichiometry, the Yps max=1 gr lactic
acid/gr glucose.
[0280] For other fermentation products a similar calculation may be
made.
[0281] SSF Mode
[0282] In SSF operation the product concentration is 25 g*Yps g/L/L
or more, 30*Yps g/L or more, 35 g*Yps/L or more, 40*Yps g/L or
more, 45*Yps g/L or more, 50*Yps g/L or more, 55*Yps g/L or more,
60*Yps g/L or more, 65*Yps g/L or more, 70*Yps g/L or more, 75*Yps
g/L or more, 80*Yps g/L or more, 85*Yps g/L or more, 90*Yps g/L or
more, 95*Yps g/L or more, 100*Yps g/L or more, 110*Yps g/L or more,
120 g/L*Yps or more or may e.g. be 25*Yps g/L-250*Yps g/L, 30*Yps
gl/L-200*Yps g/L, 40*Yps g/L-200*Yps g/L, 50*Yps g/L-200*Yps g/L,
60*Yps g/L-200*Yps g/L, 70*Yps g/L-200*Yps g/L, 80*Yps g/L-200*Yps
g/L, 90*Yps g/L, 80*Yps g/L-200*Yps g/L
[0283] Accordingly, the invention provides a method for the
preparation of a fermentation product, which method comprises:
[0284] a. degrading lignocellulose using a method as described
herein; and
[0285] b. fermenting the resulting material,
thereby to prepare a fermentation product.
[0286] Fermentation Product
[0287] The fermentation product of the invention may be any useful
product. In one embodiment, it is a product selected from the group
consisting of ethanol, n-butanol, isobutanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
fumaric acid, malic acid, itaconic acid, maleic acid, citric acid,
adipic acid, an amino acid, such as lysine, methionine, tryptophan,
threonine, and aspartic acid, 1,3-propane-diol, ethylene, glycerol,
a .beta.-lactam antibiotic and a cephalosporin, vitamins,
pharmaceuticals, animal feed supplements, specialty chemicals,
chemical feedstocks, plastics, solvents, fuels, including biofuels
and biogas or organic polymers, and an industrial enzyme, such as a
protease, a cellulase, an amylase, a glucanase, a lactase, a
lipase, a lyase, an oxidoreductases, a transferase or a xylanase.
For example the fermentation products may be produced by cells
according to the invention, following prior art cell preparation
methods and fermentation processes, which examples however should
herein not be construed as limiting. n-butanol may be produced by
cells as described in WO2008121701 or WO2008086124; lactic acid as
described in US2011053231 or US2010137551; 3-hydroxy-propionic acid
as described in WO2010010291; acrylic acid as described in
WO2009153047.
[0288] Recovery of the Fermentation Product
[0289] For the recovery of the fermentation product existing
technologies are used. For different fermentation products
different recovery processes are appropriate. Existing methods of
recovering ethanol from aqueous mixtures commonly use fractionation
and adsorption techniques. For example, a beer still can be used to
process a fermented product, which contains ethanol in an aqueous
mixture, to produce an enriched ethanol-containing mixture that is
then subjected to fractionation (e.g., fractional distillation or
other like techniques). Next, the fractions containing the highest
concentrations of ethanol can be passed through an adsorber to
remove most, if not all, of the remaining water from the
ethanol.
[0290] The following Examples illustrate the invention:
EXAMPLES
[0291] General Molecular Biology Techniques
[0292] Unless indicated otherwise, the methods used are standard
biochemical techniques. Examples of suitable general methodology
textbooks include Sambrook et al., Molecular Cloning, a Laboratory
Manual (1989) and Ausubel et al., Current Protocols in Molecular
Biology (1995), John Wiley & Sons, Inc.
[0293] Media
[0294] The media used in the experiments was either YEP-medium (10
g/l yeast extract, 20 g/l peptone) or solid YNB-medium (6.7 g/l
yeast nitrogen base, 15 g/l agar), supplemented with sugars as
indicated in the examples. For solid YEP medium, 15 g/l agar was
added to the liquid medium prior to sterilization.
[0295] In the AFM experiments, Mineral Medium was used. The
composition of Mineral Medium has been described by Verduyn et al.
(Yeast (1992), Volume 8, 501-517) and was supplemented with 2,325
g/l urea and sugars as indicated in the examples.
Transformation of Yeast Cells
[0296] Yeast transformation was done according to the method
described by Schiestl and Gietz (Current Genetics (1989), Volume
16, 339-346).
[0297] Colony PCR
[0298] Genomic DNA was extracted from single yeast colonies for PCR
according to the method described by Looke et al. (BioTechniques
(2011), Volume 50, 325-328).
[0299] AFM Procedure
[0300] The Alcohol Fermentation Monitor (AFM; Halotec, Veenendaal,
the Netherlands) is a robust and user-friendly laboratory parallel
bioreactor that allows for accurate comparisons of carbon
conversion rates and yields for six simultaneous anaerobic
fermentations.
[0301] The starting culture of the AFM experiment contained 50 mg
of yeast (dry weight). To determine this, a calibration curve was
made of the RN1001 strain of biomass vs. OD700. This calibration
curve was used in the experiment to determine the volume of cell
culture needed for 50 mg of yeast (dry weight).
[0302] Prior to the start of the AFM experiment, precultures were
grown as indicated in the examples. For each strain the OD.sub.700
was measured and 50 mg of yeast (dry weight) was inoculated in 400
ml Mineral Medium (Verduyn et al. (Yeast (1992), Volume 8,
501-517), supplemented with 2,325 g/l urea and sugars as indicated
in the examples.
[0303] RNA Isolation
[0304] RNA was isolated using the Nucleospin RNA II kit
(Machery-Nagel, GmbH & Co. KG, Duren, Germany), with a slightly
adapted manufacturer's protocol. Prior to the start of the protocol
the freshly grown yeast cells were treated in 0.2 mg lyticase/ml,
1M Sorbitol and 0.1 M EDTA buffer for 30 minutes at 30.degree. C.
After this step, the manufacturers protocol was carried out, with
exception of step 6 and 7 (desalting of silica membrane and the
DNase treatment). To remove genomic DNA, a DNase treatment was
carried out after elution of the RNA from the column. For this 10
.mu.l RNA solution was mixed with 7 .mu.l DNase and RNase free
water, 2 .mu.l 10.times. buffer and 1 .mu.l DNase (Fermentas, 68789
St. Leon-Rot/Germany). This mix was incubated for 30 minutes at
37.degree. C. For inactivation of the enzyme, 1 .mu.l 25 mM EDTA
(Fermentas, 68789 St. Leon-Rot/Germany) was added and incubated for
10 minutes at 65.degree. C.
[0305] cDNA Synthesis
[0306] cDNA synthesis was performed on the RNA using the
RevertAid.TM. H Minus First Strand cDNA Synthesis Kit (Fermentas,
68789 St. Leon-Rot/Germany).
[0307] Q-PCR
[0308] For the Q-PCR experiment the DyNAmo Colorflash SYBR Green
qPCR kit (Finnzymes, 01620 Vantaa, Finland) was used. A DNA mix was
made with 1 .mu.l cDNA, 39 .mu.l H.sub.2O and 10 .mu.l yellow
sample dye. For each reaction 5 .mu.l DNA mix was used with 10
.mu.l 2.times. DyNAmo mastermix, 7 .mu.l H.sub.2O, 1 .mu.l 10 .mu.M
forward primer and 1 .mu.l 10 .mu.M reverse primer (primer numbers
are indicated in the examples). The Q-PCR was run on the Bio-Rad
CFX96 Real-Time system (Bio-Rad, Hercules, Calif., USA), using the
program as indicated in Table 3.
TABLE-US-00003 TABLE 3 Q-PCR program PROGRAM 1 95.0.degree. C. for
5:00 2 95.0.degree. C. for 0:20 3 67.0.degree. C. for 0:20 4
72.0.degree. C. for 0:20 + Plate Read 5 GOTO 2, 49 more times 6
Melt Curve 65.0 to 95.0.degree. C., increment 0.5.degree. C., 0:05
+ Plate Read END
[0309] Strains
[0310] The strains used in the experiments are RN1001, RN1041 and
RN1216. RN1041 has been described in WO 2012067510. This strain has
the following genotype:
[0311] MAT a, ura3-52, leu2-112, his3::loxP, gre3::loxP,
loxP-pTPI1::TAL1, loxP-pTPI1::RKI1, loxP-pTPI1-TKL1,
loxP-pTPI1-RPE1, delta::pADH1-XKS1-tCYC1-LEU2,
delta::URA3-pTPI1-xylA-tCYC1
[0312] MAT a=mating type a
[0313] ura3-52, leu2-112, HIS3::loxP mutations in the URA3, LEU2
and HIS3 genes respectively. The ura3-52 mutation is complemented
by the URA3 gene on the xylA overexpression construct; the leu2-112
mutation is complemented by the LEU2 gene on the XKS1
overexpression construct. The deletion of the HIS3-gene causes a
histidine auxotrophy. For this reason, RN1041 needs histidine in
the medium for growth.
[0314] gre3::loxP is a deletion of the GRE3 gene, encoding aldose
reductase. The loxP site is left behind in the genome after marker
removal.
[0315] loxP-pTPI1 designates the overexpression of genes of, in the
experiments described herein, the non-oxidative pentose phosphate
pathway by replacement of the native promoter by the promoter of
the TPI1 gene. The loxP site upstream of the strong, constitutive
TPI1 promoter remains in the genome after marker removal (Kuyper et
al, FEMS Yeast Research 5 (2005) 925-934).
[0316] delta:: means chromosomal integration of the construct after
recombination on the long terminal repeats of the Ty1
retrotransposon.
[0317] Strain RN1001 is the parent strain of strain RN1041, i.e.
before deletion of the HIS3-gene.
[0318] Strain RN1216 has the same genotype as strain RN1041.
Example 1
Construction of a Glyoxalase Overexpression Plasmid
[0319] The GLO1 ORF was obtained from CEN.PK113-7D using primers
SEQ ID NO 25 and SEQ ID NO 26. Subsequently the PCR amplified GLO1
ORF was cloned in a TOPO blunt vector using the Zero Blunt.RTM.
TOPO.RTM. PCR Cloning kit (Invitrogen, Carlsbad, Calif., USA) and
transformed to One Shot.RTM. TOP10 chemically competent E. coli
(Invitrogen, Carlsbad, Calif., USA). Miniprep DNA isolations were
performed on several colonies. The obtained plasmids were checked
by restriction enzyme analyses. The correct plasmid was called
pRN935.
[0320] Overexpression of GLO1 was carried out on a 2.mu. plasmid.
Therefore a 2.mu. plasmid with the GLO1 expression cassette was
constructed. The construction of the GLO1 expression cassette with
the PGK1 promoter and PGI1 terminator is shown in FIG. 1. Physical
maps of the plasmids containing these three elements, present in
plasmids pRN228, pRN935 and pRN685, are given in FIGS. 2, 3 and 4
respectively. Subsequently the constructed GLO1 expression cassette
was PCR amplified using primers SEQ ID NO 1 (pPGK1-5'-F; FIG. 1)
and SEQ ID NO 2 (tPGI1-3'-R; FIG. 1). The PCR product was purified
over column by using the GeneJET.TM. Gel Extraction kit (Fermentas,
68789 St. Leon-Rot/Germany), cloned in a TOPO blunt vector using
the Zero Blunt.RTM. TOPO.RTM. PCR Cloning kit (Invitrogen,
Carlsbad, Calif., USA) and transformed to One Shot.RTM. TOP10
chemically competent E. coli(Invitrogen, Carlsbad, Calif., USA).
Miniprep DNA isolations were performed on several colonies. The
obtained plasmids were checked by restriction enzyme analyses. Two
different plasmids were obtained, one plasmid with the GLO1
expression cassette inserted in reverse orientation (pRN1048, FIG.
5) and one plasmid with the GLO1 expression cassette inserted in
forward orientation (pRN1129, FIG. 6). Subsequently the GLO1
expression cassette, from pRN1048, was cloned into the yeast
shuttle vector pRN599 (FIG. 7) between the KpnI and ApaI sites. The
resulting plasmid was transformed to One Shot.RTM. TOP10 chemically
competent E. coli (Invitrogen, Carlsbad, Calif., USA). Miniprep DNA
isolations were performed on several colonies. The obtained
plasmids were checked by restriction enzyme analyses. The correct
plasmid was called pRN1049 (FIG. 8).
[0321] Plasmid pRN228 contains the following relevant elements: the
PGK1 promoter flanked by restriction sites SpeI and PstI and a
kanamycin resistance marker.
[0322] Plasmid pRN935 contains the following relevant elements: the
GLO1 ORF flanked by restriction sites PstI and SalI and a kanamycin
resistance marker.
[0323] Plasmid pRN685 contains the following relevant elements: the
PGI1 terminator flanked by restriction sites XhoI and HindIII and a
kanamycin resistance marker.
[0324] Plasmid pRN599 contains the following relevant elements: a
2.mu. origin of replication followed by a kanMX marker (consisting
of an Ashbya gossypii TEF1 promoter, a KanMX resistance gene and
the Ashbya gossypii TEF1 terminator) and an ampicillin resistance
marker.
Primer SEQ ID NO 1 is the forward primer of the PGK1 promoter.
Primer SEQ ID NO 2 is the reverse primer of the PGI1 terminator.
Primer SEQ ID NO 25 is the forward primer for amplification of the
GLO1 gene. Primer SEQ ID NO 26 is the reverse primer for
amplification of the GLO1 gene.
Example 2
Transformation of Yeast Strain with Overexpression Plasmid
[0325] The constructed GLO1 overexpression plasmid pRN1049 was used
to transform the yeast strain RN1001. Also a control strain was
created by transforming the yeast strain RN1001 with the empty
plasmid pRN599. Correct transformants were selected on YEP plates
containing 2% glucose and 200 .mu.g/ml G418. The RN1001 strain
containing pRN1049 is named RN1001Gpl. The control strain RN1001
containing the empty plasmid pRN599 is named RN1001Epl.
[0326] The level of GLO1 expression in RN1001Gpl was determined
with Q-PCR. The control strain RN1001Epl was used as a reference.
For this experiment both strains were grown overnight in YEP medium
containing 2% glucose and 200 .mu.g/ml G418. Subsequently RNA was
isolated from the cultures and was checked for genomic DNA
contamination by PCR using primers SEQ ID NO 3 and SEQ ID NO 4. No
contamination was found. Then a cDNA synthesis was performed on the
RNA using the RevertAid kit (Fermentas, 68789 St.
Leon-Rot/Germany). Next a Q-PCR experiment was done with primers
SEQ ID NO 5 and SEQ ID NO 6. Two housekeeping genes were used as a
reference, ALG9 using primers SEQ ID NO 7 and SEQ ID NO 8 and UBC6
using primers SEQ ID NO 9 and SEQ ID NO 10. The GLO1 expression
data was normalized on the housekeeping gene with the best duplo
CT-values, in this case ALG9. The RN1001Gpl strain showed a higher
expression of GLO1 as compared to RN1001Epl (FIG. 9). As indicated
in FIG. 9, the normalized expression level of the GLO1-gene was
about 5 times higher in strain RN1001Gpl as compared to RN1001Epl,
while the normalized expression of the UBC6-gene was the same in
both strains
Primer SEQ ID NO 3 is identical to a sequence of the ACT1 gene.
Primer SEQ ID NO 4 is identical to a sequence of the ACT1 gene.
Primer SEQ ID NO 5 is identical to a sequence of the GLO1 gene.
Primer SEQ ID NO 6 is identical to a sequence of the GLO1 gene.
Primer SEQ ID NO 7 is identical to a sequence of the ALG9 gene.
Primer SEQ ID NO 8 is identical to a sequence of the ALG9 gene.
Primer SEQ ID NO 9 is identical to a sequence of the UBC6 gene.
Primer SEQ ID NO 10 is identical to a sequence of the UBC6
gene.
Example 3
AFM Experiments
[0327] After verification of higher GLO1 expression in RN1001Gpl as
compared to the control strain RN1001Epl by Q-PCR (Example 2), an
AFM experiment was started to determine the conversion rate of the
presented sugars to ethanol by measuring the CO.sub.2 production
during the experiment, since ethanol and CO.sub.2 are being
produced in equimolar amounts. During the experiment, HPLC samples
of the cultures were taken at different time points, in order to
determine the glucose and xylose consumption rate and the ethanol
production rate.
[0328] Precultures were made of both strains, by inoculating some
cell material from agar plate in 100 ml YEP containing 2% glucose
and 200 .mu.g/ml G418. The next day, 400 ml Mineral Medium
containing 2% glucose and 2% xylose was inoculated with cell
material from the precultures. No G418 was added to the Mineral
Medium in the AFM experiment.
[0329] The CO.sub.2 production curves (FIG. 10) exhibit a faster
and higher CO.sub.2 production rate in RN1001Gpl as compared to
RN1001Epl, indicating a faster glucose and xylose consumption in
the RN1001Gpl as compared to the control strain RN1001Epl. HPLC
data (Tables 4 and 5, and FIG. 11) confirmed faster xylose
consumption rates in the RN1001Gpl strain as compared to the
RN1001Epl strain. The glucose was already consumed at the second
timepoint (16.5 hours), while this was not the case in strain
RN1001Epl.
TABLE-US-00004 TABLE 4 HPLC data RN1001Gpl Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 21.4 20.2 0.0 16.5 0.1 8.8 20.0
18.5 0.0 5.2 16.1 21 0.0 1.9 16.6 22.5 0.0 0.8 17.0 24.5 0.0 0.3
17.3 40 0.0 0.0 17.6
TABLE-US-00005 TABLE 5 HPLC data RN1001Epl Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 21.4 20.3 0.0 16.5 0.3 13.4 17.7
18.5 0.0 9.1 14.4 21 0.0 4.5 15.8 22.5 0.0 2.0 16.8 24.5 0.0 0.6
17.5 40 0.0 0.1 17.9
[0330] Because no G418 was added to the AFM experiment, it was
determined how many cells still contained their plasmid. Therefore
at the end of the last AFM experiment the same amount of cell
culture was plated on YEPD agar plates and YEPD agar plates
containing G418. Four times as much colonies were obtained on the
agarplates without G418. These results showed that approximately
25% of the cells still contained their plasmid, indicating plasmid
loss in both RN1001Gpl and RN1001Epl. Therefore new transformants
were constructed which contain an extra, constitutively expressed
copy of the GLO1 gene, stably integrated in the genome.
Example 4
Construction of the Integrative Fragments
[0331] To overcome the problem of plasmid loss during the AFM
experiment, the GLO1 expression cassette was integrated into the
genome of the yeast strains together with HIS3 for selection of
correct transformants. To this end, a plasmid was constructed
containing the GLO1 expression cassette and the HIS3 expression
cassette. The GLO1 expression cassette (present in pRN1129) and the
HIS3 expression cassette (present in pRN324) were extracted from
the plasmid using restriction enzymes PvulI and SphI or ApaLI
(FIGS. 6 and 12). Subsequently both fragments were ligated together
on the PvulI site. The resulting fragment was amplified by PCR
using primers SEQ ID NO 11 and SEQ ID NO 12, cloned in a TOPO blunt
vector using the Zero Blunt.RTM. TOPO.RTM. PCR Cloning kit
(Invitrogen, Carlsbad, Calif., USA) and transformed to One
Shot.RTM. TOP10 chemically competent E. coli (Invitrogen, Carlsbad,
Calif., USA). Miniprep DNA isolations were performed on several
colonies. The obtained plasmids were checked by restriction enzyme
analyses. The correct plasmid was called pRN1142 (FIG. 13).
[0332] Subsequently the GLO1 and HIS3 fragment was PCR amplified
using primers SEQ ID NO 11 and SEQ ID NO 12 and pRN1142 as
template. To create a control strain, the HIS3 expression cassette
was PCR amplified with primers SEQ ID NO 11 and SEQ ID NO 13 and
pRN324 as template. For integration in the genome, two 500 bp
flanks needed to be obtained of integration site 1 (INT1), a map of
the integration site is given in FIG. 14 (obtained from
http://www.yeastgenome.org/). A 5' and 3' 500 bp flank, for
integration to INT1, were PCR amplified using primers SEQ ID NO 14
and SEQ ID NO 15 for the 5' flank and primers SEQ ID NO 16 and SEQ
ID NO 17 for the 3' flank. As a template genomic DNA from RN1001
was used. All PCR reactions were purified over column by using the
GeneJET.TM. Gel Extraction kit (Fermentas, 68789 St.
Leon-Rot/Germany). The nucleotide sequence of the 5' flank is
included as SEQ ID NO 18 and the 3' flank as SEQ ID NO 19.
[0333] Plasmid pRN324 contains the following relevant elements: the
HIS3 expression cassette flanked by restriction sites ApaLI and
PvulI, and an ampicillin resistance marker.
[0334] Plasmid pRN1129 contains the following relevant elements:
the GLO1 expression cassette (consisting of the PGK1 promoter, the
GLO1 ORF and the PGI1 terminator) flanked by restriction sites
PvulI and SphI, and a kanamycin resistance marker.
[0335] Primer SEQ ID NO 11 is the forward primer of the HIS3
cassette, consisting of 25 nucleotides and a tail of 50 nucleotides
on the 5'-end, identical to the 50 nucleotides of the 3'-end of the
5' 500 bp INT1 flank.
[0336] Primer SEQ ID NO 12 is the reverse primer of the GLO1
cassette, consisting of 25 nucleotides and a tail of 50 nucleotides
on the 5'-end, identical to the 50 nucleotides of the 5'-end of the
3' 500 bp INT1 flank.
[0337] Primer SEQ ID NO 13 is the reverse primer of the HIS3
cassette, consisting of 25 nucleotides and a tail of 50 nucleotides
on the 5'-end, identical to the 50 nucleotides of the 5'-end of the
3' 500 bp INT1 flank.
[0338] Primer SEQ ID NO 14 is the forward primer of the 5' 500 bp
INT1 flank.
[0339] Primer SEQ ID NO 15 is the reverse primer of the 5' 500 bp
INT1 flank.
[0340] Primer SEQ ID NO 16 is the forward primer of the 3' 500 bp
INT1 flank.
[0341] Primer SEQ ID NO 17 is the reverse primer of the 3' 500 bp
INT1 flank.
[0342] All primer sequences mentioned above are also indicated in
FIG. 15.
Example 5
Transformation of Yeast Strains with Integrative Fragments
[0343] The mechanism of integration of the PCR fragments into the
genome is schematically shown in FIG. 15. The obtained PCR
fragments (Example 4) were used to transform the strains RN1041 and
RN1216, both containing an auxotrophy for histidine. Correct
transformants were selected on YNB plates containing 2% glucose.
Transformants were checked with colony PCR, using primers SEQ ID NO
14 and SEQ ID NO 17, for integration of the expression cassettes
into the genome. The RN1041 strain containing the HIS3 and GLO1
expression cassettes is named RN1041HG, and the RN1041 control
strain containing the HIS3 expression cassette is named RN1041H.
The RN1216 strain containing the HIS3 and GLO1 expression cassettes
is named RN1216HG, and the RN1216 control strain containing the
HIS3 expression cassette is named RN1216H.
[0344] From both control strains, RN1041H and RN1216H, one colony
each was selected for Q-PCR. From RN1041 HG and RN1216HG, two
individual colonies each were selected for Q-PCR (clone 1 and clone
2).
[0345] The expression of the GLO1-gene in the transformants was
checked in a Q-PCR experiment. For the Q-PCR experiment all
selected strains were grown overnight in YEP medium containing 2%
glucose. Subsequently RNA was isolated from the cultures and was
checked for genomic DNA contamination by PCR using primers SEQ ID
NO 3 and SEQ ID NO 4. No contamination was found. Then a cDNA
synthesis was performed on the RNA using the RevertAid kit
(Fermentas, 68789 St. Leon-Rot/Germany). Next a Q-PCR experiment
was done with primers SEQ ID NO 5 and SEQ ID NO 6. Two housekeeping
genes were used as a reference, ALG9 using primers SEQ ID NO 7 and
SEQ ID NO 8 and UBC6 using primers SEQ ID NO 9 and SEQ ID NO 10.
The GLO1 expression data was normalized on the housekeeping gene
with the best duplo CT-values, in this case UBC6. All 4 strains
containing HIS3 and GLO1 showed a higher normalized expression of
GLO1 as compared to their control strain (FIG. 16), about 4-5 times
higher in the RN1041 background compared to ALG9 and about 2-3
times higher in the RN1216 background.
Example 6
AFM Experiments
[0346] After verification of higher GLO1 expression in all 4
strains containing HIS3 and GLO1 compared to their control strain
by Q-PCR (Example 5), an AFM experiment was started to determine
the conversion rate of the presented sugars into ethanol by
measuring the CO.sub.2 production during the experiment, since
ethanol and CO.sub.2 are being produced in equimolar amounts.
During the experiment, HPLC samples of the cultures were taken at
different time points, in order to determine the sugar consumption
rate and the ethanol production rate. The same 6 strains were
tested in this AFM experiment as were used in the Q-PCR experiment
for determination of GLO1 expression.
[0347] Precultures were made of all 3 RN1041 derived strains
(RN1041H, RN1041 HG-1 and RN1041HG-2), by inoculating some cell
material from plate in 100 ml YEP containing 2% glucose. The next
day 400 ml Mineral Medium containing 2% glucose and 2% xylose was
inoculated with cell material from the precultures.
[0348] The CO.sub.2 production curves of the RN1041 derived strains
(FIG. 17) showed faster and higher CO.sub.2 production rates in
RN1041HG (clone 1 and 2) as compared to RN1041H, indicating a
faster glucose and xylose consumption in RN1041HG (clone 1 and 2)
as compared to the control strain RN1041 H. The HPLC data (Tables
6, 7 and 8, and FIG. 18) indeed confirm faster xylose consumption
in the RN1041HG (clone 1 and 2) strains as compared to the RN1041H
strain, the glucose was already consumed at the second timepoint
(15 hours).
TABLE-US-00006 TABLE 6 HPLC data RN1041H Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 20.9 20.7 0.0 15 0.3 16.1 14.2 17
0.0 14.5 12.5 19 0.0 12.3 12.6 21 0.0 9.6 13.4 23 0.0 7.1 14.5 39
0.0 0.5 17.5
TABLE-US-00007 TABLE 7 HPLC data RN1041HG-1 Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 20.8 20.8 0.0 15 0.1 15.4 14.0 17
0.0 13.4 12.9 19 0.0 10.6 13.1 21 0.0 7.5 14.2 23 0.0 4.6 15.6 39
0.0 0.4 17.6
TABLE-US-00008 TABLE 8 HPLC data RN1041HG-2 Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 21.3 21.3 0.0 15 0.1 15.3 13.3 17
0.0 12.5 13.6 19 0.0 9.4 14.2 21 0.0 6.2 15.6 23 0.0 3.3 16.6 39
0.0 0.2 18.2
[0349] These results clearly showed that overexpression of the
GLO1-gene leads to a faster and more efficient fermentation of
biomass sugars, leading to a reduced fermentation time. This means
that the sugars are more efficiently being converted into
ethanol.
[0350] A new AFM experiment was started with the RN1216 derived
strains. Precultures were made of all 3 strains (RN1216H,
RN1216HG-1 and RN1216HG-2), by inoculating some cell material from
agar plate in 100 ml YEP containing 2% glucose. The next day 400 ml
Mineral Medium containing 5% glucose and 5% xylose was inoculated
with cell material from the precultures.
[0351] The CO.sub.2 production curves of RN1216 (FIG. 19) showed
faster and higher CO.sub.2 production rates in RN1216HG (clone 1
and 2) as compared to RN1216H, indicating a faster, more efficient
glucose and xylose consumption in RN1216HG (clone 1 and 2) as
compared to the control strain RN1216H. HPLC data (Tables 9, 10 and
11, and FIG. 20) confirmed a faster glucose and xylose consumption
in the RN1216HG (clone 1 and 2) strains as compared to the RN1216H
strain.
TABLE-US-00009 TABLE 9 HPLC data RN1216H Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 52.5 51.4 0.0 4 49.8 50.5 0.7 8
45.9 50.3 2.2 12 39.1 49.6 5.1 16 28.5 48.6 10.0 20 14.7 47.0 16.8
24.5 1.4 44.2 28.5 39.5 0.2 27.5 35.1 43.5 0.0 21.0 36.5 47.5 0.0
16.1 38.5 112 0.0 2.1 45.4
TABLE-US-00010 TABLE 10 HPLC data RN1216HG-1 Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 52.1 51.3 0.0 4 49.6 50.4 0.7 8
45.5 50.1 2.3 12 38.1 49.4 5.5 16 26.8 48.3 10.8 20 12.0 46.2 18.1
24.5 0.2 42.1 28.9 39.5 0.0 22.7 36.5 43.5 0.0 15.2 38.8 47.5 0.0
10.4 40.7 112 0.0 1.1 45.5
TABLE-US-00011 TABLE 11 HPLC data RN1216HG-2 Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 52.2 51.2 0.0 4 49.6 50.4 0.8 8
44.9 50.0 2.6 12 37.2 49.3 6.0 16 25.9 47.9 11.3 20 11.5 45.8 18.6
24.5 0.2 41.0 29.1 39.5 0.0 17.5 38.5 43.5 0.0 12.4 40.2 47.5 0.0
7.3 42.4 112 0.0 0.8 45.7
[0352] These results reconfirmed the results above: overexpression
of the GLO1-gene leads to a faster and more efficient fermentation
of biomass sugars, in this case xylose and glucose, leading to a
reduced overall fermentation time and more efficient conversion of
the aforementioned sugars into ethanol.
Example 7
GLO1 Variants
[0353] GLO1 homologues, from other organisms than S. cerevisiae,
are expressed in the same fashion as described in the previous
examples. To this end, gene sequences were synthesized on basis of
the protein sequences, listed as SED ID NO: 20 to SEQ ID NO: 24.
The sequence of the open reading frame was generated by the method
described in WO/2008/000632. The protein sequences are derived from
Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces
rouxii, Kluyveromyces lactis and Candida magnoliae. As a reference,
the wild-type GLO1 gene from S. cerevisiae (see previous examples)
is used.
[0354] The constructs thus obtained are used to transform strain
RN1216, as described in the previous examples.
[0355] All transformants show enhanced sugar consumption rates when
tested in an AFM experiment (see also example 10), both on basis of
carbon dioxide profiles and actual sugar concentrations during the
experiment.
[0356] In an embodiment the following signatures that are present
in some active GLO1 versions, especially in terms of enhancing
co-fermentation of mixed sugar substrates, are as follows:
TABLE-US-00012 TABLE 12 GLO1 signature patterns Sequence Position
in S. cerevisiae (SEQ ID No 20) (E,s,d)-L-X-(H,Y)-(N,s)
E242-L243-X-H245-N246 G-(F,Y)-G-H G266-Y267-G268-H269
G-X(6)-(F,i)-X(2,3)-D-X(3)-Y G301-X(6)-F308-X(2)-D311-X(3)-Y315
[0357] In table 12, X designates an amino acid (i.e. any amino
acid).
[0358] If two amino acid residues are mentioned between brackets,
either one applies (e.g. (F,Y)).
[0359] Small letters designate minor variants, e.g. (F,i) means
that in most cases, amino acid F is observed, but in a few cases
amino acid I (isoleucine).
Example 8
Construction of the Integrative Fragments
[0360] The GLO1 homologues were integrated into the genome of the
yeast strain RN1216 together with HIS3 for selection of correct
transformants. To this end, plasmids were constructed, each
containing a different GLO1 homologue together with the PGK1
promoter and PG/1 terminator. The plasmids were constructed
according to the method described in PCT/EP2013/056623. The
plasmids were called pDB1175 (FIG. 21), pDB1176 (FIG. 22), pDB1177
(FIG. 23), pDB1178 (FIG. 24) and pDB1179 (FIG. 25).
[0361] Subsequently the different GLO1 expression cassettes were
PCR amplified using primers SEQ ID NO 28 and SEQ ID NO 29 and
pDB1175, pDB1176, pDB1177, pDB1178 and pDB1179 as template. The
HIS3 expression cassette was PCR amplified using SEQ ID NO 30 and
SEQ ID NO 31 and pRN324 (FIG. 12) as template. For integration in
the genome, two 500 bp flanks needed to be obtained of integration
site 1 (INT1) (described in example 4). A 5' and 3' 500 bp flank,
for integration to INT1, were PCR amplified using primers SEQ ID NO
14 and SEQ ID NO 32 for the 5' flank and primers SEQ ID NO 33 and
SEQ ID NO 17 for the 3' flank. As a template genomic DNA from
RN1001 was used. All PCR reactions were purified over column by
using the GeneJET.TM. Gel Extraction kit (Fermentas, 68789 St.
Leon-Rot/Germany).
[0362] Plasmid pDB1175 contains the following relevant elements:
the Sc_GLO1 expression cassette (consisting of the PGK1 promoter,
the codon pair optimized GLO1 ORF from Saccharomyces cerevisiae and
the PGI1 terminator), and a kanamycin resistance marker.
[0363] Plasmid pDB1176 contains the following relevant elements:
the Cgla_GLO1 expression cassette (consisting of the PGK1 promoter,
the codon pair optimized GLO1 ORF from Candida glabrata and the
PGI1 terminator), and a kanamycin resistance marker.
[0364] Plasmid pDB1177 contains the following relevant elements:
the Zrou_GLO1 expression cassette (consisting of the PGK1 promoter,
the codon pair optimized GLO1 ORF from Zygosaccharomyces rouxii and
the PGI1 terminator), and a kanamycin resistance marker.
[0365] Plasmid pDB1178 contains the following relevant elements:
the KI_GLO1 expression cassette (consisting of the PGK1 promoter,
the codon pair optimized GLO1 ORF from Kluyveromyces lactis and the
PGI1 terminator), and a kanamycin resistance marker.
[0366] Plasmid pDB1179 contains the following relevant elements:
the Cmag_GLO1 expression cassette (consisting of the PGK1 promoter,
the codon pair optimized GLO1 ORF from Candida magnoliae and the
PGI1 terminator), and a kanamycin resistance marker.
[0367] Plasmid pRN324: described in example 4
[0368] Primer SEQ ID NO 28 is the forward primer of the homologous
GLO1 expression cassettes (including promoter and terminator).
[0369] Primer SEQ ID NO 29 is the reverse primer of the homologous
GLO1 expression cassettes (including promoter and terminator).
[0370] Primer SEQ ID NO 30 is the forward primer of the HIS3
cassette, consisting of 20 nucleotides and a tail of 50 nucleotides
on the 5'-end, identical to the 50 nucleotides of the 3'-end of the
homologous GLO1 expression cassettes.
[0371] Primer SEQ ID NO 31 is the reverse primer of the HIS3
cassette, consisting of 21 nucleotides and a tail of 50 nucleotides
on the 5'-end, identical to the 50 nucleotides of the 5'-end of the
3' 500 bp INT1 flank.
[0372] Primer SEQ ID NO 32 is the reverse primer of the 5' 500 bp
INT1 flank, consisting of 23 nucleotides and a tail of 50
nucleotides on the 5'-end, identical to the 50 nucleotides of the
5'-end of the homologous GLO1 expression cassettes.
[0373] Primer SEQ ID NO 33 is the forward primer of the 3' 500 bp
INT1 flank, consisting of 24 nucleotides and a tail of 50
nucleotides on the 5'-end, identical to the 50 nucleotides of the
3'-end of the HIS3 expression cassette.
[0374] All primer sequences mentioned above are also indicated in
FIG. 26.
Example 9
Transformation of Yeast Strain with Integrative Fragments
[0375] The mechanism of integration of the PCR fragments into the
genome is schematically shown in FIG. 26. The obtained PCR
fragments (Example 8) were used to transform strain RN1216, which
contains an auxotrophy for histidine. Correct transformants were
selected on YNB plates containing 2% glucose. Transformants were
checked with colony PCR, using primers SEQ ID NO 14 and SEQ ID NO
17, for integration of the expression cassettes into the genome.
Correct strains were named the following: RN1216 ScG_H, RN1216
CglaG_H, RN1216 ZrouG_H, RN1216 KIG_H and RN1216 CmagG_H.
[0376] Strain RN1216 ScG_H contains the Sc_GLO1 expression cassette
and the HIS3 expression cassette.
[0377] Strain RN1216 CglaG_H contains the Cgla_GLO1 expression
cassette and the HIS3 expression cassette.
[0378] Strain RN1216 ZrouG_H contains the Zrou_GLO1 expression
cassette and the HIS3 expression cassette.
[0379] Strain RN1216 KIG_H contains the KI_GLO1 expression cassette
and the HIS3 expression cassette.
[0380] Strain RN1216 CmagG_H contains the Cmag_GLO1 expression
cassette and the HIS3 expression cassette.
Example 10
AFM Experiment
[0381] An AFM experiment was started to determine the conversion
rate of the presented sugars into ethanol by measuring the CO.sub.2
production during the experiment, since ethanol and CO.sub.2 are
being produced in equimolar amounts. During the experiment, HPLC
samples of the cultures were taken at different time points, in
order to determine the sugar consumption rate and the ethanol
production rate. The strains tested in this AFM experiment are the
5 constructed strains described in example 9, and as control strain
the RN1216H strain was used which is described in example 5.
[0382] Precultures were made of all 6 strains, by inoculating some
cell material from plate in 100 ml YEP containing 2% glucose. The
next day 400 ml Mineral Medium containing 5% glucose and 5% xylose
was inoculated with cell material from the precultures.
[0383] The CO.sub.2 production curves of the RN1216 derived strains
(FIG. 27) showed faster and higher CO.sub.2 production rates in
RN1216 ScG_H, RN1216 CglaG_H, RN1216 KIG_H and RN1216 CmagG_H as
compared to RN1216H, indicating a faster glucose en xylose
consumption in these 4 GLO1 variant strains as compared to the
control strain RN1216H. Strain RN1216 ZrouG_H showed faster and
higher CO.sub.2 production rates in the first 20 hours of the
experiment as compared to RN1216H, indicating a faster glucose and
slower xylose consumption as compared to the 5 other strains tested
in this AFM experiment. The HPLC data (Tables 13, 14, 15, 16, 17,
18 and FIGS. 28, 29, 30, 31, 32, 33) indeed confirm faster glucose
and xylose consumption in the RN1216 ScG_H, RN1216 CglaG_H, RN1216
KIG_H and RN1216 CmagG_H strains as compared to the RN1216H strain.
The HPLC data also confirm faster glucose consumption in the RN1216
ZrouG_H strains as compared to the 5 other strains tested in this
AFM experiment.
TABLE-US-00013 TABLE 13 HPLC data RN1216 ScG_H Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.2 52.9 0.0 8 46.8 51.5 2.5 12
40.5 52.4 5.5 16 28.5 53.6 10.9 20 13.0 48.6 17.6 24 3.3 46.2 22.9
28 0.2 40.2 26.0 32 0.0 33.0 28.5 36 0.0 19.7 35.1 40 0.0 7.9 41.2
44 0.0 2.6 44.0 48 0.0 1.1 44.5
TABLE-US-00014 TABLE 14 HPLC data RN1216 CglaG_H Time Glucose
Xylose Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.4 53.2 0.0 4 49.6 53.1
1.3 8 49.0 55.1 2.4 16 29.1 53.4 10.5 20 9.6 46.5 19.5 24 1.2 43.6
24.1 28 0.0 35.2 28.9 32 0.0 24.8 32.7 36 0.0 15.0 37.6 40 0.0 7.7
41.7 44 0.0 3.1 44.1 48 0.0 1.2 44.8
TABLE-US-00015 TABLE 15 HPLC data RN1216 ZrouG_H Time Glucose
Xylose Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.3 53.0 0.0 4 48.6 53.1
0.7 12 38.4 51.2 5.1 16 26.1 53.1 11.3 20 3.5 46.8 21.8 24 0.4 44.0
24.1 28 0.0 36.8 28.2 32 0.0 29.4 30.6 36 0.0 21.6 34.5 40 0.0 15.4
37.9 44 0.0 10.3 40.7 48 0.0 6.7 42.3
TABLE-US-00016 TABLE 16 HPLC data RN1216 KIG_H Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.5 53.0 0.0 8 47.8 53.8 1.7 16
30.8 53.3 9.6 20 11.8 45.5 18.5 24 1.1 41.0 24.6 28 0.2 33.9 25.6
32 0.0 27.7 31.0 36 0.0 15.8 37.4 40 0.0 7.9 41.6 44 0.0 3.3 44.3
48 0.0 1.4 44.8
TABLE-US-00017 TABLE 17 HPLC data RN1216 CmagG_H Time Glucose
Xylose Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.5 52.8 0.0 4 46.5 50.3
3.2 8 40.4 49.4 7.1 12 33.3 49.6 9.0 16 31.0 52.3 9.9 24 2.3 43.6
23.8 28 0.0 35.4 28.9 32 0.0 24.6 32.7 36 0.0 14.5 37.8 40 0.0 6.8
41.6 44 0.0 2.5 44.1 48 0.0 0.9 44.8
TABLE-US-00018 TABLE 18 HPLC data RN1216H Time Glucose Xylose
Ethanol (hrs) (g/l) (g/l) (g/l) 0 50.5 52.9 0.0 8 45.7 51.1 4.2 12
34.6 51.0 7.1 16 27.8 50.0 9.9 20 16.3 50.7 15.8 24 6.8 47.8 20.7
28 1.1 44.6 24.1 32 0.0 39.2 25.8 36 0.0 28.0 31.6 40 0.0 12.8 30.8
44 0.0 8.9 41.5 48 0.0 5.3 42.7
[0384] These results show that overexpression of the GLO1-gene
derived from the strains S. cerevisiae, C. glabrata, K. lactis or
C. magnoliae leads to a faster and more efficient fermentation of
biomass sugars, in this case xylose and glucose, leading to a
reduced overall fermentation time and more efficient conversion of
the aforementioned sugars into ethanol. An exception is the
overexpression of the GLO1-gene derived from the strain Z. rouxii,
which shows a faster glucose consumption as compared to the other
strains tested in this experiment, but the overall performance of
this strain is worse.
Sequence CWU 1
1
38132DNAArtificial SequenceForward primer PGK1
promotersource(1)..(32)/organism="Artificial Sequence"
/note="Forward primer PGK1 promoter" /mol_type="unassigned DNA"
1actagtctcg agctcttcaa ctcaagacgc ac 32236DNAArtificial
SequenceReverse primer PGI1
terminatorsource(1)..(36)/organism="Artificial Sequence"
/note="Reverse primer PGI1 terminator" /mol_type="unassigned DNA"
2ttaagcttcg tacgttttaa acagttgatg agaacc 36326DNAArtificial
SequenceForward primer ACT1
genesource(1)..(26)/organism="Artificial Sequence" /note="Forward
primer ACT1 gene" /mol_type="unassigned DNA" 3ccattttgag aatcgatttg
gccggt 26428DNAArtificial SequenceReverse primer ACT1
genesource(1)..(28)/organism="Artificial Sequence" /note="Reverse
primer ACT1 gene" /mol_type="unassigned DNA" 4ggtgatttcc ttttgcattc
tttcggca 28524DNAArtificial SequenceForward primer GLO1 gene,
Q-PCRsource(1)..(24)/organism="Artificial Sequence" /note="Forward
primer GLO1 gene, Q-PCR" /mol_type="unassigned DNA" 5ggagagcctg
atgtttttag cgca 24623DNAArtificial SequenceReverse primer GLO1
gene, Q-PCRsource(1)..(23)/organism="Artificial Sequence"
/note="Reverse primer GLO1 gene, Q-PCR" /mol_type="unassigned DNA"
6tcaatccagt atccatcagg gcc 23729DNAArtificial SequenceForward
primer ALG9 gene, Q-PCRsource(1)..(29)/organism="Artificial
Sequence" /note="Forward primer ALG9 gene, Q-PCR"
/mol_type="unassigned DNA" 7cacggatagt ggctttggtg aacaattac
29832DNAArtificial SequenceReverse primer ALG9 gene,
Q-PCRsource(1)..(32)/organism="Artificial Sequence" /note="Reverse
primer ALG9 gene, Q-PCR" /mol_type="unassigned DNA" 8tatgattatc
tggcagcagg aaagaacttg gg 32929DNAArtificial SequenceForward primer
UBC6 gene, Q-PCRsource(1)..(29)/organism="Artificial Sequence"
/note="Forward primer UBC6 gene, Q-PCR" /mol_type="unassigned DNA"
9gatacttgga atcctggctg gtctgtctc 291032DNAArtificial
SequenceReverse primer UBC6 gene,
Q-PCRsource(1)..(32)/organism="Artificial Sequence" /note="Reverse
primer UBC6 gene, Q-PCR" /mol_type="unassigned DNA" 10aaagggtctt
ctgtttcatc acctgtattt gc 321175DNAArtificial SequenceForward primer
HIS3 cassette, 5' flank INT1source(1)..(75)/organism="Artificial
Sequence" /note="Forward primer HIS3 cassette, 5' flank INT1"
/mol_type="unassigned DNA" 11cagttttaaa aagtcagaga atgtagagaa
gtatggatct ttgaaaccct ttggtgagcg 60ctaggagtca ctgcc
751275DNAArtificial SequenceReverse primer GLO1 cassette, 3' flank
INT1source(1)..(75)/organism="Artificial Sequence" /note="Reverse
primer GLO1 cassette, 3' flank INT1" /mol_type="unassigned DNA"
12atcttacata gtgtcgggaa caggtcattc taaaaaaagt aaaataaaat acgttttaaa
60cagttgatga gaacc 751375DNAArtificial SequenceReverse primer HIS3
cassette, 3' flank INT1source(1)..(75)/organism="Artificial
Sequence" /note="Reverse primer HIS3 cassette, 3' flank INT1"
/mol_type="unassigned DNA" 13cataatatgt taaaagctag atcttacata
gtgtcgggaa caggtcattc ttcacaccgc 60atatgatccg tcgag
751419DNAArtificial SequenceForward primer 5' flank
INT1source(1)..(19)/organism="Artificial Sequence" /note="Forward
primer 5' flank INT1" /mol_type="unassigned DNA" 14cggcattatt
gtgtatggc 191523DNAArtificial SequenceReverse primer 5' flank
INT1source(1)..(23)/organism="Artificial Sequence" /note="Reverse
primer 5' flank INT1" /mol_type="unassigned DNA" 15agggtttcaa
agatccatac ttc 231624DNAArtificial SequenceForward primer 3' flank
INT1source(1)..(24)/organism="Artificial Sequence" /note="Forward
primer 3' flank INT1" /mol_type="unassigned DNA" 16gaatgacctg
ttcccgacac tatg 241724DNAArtificial SequenceReverse primer 3' flank
INT1source(1)..(24)/organism="Artificial Sequence" /note="Reverse
primer 3' flank INT1" /mol_type="unassigned DNA" 17cacaagctta
ttcttccaaa aatc 2418500DNASaccharomyces
cerevisiaesource(1)..(500)/organism="Saccharomyces cerevisiae"
/mol_type="unassigned DNA" 18cggcattatt gtgtatggct caataatttt
ataaaaaaag gaactattgg ttcttagtat 60tttcttgcta gaagacatat tcttaccaat
cctttcataa gctaattatg ccatccatat 120agcaagagaa tccggtgggg
gcgccatgcc tatccggcgg caacattatt actctggtat 180acgggcgtaa
ctccataata tgccaccact tacctttaac atgttcatgg taggtacccc
240acccagccat aaggaaattt tcaaaggcgt tggatcaaaa aataggcctt
tatttcatcg 300cgtgattgag gagcataaca tgtttagtga aggtttcttt
tggaaaactt cagtcgctca 360ttattagaac cagggaggtc caggctttgc
tggtgggaga gaaagcttat gaagctgggg 420ttgcagattt gtcgattggt
cgccagtaca cagttttaaa aagtcagaga atgtagagaa 480gtatggatct
ttgaaaccct 50019478DNASaccharomyces
cerevisiaesource(1)..(478)/organism="Saccharomyces cerevisiae"
/mol_type="unassigned DNA" 19gaatgacctg ttcccgacac tatgtaagat
ctagctttta acatattatg gaaacctgaa 60atgtaaaatc tgaatttttg tatatgtgtt
tatatttggg tagttctttt gaggaaagca 120tgcatagact tgctgtacga
actttatgtg acttgtagtg acgctgtttc atgagacttt 180agccctttga
acatattatc atatctcagc ttgaaatact atagatttac ttttgcagcc
240atttcttggt gctccaaggt tgtgcgtatc tattacttaa tttctgtcct
tgccaagttt 300tgcagcaggg cggtcacaag actcctctgc cgtcattcct
tagtccttcg ggaacacact 360tatttatgta tttgtattct acaattctac
ggtgcacaag ggttgggcac tgttgagctc 420agcacgcaac tattgctggc
atgaagataa gattgatttt tggaagaata agcttgtg 47820326PRTSaccharomyces
cerevisiae 20Met Ser Thr Asp Ser Thr Arg Tyr Pro Ile Gln Ile Glu
Lys Ala Ser1 5 10 15Asn Asp Pro Thr Leu Leu Leu Asn His Thr Cys Leu
Arg Val Lys Asp 20 25 30Pro Ala Arg Thr Val Lys Phe Tyr Thr Glu His
Phe Gly Met Lys Leu 35 40 45Leu Ser Arg Lys Asp Phe Glu Glu Ala Lys
Phe Ser Leu Tyr Phe Leu 50 55 60Ser Phe Pro Lys Asp Asp Ile Pro Lys
Asn Lys Asn Gly Glu Pro Asp65 70 75 80Val Phe Ser Ala His Gly Val
Leu Glu Leu Thr His Asn Trp Gly Thr 85 90 95Glu Lys Asn Pro Asp Tyr
Lys Ile Asn Asn Gly Asn Glu Glu Pro His 100 105 110Arg Gly Phe Gly
His Ile Cys Phe Ser Val Ser Asp Ile Asn Lys Thr 115 120 125Cys Glu
Glu Leu Glu Ser Gln Gly Val Lys Phe Lys Lys Arg Leu Ser 130 135
140Glu Gly Arg Gln Lys Asp Ile Ala Phe Ala Leu Gly Pro Asp Gly
Tyr145 150 155 160Trp Ile Glu Leu Ile Thr Tyr Ser Arg Glu Gly Gln
Glu Tyr Pro Lys 165 170 175Gly Ser Val Gly Asn Lys Phe Asn His Thr
Met Ile Arg Ile Lys Asn 180 185 190Pro Thr Arg Ser Leu Glu Phe Tyr
Gln Asn Val Leu Gly Met Lys Leu 195 200 205Leu Arg Thr Ser Glu His
Glu Ser Ala Lys Phe Thr Leu Tyr Phe Leu 210 215 220Gly Tyr Gly Val
Pro Lys Thr Asp Ser Val Phe Ser Cys Glu Ser Val225 230 235 240Leu
Glu Leu Thr His Asn Trp Gly Thr Glu Asn Asp Pro Asn Phe His 245 250
255Tyr His Asn Gly Asn Ser Glu Pro Gln Gly Tyr Gly His Ile Cys Ile
260 265 270Ser Cys Asp Asp Ala Gly Ala Leu Cys Lys Glu Ile Glu Val
Lys Tyr 275 280 285Gly Asp Lys Ile Gln Trp Ser Pro Lys Phe Asn Gln
Gly Arg Met Lys 290 295 300Asn Ile Ala Phe Leu Lys Asp Pro Asp Gly
Tyr Ser Ile Glu Val Val305 310 315 320Pro His Gly Leu Ile Ala
32521319PRTCandida glabrata 21Met Ser Tyr Pro His Lys Ile Ala Ala
Ala His Asp Asp Pro Thr Leu1 5 10 15Met Phe Asn His Thr Cys Leu Arg
Ile Lys Asp Pro Ala Lys Ser Ile 20 25 30Pro Phe Tyr Gln Lys His Phe
Gly Met Glu Leu Leu Asn Lys Leu Asp 35 40 45Phe Pro Glu Met Lys Phe
Ser Leu Phe Phe Leu Ser Phe Pro Lys Asp 50 55 60Asn Val Ala Lys Asn
Ser Glu Gly Lys Asn Asp Val Phe Ser Thr Ser65 70 75 80Gly Ile Leu
Glu Leu Thr His Asn Trp Gly Ser Glu Asn Asp Ala Asp 85 90 95Phe Lys
Ile Cys Asn Gly Asn Glu Glu Pro His Arg Gly Phe Gly His 100 105
110Ile Cys Phe Ser Tyr Ala Asp Ile Asn Ala Ala Cys Ser Lys Leu Glu
115 120 125Ala Glu Gly Val Ser Phe Lys Lys Arg Leu Thr Asp Gly Arg
Met Lys 130 135 140Asp Ile Ala Phe Ala Leu Asp Pro Asp Gly Tyr Trp
Ile Glu Leu Ile145 150 155 160Arg Tyr Asp Arg Glu Asn Ser Pro Lys
Lys Asp Val Gly Ser Arg Phe 165 170 175Asn His Thr Met Val Arg Val
Lys Asp Pro Lys Ala Ser Leu Glu Phe 180 185 190Tyr Gln Asn Val Leu
Gly Met Lys Leu Leu Arg Thr Ser Glu His Glu 195 200 205Ala Ala Lys
Phe Thr Leu Tyr Phe Leu Gly Tyr Lys Val Ser Ser Glu 210 215 220Asp
Asn Glu Phe Ser His Glu Gly Val Leu Glu Leu Thr His Asn Trp225 230
235 240Gly Thr Glu Asn Glu Ala Asp Phe Lys Tyr His Asn Gly Asn Asp
Lys 245 250 255Pro Gln Gly Tyr Gly His Ile Cys Val Ser Cys Lys Asp
Pro Ala Lys 260 265 270Leu Cys Asn Glu Ile Glu Gln Thr Tyr Gly Asp
Lys Ile Gln Trp Ala 275 280 285Pro Lys Phe Asn Gln Gly Lys Leu Lys
Asn Ile Ala Phe Leu Lys Asp 290 295 300Pro Asp Gly Tyr Ser Ile Glu
Val Val Pro His Gly Leu Ile Val305 310 31522347PRTZygosaccharomyces
rouxii 22Met Phe Ser Arg Val Phe Ser Arg Leu Gly Leu Ile Lys Gln
His Ile1 5 10 15Arg Thr Met Ser Thr Lys Thr Ser Glu Ala Gln Tyr Tyr
Thr Lys Lys 20 25 30Ile Ala Ser Ala Val Gly Asp Pro Ser Leu Arg Phe
Asn His Thr Cys 35 40 45Leu Arg Ile Lys Asp Pro Ser Ala Ser Val Glu
Phe Tyr Lys Lys His 50 55 60Phe Asn Met Thr Leu Leu Ser Lys Lys Asp
Phe Pro Asp Met Lys Phe65 70 75 80Ser Leu Tyr Phe Leu Val Met Thr
Lys Glu Asn Leu Pro Lys Asn Glu 85 90 95Lys Gly Glu Asn Leu Val Phe
Ala Asn Arg Gly Ile Leu Glu Leu Thr 100 105 110His Asn Trp Gly Thr
Glu Ala Asp Pro Glu Tyr Lys Val Asn Asn Gly 115 120 125Asn Val Glu
Pro His Arg Gly Phe Gly His Ile Cys Phe Ser Val Ala 130 135 140Asn
Val Glu Ser Thr Cys Gln Arg Leu Glu Ser Glu Gly Val Lys Phe145 150
155 160Gln Lys Arg Leu Val Asp Gly Arg Gln Lys Asn Ile Ala Phe Ala
Leu 165 170 175Asp Pro Asp Gly Tyr Trp Ile Glu Leu Ile Gln Tyr Ile
Asn Glu Ser 180 185 190Gly Glu Gly Pro Lys Thr Asp Leu Gly Asn Arg
Phe Asn His Thr Met 195 200 205Val Arg Val Lys Asp Pro Val Lys Ser
Leu Glu Phe Tyr Gln Asn Val 210 215 220Leu Gly Met Thr Leu His Arg
Val Ser Glu His Ala Asn Ala Lys Phe225 230 235 240Thr Leu Tyr Phe
Leu Gly Tyr Asp Ile Pro Gln Gly Asp Ser Thr Gly 245 250 255Ser Ala
Glu Thr Leu Leu Glu Leu Thr His Asn Trp Gly Thr Glu Asn 260 265
270Asp Pro Asp Phe His Tyr His Asn Gly Asn Ala Gln Pro Gln Gly Tyr
275 280 285Gly His Ile Cys Ile Thr Cys Lys Asp Pro Gly Ala Leu Cys
Glu Glu 290 295 300Ile Glu Lys Lys Tyr Asn Glu Gln Val Val Trp Ser
Pro Lys Trp Asn305 310 315 320His Gly Lys Met Lys Asn Leu Ala Phe
Ile Lys Asp Pro Asp Gly Tyr 325 330 335Ser Ile Glu Ile Val Pro Ala
Glu Leu Val Leu 340 34523338PRTKluyveromyces lactis 23Met Phe Lys
Glu Arg Leu Leu Asn Ile Leu Lys His Ile Arg Pro Met1 5 10 15Ser Thr
Glu Thr Thr Ala Lys Tyr Tyr Pro Lys Ile Val Glu Ser Ala 20 25 30Gln
Ala Asp Gln Ser Leu Lys Leu Asn His Thr Cys Phe Arg Val Lys 35 40
45Asp Pro Lys Val Thr Val Ala Phe Tyr Gln Glu Gln Phe Gly Met Lys
50 55 60Leu Leu Asp His Lys Lys Phe Pro Asp Met Lys Phe Asp Leu Tyr
Phe65 70 75 80Leu Ser Phe Pro Asn Lys Gln Phe Ser Asn Asn Ser Gln
Gly Ala Ile 85 90 95Asp Val Phe Arg Glu Asn Gly Ile Leu Glu Leu Thr
His Asn Tyr Gly 100 105 110Thr Glu Ser Asp Pro Ala Tyr Lys Val Asn
Asn Gly Asn Glu Glu Pro 115 120 125His Arg Gly Phe Gly His Ile Cys
Phe Ser Val Ser Asn Leu Glu Ala 130 135 140Glu Cys Glu Arg Leu Glu
Ser Asn Gly Val Lys Phe Lys Lys Arg Leu145 150 155 160Thr Asp Gly
Ser Gln Arg Asn Ile Ala Phe Ala Leu Asp Pro Asn Gly 165 170 175Tyr
Trp Ile Glu Leu Ile Gln Asn Asn Glu Ser Gly Glu Gly Asn Asn 180 185
190Tyr Lys Phe Asn His Thr Met Val Arg Val Lys Asp Pro Ile Lys Ser
195 200 205Leu Glu Phe Tyr Gln Asn Val Leu Gly Met Lys Ile Leu Asp
Val Ser 210 215 220Asp His Ser Asn Ala Lys Phe Thr Leu Tyr Phe Leu
Gly Tyr Glu Asn225 230 235 240Asp Gln Lys Gly Ile Ala Arg Gly Ser
Arg Glu Ser Ile Leu Glu Leu 245 250 255Thr His Asn Trp Gly Thr Glu
Asn Asp Pro Asp Phe Ala Tyr His Thr 260 265 270Gly Asn Thr Glu Pro
Gln Gly Tyr Gly His Ile Cys Ile Ser Asn Lys 275 280 285Asp Pro Ala
Thr Leu Cys Ala Glu Ile Glu Lys Leu Tyr Pro Asp Ile 290 295 300Gln
Trp Ser Pro Lys Phe Asn Gln Gly Lys Met Lys Asn Leu Ala Phe305 310
315 320Ile Lys Asp Pro Asp Gly Tyr Ser Ile Glu Val Val Pro Tyr Gly
Leu 325 330 335Gly Val24315PRTCandida magnoliae 24Met Leu Gly Lys
Val Ala Gln Lys Phe Leu Asn His Thr Cys Ile Arg1 5 10 15Ile Ala Asp
Pro Ala Arg Ser Leu Ala Phe Tyr Glu Lys Asn Phe Gly 20 25 30Met Lys
Leu Val Thr Gln Leu Asp Val Lys Glu Val Gly Phe Thr Leu 35 40 45Tyr
Tyr Leu Gly Phe Thr Gly Pro Lys Ser Leu Tyr Lys Asp Thr Pro 50 55
60Trp Tyr Lys Arg Gly Gly Leu Leu Glu Leu Thr His Asn His Gly Ala65
70 75 80Thr Pro Glu Asn Phe Glu Ala Asn Asn Gly Asn Lys Glu Pro His
Arg 85 90 95Gly Phe Gly His Ile Cys Phe Ser Val Ser Asp Leu Glu Lys
Thr Cys 100 105 110Glu Lys Leu Glu Gly Asn Gly Val Gly Phe Gln Lys
Arg Leu Thr Asp 115 120 125Gly Arg Gln Lys Asn Ile Ala Phe Ala Leu
Asp Pro Asp Gly Tyr Trp 130 135 140Ile Glu Leu Ile Arg Asn Gly Asn
Glu Gly Ala Glu Ser Pro Glu Thr145 150 155 160Cys Thr Thr Arg Phe
Asn His Ser Met Ile Arg Val Lys Asp Lys Asp 165 170 175Ala Ala Leu
Asp Phe Tyr Thr Asn Lys Leu Gly Met Thr Leu Val Asp 180 185 190Thr
Ser Asp Phe Pro Glu Ala Lys Phe Thr Leu Phe Phe Leu Ser Phe 195 200
205Asp Pro Thr Ser Val Lys Glu Arg Ser Arg Gly Gly Thr Glu Gly Leu
210 215 220Ile Glu Leu Thr Tyr Asn Tyr Gly Ser Glu Gln Asp Val Asn
Leu His225 230 235 240Tyr His Asn Gly Asn Thr Asp Pro Gln Gly
Phe Gly His Phe Gly Val 245 250 255Thr Val Pro Asp Ala Lys Ala Phe
Leu Ser Glu Leu Glu Ser Lys Gly 260 265 270Val Arg Val Thr Lys Gln
Leu Thr Glu Gly Lys Met Lys Phe Met Gly 275 280 285Phe Val Ser Asp
Pro Asp Gly Tyr Leu Ile Glu Val Leu Pro Gln Arg 290 295 300Asp Phe
Pro Lys Asp Leu Phe Ser Pro Ser Leu305 310 3152532DNAArtificial
SequenceForward primer of the GLO1
ORFsource(1)..(32)/organism="Artificial Sequence" /note="Forward
primer of the GLO1 ORF" /mol_type="unassigned DNA" 25agctgcagaa
aatgtccact gatagtacac gc 322635DNAArtificial SequenceReverse primer
of the GLO1 ORFsource(1)..(35)/organism="Artificial Sequence"
/note="Reverse primer of the GLO1 ORF" /mol_type="unassigned DNA"
26aagtcgactt aggcaatcaa accatgagga acgac 3527981DNASaccharomyces
cerevisiaesource(1)..(981)/organism="Saccharomyces cerevisiae"
/mol_type="unassigned DNA" 27atgtccactg atagtacacg ctatccaatt
cagattgaga aagcctcgaa tgatccaacc 60cttctgctta atcacacatg tttaagagtc
aaggatccag caaggaccgt taagttctac 120accgaacact tcggtatgaa
gctattaagc agaaaggatt ttgaagaagc aaaatttagc 180ttgtactttt
taagctttcc aaaagacgac atacccaaaa ataagaatgg agagcctgat
240gtttttagcg cacacggtgt cttagaacta actcacaatt ggggtactga
aaaaaaccca 300gactacaaga tcaacaacgg gaatgaggaa cctcatcgtg
gatttgggca catctgtttt 360tctgtatccg atatcaataa aacctgcgaa
gagctagaat ctcagggtgt caaattcaag 420aagagactct ctgaaggaag
acagaaggac attgcgtttg ctttaggccc tgatggatac 480tggattgagt
tgatcacata ttctagagag ggtcaggaat acccaaaggg ctcagtaggt
540aacaagttca atcataccat gattcgtatt aaaaacccaa cccggtcttt
agaattctac 600cagaatgtgt tgggcatgaa attattaaga actagtgagc
acgaaagtgc aaaatttacg 660ttatactttc ttggttatgg cgttccaaag
accgacagcg ttttttcatg tgaaagtgtg 720ttggagttaa ctcataattg
gggaactgag aatgatccaa acttccacta tcataacggt 780aactcagagc
cccagggtta tggtcacatc tgcataagtt gtgatgacgc tggcgccctt
840tgtaaagaaa ttgaagtgaa atacggcgat aagatccaat ggtctcctaa
atttaaccaa 900ggcagaatga agaatattgc ctttttgaag gatcctgatg
gttattccat tgaagtcgtt 960cctcatggtt tgattgccta a
9812822DNAArtificial SequenceForward primer homologous GLO1
expression cassettessource(1)..(22)/organism="Artificial Sequence"
/note="Forward primer homologous GLO1 expression cassettes"
/mol_type="unassigned DNA" 28aagcgacttc caatcgcttt gc
222921DNAArtificial SequenceReverse primer homologous GLO1
expression cassettessource(1)..(21)/organism="Artificial Sequence"
/note="Reverse primer homologous GLO1 expression cassettes"
/mol_type="unassigned DNA" 29aaagcaaagg aaggagagaa c
213070DNAArtificial SequenceForward primer HIS3 cassette,
homologous GLO1 expression
cassettessource(1)..(70)/organism="Artificial Sequence"
/note="Forward primer HIS3 cassette, homologous GLO1 expression
cassettes" /mol_type="unassigned DNA" 30ttgcccatcg aacgtacaag
tactcctctg ttctctcctt cctttgcttt taactatgcg 60gcatcagagc
703171DNAArtificial SequenceReverse primer HIS3 cassette, 3a flank
INT1source(1)..(71)/organism="Artificial Sequence" /note="Reverse
primer HIS3 cassette, 3 flank INT1" /mol_type="unassigned DNA"
31acttagtatg gtctgttgga aaggattgtg gcttcgcata caggctttct tcctgatgcg
60gtattttctc c 713273DNAArtificial SequenceReverse primer INT1 5a
flank, homologous GLO1 expression
cassettessource(1)..(73)/organism="Artificial Sequence"
/note="Reverse primer INT1 5 flank, homologous GLO1 expression
cassettes" /mol_type="unassigned DNA" 32aaacgcctgt gggtgtggta
ctggatatgc aaagcgattg gaagtcgctt agggtttcaa 60agatccatac ttc
733374DNAArtificial SequenceForward primer INT1 3a flank, HIS3
cassettesource(1)..(74)/organism="Artificial Sequence"
/note="Forward primer INT1 3 flank, HIS3 cassette"
/mol_type="unassigned DNA" 33agaaagcctg tatgcgaagc cacaatcctt
tccaacagac catactaagt gaatgacctg 60ttcccgacac tatg
7434981DNAArtificial SequenceGLO1 S. cerevisiae codon-pair
optimized sequencesource(1)..(981)/organism="Artificial Sequence"
/note="GLO1 S. cerevisiae codon-pair optimized sequence"
/mol_type="unassigned DNA" 34atgtccactg actctaccag atacccaatt
caaattgaaa aggcctccaa cgacccaact 60ctattattga accacacctg tttgagagtc
aaggacccag ctagaactgt caaattctac 120actgaacact tcggtatgaa
attgttgtcc agaaaggact ttgaagaagc taagttctct 180ttgtacttct
tgtctttccc aaaggatgac attccaaaga acaagaacgg tgaaccagat
240gttttctctg ctcacggtgt cttggaatta acccacaact ggggcactga
aaagaaccca 300gactacaaga tcaacaacgg taacgaagaa cctcaccgtg
gtttcggtca tatctgtttc 360tctgtttctg acatcaacaa gacctgtgaa
gaattggaat ctcaaggtgt caagttcaag 420aagagattat ctgaaggtcg
tcaaaaggat atcgcctttg ctttgggtcc agatggttac 480tggattgaat
tgatcaccta ctccagagaa ggtcaagaat acccaaaggg ttccgttggt
540aacaaattca accacaccat gatcagaatc aagaacccaa ccagatcttt
ggaattctac 600caaaacgttt tgggtatgaa gttgttgaga acttctgaac
acgaatctgc caagttcact 660ttatacttct tgggttacgg tgttccaaag
accgattctg tcttttcctg tgaatccgtt 720ttggaattga cccataactg
gggtactgaa aatgacccaa acttccacta ccacaatggt 780aactctgaac
ctcaaggtta cggtcacatc tgtatctctt gtgatgatgc tggtgctttg
840tgtaaggaaa ttgaagtcaa atacggtgac aagattcaat ggtccccaaa
gttcaaccaa 900ggtagaatga agaacattgc tttcttgaaa gacccagacg
gttactccat cgaagttgtt 960ccacacggtt tgatcgctta a
98135960DNAArtificial SequenceGLO1 C. glabrata codon-pair optimized
sequencesource(1)..(960)/organism="Artificial Sequence" /note="GLO1
C. glabrata codon-pair optimized sequence" /mol_type="unassigned
DNA" 35atgtcttacc cacacaagat tgctgctgcc cacgatgacc caactttgat
gttcaaccac 60acctgtttga gaatcaagga cccagctaag tccattccat tttaccaaaa
gcatttcggt 120atggaattgt tgaacaaatt ggacttccca gaaatgaaat
tctccttgtt tttcttatct 180ttcccaaagg acaacgttgc caagaactct
gaaggtaaga acgatgtctt ctccacttct 240ggtatcttgg aattgaccca
caactggggt tctgaaaacg atgctgattt caagatctgt 300aacggtaacg
aagaaccaca ccgtggtttc ggtcacatct gtttctctta cgctgatatc
360aacgctgctt gttccaaatt ggaagctgaa ggtgtttctt tcaagaagag
attgaccgat 420ggtagaatga aggatatcgc ctttgctttg gacccagacg
gttactggat cgaattaatc 480agatacgaca gagaaaactc tccaaagaag
gacgtcggtt ccagattcaa ccataccatg 540gtcagagtta aggacccaaa
ggcttctttg gaattctacc aaaacgttct aggtatgaaa 600ttgttgagaa
cctctgaaca cgaagctgcc aagttcactt tgtacttctt aggttacaag
660gtttcctctg aagacaacga attctctcac gaaggtgtct tagaattgac
tcataactgg 720ggtactgaaa atgaagctga cttcaaatac cacaacggta
atgacaagcc acaaggttac 780ggtcacattt gtgtctcctg taaggatcca
gccaagttgt gtaacgaaat tgaacaaacc 840tacggtgaca agatccaatg
ggctccaaag ttcaaccaag gtaaattgaa gaacattgct 900ttcttgaagg
accctgatgg ttactccatc gaagttgttc cacacggttt gattgtataa
96036948DNAArtificial SequenceGLO1 C. magnoliae codon-pair
optimized sequencesource(1)..(948)/organism="Artificial Sequence"
/note="GLO1 C. magnoliae codon-pair optimized sequence"
/mol_type="unassigned DNA" 36atgttgggta aggttgctca aaagttcttg
aaccacacct gtatcagaat tgctgaccca 60gctcgttctt tggctttcta cgaaaagaac
tttggtatga aattggttac tcaattggat 120gtcaaggaag tcggtttcac
tttgtactac ttgggtttca ccggtccaaa gtctttatac 180aaggacactc
catggtacaa gagaggtggt ctattggaat tgactcacaa ccatggtgcc
240actccagaaa actttgaagc taacaatggt aacaaggaac cacacagagg
ttttggtcac 300atctgtttct ccgtttctga tttggaaaag acctgtgaaa
aattggaagg taacggtgtc 360ggtttccaaa agagattgac cgatggtaga
caaaagaaca ttgcctttgc tttggaccca 420gacggttact ggattgaatt
gatcagaaac ggtaacgaag gtgctgaatc tccagaaacc 480tgtaccacca
gattcaacca ctccatgatc agagttaagg acaaggacgc tgctttggat
540ttctacacca acaaattggg tatgactttg gttgacacct ctgacttccc
agaagccaag 600ttcactttat tcttcttgtc tttcgaccca acttccgtca
aggaaagatc cagaggtggt 660actgaaggtt taatcgaatt gacctacaac
tacggttctg aacaagatgt caacttgcac 720taccacaacg gtaacactga
cccacaaggt ttcggtcact tcggtgtcac cgttccagat 780gctaaggctt
tcttgtccga attggaatcc aagggtgtcc gtgtcaccaa gcaattgact
840gaaggtaaga tgaaattcat gggtttcgtt tctgacccag atggttactt
gattgaagtt 900ttgcctcaaa gagatttccc aaaggactta ttctctccat cgctctaa
948371017DNAArtificial SequenceGLO1 K. lactis codon-pair optimized
sequencesource(1)..(1017)/organism="Artificial Sequence"
/note="GLO1 K. lactis codon-pair optimized sequence"
/mol_type="unassigned DNA" 37atgttcaagg aaagattatt gaacatcttg
aagcacatca gaccaatgtc tactgaaacc 60accgccaagt actacccaaa gattgtcgaa
tctgctcaag ctgaccaatc tttgaaatta 120aaccatacct gtttcagagt
caaggatcca aaggttaccg ttgctttcta ccaagaacaa 180tttggtatga
aattgttgga ccacaagaag ttcccagaca tgaagttcga cttgtacttc
240ttgtctttcc caaacaagca attctccaac aactctcaag gtgccattga
tgttttcaga 300gaaaacggta tcttggaatt gacccacaac tacggtactg
aatctgaccc agcttacaag 360gtcaacaacg gtaacgaaga accacaccgt
ggtttcggtc acatctgttt ctccgtttcc 420aacttggaag ctgaatgtga
aagattggaa tccaacggtg tcaagttcaa gaagagattg 480accgatggtt
ctcaaagaaa cattgctttc gctttggacc caaacggtta ctggattgaa
540ttgatccaaa acaacgaatc tggtgaaggt aacaactaca aattcaacca
caccatggtt 600cgtgttaagg acccaatcaa atctttggaa ttctaccaaa
acgtcttggg tatgaagatc 660ttggatgtct ctgaccattc caatgctaag
ttcactttat acttcttggg ttacgaaaat 720gaccaaaagg gtattgccag
aggttccaga gaatccatct tagaattgac tcacaactgg 780ggtactgaaa
acgacccaga ttttgcttac cacaccggta acactgaacc tcaaggttac
840ggtcacattt gtatctctaa caaagaccca gctactctat gtgctgaaat
tgaaaagttg 900tacccagata tccaatggtc cccaaagttc aaccaaggta
agatgaagaa cttggccttt 960atcaaggacc cagatggtta ctctattgaa
gttgttccat acggtttagg ggtgtaa 1017381044DNAArtificial SequenceGLO1
Z. rouxii codon-pair optimized
sequencesource(1)..(1044)/organism="Artificial Sequence"
/note="GLO1 Z. rouxii codon-pair optimized sequence"
/mol_type="unassigned DNA" 38atgttctccc gtgttttctc cagattaggt
ttgatcaagc aacacatcag aaccatgtcc 60accaagactt ctgaagccca atactacacc
aagaagattg cttctgctgt cggtgaccca 120tctttgcgtt tcaaccacac
ctgtttgaga atcaaggacc catctgcttc cgtcgaattc 180tacaagaagc
acttcaacat gactttgttg tccaagaagg acttcccaga catgaaattc
240tctctatact ttttggttat gaccaaggaa aacttgccaa agaacgaaaa
gggtgaaaac 300ttggtttttg ctaacagagg tatcttggaa ttaacccaca
actggggcac tgaagctgac 360ccagaataca aggtcaacaa tggtaacgtt
gaacctcaca gaggtttcgg tcacatttgt 420ttctctgttg ctaacgtcga
atccacttgt caaagattgg aatctgaagg tgttaagttc 480caaaagagat
tagtcgatgg tagacaaaag aacattgcct ttgctttgga cccagatggt
540tactggatcg aattgattca atacatcaac gaatctggtg aaggtccaaa
gaccgatttg 600ggtaacagat tcaaccatac catggttaga gtcaaggatc
cagttaagtc tttggaattc 660taccaaaatg tcttaggtat gactttgcac
agagtttctg aacacgctaa cgccaaattc 720actttgtact tcttgggtta
cgatatccca caaggtgact ctactggttc tgctgaaact 780ttgttggaat
tgacccataa ctggggtact gaaaacgacc cagatttcca ctaccacaac
840ggtaacgccc aaccacaagg ttacggtcac atctgtatca cctgtaagga
cccaggtgct 900ttgtgtgaag aaattgaaaa gaaatacaac gaacaagttg
tctggtcccc aaaatggaac 960cacggtaaga tgaagaactt ggctttcatc
aaagatccag acggttactc cattgaaatt 1020gttccagctg aattggtctt ataa
1044
* * * * *
References