U.S. patent application number 14/510582 was filed with the patent office on 2015-01-22 for methods for improving malic acid production in filamentous fungi.
The applicant listed for this patent is Novozymes, Inc.. Invention is credited to Alan Berry, Stephen Brown, Sheryl Luttringer, Debbie Yaver.
Application Number | 20150024446 14/510582 |
Document ID | / |
Family ID | 43017152 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024446 |
Kind Code |
A1 |
Brown; Stephen ; et
al. |
January 22, 2015 |
Methods for improving malic acid production in filamentous
fungi
Abstract
The present invention relates to methods of producing a C4
dicarboxylic acid, comprising: (a) cultivating a filamentous fungal
host cell comprising a polynucleotide selected from the group
consisting of a heterologous first polynucleotide encoding a C4
dicarboxylic acid transporter, a heterologous second polynucleotide
encoding a malate dehydrogenase, and a heterologous third
polynucleotide encoding a pyruvate carboxylase; wherein the
filamentous fungal host cell is capable of secreting increased
levels of the C4 dicarboxylic acid compared to the filamentous
fungal host cell without the heterologous polynucleotide when
cultivated under the same conditions; and (b) recovering the C4
dicarboxylic acid. The present invention also relates to methods
for increasing C4 dicarboxylic acid production, filamentous fungal
host cells and malate dehydrogenase variants.
Inventors: |
Brown; Stephen; (Davis,
CA) ; Luttringer; Sheryl; (Loomis, CA) ;
Yaver; Debbie; (Davis, CA) ; Berry; Alan;
(Granite Bay, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes, Inc. |
Davis |
CA |
US |
|
|
Family ID: |
43017152 |
Appl. No.: |
14/510582 |
Filed: |
October 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14269591 |
May 5, 2014 |
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14510582 |
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12870523 |
Aug 27, 2010 |
8741611 |
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14269591 |
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61238962 |
Sep 1, 2009 |
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61327224 |
Apr 23, 2010 |
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61356971 |
Jun 21, 2010 |
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Current U.S.
Class: |
435/145 ;
435/254.3 |
Current CPC
Class: |
C12N 15/80 20130101;
C12P 7/46 20130101; C07K 14/38 20130101 |
Class at
Publication: |
435/145 ;
435/254.3 |
International
Class: |
C12P 7/46 20060101
C12P007/46; C12N 15/80 20060101 C12N015/80 |
Claims
1. An Aspergillus oryzae host cell comprising a heterologous
polynucleotide encoding a malic acid transporter, wherein the malic
acid transporter is selected from: (a) a malic acid transporter
comprising an amino acid sequence having at least 95% sequence
identity with SEQ ID NO: 36; (b) a malic acid transporter encoded
by a polynucleotide that hybridizes under very high stringency
conditions with the full-length complementary strand to SEQ ID NO:
35 or SEQ ID NO: 37; and (c) a malic acid transporter encoded by a
polynucleotide comprising a nucleotide sequence having at least 95%
sequence identity with SEQ ID NO: 35 or SEQ ID NO: 37; and wherein
the host cell is capable of secreting increased levels of malic
acid compared to the host cell without the heterologous
polynucleotide encoding the malic acid transporter when cultivated
under the same conditions.
2. The host cell of claim 1, wherein the malic acid transporter
comprises an amino acid sequence having at least 95% sequence
identity sequence identity with SEQ ID NO: 36.
3. The host cell of claim 1, wherein the malic acid transporter
comprises an amino acid sequence having at least 97% sequence
identity with SEQ ID NO: 36.
4. The host cell of claim 1, wherein the malic acid transporter
comprises an amino acid sequence having at least 98% sequence
identity with SEQ ID NO: 36.
5. The host cell of claim 1, wherein the malic acid transporter
comprises an amino acid sequence having at least 99% sequence
identity with SEQ ID NO: 36.
6. The host cell of claim 1, wherein the malic acid transporter is
encoded by a polynucleotide that hybridizes under very high
stringency conditions with the full-length complementary strand to
SEQ ID NO: 35 or SEQ ID NO: 37.
7. The host cell of claim 1, wherein the malic acid transporter is
encoded by a polynucleotide comprising a nucleotide sequence having
at least 95% sequence identity with SEQ ID NO: 35 or SEQ ID NO:
37.
8. The host cell of claim 1, wherein the malic acid transporter
comprises or consists of SEQ ID NO: 36.
9. The host cell of claim 1, wherein the heterologous
polynucleotide encoding the malic acid transporter is operably
linked to a promoter foreign to the polynucleotide encoding the
malic acid transporter.
10. The host cell of claim 1, further comprising a heterologous
polynucleotide encoding a malate dehydrogenase.
11. The host cell of claim 10, wherein the malate dehydrogenase
comprises or consists of SEQ ID NO: 18 or SEQ ID NO: 20.
12. The host cell of claim 1, further comprising a heterologous
polynucleotide encoding a pyruvate carboxylase.
13. The host cell of claim 12, wherein the pyruvate carboxylase
comprises or consists of SEQ ID NO: 27.
14. The host cell of claim 1, wherein host cell is capable of
secreting at least 50% more malic acid compared to the filamentous
fungal host cell without the polynucleotide encoding the malic acid
transporter when cultivated under the same conditions.
15. The host cell of claim 1, wherein the host cell is capable of
producing malic acid at a level greater than 120 g/L.
16. A method of producing malic acid, comprising: (a) cultivating
the host cell of claim 1 in a medium; and (b) recovering the malic
acid.
17. An Aspergillus oryzae host cell comprising a heterologous
polynucleotide encoding a malic acid transporter, wherein the malic
acid transporter differs by no more than ten amino acids from SEQ
ID NO: 36; and wherein the host cell is capable of secreting more
malic acid compared to the host cell without the heterologous
polynucleotide encoding the malic acid transporter when cultivated
under the same conditions.
18. The host cell of claim 17, wherein the malic acid transporter
differs by conservative substitution(s) of SEQ ID NO: 36.
19. The host cell of claim 17, wherein the malic acid transporter
differs by no more than five amino acids from SEQ ID NO: 36.
20. The host cell of claim 19, wherein the malic acid transporter
differs by conservative substitution(s) of SEQ ID NO: 36.
21. The host cell of claim 17, wherein the cell is capable of
secreting at least 50% more malic acid compared to the host cell
without the heterologous polynucleotide encoding the malic acid
transporter when cultivated under the same conditions.
22. A method of producing malic acid, comprising: (a) cultivating
the host cell of claim 20 in a medium; and (b) recovering the malic
acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/269,591, filed May 5, 2014, which is a divisional of
U.S. patent application Ser. No. 12/870,523, filed Aug. 27, 2010,
now U.S. Pat. No. 8,741,611, which claims priority benefit of U.S.
Provisional Application No. 61/238,962, filed Sep. 1, 2009; U.S.
Provisional Application No. 61/327,224, filed Apr. 23, 2010; and
U.S. Application No. 61/356,971, filed Jun. 21, 2010. The entire
contents of those applications are hereby incorporated by reference
herein.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods for improving C4
dicarboxylic acid production in filamentous fungi.
[0005] 2. Description of the Related Art
[0006] Organic acids have a long history of commercial use in a
variety of industries. For example, organic acids are used in the
food and feed industries (citric acid, ascorbic acid, lactic acid,
acetic acid, and gluconic acid) as monomers for the production of
various polymers (adipic acid, lactic acid, acrylic acid, and
itaconic acid), as metal chelators (gluconic acid), and as "green"
solvents (acetic acid) (Sauer et al., 2008, Trends in Biotechnology
26: 100-108). Organic acids may themselves be commercial products
or they may be chemical building blocks used in the manufacture of
other chemicals. In addition to specialty applications, it has long
been recognized that C4 dicarboxylic acids can also serve as
building block compounds for the production of large volume
industrial chemicals, such as 1,4-butanediol, tetrahydrofuran, and
gamma-butyrolactone. The cost of producing these large volume
industrial chemicals by traditional petrochemical routes has
increased significantly due to the high cost of petroleum derived
building blocks.
[0007] Organic acids are produced commercially either by chemical
synthesis from petroleum derived feedstocks (e.g., fumaric acid,
malic acid, acrylic acid, and adipic acid) or by microbial
fermentation (e.g., citric acid, lactic acid, gluconic acid, and
itaconic acid).
[0008] Some organic acids such as fumaric acid and malic acid can
also be produced by microbial fermentation, but are currently
produced commercially by chemical synthesis from petrochemical
feedstocks due to lower production costs. However, the rising cost
of petroleum derived building block chemicals, the geopolitical
instability affecting crude oil prices, and the desire to implement
manufacturing processes that utilize feedstocks derived from
renewable resources have stimulated a renewed interest in producing
organic acids and other chemicals by microbial fermentation.
[0009] While malic acid is produced commercially today by chemical
synthesis from petrochemical feedstocks, it can also be produced by
microbial fermentation. Malic acid has been produced at high levels
in genetically engineered yeast (Saccharomyces cerevisiae) (Zelle
et al., 2008, Appl. Environ. Microbiol. 74: 2766-2777) and
naturally occurring filamentous fungi such as Aspergillus spp.
(U.S. Pat. No. 3,063,910; Bercovitz et al., 1990, Appl. Environ.
Microbiol. 56: 1594-1597). Abe et al. (U.S. Pat. No. 3,063,910) and
Bercovitz et al. (1990, Appl. Environ. Microbiol. 56: 1594-1597)
reported high levels of malic acid production in several species of
Aspergillus. Moreover, Battat et al. (1991, Biotechnol.
Bioengineering 37: 1108-1116) reported malic acid production as
high as 113 g/L by Aspergillus flavus in a stirred fermentor under
optimized conditions. Dicarboxylic acid production by microbial
fermentation in yeast is described in WO 2010/003728. Malic acid
production by microbial fermentation is also described in WO
2009/011974 and WO 2009/155382. Improvement of malic acid
production by genetic engineering of Aspergillus will enable
economical commercial malic acid production by fermentation.
[0010] Malic acid overproduction in Aspergillus spp. occurs under
specific culture conditions (aerobic conditions and high C:N ratio;
calcium carbonate is also added as a neutralizing agent and as
source of CO.sub.2 for malic acid biosynthesis). Under these
conditions, overflow metabolism via the cytosolic, reductive
tricarboxylic acid (TCA) cycle results in increased malic acid
biosynthesis and secretion into the culture medium. Increased malic
acid production has been reported in Saccharomyces cerevisiae by
increasing the level of pyruvate carboxylase (Bauer et al., 1999,
FEMS Microbiol Lett. 179: 107-113) or malate dehydrogenase (Pines
et al., 1997, Appl. Microbiol. Biotechnol. 48: 248-255) using
genetic engineering and increasing expression of a malic acid
transporter (Zelle et al., 2008, supra. It has been suggested,
based on biochemical evidence, that malate dehydrogenase activity
is limiting malic acid production in Aspergillus flavus strain ATCC
13697 (Peleg et al., 1988, Appl. Microbiol. Biotechnol. 28: 69-75).
However, no direct improvement in malic acid production has been
shown in Aspergillus as a result of genetic engineering using
recombinant DNA techniques.
[0011] The present invention relates to methods for improving C4
dicarboxylic acid production, such as malic acid production, in
filamentous fungi.
SUMMARY OF THE INVENTION
[0012] The present invention relates to methods of producing a C4
dicarboxylic acid (e.g., malic acid), comprising: (a) cultivating a
filamentous fungal host cell comprising one or more (several)
polynucleotides selected from the group consisting of a
heterologous first polynucleotide encoding a C4 dicarboxylic acid
transporter, a heterologous second polynucleotide encoding a malate
dehydrogenase, and a heterologous third polynucleotide encoding a
pyruvate carboxylase; wherein the filamentous fungal host cell
secretes (or is capable of secreting) increased levels of the C4
dicarboxylic acid compared to the filamentous fungal host cell
without the one or more (several) heterologous polynucleotides
encoding the C4 dicarboxylic acid transporter, the malate
dehydrogenase, and the pyruvate carboxylase when cultivated under
the same conditions; and (b) recovering the C4 dicarboxylic
acid.
[0013] The present invention also relates to methods for increasing
C4 dicarboxylic acid production (e.g., malic acid production)
relative to a parent host cell, comprising: (a) transforming into a
filamentous fungal host cell one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase, wherein the filamentous fungal host cell secretes (or
is capable of secreting) increased levels of the C4 dicarboxylic
acid compared to the filamentous fungal host cell without the one
or more (several) heterologous polynucleotides encoding the C4
dicarboxylic acid transporter, the malate dehydrogenase, and the
pyruvate carboxylase when cultivated under the same conditions; (b)
cultivating the transformed filamentous fungal host cell in a
medium; and (c) recovering the C4 dicarboxylic acid.
[0014] The present invention also relates to filamentous fungal
host cells, comprising one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase, wherein the filamentous fungal host cells secrete
increased levels of a C4 dicarboxylic acid (e.g., malic acid)
compared to the filamentous fungal host cells without the one or
more (several) heterologous polynucleotides encoding the C4
dicarboxylic acid transporter, the malate dehydrogenase, and the
pyruvate carboxylase when cultivated under the same conditions.
[0015] In one aspect, the heterologous second polynucleotide
encodes a variant of a parent malate dehydrogenase comprising (i) a
deletion at positions equivalent to positions 2 to 17 or a portion
thereof of SEQ ID NO: 18, and (ii) a substitution at a position
equivalent to position 48 of SEQ ID NO: 18; wherein the deletion
and the substitution reduce mitochondrial import in vivo of the
malate dehydrogenase variant thereby increasing the level of the
malate dehydrogenase variant in the cytosol, and wherein the
filamentous fungal host cell secretes (or is capable of secreting)
increased levels of the malic acid compared to the filamentous
fungal host cell without the polynucleotide encoding the malate
dehydrogenase variant when cultivated under the same
conditions.
[0016] The present invention also relates to variants of a parent
malate dehydrogenase comprising (i) a deletion at positions
equivalent to positions 2 to 17 or a portion thereof of SEQ ID NO:
18, and (ii) a substitution at a position equivalent to position 48
of SEQ ID NO: 18, wherein the variant has malate dehydrogenase
activity.
[0017] The present invention also relates to isolated
polynucleotides encoding the malate dehydrogenase variants, and
nucleic acid constructs, vectors, and host cells comprising the
polynucleotides.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows a restriction map of pShTh60.
[0019] FIG. 2 shows a restriction map of pShTh104.
[0020] FIG. 3 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus oryzae NRRL 3488 C4 dicarboxylic
acid transporter gene (mae3) (SEQ ID NOs: 7 and 8,
respectively).
[0021] FIG. 4 shows a restriction map of pShTh73.
[0022] FIG. 5 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus oryzae NRRL 3488 malate
dehydrogenase 1 gene (mdh1) (SEQ ID NOs: 17 and 18,
respectively).
[0023] FIG. 6 shows a restriction map of pShTh71.
[0024] FIG. 7 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus oryzae NRRL 3488 malate
dehydrogenase 3 gene (mdh3) (SEQ ID NOs: 19 and 20,
respectively).
[0025] FIG. 8 shows a restriction map of pSaMF21.
[0026] FIGS. 9A and 9B show the genomic DNA sequence and the
deduced amino acid sequence of an Aspergillus oryzae NRRL 3488
pyruvate carboxylase gene (pyc) (SEQ ID NOs: 26 and 27,
respectively).
[0027] FIG. 10 shows a restriction map of pRYAN1.
[0028] FIG. 11 shows a restriction map of pAtC4T.
[0029] FIG. 12 shows a restriction map of pShTh122AtC4t.
[0030] FIG. 13 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus terreus C4 dicarboxylic acid
transporter gene (atc4t) (SEQ ID NOs: 33 and 34, respectively).
[0031] FIG. 14 shows a restriction map of p0941304_sspMAE1_pMK.
[0032] FIG. 15 shows a restriction map of pSaMF27.
[0033] FIGS. 16A and 16B show the genomic codon-optimized DNA
sequence (CO), the deduced amino acid sequence, and the genomic
wild-type DNA sequence (WT) of a Schizosaccharomyces pombe C4
dicarboxylic acid transporter gene (mae1) (SEQ ID NOs: 35, 36, and
37, respectively).
DEFINITIONS
[0034] C4 dicarboxylic acid transporter: The term "C4 dicarboxylic
acid transporter" is defined herein as a dicarboxylic acid permease
that can transport malic acid, succinic acid, oxaloacetic acid,
malonic acid, and/or fumaric acid outside a cell (Grobler et al.,
1995, Yeast 11: 1485-1491; Camarasa et al., 2001, Applied and
Environmental Microbiology 67: 4144-4151). A computational method
to predict mitochondrially imported proteins and their targeting
sequences is described by Claros and Vincens, 1996, Eur. J.
Biochem. 241: 779-786.
[0035] The C4 dicarboxylic acid transporters have at least 20%,
e.g., at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% of the malic acid transporter
activity of SEQ ID NO: 8, SEQ ID NO: 34, and/or SEQ ID NO: 36.
[0036] Malate dehydrogenase: The term "malate dehydrogenase" is
defined herein as a malate:NAD.sup.+ oxidoreductase (EC 1.1.1.37)
that catalyzes the reduction of oxaloacetate in the presence of
NADH+H.sup.+ to malate and NAD.sup.+. For purposes of the present
invention, malate dehydrogenase activity is determined according to
the following procedure. The assay solution consists of 1 mM
oxaloacetic acid, 100 mM Tris pH 8.0, 10 mM NaHCO.sub.3, 5 mM
MgCl.sub.2, and 0.1 mM NADH (Sigma Chemical Co., St. Louis, Mo.,
USA). The assay solution without oxaloacetic acid as substrate is
run as a control to measure background NADH degradation rates.
Dilutions of 1/100, 1/500, 1/2500, and 1/12500 of each supernatant
are prepared with double-distilled water. Aliquots of 270 .mu.l of
the assay solution are dispensed into 96 well polystyrene flat
bottom plates. A 30 .mu.l sample of each diluted supernatant is
added to initiate the assay. The reactions are monitored using a
SPECTRAMAX.RTM. 340PC plate reader (Molecular Devices, Sunnyvale,
Calif., USA) with the following settings: 340 nm, kinetic reading.
A concentration series of NADH is used to construct a standard
curve and a dilution series of purified malic dehydrogenase (Sigma
Chemical Co., St. Louis, Mo., USA) is used as a positive control.
One unit of malate dehydrogenase activity equals the amount of
enzyme capable of converting 1 .mu.mole of oxaloacetate and
NADH+H.sup.+ to malate and NAD.sup.+ per minute at pH 8.0,
25.degree. C. The malate dehydrogenases have at least 20%, e.g., at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% of the malate dehydrogenase activity of
SEQ ID NO: 18 or SEQ ID NO: 20.
[0037] Pyruvate carboxylase: The term "pyruvate carboxylase" is
defined herein as a pyruvate:carbon-dioxide ligase (ADP-forming)
(EC 6.4.1.1) that catalyzes the carboxylation of pyruvate in the
presence of ATP and HCO.sub.3.sup.- to oxaloacetate, ADP, and
phosphate. For purposes of the present invention, pyruvate
carboxylase activity is determined according to the procedure of
the SIGMA.RTM. Quality Control Test procedure for pyruvate
carboxylase (Sigma Chemical Co., St. Louis, Mo., USA) except the
assay uses Tris buffer at pH 8.0. One unit of pyruvate carboxylase
activity equals the amount of enzyme capable of converting 1
.mu.mole of pyruvate and CO.sub.2 to oxaloacetate per minute at pH
7.8, 30.degree. C.
[0038] The pyruvate carboxylases have at least 20%, e.g., at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100% of the pyruvate carboxylase activity of SEQ
ID NO: 27.
[0039] Heterologous polynucleotide: The term "heterologous
polynucleotide" is defined herein as a polynucleotide that is not
native to the host cell; a native polynucleotide in which
structural modifications have been made to the coding region; a
native polynucleotide whose expression is quantitatively altered as
a result of a manipulation of the DNA by recombinant DNA
techniques, e.g., a different (foreign) promoter; or a native
polynucleotide whose expression is quantitatively altered by the
introduction of one or more (several) extra copies of the
polynucleotide into the host cell.
[0040] Isolated polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide that is isolated from a source.
In a preferred aspect, the polypeptide is at least 1% pure, e.g.,
at least 5% pure, at least 10% pure, at least 20% pure, at least
40% pure, at least 60% pure, at least 80% pure, at least 90% pure,
at least 95% pure, at least 96% pure, at least 97% pure, at least
98% pure, at least 99% pure, at least 99.5% pure, or 100% pure as
determined by SDS-PAGE.
[0041] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation that contains
at most 10%, e.g., at most 8%, at most 6%, at most 5%, at most 4%,
at most 3%, at most 2%, at most 1%, or at most 0.5% by weight of
other polypeptide material with which it is natively or
recombinantly associated. It is, therefore, preferred that the
substantially pure polypeptide is at least 92% pure, e.g., at least
94% pure, at least 95% pure, at least 96% pure, at least 97% pure,
at least 98% pure, at least 99% pure, at least 99.5% pure, or 100%
pure by weight of the total polypeptide material present in the
preparation. The polypeptides are preferably in a substantially
pure form, i.e., that the polypeptide preparation is essentially
free of other polypeptide material with which it is natively or
recombinantly associated. This can be accomplished, for example, by
preparing the polypeptide by well-known recombinant methods or by
classical purification methods.
[0042] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0043] For purposes of the present invention, the degree of
identity between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277),
preferably version 3.0.0 or later. The optional parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0044] For purposes of the present invention, the degree of
identity between two deoxyribonucleotide sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
supra) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, supra), preferably version 3.0.0 or later. The
optional parameters used are gap open penalty of 10, gap extension
penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)
substitution matrix. The output of Needle labeled "longest
identity" (obtained using the -nobrief option) is used as the
percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0045] Homologous sequence: The term "homologous sequence" is
defined herein as a predicted protein that gives an E value (or
expectancy score) of less than 0.001 in a tfasty search (Pearson,
W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener
and S. A. Krawetz, ed., pp. 185-219) with the Aspergillus oryzae C4
dicarboxylic acid transporter of SEQ ID NO: 8, SEQ ID NO: 34, or
SEQ ID NO: 36, the Aspergillus oryzae malate dehydrogenase of SEQ
ID NO. 18 or SEQ ID NO: 20, or the Aspergillus oryzae pyruvate
carboxylase of SEQ ID NO. 27.
[0046] Polypeptide fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more (several) amino
acids deleted from the amino and/or carboxyl terminus of SEQ ID NO:
8, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 34, or
SEQ ID NO: 36; or a homologous sequence thereof; wherein the
fragment has C4 dicarboxylic acid transporter activity (for a
fragment of SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO: 36), malate
dehydrogenase activity (for a fragment of SEQ ID NO: 18 or SEQ ID
NO: 20), or pyruvate carboxylase activity (for a fragment of SEQ ID
NO: 27).
[0047] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more (several) nucleotides
deleted from the 5' and/or 3' end of SEQ ID NO: 7, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ
ID NO: 37; or a homologous sequence thereof; wherein the
subsequence encodes a polypeptide fragment having C4 dicarboxylic
acid transporter activity (for a subsequence of SEQ ID NO: 7, SEQ
ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37), malate dehydrogenase
activity (for a subsequence of SEQ ID NO: 17 or SEQ ID NO: 19), or
pyruvate carboxylase activity (for a subsequence of SEQ ID NO:
26).
[0048] Allelic variant: The term "allelic variant" denotes herein
any of two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in polymorphism within populations. Gene
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An
allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a gene.
[0049] Isolated polynucleotide: The term "isolated polynucleotide"
as used herein refers to a polynucleotide that is isolated from a
source. In one aspect, the polynucleotide is at least 1% pure,
e.g., at least 5% pure, at least 10% pure, at least 20% pure, at
least 40% pure, at least 60% pure, at least 80% pure, at least 90%
pure, at least 95% pure, at least 96% pure, at least 97% pure, at
least 98% pure, at least 99% pure, at least 99.5% pure, or 100%
pure, as determined by agarose electrophoresis.
[0050] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, e.g., at most 8%, at most 6%, at most 5%, at
most 4%, at most 3%, at most 2%, at most 1%, or at most 0.5% by
weight of other polynucleotide material with which it is natively
or recombinantly associated. A substantially pure polynucleotide
may, however, include naturally occurring 5' and 3' untranslated
regions, such as promoters and terminators. In one aspect, the
substantially pure polynucleotide is at least 90% pure, e.g., at
least 92% pure, at least 94% pure, at least 95% pure, at least 96%
pure, at least 97% pure, at least 98% pure, at least 99% pure, at
least 99.5%, or 100% pure by weight of the total polynucleotide
material present in the preparation. The polynucleotides are
preferably in a substantially pure form, i.e., that the
polynucleotide preparation is essentially free of other
polynucleotide material with which it is natively or recombinantly
associated. The polynucleotides may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.
[0051] Coding sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG and ends with a stop codon such as TAA, TAG,
and TGA. The coding sequence may be a DNA, cDNA, synthetic, or
recombinant nucleotide sequence.
[0052] cDNA: The term "cDNA" is defined herein as a DNA molecule
that can be prepared by reverse transcription from a mature,
spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks
intron sequences that may be present in the corresponding genomic
DNA. The initial, primary RNA transcript is a precursor to mRNA
that is processed through a series of steps before appearing as
mature spliced mRNA. These steps include the removal of intron
sequences by a process called splicing. cDNA derived from mRNA
lacks, therefore, any intron sequences. In some instances, a cDNA
sequence may be identical to a genomic DNA sequence.
[0053] Nucleic acid construct: The term "nucleic acid construct" as
used herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which is modified to contain segments of nucleic acids in a
manner that would not otherwise exist in nature or which is
synthetic. The term nucleic acid construct is synonymous with the
term "expression cassette" when the nucleic acid construct contains
the control sequences required for expression of a coding
sequence.
[0054] Control sequences: The term "control sequences" is defined
herein to include all components necessary for the expression of a
polynucleotide encoding a polypeptide. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
polypeptide or native or foreign to each other. Such control
sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the nucleotide sequence encoding a
polypeptide.
[0055] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0056] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0057] Expression vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide and is operably linked to
additional nucleotides that provide for its expression.
[0058] Host cell: The term "host cell", as used herein, includes
any cell type that is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct or
expression vector comprising a polynucleotide encoding a malate
dehydrogenase, a polynucleotide encoding a pyruvate carboxylase,
and/or a polynucleotide encoding a C4 dicarboxylic acid
transporter.
[0059] Artificial variant: When used herein, the term "artificial
variant" means a polypeptide having malate dehydrogenase activity
produced by an organism expressing a modified polynucleotide
sequence (e.g., a modified polynucleotide sequence comprising or
consisting of SEQ ID NO: 17); or a homologous sequence thereof. The
modified nucleotide sequence is obtained through human intervention
by modification of the polynucleotide sequence comprising or
consisting of SEQ ID NO: 17; or a homologous sequence thereof.
[0060] Variant: The term "variant" means a polypeptide having
activity, e.g., C4 dicarboxylic acid transporter activity,
comprising an alteration/modification, i.e., a substitution,
insertion, and/or deletion of one or more (several) amino acid
residues at one or more (several) positions. A substitution means a
replacement of an amino acid occupying a position with a different
amino acid; a deletion means removal of an amino acid occupying a
position; and an insertion means adding one or more (several),
e.g., 1-3 amino acids, adjacent to an amino acid occupying a
position.
[0061] Reference to "about" a value or parameter herein includes
aspects that are directed to that value or parameter per se. For
example, description referring to "about X" includes the aspect
"X".
[0062] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise. It is understood that the
aspects of the invention described herein include "consisting"
and/or "consisting essentially of" aspects.
[0063] Unless defined otherwise or clearly indicated by context,
all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The present invention describes the overexpression of
specific genes in a filamentous fungus, e.g., Aspergillus, to
enhance C4 dicarboxylic acid production, such as malic acid
production, that encompasses carboxylation of pyruvate to
oxaloacetate by a pyruvate carboxylase, reduction of oxaloacetate
to malate by a malate dehydrogenase, and/or transport of the C4
dicarboxylic acid out of the cell via a C4 dicarboxylic acid
transporter.
[0065] The present invention relates to methods of producing C4
dicarboxylic acid (e.g., malic acid), comprising: (a) cultivating
in a medium a filamentous fungal host cell comprising one or more
(several) polynucleotides selected from the group consisting of a
heterologous first polynucleotide encoding a C4 dicarboxylic acid
transporter, a heterologous second polynucleotide encoding a malate
dehydrogenase, and a heterologous third polynucleotide encoding a
pyruvate carboxylase; wherein the filamentous fungal host cell
secretes (or is capable of secreting) increased levels of the C4
dicarboxylic acid compared to the filamentous fungal host cell
without the one or more (several) heterologous polynucleotides
encoding the C4 dicarboxylic acid transporter, the malate
dehydrogenase, and the pyruvate carboxylase when cultivated under
the same conditions; and (b) recovering the C4 dicarboxylic
acid.
[0066] The present invention also relates to methods for increasing
C4 dicarboxylic acid production (e.g., malic acid production)
relative to a parent host cell, comprising: (a) transforming into a
filamentous fungal host cell one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase, wherein the filamentous fungal host cell secretes (or
is capable of secreting) increased levels of malic acid compared to
the filamentous fungal host cell without the one or more (several)
heterologous polynucleotides encoding the C4 dicarboxylic acid
transporter, the malate dehydrogenase, and the pyruvate carboxylase
when cultivated under the same conditions; (b) cultivating the
transformed filamentous fungal host cell in a medium; and (c)
recovering the C4 dicarboxylic acid.
[0067] The present invention also relates to filamentous fungal
host cells, comprising one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase, wherein the filamentous fungal host cell secretes (or
is capable of secreting) increased levels of the C4 dicarboxylic
acid (e.g., malic acid) compared to the filamentous fungal host
cell without the one or more (several) heterologous polynucleotides
encoding the C4 dicarboxylic acid transporter, the malate
dehydrogenase, and the pyruvate carboxylase when cultivated under
the same conditions.
[0068] In any of these aspects, the C4 dicarboxylic acid is malic
acid, succinic acid, oxaloacetic acid, malonic acid, or fumaric
acid, or combinations thereof. In some aspects, the C4 dicarboxylic
acid is malic acid, succinic acid, or fumaric acid, or combinations
thereof. In some aspects, the C4 dicarboxylic acid is malic acid or
fumaric acid, or a combination of malic acid and fumaric acid. In
some aspects, the C4 dicarboxylic acid is malic acid.
[0069] In one aspect, the filamentous fungal host cell comprises a
heterologous polynucleotide encoding a C4 dicarboxylic acid
transporter. In another aspect, the filamentous fungal host cell
comprises a heterologous polynucleotide encoding a malate
dehydrogenase. In another aspect, the filamentous fungal host cell
comprises a heterologous polynucleotide encoding a pyruvate
carboxylase. In another aspect, the filamentous fungal host cell
comprises a heterologous polynucleotide encoding a C4 dicarboxylic
acid transporter and a heterologous polynucleotide encoding a
malate dehydrogenase. In another aspect, the filamentous fungal
host cell comprises a heterologous polynucleotide encoding a C4
dicarboxylic acid transporter and a heterologous polynucleotide
encoding a pyruvate carboxylase. In another aspect, the filamentous
fungal host cell comprises a heterologous polynucleotide encoding a
malate dehydrogenase and a heterologous polynucleotide encoding a
pyruvate carboxylase. In another aspect, the filamentous fungal
host cell comprises a heterologous polynucleotide encoding a C4
dicarboxylic acid transporter, a heterologous polynucleotide
encoding a malate dehydrogenase, and a heterologous polynucleotide
encoding a pyruvate carboxylase.
[0070] In the methods of the present invention, the recombinant
filamentous fungal host cell is cultivated in a nutrient medium
suitable for production of the C4 dicarboxylic acid transporter,
the malate dehydrogenase, and/or the pyruvate carboxylase using
methods well known in the art. For example, the cell may be
cultivated by shake flask cultivation, and small-scale or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermentors performed in a suitable medium and under conditions
allowing the polypeptide(s) to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures
known in the art. Suitable media are available from commercial
suppliers, may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection), or
may be prepared from commercially available ingredients.
[0071] The C4 dicarboxylic acid transporter, malate dehydrogenase,
and pyruvate carboxylase can be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the malate
dehydrogenase and the pyruvate carboxylase, as described
herein.
[0072] C4 dicarboxylic acids such as malic acid can be recovered
using any method known in the art. See, for example, WO 1998/022611
and U.S. Pat. No. 7,601,865.
[0073] In a one aspect, the level of secreted C4 dicarboxylic acid
produced by a filamentous fungal host cell comprising a
heterologous first polynucleotide encoding a C4 dicarboxylic acid
transporter, a heterologous second polynucleotide encoding a malate
dehydrogenase, and/or a heterologous third polynucleotide encoding
a pyruvate carboxylase is increased at least 25%, e.g., at least
50%, at least 100%, at least 200%, or at 500% compared to the
filamentous fungal host cell without the heterologous first
polynucleotide, heterologous second polynucleotide, and/or
heterologous third polynucleotide when cultivated under the same
conditions.
[0074] In one aspect, a cultivated preparation of the filamentous
fungal host cell produces (or is capable of producing) C4
dicarboxylic acid (e.g., malic acid) at a level greater than about
any of 30 g/L, 60 g/L, 90 g/L, 120 g/L, 150 g/L, 175 g/L, 200 g/L,
225 g/L, or 250 g/L. In another aspect, a cultivated preparation of
the filamentous fungal host cell produces (or is capable of
producing) C4 dicarboxylic acid (e.g., malic acid) at a level
greater than about any of 30 g/L, 60 g/L, 90 g/L, 120 g/L, 150 g/L,
175 g/L, 200 g/L, 225 g/L, or 250 g/L within 24, 48, 72, 96, 120,
144, 165, or 192 hours. For example, in one aspect, the preparation
produces (or is capable of producing) C4 dicarboxylic acid (e.g.,
malic acid) at a level greater than about any of 30 g/L, 60 g/L, 90
g/L, 120 g/L, 150 g/L, 175 g/L, 200 g/L, 225 g/L, or 250 g/L within
48 hours. In another aspect, the preparation produces (or is
capable of producing) C4 dicarboxylic acid (e.g., malic acid) at a
level greater than about any of 30 g/L, 60 g/L, 90 g/L, 120 g/L,
150 g/L, 175 g/L, 200 g/L, 225 g/L, or 250 g/L within 144 hours. In
any of these aspects or aspects of the methods herein, a cultivated
preparation of the filamentous fungal host cell produces (or is
capable of producing) the C4 dicarboxylic acid (e.g., malic acid)
at a pH of less than or equal to about 7.0, such as 6.0 to 7.0, 6.0
to 6.5, 6.5 to 7.0, about 6.5, or less than about 6.5.
C4 Dicarboxylic Acid Transporters and Polynucleotides Encoding the
C4 Dicarboxylic Acid Transporters
[0075] In the present invention, the C4 dicarboxylic acid
transporter can be any C4 dicarboxylic acid transporter that is
suitable for practicing the present invention. In one aspect, the
C4 dicarboxylic acid transporter is a transporter that is
overexpressed under culture conditions that produces malic acid in
high titers.
[0076] In one aspect, the C4 dicarboxylic acid transporter is (a) a
C4 dicarboxylic acid transporter comprising an amino acid sequence
having at least 60% sequence identity with SEQ ID NO: 8, SEQ ID NO:
34, and/or SEQ ID NO: 36; (b) a C4 dicarboxylic acid transporter
encoded by a polynucleotide that hybridizes under low stringency
conditions with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35 and/or
SEQ ID NO: 37; or a full-length complementary strand thereof; (c) a
C4 dicarboxylic acid transporter encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35 and/or SEQ
ID NO: 37; (d) a C4 dicarboxylic acid transporter variant
comprising a substitution, deletion, and/or insertion of one or
more (several) amino acids of the mature polypeptide of SEQ ID NO:
8, SEQ ID NO: 34, and/or SEQ ID NO: 36; or (e) a fragment of the C4
dicarboxylic acid transporter of (a), (b), (c), or (d) that has C4
dicarboxylic acid transporter activity.
[0077] In a first aspect, the C4 dicarboxylic acid transporter is
(a) a C4 dicarboxylic acid transporter comprising an amino acid
sequence having at least 60% sequence identity with SEQ ID NO: 8;
(b) a C4 dicarboxylic acid transporter encoded by a polynucleotide
that hybridizes under low stringency conditions with SEQ ID NO: 7
or the full-length complementary strand thereof; (c) a C4
dicarboxylic acid transporter encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity with SEQ ID NO: 7; (d) a C4 dicarboxylic acid transporter
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 8; or (e) a fragment of the C4 dicarboxylic acid transporter
of (a), (b), (c), or (d) that has C4 dicarboxylic acid transporter
activity.
[0078] In one aspect, the C4 dicarboxylic acid transporter
comprises an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 8 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which have C4 dicarboxylic acid transporter activity (hereinafter
"homologous C4 dicarboxylic acid transporters"). In one aspect, the
homologous C4 dicarboxylic acid transporter comprises an amino acid
sequence that differs by no more than ten amino acids, e.g., by no
more than five amino acids, by no more than four amino acids, by no
more than three amino acids, by no more than two amino acids, or by
one amino acid from SEQ ID NO: 8.
[0079] In one aspect, the C4 dicarboxylic acid transporter
comprises the amino acid sequence of SEQ ID NO: 8 or an allelic
variant thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter comprises the amino acid sequence of SEQ ID NO: 8. In
another aspect, the C4 dicarboxylic acid transporter consists of
the amino acid sequence of SEQ ID NO: 8 or an allelic variant
thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter consists of the amino acid sequence of SEQ ID NO:
8.
[0080] In one aspect, the C4 dicarboxylic acid transporter is a
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 8. Preferably, amino acid changes are of a minor nature,
that is conservative amino acid substitutions or insertions that do
not significantly affect the folding and/or activity of the
protein; small deletions, typically of one to about 30 amino acids;
small amino-terminal or carboxyl-terminal extensions, such as an
amino-terminal methionine residue; a small linker peptide of up to
about 20-25 residues; or a small extension that facilitates
purification by changing net charge or another function, such as a
poly-histidine tract, an antigenic epitope or a binding domain.
[0081] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0082] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0083] Essential amino acids in a parent polypeptide can be
identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter
technique, single alanine mutations are introduced at every residue
in the molecule, and the resultant mutant molecules are tested for
C4 dicarboxylic acid transporter activity to identify amino acid
residues that are critical to the activity of the molecule. See
also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The
active site of the enzyme or other biological interaction can also
be determined by physical analysis of structure, as determined by
such techniques as nuclear magnetic resonance, crystallography,
electron diffraction, or photoaffinity labeling, in conjunction
with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett.
309: 59-64. The identities of essential amino acids can also be
inferred from analysis of identities with polypeptides that are
related to the parent polypeptide.
[0084] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0085] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0086] In some aspects, the total number of amino acid
substitutions, deletions and/or insertions of the mature
polypeptide of SEQ ID NO: 8 is not more than 10, e.g., not more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9. In another aspect, the total
number of amino acid substitutions, deletions and/or insertions of
the mature polypeptide of SEQ ID NO: 8 is 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10.
[0087] In another aspect, the C4 dicarboxylic acid transporter is a
fragment of SEQ ID NO: 8, wherein the fragment has C4 dicarboxylic
acid transporter activity. A fragment of SEQ ID NO: 8 is a
polypeptide having one or more (several) amino acids deleted from
the amino and/or carboxyl terminus of this amino acid sequence. In
one aspect, the fragment contains at least 320 amino acid residues,
e.g., preferably at least 340 amino acid residues, or at least 360
amino acid residues of SEQ ID NO: 8.
[0088] The C4 dicarboxylic acid transporter may be a fused
polypeptide or cleavable fusion polypeptide in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide of the present invention. A fused polypeptide is
produced by fusing a polynucleotide encoding another polypeptide to
a polynucleotide of the present invention. Techniques for producing
fusion polypeptides are known in the art, and include ligating the
coding sequences encoding the polypeptides so that they are in
frame and that expression of the fused polypeptide is under control
of the same promoter(s) and terminator. Fusion proteins may also be
constructed using intein technology in which fusions are created
post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583;
Dawson et al., 1994, Science 266: 776-779).
[0089] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
[0090] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under at least very low
stringency conditions, e.g., low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with SEQ
ID NO: 7, a subsequence thereof, or a full-length complementary
strand of the foregoing (J. Sambrook, E. F. Fritsch, and T.
Maniatis, 1989, supra). The subsequence may encode a polypeptide
fragment having C4 dicarboxylic acid transporter activity. A
subsequence of SEQ ID NO: 7, or a homolog thereof, is a nucleotide
sequence where one or more (several) nucleotides have been deleted
from the 5'- and/or 3'-end. In one aspect, a subsequence contains
at least 960 nucleotides, e.g., at least 1020 nucleotides or at
least 1080 nucleotides SEQ ID NO: 7.
[0091] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising or consisting of a
nucleotide sequence having a degree of sequence identity to SEQ ID
NO: 7 of at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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%, which encode
polypeptides having C4 dicarboxylic acid transporter activity.
[0092] In a second aspect, the C4 dicarboxylic acid transporter is
(a) a C4 dicarboxylic acid transporter comprising an amino acid
sequence having at least 60% sequence identity with SEQ ID NO: 34;
(b) a C4 dicarboxylic acid transporter encoded by a polynucleotide
that hybridizes under low stringency conditions with SEQ ID NO: 33
or the full-length complementary strand thereof; (c) a C4
dicarboxylic acid transporter encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity with SEQ ID NO: 33; (d) a C4 dicarboxylic acid transporter
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 34; or (e) a fragment of the C4 dicarboxylic acid
transporter of (a), (b), (c), or (d) that has C4 dicarboxylic acid
transporter activity.
[0093] In one aspect, the C4 dicarboxylic acid transporter
comprises an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 34 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which have C4 dicarboxylic acid transporter activity (hereinafter
"homologous C4 dicarboxylic acid transporters"). In one aspect, the
homologous C4 dicarboxylic acid transporter comprises an amino acid
sequence that differs by no more than ten amino acids, e.g., by no
more than five amino acids, by no more than four amino acids, by no
more than three amino acids, by no more than two amino acids, or by
one amino acid from SEQ ID NO: 34.
[0094] In one aspect, the C4 dicarboxylic acid transporter
comprises the amino acid sequence of SEQ ID NO: 34 or an allelic
variant thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter comprises the amino acid sequence of SEQ ID NO: 34. In
another aspect, the C4 dicarboxylic acid transporter consists of
the amino acid sequence of SEQ ID NO: 34 or an allelic variant
thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter consists of the amino acid sequence of SEQ ID NO:
34.
[0095] In one aspect, the C4 dicarboxylic acid transporter is a
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 34, as described supra. In some aspects, the total number of
amino acid substitutions, deletions and/or insertions of the mature
polypeptide of SEQ ID NO: 34 is not more than 10, e.g., not more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9. In another aspect, the total
number of amino acid substitutions, deletions and/or insertions of
the mature polypeptide of SEQ ID NO: 34 is 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10.
[0096] In another aspect, the C4 dicarboxylic acid transporter is a
fragment of SEQ ID NO: 34, wherein the fragment has C4 dicarboxylic
acid transporter activity. In one aspect, a fragment contains at
least 334 amino acid residues, e.g., at least 354 amino acid
residues or at least 374 amino acid residues of SEQ ID NO: 34.
[0097] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under at least very low
stringency conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 33,
a subsequence of thereof, or a full-length complementary strand of
the foregoing, (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
supra). The subsequence may encode a polypeptide fragment having C4
dicarboxylic acid transporter activity. A subsequence of SEQ ID NO:
33, or a homolog thereof, is a nucleotide sequence where one or
more (several) nucleotides have been deleted from the 5'- and/or
3'-end. In one aspect, a subsequence contains at least 1002
nucleotides, e.g., at least 1062 nucleotides or at least 1122
nucleotides SEQ ID NO: 33.
[0098] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising or consisting of a
nucleotide sequence having a degree of sequence identity to SEQ ID
NO: 33 of at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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%, which encode
polypeptides having C4 dicarboxylic acid transporter activity.
[0099] In a third aspect, the C4 dicarboxylic acid transporter is
(a) a C4 dicarboxylic acid transporter comprising an amino acid
sequence having at least 60% sequence identity with SEQ ID NO: 36;
(b) a C4 dicarboxylic acid transporter encoded by a polynucleotide
that hybridizes under low stringency conditions with SEQ ID NO: 35
or SEQ ID NO: 37, or a full-length complementary strand thereof; or
(c) a C4 dicarboxylic acid transporter encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity with SEQ ID NO: 35 or SEQ ID NO: 37; (d) a C4 dicarboxylic
acid transporter variant comprising a substitution, deletion,
and/or insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 36; or (e) a fragment of the C4
dicarboxylic acid transporter of (a), (b), (c), or (d) that has C4
dicarboxylic acid transporter activity.
[0100] In one aspect, the C4 dicarboxylic acid transporter
comprises an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 36 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which have C4 dicarboxylic acid transporter activity (hereinafter
"homologous C4 dicarboxylic acid transporters"). In one aspect, the
homologous C4 dicarboxylic acid transporter comprises an amino acid
sequence that differs by no more than ten amino acids, e.g., by no
more than five amino acids, by no more than four amino acids, by no
more than three amino acids, by no more than two amino acids, or by
one amino acid from SEQ ID NO: 36.
[0101] In one aspect, the C4 dicarboxylic acid transporter
comprises the amino acid sequence of SEQ ID NO: 36 or an allelic
variant thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter comprises the amino acid sequence of SEQ ID NO: 36. In
another aspect, the C4 dicarboxylic acid transporter consists of
the amino acid sequence of SEQ ID NO: 36 or an allelic variant
thereof; or a fragment thereof having C4 dicarboxylic acid
transporter activity. In another aspect, the C4 dicarboxylic acid
transporter consists of the amino acid sequence of SEQ ID NO:
36.
[0102] In one aspect, the C4 dicarboxylic acid transporter is a
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 36, as described supra. In some aspects, the total number of
amino acid substitutions, deletions and/or insertions of the mature
polypeptide of SEQ ID NO: 36 is not more than 10, e.g., not more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9. In another aspect, the total
number of amino acid substitutions, deletions and/or insertions of
the mature polypeptide of SEQ ID NO: 36 is 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10.
[0103] In another aspect, the C4 dicarboxylic acid transporter is a
fragment of SEQ ID NO: 36, wherein the fragment has C4 dicarboxylic
acid transporter activity. In one aspect, a fragment contains at
least 375 amino acid residues, e.g., at least 395 amino acid
residues or at least 415 amino acid residues of SEQ ID NO: 36.
[0104] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under at least very low
stringency conditions, e.g., low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with SEQ
ID NO: 35 or SEQ ID NO: 37, a subsequence thereof, or a full-length
complementary strand of the foregoing (J. Sambrook, E. F. Fritsch,
and T. Maniatis, 1989, supra). The subsequence may encode a
polypeptide fragment having C4 dicarboxylic acid transporter
activity. A subsequence of SEQ ID NO: 35, SEQ ID NO: 37, or a
homolog thereof, is a nucleotide sequence where one or more
(several) nucleotides have been deleted from the 5'- and/or 3'-end.
In one aspect, a subsequence contains at least 1125 nucleotides,
e.g., at least 1185 nucleotides or at least 1245 nucleotides of SEQ
ID NO: 35 or SEQ ID NO: 37.
[0105] In one aspect, the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising or consisting of a
nucleotide sequence having a degree of sequence identity to SEQ ID
NO: 35 or SEQ ID NO: 37 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which encode polypeptides having C4 dicarboxylic acid transporter
activity.
[0106] The polynucleotide of SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID
NO: 35, or SEQ ID NO: 37; or a subsequence thereof; as well as the
amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO:
36; or a fragment thereof; may be used to design nucleic acid
probes to identify and clone DNA encoding C4 dicarboxylic acid
transporters from strains of different genera or species according
to methods well known in the art. In particular, such probes can be
used for hybridization with the genomic or cDNA of the genus or
species of interest, following standard Southern blotting
procedures, in order to identify and isolate the corresponding gene
therein. Such probes can be considerably shorter than the entire
sequence, but should be at least 14, e.g., at least 25, at least
35, at least 70 nucleotides in length. In one aspect, the nucleic
acid probe is at least 100 nucleotides in length, e.g., at least
200 nucleotides, at least 300 nucleotides, at least 400
nucleotides, or at least 500 nucleotides in length. Even longer
probes may be used, e.g., nucleic acid probes that are at least 600
nucleotides, at least 700 nucleotides, at least 800 nucleotides, or
at least 900 nucleotides in length. Both DNA and RNA probes can be
used. The probes are typically labeled for detecting the
corresponding gene (for example, with .sup.32P, .sup.3H, .sup.35S,
biotin, or avidin). Such probes are encompassed by the present
invention.
[0107] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a C4 dicarboxylic acid transporter.
Genomic or other DNA from such other organisms may be separated by
agarose or polyacrylamide gel electrophoresis, or other separation
techniques. DNA from the libraries or the separated DNA may be
transferred to and immobilized on nitrocellulose or other suitable
carrier material. In order to identify a clone or DNA that is
homologous with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ
ID NO: 37; or a subsequence thereof; the carrier material is used
in a Southern blot. For purposes of the present invention,
hybridization indicates that the polynucleotide hybridizes to a
labeled nucleotide probe corresponding to the polynucleotide shown
in SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37; a
complementary strand thereof; or a subsequence of the foregoing,
under low to very high stringency conditions. Molecules to which
the probe hybridizes can be detected using, for example, X-ray film
or any other detection means known in the art.
[0108] In one aspect, the nucleic acid probe is SEQ ID NO: 7, SEQ
ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or a subsequence thereof.
In another aspect, the nucleic acid probe is a polynucleotide
sequence that encodes SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO:
36; or a subsequence thereof. In one aspect, the nucleic acid probe
is SEQ ID NO: 7. In another aspect, the nucleic acid probe is a
polynucleotide sequence that encodes SEQ ID NO: 8, or a subsequence
thereof. In another aspect, the nucleic acid probe is SEQ ID NO:
33. In another aspect, the nucleic acid probe is a polynucleotide
sequence that encodes SEQ ID NO: 34, or a subsequence thereof. In
one aspect, the nucleic acid probe is SEQ ID NO: 35 or SEQ ID NO:
37. In one aspect, the nucleic acid probe is SEQ ID NO: 35. In
another aspect, the nucleic acid probe is SEQ ID NO: 37. In another
aspect, the nucleic acid probe is a polynucleotide sequence that
encodes SEQ ID NO: 36, or a subsequence thereof.
[0109] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0110] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS at 45.degree. C. (very low stringency),
at 50.degree. C. (low stringency), at 55.degree. C. (medium
stringency), at 60.degree. C. (medium-high stringency), at
65.degree. C. (high stringency), or at 70.degree. C. (very high
stringency).
[0111] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0112] For short probes of about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0113] The C4 dicarboxylic acid transporter may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the C4 dicarboxylic acid transporter encoded
by a polynucleotide is produced by the source or by a cell in which
the polynucleotide from the source has been inserted. In one
aspect, the C4 dicarboxylic acid transporter is transported to the
outer membrane.
[0114] The C4 dicarboxylic acid transporter may be a bacterial C4
dicarboxylic acid transporter. For example, the C4 dicarboxylic
acid transporter may be a Gram positive bacterial polypeptide such
as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,
Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,
or Oceanobacillus C4 dicarboxylic acid transporter, or a Gram
negative bacterial polypeptide such as an E. coli, Pseudomonas,
Salmonella, Campylobacter, Helicobacter, Flavobacterium,
Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma C4 dicarboxylic
acid transporter.
[0115] In one aspect, the C4 dicarboxylic acid transporter is a
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,
Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus
firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,
Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,
Bacillus subtilis, or Bacillus thuringiensis C4 dicarboxylic acid
transporter.
[0116] In another aspect, the C4 dicarboxylic acid transporter is a
Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus
uberis, or Streptococcus equi subsp. Zooepidemicus C4 dicarboxylic
acid transporter.
[0117] In another aspect, the C4 dicarboxylic acid transporter is a
Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces
coelicolor, Streptomyces griseus, or Streptomyces lividans C4
dicarboxylic acid transporter.
[0118] The C4 dicarboxylic acid transporter may be a fungal C4
dicarboxylic acid transporter. In one aspect, the fungal C4
dicarboxylic acid transporter is a yeast C4 dicarboxylic acid
transporter such as a Candida, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, or Yarrowia C4 dicarboxylic
acid transporter.
[0119] In one aspect, the C4 dicarboxylic acid transporter is a
Schizosaccharomyces C4 dicarboxylic acid transporter, e.g., a
Schizosaccharomyces pombe C4 dicarboxylic acid transporter, such as
a Schizosaccharomyces pombe C4 dicarboxylic acid transporter of SEQ
ID NO: 36.
[0120] In another aspect, the fungal C4 dicarboxylic acid
transporter is a filamentous fungal C4 dicarboxylic acid
transporter such as an Acremonium, Agaricus, Alternaria,
Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,
Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,
Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia,
Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,
Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe,
Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix,
Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces,
Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,
Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella,
or Xylaria C4 dicarboxylic acid transporter.
[0121] In another aspect, the C4 dicarboxylic acid transporter is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis C4
dicarboxylic acid transporter.
[0122] In another aspect, the C4 dicarboxylic acid transporter is
an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus
awamori, Aspergillus flavus, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger, Aspergillus oryzae, Aspergillus sojae, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,
Chrysosporium merdarium, Chrysosporium inops, Chrysosporium
pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum,
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium
sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium
venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,
Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillium funiculosum, Penicillium purpurogenum,
Phanerochaete chrysosporium, Thielavia achromatica, Thielavia
albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia
fimeti, Thielavia microspora, Thielavia ovispora, Thielavia
peruviana, Thielavia spededonium, Thielavia setosa, Thielavia
subthermophila, Thielavia terrestris, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride C4 dicarboxylic acid transporter.
[0123] In one aspect, the C4 dicarboxylic acid transporter is an
Aspergillus C4 dicarboxylic acid transporter. In another aspect,
the C4 dicarboxylic acid transporter is an Aspergillus oryzae C4
dicarboxylic acid transporter, such as an Aspergillus oryzae C4
dicarboxylic acid transporter of SEQ ID NO: 8. In another aspect,
the C4 dicarboxylic acid transporter is an Aspergillus terreus C4
dicarboxylic acid transporter, such as an Aspergillus terreus C4
dicarboxylic acid transporter of SEQ ID NO: 34.
[0124] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0125] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0126] The C4 dicarboxylic acid transporter may also be identified
and obtained from other sources including microorganisms isolated
from nature (e.g., soil, composts, water, etc.) or DNA samples
obtained directly from natural materials (e.g., soil, composts,
water, etc,) using the above-mentioned probes. Techniques for
isolating microorganisms and DNA directly from natural habitats are
well known in the art. The polynucleotide encoding a C4
dicarboxylic acid transporter may then be derived by similarly
screening a genomic or cDNA library of another microorganism or
mixed DNA sample. Once a polynucleotide encoding a C4 dicarboxylic
acid transporter has been detected with suitable probe(s) as
described herein, the sequence may be isolated or cloned by
utilizing techniques that are known to those of ordinary skill in
the art (see, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatus,
1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold
Spring Harbor, N.Y.).
[0127] The C4 dicarboxylic acid transporter can also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a polynucleotide (or a portion thereof) encoding another
polypeptide to a polynucleotide (or a portion thereof). Techniques
for producing fusion polypeptides are known in the art, and include
ligating the coding sequences encoding the polypeptides so that
they are in frame and that expression of the fused polypeptide is
under control of the same promoter(s) and terminator. Fusion
proteins may also be constructed using intein technology in which
fusions are created post-translationally (Cooper et al., 1993, EMBO
J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
[0128] Techniques used to isolate or clone a polynucleotide
encoding a C4 dicarboxylic acid transporter are known in the art
and include isolation from genomic DNA, preparation from cDNA, or a
combination thereof. The cloning of the polynucleotides from such
genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR) or antibody screening of expression
libraries to detect cloned DNA fragments with shares structural
features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application, Academic Press, New York. Other nucleic acid
amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of Aspergillus, or another or related organism
and thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.
[0129] In one aspect, the isolated polynucleotide comprises or
consists of SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID
NO: 37. In another aspect, the isolated polynucleotide comprises or
consists of SEQ ID NO: 7. In another aspect, the isolated
polynucleotide comprises or consists of SEQ ID NO: 33. In another
aspect, the isolated polynucleotide comprises or consists of SEQ ID
NO: 35 or SEQ ID NO: 37. In another aspect, the isolated
polynucleotide encodes a C4 dicarboxylic acid transporter
comprising or consisting of SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID
NO: 36. In another aspect, the isolated polynucleotide encodes a C4
dicarboxylic acid transporter comprising or consisting of SEQ ID
NO: 8. In another aspect, the isolated polynucleotide encodes a C4
dicarboxylic acid transporter comprising or consisting of SEQ ID
NO: 34. In another aspect, the isolated polynucleotide encodes a C4
dicarboxylic acid transporter comprising or consisting of SEQ ID
NO: 36. Thus, the present invention encompasses nucleotide
sequences that encode polypeptides comprising or consisting of the
amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO:
36, but which differ from SEQ ID NO: 7, SEQ ID NO: 33, or SEQ ID
NO: 35 or SEQ ID NO: 37, respectively, by virtue of the degeneracy
of the genetic code. The present invention also relates to
subsequences of SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ
ID NO: 37 that encode fragments of SEQ ID NO: 8, SEQ ID NO: 34, or
SEQ ID NO: 36, respectively, that have C4 dicarboxylic acid
transporter activity.
[0130] In another aspect, the isolated polynucleotide can be a
mutant polynucleotide comprising or consisting of at least one
mutation in SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID
NO: 37 in which the mutant nucleotide sequence encodes SEQ ID NO:
8, SEQ ID NO: 34, or SEQ ID NO: 36, respectively. In another
aspect, the isolated polynucleotide can be a mutant polynucleotide
comprising or consisting of at least one mutation in SEQ ID NO: 7,
in which the mutant nucleotide sequence encodes SEQ ID NO: 8. In
another aspect, the isolated polynucleotide can be a mutant
polynucleotide comprising or consisting of at least one mutation in
SEQ ID NO: 33, in which the mutant nucleotide sequence encodes SEQ
ID NO: 34. In another aspect, the isolated polynucleotide can be a
mutant polynucleotide comprising or consisting of at least one
mutation in SEQ ID NO: 35 or SEQ ID NO: 37, in which the mutant
nucleotide sequence encodes SEQ ID NO: 36.
[0131] In another aspect, the isolated polynucleotide comprises or
consists of a nucleotide sequence having a degree of sequence
identity to SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID
NO: 37 of at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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,
which encode active C4 dicarboxylic acid transporters.
[0132] For example, in one aspect, the isolated polynucleotide
comprises or consists of a nucleotide sequence having a degree of
sequence identity to SEQ ID NO: 7 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 91%, 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, which encode active C4 dicarboxylic acid
transporters. In another aspect, the isolated polynucleotide
comprises or consists of a nucleotide sequence having a degree of
sequence identity to SEQ ID NO: 33 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 91%, 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, which encode active C4 dicarboxylic acid
transporters. In another aspect, the isolated polynucleotide
comprises or consists of a nucleotide sequence having a degree of
sequence identity to SEQ ID NO: 35 or SEQ ID NO: 37 of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 91%, 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, which encode active
C4 dicarboxylic acid transporters.
[0133] In another aspect, the isolated polynucleotide encoding a C4
dicarboxylic acid transporter hybridizes under at least very low
stringency conditions, e.g., low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with (i)
SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, an
allelic variant or subsequence thereof, or a full-length
complementary strand of the foregoing (Sambrook et al., 1989,
supra), as defined herein.
[0134] For example, in one aspect, the isolated polynucleotide
encoding a C4 dicarboxylic acid transporter hybridizes under at
least very low stringency conditions, e.g., low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with SEQ ID NO: 7, an allelic variant or subsequence
thereof, or a full-length complementary strand of the foregoing,
(Sambrook et al., 1989, supra), as defined herein. In another
aspect, the isolated polynucleotide encoding a C4 dicarboxylic acid
transporter hybridizes under at least very low stringency
conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 33,
an allelic variant or subsequence thereof, or a full-length
complementary strand of the foregoing (Sambrook et al., 1989,
supra), as defined herein. In another aspect, the isolated
polynucleotide encoding a C4 dicarboxylic acid transporter
hybridizes under at least very low stringency conditions, e.g., low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 35 or SEQ ID NO: 37, an
allelic variant or subsequence thereof, or a full-length
complementary strand of the foregoing (Sambrook et al., 1989,
supra), as defined herein.
[0135] In one aspect, the isolated polynucleotide encoding a C4
dicarboxylic acid transporter is obtained by (a) hybridizing a
population of DNA under very low, low, medium, medium-high, high,
or very high stringency conditions with SEQ ID NO: 7, SEQ ID NO:
33, SEQ ID NO: 35 or SEQ ID NO: 37, or a full-length complementary
strand thereof; and (b) isolating the hybridizing polynucleotide,
which encodes a polypeptide having C4 dicarboxylic acid transporter
activity.
[0136] For example, in one aspect, the isolated polynucleotide
encoding a C4 dicarboxylic acid transporter is obtained by (a)
hybridizing a population of DNA under very low, low, medium,
medium-high, high, or very high stringency conditions with SEQ ID
NO: 7, or the full-length complementary strand thereof; and (b)
isolating the hybridizing polynucleotide, which encodes a
polypeptide having C4 dicarboxylic acid transporter activity. In
another aspect, the isolated polynucleotide encoding a C4
dicarboxylic acid transporter is obtained by (a) hybridizing a
population of DNA under very low, low, medium, medium-high, high,
or very high stringency conditions with SEQ ID NO: 33 or the
full-length complementary strand thereof; and (b) isolating the
hybridizing polynucleotide, which encodes a polypeptide having C4
dicarboxylic acid transporter activity. In another aspect, the
isolated polynucleotide encoding a C4 dicarboxylic acid transporter
is obtained by (a) hybridizing a population of DNA under very low,
low, medium, medium-high, high, or very high stringency conditions
with SEQ ID NO: 35 or SEQ ID NO: 37, or a full-length complementary
strand thereof; and (b) isolating the hybridizing polynucleotide,
which encodes a polypeptide having C4 dicarboxylic acid transporter
activity.
[0137] Other C4 dicarboxylic acid transporters that can be used to
practice the present invention include, e.g., the Aspergillus
flavus C4 dicarboxylic acid transporter (AFLA.sub.--107340).
Malate Dehydrogenases and Polynucleotides Encoding the Malate
Dehydrogenases
[0138] In the present invention, the malate dehydrogenase can be
any malate dehydrogenase that is suitable for practicing the
present invention. In one aspect, the malate dehydrogenase is an
enzyme that is present in the cytosol of the host cell. Malate
dehydrogenases that can be used to practice the present invention
include, but are not limited to, an Aspergillus fumigatus malate
dehydrogenase (AFUA.sub.--2G13800; Nierman et al., 2005, Nature
438: 1151-1156); Aspergillus nidulans malate dehydrogenase
(AN5031.1, AN6499.1; Sims et al., 2004, Mycol. Res. 108: 853-857);
Aspergillus niger malate dehydrogenase (An11g07190, An12g00160,
An15g00070; Pel et al., 2007, Nature Biotechnology 25: 221-231);
Aspergillus oryzae NRRL 3488 malate dehydrogenase (genomic DNA
sequence of SEQ ID NO: 19 and the deduced amino acid sequence of
SEQ ID NO: 20); Phytophthora infestans malate dehydrogenase (PITG
15476.1; Calcagno et al., 2009, Mycological Research 113: 771-781);
and Saccharomyces cerevisiae malate dehydrogenase (YOL126C; Minard
and McAlister-Henn, 1991, Mol. Cell. Biol. 11: 370-380; YDL078C;
McAlister-Henn et al., 1995, Journal of Biological Chemistry 270:
21220-21225).
[0139] In one aspect, the malate dehydrogenase is (a) a malate
dehydrogenase comprising an amino acid sequence having at least 60%
sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20; (b) a malate
dehydrogenase encoded by a polynucleotide that hybridizes under at
least low stringency conditions with (i) SEQ ID NO: 17 or SEQ ID
NO: 19, (ii) the cDNA sequence contained in SEQ ID NO: 17 or SEQ ID
NO: 19, or (iii) a full-length complementary strand of (i) or (ii);
(c) a malate dehydrogenase encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity with SEQ
ID NO: 17 or SEQ ID NO: 19; (d) a malate dehydrogenase variant
comprising a substitution, deletion, and/or insertion of one or
more (several) amino acids of the mature polypeptide of SEQ ID NO:
18 or SEQ ID NO: 20; or (e) a fragment of the malate dehydrogenase
of (a), (b), (c), or (d) that has malate dehydrogenase
activity.
[0140] In a one aspect, the malate dehydrogenase comprises or
consists of an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 18 or SEQ ID NO: 20 of at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99%, which has malate dehydrogenase activity (hereinafter
"homologous malate dehydrogenases"). In one aspect, the homologous
malate dehydrogenase comprises an amino acid sequence that differs
by no more than ten amino acids, e.g., by no more than five amino
acids, by no more than four amino acids, by no more than three
amino acids, by no more than two amino acids, or by one amino acid
from SEQ ID NO: 18 or SEQ ID NO: 20.
[0141] In one aspect, the malate dehydrogenase comprises or
consists of an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 18 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99%. In one aspect,
the homologous malate dehydrogenase comprises an amino acid
sequence that differs by no more than ten amino acids, e.g., by no
more than five amino acids, by no more than four amino acids, by no
more than three amino acids, by no more than two amino acids, or by
one amino acid from SEQ ID NO: 18.
[0142] In another aspect, the malate dehydrogenase comprises or
consists of an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 20 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99%, which has malate
dehydrogenase activity. In one aspect, the homologous malate
dehydrogenase comprises an amino acid sequence that differs by no
more than ten amino acids, e.g., by no more than five amino acids,
by no more than four amino acids, by no more than three amino
acids, by no more than two amino acids, by one amino acid from SEQ
ID NO: 20.
[0143] A substantially homologous malate dehydrogenase may have one
or more (several) amino acid substitutions, deletions and/or
insertions, as described supra.
[0144] In one aspect, the malate dehydrogenase comprises the amino
acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20 or an allelic
variant thereof; or a fragment thereof having malate dehydrogenase
activity. In another aspect, the malate dehydrogenase comprises the
amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In another
aspect, the malate dehydrogenase comprises the amino acid sequence
of SEQ ID NO: 18. In another aspect, the malate dehydrogenase
comprises the amino acid sequence of SEQ ID NO: 20. In another
aspect, the malate dehydrogenase consists of the amino acid
sequence of SEQ ID NO: 18 or SEQ ID NO: 20 or an allelic variant
thereof; or a fragment thereof having malate dehydrogenase
activity. In another aspect, the malate dehydrogenase consists of
the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In
another aspect, the malate dehydrogenase consists of the amino acid
sequence of SEQ ID NO: 18. In another aspect, the malate
dehydrogenase consists of the amino acid sequence of SEQ ID NO:
20.
[0145] In another aspect, the malate dehydrogenase is a variant
comprising a substitution, deletion, and/or insertion of one or
more (several) amino acids of the mature polypeptide of SEQ ID NO:
18 or SEQ ID NO: 20, as described supra. In some aspects, the total
number of amino acid substitutions, deletions and/or insertions of
the mature polypeptide of SEQ ID NO: 18 or SEQ ID NO: 20 is not
more than 10, e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8 or 9. In
another aspect, the total number of amino acid substitutions,
deletions and/or insertions of the mature polypeptide of SEQ ID NO:
18 or SEQ ID NO: 20 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0146] In another aspect, the malate dehydrogenase is a fragment of
SEQ ID NO: 18 or SEQ ID NO: 20, wherein the fragment has malate
dehydrogenase activity. In one aspect, a fragment of SEQ ID NO: 18
contains at least 290 amino acid residues, e.g., at least 305 amino
acid residues or at least 320 amino acid residues. In one aspect, a
fragment of SEQ ID NO: 20 contains at least 280 amino acid
residues, e.g., at least 295 amino acid residues or at least 310
amino acid residues.
[0147] In a another aspect, the malate dehydrogenase is encoded by
a polynucleotide that hybridizes under at least very low stringency
conditions, e.g., low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
17 or SEQ ID NO: 19, (ii) the cDNA sequence contained in SEQ ID NO:
17 or SEQ ID NO: 19, (iii) a subsequence of (i) or (ii), or (iv) a
full-length complementary strand of (i), (ii), or (iii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, supra). The
subsequence may encode a polypeptide fragment having malate
dehydrogenase activity.
[0148] A subsequence of SEQ ID NO: 17 or SEQ ID NO: 19, or a
homolog thereof, is a nucleotide sequence where one or more
(several) nucleotides have been deleted from the 5'- and/or 3'-end.
In one aspect, a subsequence of SEQ ID NO: 17 contains at least 870
nucleotides, e.g., at least 915 nucleotides or at least 960
nucleotides. In another aspect, a subsequence of SEQ ID NO: 19
contains at least 840 nucleotides, e.g., at least 885 nucleotides
or at least 930 nucleotides.
[0149] The polynucleotide of SEQ ID NO: 17 or SEQ ID NO: 19; or a
subsequence thereof; as well as the amino acid sequence of SEQ ID
NO: 18 or SEQ ID NO: 20; or a fragment thereof; may be used to
design nucleic acid probes to identify and clone DNA encoding
malate dehydrogenases from strains of different genera or species,
as described supra. Such probes are encompassed by the present
invention.
[0150] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a malate dehydrogenase, as described
supra.
[0151] In one aspect, the nucleic acid probe is SEQ ID NO: 17 or
SEQ ID NO: 19. In another aspect, the nucleic acid probe is SEQ ID
NO: 17. In another aspect, the nucleic acid probe is SEQ ID NO: 19.
In another aspect, the nucleic acid probe is a polynucleotide
sequence that encodes SEQ ID NO: 18 or SEQ ID NO: 20, or a
subsequence thereof. In another aspect, the nucleic acid probe is a
polynucleotide sequence that encodes SEQ ID NO: 18, or a
subsequence thereof. In another aspect, the nucleic acid probe is a
polynucleotide sequence that encodes SEQ ID NO: 20, or a
subsequence thereof.
[0152] For long probes of at least 100 nucleotides in length, very
low to very high stringency and washing conditions are defined as
described supra.
[0153] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency and washing conditions are
defined as described supra.
[0154] In another aspect, the malate dehydrogenase is encoded by a
polynucleotide comprising or consisting of a nucleotide sequence
having a degree of sequence identity to SEQ ID NO: 17 or SEQ ID NO:
19 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 91%, 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%, which encodes a
polypeptide having malate dehydrogenase activity.
[0155] The malate dehydrogenase may be obtained from microorganisms
of any genus. For purposes of the present invention, the term
"obtained from" as used herein in connection with a given source
shall mean that the malate dehydrogenase encoded by a
polynucleotide is produced by the source or by a cell in which the
polynucleotide from the source has been inserted.
[0156] In one aspect, the malate dehydrogenase may be a bacterial,
yeast, or filamentous fungal malate dehydrogenase obtained from the
microorganisms described herein.
[0157] In another aspect, the malate dehydrogenase is an
Aspergillus oryzae malate dehydrogenase, e.g., the Aspergillus
oryzae malate dehydrogenase of SEQ ID NO: 18 or SEQ ID NO: 20.
[0158] The malate dehydrogenase may also be identified and obtained
from other sources including microorganisms isolated from nature
(e.g., soil, composts, water, etc.) or DNA samples obtained
directly from natural materials (e.g., soil, composts, water, etc,)
as described supra.
[0159] The malate dehydrogenase can also include fused polypeptides
or cleavable fusion polypeptides, as described supra.
[0160] Techniques used to isolate or clone a polynucleotide
encoding a malate dehydrogenase are described supra.
[0161] In another aspect, the malate dehydrogenase is a variant of
a parent malate dehydrogenase that comprises one or more (several)
modifications of the amino acid sequence, which reduces
mitochondrial import in vivo of the malate dehydrogenase.
[0162] In another aspect, the malate dehydrogenase is a variant of
a parent malate dehydrogenase comprising (i) a deletion at
positions equivalent or corresponding to positions 2 to 17 or a
portion thereof of SEQ ID NO: 18, and (ii) a substitution at a
position equivalent to position 48 of SEQ ID NO: 18; wherein the
deletion and the substitution reduce mitochondrial import in vivo
of the malate dehydrogenase variant thereby increasing the level of
the malate dehydrogenase variant in the cytosol. Such variants are
described in detail below.
Conventions for Designation of Malate Dehydrogenase Variants
[0163] For purposes of the present invention, the amino acid
sequence of the malate dehydrogenase disclosed in SEQ ID NO: 18 is
used to determine the corresponding or equivalent amino acid
residue in another malate dehydrogenase. The amino acid sequence of
another malate dehydrogenase is aligned with the amino acid
sequence of the malate dehydrogenase of SEQ ID NO: 18, and based on
the alignment the amino acid position number corresponding to any
amino acid residue in the amino acid sequence of the malate
dehydrogenase of SEQ ID NO: 18 can be determined.
[0164] An alignment of polypeptide sequences may be made, for
example, using "ClustalW" (Thompson, J. D., Higgins, D. G. and
Gibson, T. J., 1994, CLUSTAL W: Improving the sensitivity of
progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix choice, Nucleic
Acids Research 22: 4673-4680). An alignment of DNA sequences may be
done using the polypeptide alignment as a template, replacing the
amino acids with the corresponding codon from the DNA sequence.
[0165] Pairwise sequence comparison algorithms in common use are
adequate to detect similarities between polypeptide sequences that
have not diverged beyond the point of approximately 20-30% sequence
identity (Doolittle, 1992, Protein Sci. 1: 191-200; Brenner et al.,
1998, Proc. Natl. Acad. Sci. USA 95, 6073-6078). However, truly
homologous polypeptides with the same fold and similar biological
function have often diverged to the point where traditional
sequence-based comparison fails to detect their relationship
(Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Greater
sensitivity in sequence-based searching can be attained using
search programs that utilize probabilistic representations of
polypeptide families (profiles) to search databases. For example,
the PSI-BLAST program generates profiles through an iterative
database search process and is capable of detecting remote homologs
(Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even
greater sensitivity can be achieved if the family or superfamily
for the polypeptide of interest has one or more (several)
representatives in the protein structure databases. Programs such
as GenTHREADER (Jones 1999, J. Mol. Biol. 287: 797-815; McGuffin
and Jones, 2003, Bioinformatics 19: 874-881) utilize information
from a variety of sources (PSI-BLAST, secondary structure
prediction, structural alignment profiles, and solvation
potentials) as input to a neural network that predicts the
structural fold for a query sequence. Similarly, the method of
Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to
align a sequence of unknown structure with the superfamily models
present in the SCOP database. These alignments can in turn be used
to generate homology models for the polypeptide of interest, and
such models can be assessed for accuracy using a variety of tools
developed for that purpose.
[0166] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example, the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. Two or more protein structures can be aligned using a
variety of algorithms such as the distance alignment matrix (Holm
and Sander, 1998, Proteins 33: 88-96) or combinatorial extension
(Shindyalov and Bourne, 1998, Protein Eng. 11: 739-747), and
implementations of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567). These structural alignments can be
used to predict the structurally and functionally corresponding
amino acid residues in proteins within the same structural
superfamily. This information, along with information derived from
homology modeling and profile searches, can be used to predict
which residues to mutate when moving mutations of interest from one
protein to a close or remote homolog.
[0167] In describing the malate dehydrogenase variants of the
present invention, the nomenclature described below is adapted for
ease of reference. In all cases, the accepted IUPAC single letter
or triple letter amino acid abbreviation is employed.
[0168] Substitutions. For an amino acid substitution, the following
nomenclature is used: Original amino acid, position, substituted
amino acid. Accordingly, the substitution of threonine with alanine
at position 226 is designated as "Thr226Ala" or "T226A". Multiple
mutations are separated by addition marks ("+"), e.g.,
"Gly205Arg+Ser411Phe" or "G205R+S411F", representing mutations at
positions 205 and 411 substituting glycine (G) with arginine (R),
and serine (S) with phenylalanine (F), respectively.
[0169] Deletions. For an amino acid deletion, the following
nomenclature is used: Original amino acid, position*. Accordingly,
the deletion of glycine at position 195 is designated as "Gly195*"
or "G195*". Multiple deletions are separated by addition marks
("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".
[0170] Insertions. For an amino acid insertion, the following
nomenclature is used: Original amino acid, position, original amino
acid, new inserted amino acid. Accordingly the insertion of lysine
after glycine at position 195 is designated "Gly195GlyLys" or
"G195GK". Multiple insertions of amino acids are designated
[Original amino acid, position, original amino acid, new inserted
amino acid #1, new inserted amino acid #2; etc.]. For example, the
insertion of lysine and alanine after glycine at position 195 is
indicated as "Gly195GlyLysAla" or "G195GKA".
[0171] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example the sequences would thus be:
TABLE-US-00001 Parent: Variant: 195 195 195a 195b G G - K - A
Parent Malate Dehydrogenases and Polynucleotides Encoding Parent
Malate Dehydrogenases
[0172] In the present invention, the parent malate dehydrogenase
can be any malate dehydrogenase that is imported in vivo into the
mitochondria of the host cell.
[0173] In one aspect, the parent malate dehydrogenase is (a) a
malate dehydrogenase comprising an amino acid sequence having at
least 60% sequence identity with SEQ ID NO: 18; (b) a malate
dehydrogenase encoded by a polynucleotide that hybridizes under at
least low stringency conditions with (i) SEQ ID NO: 17, (ii) the
cDNA sequence contained in SEQ ID NO: 17, or (iii) the full-length
complementary strand of (i) or (ii); or (c) a malate dehydrogenase
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60% sequence identity with SEQ ID NO: 17, wherein the
parent malate dehydrogenase comprises one or more (several)
mitochondrial targeting sequences (Claros and Vincens, 1996,
supra).
[0174] In one aspect, the parent malate dehydrogenase comprises or
consists of an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 18 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which have malate dehydrogenase activity (hereinafter "homologous
dehydrogenases"). In one aspect, the homologous dehydrogenase
comprises an amino acid sequence that differs by no more than ten
amino acids, e.g., by no more than five amino acids, by no more
than four amino acids, by no more than three amino acids, by no
more than two amino acids, or by one amino acid from SEQ ID NO:
18.
[0175] A substantially homologous parent malate dehydrogenase may
have one or more (several) amino acid substitutions, deletions
and/or insertions, as described supra.
[0176] In one aspect, the parent malate dehydrogenase comprises the
amino acid sequence of SEQ ID NO: 18 or an allelic variant thereof;
or a fragment thereof having malate dehydrogenase activity. In
another aspect, the parent malate dehydrogenase comprises the amino
acid sequence of SEQ ID NO: 18. In another aspect, the parent
malate dehydrogenase consists of the amino acid sequence of SEQ ID
NO: 18 or an allelic variant thereof; or a fragment thereof having
malate dehydrogenase activity. In another aspect, the parent malate
dehydrogenase consists of the amino acid sequence of SEQ ID NO:
18.
[0177] A fragment of SEQ ID NO: 18 is a polypeptide having one or
more (several) amino acids deleted from the amino and/or carboxyl
terminus of this amino acid sequence. In one aspect, a fragment
contains at least 295 amino acid residues, e.g., at least 310 amino
acid residues or at least 325 amino acid residues of SEQ ID NO:
18.
[0178] In another aspect, the parent malate dehydrogenase is
encoded by a polynucleotide that hybridizes under at least very low
stringency conditions, e.g., low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with (i)
SEQ ID NO: 17, (ii) the cDNA sequence contained in SEQ ID NO: 17,
(iii) a subsequence of (i) or (ii), or (iv) the full-length
complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory
Manual, 2d edition, Cold Spring Harbor, N.Y.). The subsequence may
encode a polypeptide fragment having malate dehydrogenase
activity.
[0179] A subsequence of SEQ ID NO: 17, or a homolog thereof, is a
nucleotide sequence where one or more (several) nucleotides have
been deleted from the 5'- and/or 3'-end. In one aspect, a
subsequence contains at least 885 nucleotides, e.g., at least 930
nucleotides or at least 975 nucleotides of SEQ ID NO: 17.
[0180] The parent enzyme may also be an allelic variant or
artificial variant of a malate dehydrogenase.
[0181] The polynucleotide of SEQ ID NO: 17; or a subsequence
thereof; as well as the amino acid sequence of SEQ ID NO: 18; or a
fragment thereof; may be used to design nucleic acid probes to
identify and clone DNA encoding parent malate dehydrogenases from
strains of different genera or species, as described supra.
[0182] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a malate dehydrogenase, as described
supra.
[0183] In one aspect, the nucleic acid probe is SEQ ID NO: 17. In
another aspect, the nucleic acid probe is a polynucleotide sequence
that encodes SEQ ID NO: 18, or a subsequence thereof.
[0184] For long probes of at least 100 nucleotides in length, very
low to very high stringency and washing conditions are defined as
described supra.
[0185] For short probes that are about 15 nucleotides to about 70
nucleotides in length, stringency and washing conditions are
defined as described supra.
[0186] In another aspect, the parent malate dehydrogenase is
encoded by a polynucleotide comprising or consisting of a
nucleotide sequence having a degree of sequence identity to SEQ ID
NO: 17 of at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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%, which encodes a
polypeptide having malate dehydrogenase activity.
[0187] The parent malate dehydrogenase may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the parent malate dehydrogenase encoded by a
polynucleotide is produced by the source or by a cell in which the
polynucleotide from the source has been inserted.
[0188] In one aspect, the parent malate dehydrogenase may be a
bacterial, a yeast, or a filamentous fungal malate dehydrogenase
obtained from the microorganisms described herein in the C4
dicarboxylic acid transporter section.
[0189] In another aspect, the parent malate dehydrogenase is an
Aspergillus oryzae malate dehydrogenase, e.g., the Aspergillus
oryzae malate dehydrogenase of SEQ ID NO: 18.
[0190] The parent malate dehydrogenase may also be identified and
obtained from other sources including microorganisms isolated from
nature (e.g., soil, composts, water, etc.) or DNA samples obtained
directly from natural materials (e.g., soil, composts, water, etc,)
as described supra.
[0191] The parent malate dehydrogenase can also include fused
polypeptides or cleavable fusion polypeptides, as described
supra.
[0192] Techniques used to isolate or clone a polynucleotide
encoding a parent malate dehydrogenase are described supra.
[0193] In one aspect, the isolated polynucleotide comprises or
consists of SEQ ID NO: 17. In another aspect, the isolated
polynucleotide encodes a parent malate dehydrogenase comprising or
consisting of SEQ ID NO: 18. The present invention also encompasses
nucleotide sequences that encode polypeptides comprising or
consisting of the amino acid sequence of SEQ ID NO: 18, which
differ from SEQ ID NO: 17 by virtue of the degeneracy of the
genetic code. The present invention also relates to subsequences of
SEQ ID NO: 17 that encode fragments of SEQ ID NO: 18 that have
malate dehydrogenase activity.
[0194] In another aspect, the isolated polynucleotide can be a
mutant polynucleotide comprising or consisting of at least one
mutation in SEQ ID NO: 17, in which the mutant nucleotide sequence
encodes SEQ ID NO: 18.
[0195] In another aspect, the isolated polynucleotide comprises or
consists of a nucleotide sequence that has a degree of sequence
identity to SEQ ID NO: 17 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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, which encodes a parent polypeptide having malate
dehydrogenase activity.
[0196] In another aspect, the isolated polynucleotide encoding a
parent malate dehydrogenase hybridizes under at least very low
stringency conditions, e.g., low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with (i)
SEQ ID NO: 17, (ii) the cDNA sequence contained in SEQ ID NO: 17,
or (iii) the full-length complementary strand of (i) or (ii); or
allelic variants and subsequences thereof (Sambrook et al., 1989,
supra), as defined herein.
[0197] In another aspect, the isolated polynucleotide encoding a
parent malate dehydrogenase is obtained by (a) hybridizing a
population of DNA under very low, low, medium, medium-high, high,
or very high stringency conditions with (i) SEQ ID NO: 17, (ii) the
cDNA sequence contained in SEQ ID NO: 17, or (iii) the full-length
complementary strand of (i) or (ii); and (b) isolating the
hybridizing polynucleotide, which encodes the parent polypeptide
having malate dehydrogenase activity.
[0198] Other parent malate dehydrogenases that can be used to
practice the present invention include, but are not limited to, a
Aspergillus nidulans malate dehydrogenase (AN6717.1; SIMS et al.,
2004, Mycol. Res. 108: 853-857); Aspergillus niger malate
dehydrogenase (An16g00120; Pel et al., 2007, Nature Biotechnology
25: 221-231); Phytophthora infestans malate dehydrogenase (PITG
13614.1; Calcagno et al., 2009, Mycological Research 113: 771-781);
Saccharomyces cerevisiae malate dehydrogenase (YKL085W;
McAlister-Henn and Thompson, 1987, J Bacteriol. 169: 5157-5166);
Talaromyces emersonii malate dehydrogenase (AF439996, AF487682;
Maloney et al., 2004, Eur. J. Biochem. 271: 3115-3126); and
Ustilago maydis malate dehydrogenase (um00403, um11161; McCann and
Snetselaar, 2008, Fungal Genetics and Biology 45: S77-S87).
Preparation of Variants
[0199] Variants of a parent malate dehydrogenase can be prepared
according to any mutagenesis procedure known in the art, such as
site-directed mutagenesis, synthetic gene construction,
semi-synthetic gene construction, etc.
[0200] Site-directed mutagenesis is a technique in which one or
several mutations are created at a defined site in a polynucleotide
molecule encoding the parent malate dehydrogenase. The technique
can be performed in vitro or in vivo.
[0201] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide molecule
of interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian, et. al., (Tian, et. al., Nature 432:1050-1054)
and similar technologies wherein oligonucleotides are synthesized
and assembled upon photo-programmable microfluidic chips.
[0202] Site-directed mutagenesis can be accomplished in vitro by
PCR involving the use of oligonucleotide primers containing the
desired mutation. Site-directed mutagenesis can also be performed
in vitro by cassette mutagenesis involving the cleavage by a
restriction enzyme at a site in the plasmid comprising a
polynucleotide encoding the parent malate dehydrogenase and
subsequent ligation of an oligonucleotide containing the mutation
in the polynucleotide. Usually the restriction enzyme that digests
at the plasmid and the oligonucleotide is the same, permitting
sticky ends of the plasmid and insert to ligate to one another.
See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci.
USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Research
18: 7349-4966.
[0203] Site-directed mutagenesis can be accomplished in vivo by
methods known in the art. See, for example, U.S. Pat. No.
7,314,712; Storici et al., 2001, Nature Biotechnology 19: 773-776;
Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino,
1996, Fungal Genet. Newslett. 43: 15-16.
[0204] Any site-directed mutagenesis procedure can be used in the
present invention. There are many commercial kits available that
can be used to prepare variants of a parent malate
dehydrogenase.
[0205] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis followed by a relevant screening procedure, such as
those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:
53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:
2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be
used include error-prone PCR, phage display (e.g., Lowman et al.,
1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO
92/06204) and region-directed mutagenesis (Derbyshire et al., 1986,
Gene 46:145; Ner et al., 1988, DNA 7:127).
[0206] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error-prone PCR
implication.
[0207] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of
interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al. (2004, Nature 432: 1050-1054) and similar
technologies wherein oligonucleotide are synthesized and assembled
upon photo-programmable microfluidic chips.
[0208] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
Variants and Polynucleotides Encoding Malate Dehydrogenase
Variants
[0209] In the present invention, variants of a parent malate
dehydrogenase may comprise (i) a deletion at positions equivalent
or corresponding to positions 2 to 17 or a portion thereof of SEQ
ID NO: 18, and (ii) a substitution at a position equivalent to
position 48 of SEQ ID NO: 18. In a preferred aspect, the variant
having malate dehydrogenase activity comprises an amino acid
sequence having a degree of sequence identity of at least 60%,
e.g., at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least about 97%, at
least 98%, at least 99% to the amino acid sequence of the parent
malate dehydrogenase, e.g., SEQ ID NO: 18. In a preferred aspect,
the parent malate dehydrogenase is SEQ ID NO: 18.
[0210] In one aspect, a variant of the present invention comprises
deletions at positions equivalent to positions 2 to 17 of SEQ ID
NO: 18 and a substitution at a position equivalent to position 48
of SEQ ID NO: 18. In another aspect, a variant of the present
invention comprises a deletion at one or more (several) positions
equivalent to positions 2 to 17 of SEQ ID NO: 18 and a substitution
at a position equivalent to position 48 of SEQ ID NO: 18. In
another aspect, a variant of the present invention comprises
deletions at positions 2 to 17 of SEQ ID NO: 18 and a substitution
at position 48 of SEQ ID NO: 18. In another aspect, a variant of
the present invention comprises a deletion at one or more (several)
positions 2 to 17 of SEQ ID NO: 18 and a substitution at position
48 of SEQ ID NO: 18. In another aspect, the variant comprises a
deletion of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at positions
corresponding to positions 2 to 17 and a substitution with Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val at a position equivalent to
position 48 of SEQ ID NO: 18. In another aspect, the variant
comprises a deletion of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at
one or more (several) positions corresponding to positions 2 to 17
and a substitution with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at a
position equivalent to position 48 of SEQ ID NO: 18. In another
aspect, the variant comprises deletions of Phe, Ala, Ala, Arg, Gln,
Ser, Phe, Asn, Leu, Leu, Gln, Lys, Arg, Ala, Phe, or Ser at
positions equivalent to positions 2 to 17 of SEQ ID NO: 18, and Tyr
as a substitution at a position equivalent to position 48 of SEQ ID
NO: 18. In another aspect, the variant comprises deletions of Phe,
Ala, Ala, Arg, Gln, Ser, Phe, Asn, Leu, Leu, Gln, Lys, Arg, Ala,
Phe, or Ser at one or more (several) positions equivalent to
positions 2 to 17 of SEQ ID NO: 18, and Tyr as a substitution at a
position equivalent to position 48 of SEQ ID NO: 18. In another
aspect, the variant comprises the deletions
Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18. In
another aspect, the variant comprises one or more (several) of the
deletions Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
[0211] The variants may further comprise one or more (several)
deletions, substitutions, and/or insertions of the amino acid
sequence.
[0212] The present invention also relates to isolated
polynucleotides that encode variants of a parent malate
dehydrogenase, wherein the polynucleotides encode malate
dehydrogenase variants comprising (i) a deletion at positions
equivalent to positions 2 to 17 or a portion thereof of SEQ ID NO:
18, and (ii) a substitution at a position equivalent to position 48
of SEQ ID NO: 18, wherein the parent malate dehydrogenase is (a) a
malate dehydrogenase comprising an amino acid sequence having at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 91%, 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 SEQ ID NO: 18;
(ii) a malate dehydrogenase encoded by a polynucleotide that
hybridizes under at least very low stringency conditions, e.g., low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with (i) SEQ ID NO: 17, (ii) the cDNA
sequence contained in SEQ ID NO: 17, or (iii) the full-length
complementary strand of (i) or (ii), wherein the variant has malate
dehydrogenase activity.
[0213] In one aspect, the isolated polynucleotide encodes a malate
dehydrogenase variant comprising deletions at positions equivalent
to positions 2 to 17 of SEQ ID NO: 18 and a substitution at a
position equivalent to position 48 of SEQ ID NO: 18. In another
aspect, the isolated polynucleotide encodes a malate dehydrogenase
variant comprising a deletion at one or more (several) positions
equivalent to positions 2 to 17 of SEQ ID NO: 18 and a substitution
at a position equivalent to position 48 of SEQ ID NO: 18. In
another aspect, the isolated polynucleotide encodes a malate
dehydrogenase variant comprising deletions at positions 2 to 17 of
SEQ ID NO: 18 and a substitution at position 48 of SEQ ID NO: 18.
In another aspect, the isolated polynucleotide encodes a malate
dehydrogenase variant comprising a deletion at one or more
(several) positions 2 to 17 of SEQ ID NO: 18 and a substitution at
position 48 of SEQ ID NO: 18. In another aspect, the isolated
polynucleotide encodes a malate dehydrogenase variant comprising a
deletion of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at positions
corresponding to positions 2 to 17 and a substitution with Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val at a position equivalent to
position 48 of SEQ ID NO: 18. In another aspect, the isolated
polynucleotide encodes a malate dehydrogenase variant comprising a
deletion of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at of one or more
(several) positions corresponding to positions 2 to 17 and a
substitution with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at a position
equivalent to position 48 of SEQ ID NO: 18. In another aspect, the
isolated polynucleotide encodes a malate dehydrogenase variant
comprising deletions of Phe, Ala, Ala, Arg, Gln, Ser, Phe, Asn,
Leu, Leu, Gln, Lys, Arg, Ala, Phe, or Ser at positions equivalent
to positions 2 to 17 of SEQ ID NO: 18, and Tyr as a substitution at
a position equivalent to position 48 of SEQ ID NO: 18. In another
aspect, the isolated polynucleotide encodes a malate dehydrogenase
variant comprising deletions of Phe, Ala, Ala, Arg, Gln, Ser, Phe,
Asn, Leu, Leu, Gln, Lys, Arg, Ala, Phe, or Ser at one or more
(several) positions equivalent to positions 2 to 17 of SEQ ID NO:
18, and Tyr as a substitution at a position equivalent to position
48 of SEQ ID NO: 18. In another aspect, the isolated polynucleotide
encodes a malate dehydrogenase variant comprising the deletions
Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18. In
another aspect, the isolated polynucleotide encodes a malate
dehydrogenase variant comprising one or more (several) of the
deletions Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
Pyruvate Carboxylases and Polynucleotides Encoding Pyruvate
Carboxylases
[0214] In the present invention, the pyruvate carboxylase can be
any pyruvate carboxylase that is suitable for practicing the
present invention. In one aspect, the pyruvate carboxylase is an
enzyme that is present in the cytosol of the host cell.
[0215] In one aspect, the pyruvate carboxylase is (a) a pyruvate
carboxylase comprising an amino acid sequence having at least 60%
sequence identity with SEQ ID NO: 27; (b) a pyruvate carboxylase
encoded by a polynucleotide that hybridizes under low stringency
conditions with (i) SEQ ID NO: 26, (ii) the cDNA sequence contained
in SEQ ID NO: 26, or (iii) the full-length complementary strand of
(i) or (ii); (c) a pyruvate carboxylase encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity with SEQ ID NO: 26; (d) a pyruvate carboxylase variant
comprising a substitution, deletion, and/or insertion of one or
more (several) amino acids of the mature polypeptide of SEQ ID NO:
26; or (e) a fragment of the pyruvate carboxylase of (a), (b), (c),
or (d) that has pyruvate carboxylase activity.
[0216] In one aspect, the pyruvate carboxylase comprises or
consists of an amino acid sequence having a degree of sequence
identity to SEQ ID NO: 27 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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%,
which has pyruvate carboxylase activity (hereinafter "homologous
pyruvate carboxylases"). In one aspect, the homologous pyruvate
carboxylase comprises an amino acid sequence that differs by no
more than ten amino acids, e.g., by no more than five amino acids,
by no more than four amino acids, by no more than three amino
acids, by no more than two amino acids, or by one amino acid from
SEQ ID NO: 27.
[0217] A substantially homologous pyruvate carboxylase may have one
or more (several) amino acid substitutions, deletions and/or
insertions, as described supra.
[0218] In one aspect, the pyruvate carboxylase comprises the amino
acid sequence of SEQ ID NO: 27 or an allelic variant thereof; or a
fragment thereof having pyruvate carboxylase activity. In another
aspect, the pyruvate carboxylase comprises the amino acid sequence
of SEQ ID NO: 27. In another aspect, the pyruvate carboxylase
consists of the amino acid sequence of SEQ ID NO: 27 or an allelic
variant thereof; or a fragment thereof having pyruvate carboxylase
activity. In another aspect, the pyruvate carboxylase consists of
the amino acid sequence of SEQ ID NO: 27.
[0219] In one aspect, the pyruvate carboxylase is a variant
comprising a substitution, deletion, and/or insertion of one or
more (several) amino acids of the mature polypeptide of SEQ ID NO:
27, as described supra. In some aspects, the total number of amino
acid substitutions, deletions and/or insertions of the mature
polypeptide of SEQ ID NO: 27 is not more than 10, e.g., not more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9. In another aspect, the total
number of amino acid substitutions, deletions and/or insertions of
the mature polypeptide of SEQ ID NO: 27 is 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10.
[0220] In another aspect, the pyruvate carboxylase is a fragment of
SEQ ID NO: 27, wherein the fragment has pyruvate carboxylase
activity. In one aspect, a fragment contains at least 1020 amino
acid residues, e.g., at least 1080 amino acid residues or at least
1140 amino acid residues SEQ ID NO: 27.
[0221] In a another aspect, the pyruvate carboxylase is encoded by
a polynucleotide that hybridizes under at least very low stringency
conditions, e.g., low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
26, (ii) the cDNA sequence contained in SEQ ID NO: 26, (iii) a
subsequence of (i) or (ii), or (iv) the full-length complementary
strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T.
Maniatis, 1989, supra). The subsequence may encode a polypeptide
fragment having pyruvate carboxylase activity.
[0222] A subsequence of SEQ ID NO: 26, or a homolog thereof, is a
nucleotide sequence where one or more (several) nucleotides have
been deleted from the 5'- and/or 3'-end. In one aspect, a
subsequence contains at least 3060 nucleotides, e.g., at least 3240
nucleotides or at least 3420 nucleotides of SEQ ID NO: 26.
[0223] The polynucleotide of SEQ ID NO: 26; or a subsequence
thereof; as well as the amino acid sequence of SEQ ID NO: 27; or a
fragment thereof; may be used to design nucleic acid probes to
identify and clone DNA encoding pyruvate carboxylases from strains
of different genera or species, as described supra. Such probes are
encompassed by the present invention.
[0224] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a pyruvate carboxylase, as described
supra.
[0225] In one aspect, the nucleic acid probe is SEQ ID NO: 26. In
another aspect, the nucleic acid probe is a polynucleotide sequence
that encodes SEQ ID NO: 27, or a subsequence thereof.
[0226] For long probes of at least 100 nucleotides in length, very
low to very high stringency and washing conditions are defined as
described supra.
[0227] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency and washing conditions are
defined as described supra.
[0228] In another aspect, the pyruvate carboxylase is encoded by a
polynucleotide comprising or consisting of a nucleotide sequence
having a degree of sequence identity to SEQ ID NO: 26 of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 91%, 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%, which encodes a polypeptide having
pyruvate carboxylase activity.
[0229] The pyruvate carboxylases may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the pyruvate carboxylase encoded by a
polynucleotide is produced by the source or by a cell in which the
polynucleotide from the source has been inserted.
[0230] In one aspect, the pyruvate carboxylase may be a bacterial,
a yeast, or a filamentous fungal pyruvate carboxylase obtained from
the microorganisms described herein.
[0231] In another aspect, the pyruvate carboxylase is an
Aspergillus oryzae pyruvate carboxylase, e.g., the Aspergillus
oryzae pyruvate carboxylase of SEQ ID NO: 27.
[0232] The pyruvate carboxylase may also be identified and obtained
from other sources including microorganisms isolated from nature
(e.g., soil, composts, water, etc.) or DNA samples obtained
directly from natural materials (e.g., soil, composts, water, etc,)
as described supra.
[0233] The pyruvate carboxylase can also include fused polypeptides
or cleavable fusion polypeptides, as described supra.
[0234] The pyruvate carboxylase can also be a variant of a
mitochondrial pyruvate carboxylase, such that in vivo importation
into the mitochondria is reduced thereby increasing the level of
the pyruvate carboxylase variant in the cytosol.
[0235] Techniques used to isolate or clone a polynucleotide
encoding a pyruvate carboxylase are described supra.
[0236] In one aspect, the isolated polynucleotide comprises or
consists of SEQ ID NO: 26. In another aspect, the isolated
polynucleotide encodes a pyruvate carboxylase comprising or
consisting of SEQ ID NO: 27. The present invention also encompasses
nucleotide sequences that encode polypeptides comprising or
consisting of the amino acid sequence of SEQ ID NO: 27, which
differ from SEQ ID NO: 26 by virtue of the degeneracy of the
genetic code. The present invention also relates to subsequences of
SEQ ID NO: 26 that encode fragments of SEQ ID NO: 27 that have
pyruvate carboxylase activity.
[0237] In another aspect, the isolated polynucleotide can be a
mutant polynucleotide comprising or consisting of at least one
mutation in SEQ ID NO: 26, in which the mutant nucleotide sequence
encodes SEQ ID NO: 27.
[0238] In another aspect, the isolated polynucleotide comprises or
consists of nucleotide sequences that have a degree of sequence
identity to SEQ ID NO: 26 of at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 91%, 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, which encode an active pyruvate carboxylase.
[0239] In another aspect, the isolated polynucleotide encoding a
pyruvate carboxylase hybridizes under at least very low stringency
conditions, e.g., low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
26, (ii) the cDNA sequence contained in SEQ ID NO: 26, or (iii) the
full-length complementary strand of (i) or (ii); or allelic
variants and subsequences thereof (Sambrook et al., 1989, supra),
as defined herein.
[0240] In another aspect, the isolated polynucleotide encoding a
pyruvate carboxylase is obtained by (a) hybridizing a population of
DNA under very low, low, medium, medium-high, high, or very high
stringency conditions with (i) SEQ ID NO: 26, (ii) the cDNA
sequence contained in SEQ ID NO: 26, or (iii) the full-length
complementary strand of (i) or (ii); and (b) isolating the
hybridizing polynucleotide, which encodes a polypeptide having
pyruvate carboxylase activity.
[0241] Other pyruvate carboxylases that can be used to practice the
present invention include, but are not limited to, a Aspergillus
ciavatus NRRL 1 pyruvate carboxylase (XP.sub.--001271664; Direct
Submission, Submitted (26 Oct. 2006), The Institute for Genomic
Research, 9712 Medical Center Drive, Rockville, Md. 20850, USA);
Aspergillus fumigatus Af293 pyruvate carboxylase (XP.sub.--752054;
Nierman et al., 2005, Nature 438: 1151-1156); Aspergillus nidulans
FGSC A4 pyruvate carboxylase (XP.sub.--662066; Galagan et al.,
2005, Nature 438: 1105-1115); Aspergillus niger pyruvate
carboxylase (An15g02820; Pel et al., 2007, Nature Biotechnology 25:
221-231; ASPNG 5061; Panneman et al., Submitted (July 1998) to the
EMBL/GenBank/DDBJ databases); Aspergillus terreus pyruvate
carboxylase (O93918; Direct Submission, Submitted (October 1998)
The Institute for Genomic Research, 9712 Medical Center Drive,
Rockville, Md. 20850, USA); Magnaporthe grisea 70-15 pyruvate
carboxylase (XP.sub.--367852; Direct Submission, Submitted (26 Sep.
2005) Broad Institute of MIT and Harvard, 320 Charles Street,
Cambridge, Mass. 02142, USA); Neurospora crassa OR74A pyruvate
carboxylase (XP.sub.--965636; Galagan et al., 2003, Nature 422:
859-868); Rhizopus oryzaepyruvate carboxylase (RO3G.sub.--06931.1);
Saccharomyces cerevisiae pyruvate carboxylase (NP.sub.--009777;
Gaffeau et al., 1996, Science 274: 546-547); Schizosaccharomyces
pompe pyruvate carboxylase (NP.sub.--595900; Direct Submission,
Submitted (29 Jun. 2007) European Schizosaccharomyces genome
sequencing project, Sanger Institute, The Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SA); and Ustilago maydis pyruvate
carboxylase (um01054; McCann and Snetselaar, 2008, Fungal Genetics
and Biology 45: S77-S87).
Nucleic Acid Constructs
[0242] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide encoding a C4
dicarboxylic acid transporter, an isolated polynucleotide encoding
a malate dehydrogenase, and/or an isolated polynucleotide encoding
a pyruvate carboxylase operably linked to one or more (several)
control sequences that direct the expression of the coding sequence
in a suitable host cell under conditions compatible with the
control sequences.
[0243] An isolated polynucleotide encoding a C4 dicarboxylic acid
transporter, a malate dehydrogenase, and/or a pyruvate carboxylase
may be manipulated in a variety of ways to provide for expression
of the polypeptide(s). Manipulation of a polynucleotide's sequence
prior to its insertion into a vector may be desirable or necessary
depending on the expression vector. The techniques for modifying
polynucleotide sequences utilizing recombinant DNA methods are well
known in the art.
[0244] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence that is recognized by a host cell
for expression of a polynucleotide encoding a C4 dicarboxylic acid
transporter, malate dehydrogenase, and/or a pyruvate carboxylase.
The promoter sequence contains transcriptional control sequences
that mediate the expression of the polypeptide. The promoter may be
any nucleotide sequence that shows transcriptional activity in the
host cell of choice including mutant, truncated, and hybrid
promoters, and may be obtained from genes encoding extracellular or
intracellular polypeptides either homologous or heterologous to the
host cell.
[0245] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs in a filamentous
fungal host cell are promoters obtained from the genes for
Aspergillus oryzae TAKA amylase, Aspergillus oryzae translation
elongation factor, Aspergillus oryzae phosphoglycerate kinase,
Aspergillus oryzae glycerol-3-phosphate dehydrogenase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase and Aspergillus nidulans triose phosphate
isomerase); and mutant, truncated, and hybrid promoters
thereof.
[0246] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator that is functional in the host cell of
choice may be used in the present invention.
[0247] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0248] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA that is important for translation
by the host cell. The leader sequence is operably linked to the 5'
terminus of the nucleotide sequence encoding the polypeptide. Any
leader sequence that is functional in the host cell of choice may
be used in the present invention.
[0249] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0250] The control sequence may also be a polyadenylation sequence;
a sequence operably linked to the 3' terminus of the nucleotide
sequence and, when transcribed, is recognized by the host cell as a
signal to add polyadenosine residues to transcribed mRNA. Any
polyadenylation sequence that is functional in the host cell of
choice may be used in the present invention.
[0251] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0252] The control sequence may also be a signal peptide coding
sequence that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
a cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding sequence naturally linked in translation reading frame with
the segment of the coding sequence that encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding sequence that is foreign to the
coding sequence. The foreign signal peptide coding sequence may be
required where the coding sequence does not naturally contain a
signal peptide coding sequence. Alternatively, the foreign signal
peptide coding sequence may simply replace the natural signal
peptide coding sequence in order to enhance secretion of the
polypeptide. However, any signal peptide coding sequence that
directs the expressed polypeptide into the secretory pathway of a
host cell of choice, i.e., secreted into a culture medium, may be
used in the present invention.
[0253] The control sequence may also be a propeptide coding
sequence that codes for an amino acid sequence positioned at the
amino terminus of a polypeptide. The resultant polypeptide is known
as a proenzyme or propolypeptide (or a zymogen in some cases). A
propeptide is generally inactive and can be converted to a mature
active polypeptide by catalytic or autocatalytic cleavage of the
propeptide from the propolypeptide. The propeptide coding sequence
may be obtained from the genes for Saccharomyces cerevisiae
alpha-factor, Rhizomucor miehei aspartic proteinase, and
Myceliophthora thermophila laccase (WO 95/33836).
[0254] Where both signal peptide and propeptide sequences are
present at the amino terminus of a polypeptide, the propeptide
sequence is positioned next to the amino terminus of a polypeptide
and the signal peptide sequence is positioned next to the amino
terminus of the propeptide sequence.
[0255] It may also be desirable to add regulatory sequences that
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those that cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
filamentous fungi include the TAKA alpha-amylase promoter,
Aspergillus niger glucoamylase promoter, and Aspergillus oryzae
glucoamylase promoter may be used as regulatory sequences. Other
examples of regulatory sequences are those that allow for gene
amplification. In eukaryotic systems, these regulatory sequences
include the dihydrofolate reductase gene that is amplified in the
presence of methotrexate, and the metallothionein genes that are
amplified with heavy metals. In these cases, the nucleotide
sequence encoding the polypeptide would be operably linked with the
regulatory sequence.
Expression Vectors
[0256] The present invention also relates to recombinant expression
vectors comprising a an isolated polynucleotide encoding a C4
dicarboxylic acid transporter, an isolated polynucleotide encoding
a malate dehydrogenase, and/or an isolated polynucleotide encoding
a pyruvate carboxylase, a promoter, and transcriptional and
translational stop signals. The various nucleic acids and control
sequences described herein may be joined together to produce a
recombinant expression vector that may include one or more
(several) convenient restriction sites to allow for insertion or
substitution of the nucleotide sequence encoding the polypeptide at
such sites. Alternatively, a polynucleotide sequence may be
expressed by inserting the nucleotide sequence or a nucleic acid
construct comprising the sequence into an appropriate vector for
expression. In creating the expression vector, the coding sequence
is located in the vector so that the coding sequence is operably
linked with the appropriate control sequences for expression.
[0257] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about expression of the nucleotide
sequence. The choice of the vector will typically depend on the
compatibility of the vector with the host cell into which the
vector is to be introduced. The vectors may be linear or closed
circular plasmids.
[0258] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0259] The vectors may contain one or more (several) selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0260] Examples of selectable markers for use in a filamentous
fungal host cell include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus.
[0261] The vectors may contain an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0262] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may contain additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements may contain a sufficient number of nucleic acids, such as
100 to 10,000 base pairs, e.g., 400 to 10,000 base pairs or 800 to
10,000 base pairs, which have a high degree of sequence identity to
the corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding nucleotide sequences. On the other hand,
the vector may be integrated into the genome of the host cell by
non-homologous recombination.
[0263] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0264] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0265] More than one copy of a polynucleotide may be inserted into
a host cell to increase production of the gene product. An increase
in the copy number of the polynucleotide can be obtained by
integrating at least one additional copy of the sequence into the
host cell genome or by including an amplifiable selectable marker
gene with the polynucleotide where cells containing amplified
copies of the selectable marker gene, and thereby additional copies
of the polynucleotide, can be selected for by cultivating the cells
in the presence of the appropriate selectable agent.
[0266] The procedures used to ligate the elements described above
to construct the recombinant expression vectors are well known to
one skilled in the art (see, e.g., Sambrook et al., 1989,
supra).
Host Cells
[0267] The present invention also relates to recombinant
filamentous fungal host cells, comprising an isolated
polynucleotide encoding a C4 dicarboxylic acid transporter, an
isolated polynucleotide encoding a malate dehydrogenase, and/or an
isolated polynucleotide encoding a pyruvate carboxylase, a
promoter, and transcriptional and translational stop signals, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising such a polynucleotide(s) is
introduced into a host cell so that the vector is maintained as a
chromosomal integrant or as a self-replicating extra-chromosomal
vector as described earlier. The term "host cell" encompasses any
progeny of a parent cell that is not identical to the parent cell
due to mutations that occur during replication. The choice of a
host cell will to a large extent depend upon the gene encoding the
polypeptide and its source.
[0268] The host cell may be any filamentous fungal cell useful in
the recombinant production of C4 dicarboxylic acids.
[0269] "Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK). The
filamentous fungi are generally characterized by a mycelial wall
composed of chitin, cellulose, glucan, chitosan, mannan, and other
complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.
[0270] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus,
Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trametes, or Trichoderma cell.
[0271] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus flavus, Aspergillus
fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus
nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus
parasiticus, or Aspergillus sojae cell. In another most preferred
aspect, the filamentous fungal host cell is a Fusarium
bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium
culmorum, Fusarium graminearum, Fusarium graminum, Fusarium
heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium
reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium
sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,
Fusarium torulosum, Fusarium trichothecioides, or Fusarium
venenatum cell. In another most preferred aspect, the filamentous
fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis
gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa,
Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,
Chrysosporium merdarium, Chrysosporium inops, Chrysosporium
pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum,
Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola
lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillium purpurogenum, Phanerochaete chrysosporium,
Phiebia radiata, Pleurotus eryngii, Rhizopus oryzae, Thielavia
terrestris, Trametes villosa, Trametes versicolor, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride cell.
[0272] In a preferred aspect, the Aspergillus host cell is
Aspergillus oryzae.
[0273] Filamentous fungal cells may be transformed by a process
involving protoplast formation, transformation of the protoplasts,
and regeneration of the cell wall in a manner known per se.
Suitable procedures for transformation of Aspergillus and
Trichoderma host cells are described in U.S. Pat. No. 5,536,661 and
Yelton et al., 1984, Proceedings of the National Academy of
Sciences USA 81: 1470-1474. Suitable methods for transforming
Fusarium species are described by Malardier et al., 1989, Gene 78:
147-156, and U.S. Pat. No. 5,837,847.
[0274] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
[0275] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Fungal Strains
[0276] Aspergillus oryzae NRRL 3488 (or ATCC 56747) was used as
source of a C4 dicarboxylic acid transporter gene, a pyruvate
carboxylase gene, and malate dehydrogenase genes, mdh1 and mdh3,
and for production of malic acid.
Media
[0277] YEG medium was composed of 20 g glucose, 5 g yeast extract,
and deionized water to 1 liter.
[0278] COVE plates were composed of 1 M sucrose, 2% COVE salt
solution, 10 mM acetamide, 15 mM CsCl, and 25 g/l Agar Noble.
[0279] COVE salt solution was composed of 26 g KCl, 26 g
MgSO.sub.4.7H.sub.2O, 76 g KH.sub.2PO.sub.4, 50 ml of COVE trace
elements solution, and deionized water to 1 liter.
[0280] COVE trace elements solution was composed of 0.04 g
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.04 g CuSO.sub.4.5H.sub.2O, 1.2
g FeSO.sub.4.7H.sub.2O, 0.7 g MnSO.sub.4.H.sub.2O, 0.8 g
Na.sub.2MoO.sub.2.2H.sub.2O, 10 g ZnSO.sub.4.7H.sub.2O and
deionized water to 1 liter.
[0281] Seed medium B was composed of 30 g glucose, 3 g Bacto
Peptone, 560 mg KH.sub.2PO.sub.4, 560 mg K.sub.2HPO.sub.4, 925 mg
NaH.sub.2PO.sub.4.H.sub.2O, 820 mg Na.sub.2HPO.sub.4, 75 mg
MgSO.sub.4.7H.sub.2O, 75 mg CaCl.sub.2.H.sub.2O, 0.75 ml of
1000.times. Micronutrient Solution, and deionized water to 1
liter.
[0282] Acid production medium C was composed of 100 g glucose, 80 g
CaCO.sub.3, 6 g Bacto Peptone, 150 mg KH.sub.2PO.sub.4, 150 mg
K.sub.2HPO.sub.4, 100 mg MgSO.sub.4.7H.sub.2O, 100 mg
CaCl.sub.2.H.sub.2O, 1 ml 1000.times. Micronutrient Solution, and
deionized water to 1 liter.
[0283] 1000.times. Micronutrient Solution was composed of 5 g NaCl,
5 g FeSO.sub.4.7H.sub.2O, 1 g citric acid, and deionized water to 1
liter.
[0284] PDA plates were composed of 39 g/l potato dextrose agar.
[0285] 2XYT+amp plates were composed of 16 g tryptone, 10 g yeast
extract, 5 g NaCl, 100 mg ampicillin, 15 g Bacto agar, and
deionized water to 1 liter.
Example 1
Cloning of the Aspergillus oryzae NRRL 3488 C4 Dicarboxylic Acid
Transporter Gene Mae3 and Construction of Expression Vectors
pShTh104/mae3
[0286] The malic acid transporter gene, mae3, was cloned from
Aspergillus oryzae NRRL 3488 genomic DNA by PCR amplification using
primers homologous to Aspergillus oryzae ATCC 42149 predicted C4
dicarboxylic acid transporter gene model number AO090023000318
(Galagan et al., 2005, Nature 438: 1105-1115).
[0287] Genomic DNA from Aspergillus oryzae NRRL 3488 was isolated
by inoculating 100 ml of YEG medium in a shake flask with
2.times.10.sup.6 spores and incubating the flask at 37.degree. C.
overnight with shaking at 200 rpm. The mycelia were harvested by
filtration using a MIRACLOTH.RTM. (Calbiochem, San Diego, Calif.,
USA) lined funnel and approximately 2 g of mycelia were recovered
and frozen in liquid nitrogen. The mycelia were disrupted by
grinding in a cold mortar and pestle. Genomic DNA was isolated from
the powdered mycelia using a DNeasy.RTM. Plant Maxi Kit (QIAGEN
Inc., Valencia, Calif., USA) according to the manufacturer's
instructions. The Aspergillus oryzae mae3 gene was amplified using
primer 065266 and primer 065267 shown below.
TABLE-US-00002 Primer 065266: (SEQ ID NO: 1)
5'-GTGATAGAACATCGTCCATAATGCTGACACCTCCCAAGTT-3' Primer 065267: (SEQ
ID NO: 2) 5'-AGTCACCTCTAGTTAATTAATTACTAATCAGATACATCCTCAT-3'
[0288] The amplification reactions were performed using an
EXPAND.RTM. High Fidelity PCR System (Roche, Indianapolis, Ind.,
USA) according to manufacturer's instructions. Each PCR reaction
contained 47 ng of Aspergillus oryzae NRRL 3488 genomic DNA, 200
.mu.M dNTPs, 50 pM of primer 065266, 50 pM primer 065267, 1.times.
EXPAND.RTM. reaction buffer (Roche, Indianapolis, Ind., USA), and
2.6 units of EXPAND.RTM. High Fidelity enzyme mix (Roche,
Indianapolis, Ind., USA) in a final volume of 50 .mu.l. The
amplification reactions were incubated in an EPPENDORF.RTM.
MASTERCYCLER.RTM. (Eppendorf Scientific Inc., Westbury, N.Y., USA)
programmed for 1 cycle at 94.degree. C. for 2 minutes; 30 cycles
each at 94.degree. C. for 15 seconds, 60.degree. C. for 30 seconds,
and 72.degree. C. for 1 minute; and 1 cycle at 72.degree. C. for 7
minutes.
[0289] The PCR product was purified by 1% agarose gel
electrophoresis using 50 mM Tris base-50 mM acetate-0.1 mM disodium
EDTA (TAE) buffer. A fragment of approximately 1.1 kb was excised
from the gel and extracted from the agarose using a QIAQUICK.RTM.
Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA). DNA
sequence analysis was used to confirm the integrity of the mae3
coding sequence using primers 996270, 065067, 065130, 065129 shown
below.
TABLE-US-00003 Primer 996270: (SEQ ID NO: 3)
5'-CTATAGCGAAATGGATTGATTGTCT-3' Primer 065067: (SEQ ID NO: 4)
5'-TGACCTTCCACGCTGACCAC-3' Primer 065130: (SEQ ID NO: 5)
5'-CTAATCAGATACATCCTCA-3' Primer 065129: (SEQ ID NO: 6)
5'-ATGCTGACACCTCCCAAGT-3'
[0290] The 1.1 kb fragment was then cloned into Sex AI/Mung bean
nuclease and Pac I digested pShTh60 (FIG. 1) using an IN-FUSION.TM.
Cloning Kit (Clontech, Mountain View, Calif., USA) according to the
manufacturer's instructions resulting in plasmid pShTh104 (FIG. 2).
Plasmid pShTh60 is an expression vector comprising the Aspergillus
oryzae Pgk promoter and the Aspergillus niger glucoamylase
terminator. Plasmid pShTh104 was isolated using a QIAfilter Maxi
Plasmid Isolation Kit (QIAGEN Inc., Valencia, Calif., USA).
Example 2
Characterization of Aspergillus oryzae NRRL 3488 mae3 C4
Dicarboxylic Acid Transporter Gene
[0291] DNA sequencing of the Aspergillus oryzae NRRL 3488 C4
dicarboxylic acid transporter gene mae3 was performed with an
ABI3130XL DNA Analyzer (Applied Biosystems, Inc., Foster City,
Calif., USA) using the primer walking technique with dye-terminator
chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60).
[0292] The nucleotide sequence (SEQ ID NO: 7) and deduced amino
acid sequence (SEQ ID NO: 8) of the Aspergillus oryzae NRRL 3488 C4
dicarboxylic acid transporter mae3 gene are shown in FIG. 3. The
genomic coding sequence of 1143 bp (including stop codon) encodes a
polypeptide of 380 amino acids with a predicted mass of 42 kDa. The
gene contains no introns.
[0293] A comparative pairwise global alignment of amino acid
sequences in public databases was determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of EMBOSS
with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix. The alignment showed that the deduced amino acid
sequence of the Aspergillus oryzae NRRL 3488 C4 dicarboxylic acid
transporter mae3 gene shares 29.5% sequence identity to the deduced
amino acid sequence of the Schizosaccharomyces pombe C4
dicarboxylic acid transporter gene (mae1; GenBank accession number
U21002).
Example 3
Transformation of pShTh104 into Aspergillus oryzae NRRL 3488
[0294] Protoplasts of Aspergillus oryzae NRRL 3488 were prepared by
inoculating approximately 2.times.10.sup.7 spores into 100 ml of
YEG medium and incubating the flask at 27.degree. C. for 16-18
hours at 140 rpm. Mycelia were collected by pouring the culture
through a sterile funnel lined with MIRACLOTH.RTM. and rinsing with
50 ml of 0.7 M KCl. The washed mycelia were resuspended in a 125 ml
flask containing 20 ml of protoplasting solution composed of 5 mg
of GLUCANEX.TM. (Novozymes A/S, Bagsv.ae butted.rd, Denmark) and
0.5 mg of chitinase (Sigma Chemical Co., St. Louis, Mo., USA) per
ml of 0.7 M KCl (filter sterilized) and incubated at 34.degree. C.
for 30 minutes with mixing at 80 rpm. The protoplasting solution
was poured through a sterile funnel lined with MIRACLOTH.RTM. and
rinsed with 50 ml of STC buffer (1 M sorbitol-10 mM Tris-HCl pH
6.5-10 mM CaCl.sub.2). The flow-through was collected in two 50 ml
polypropylene tubes, which were centrifuged at 1300.times.g for 10
minutes at room temperature. The supernatants were discarded and
the protoplast pellets were resuspended in 20 ml of STC buffer. The
protoplasts were washed by two rounds of resuspending the pellets
in 20 ml of STC buffer and centrifugation at 1300.times.g for 10
minutes at room temperature. The final pellets were resuspended in
2 ml of STC buffer. The protoplasts were counted by removing a 10
.mu.l sample and counting them in a hemacytometer (VWR, West
Chester, Pa., USA). The volume was adjusted with STC buffer to
obtain a protoplast concentration of 2.times.10.sup.7 per ml.
[0295] Six transformation reactions were prepared for each
expression vector. For each transformation reaction, 100 .mu.l of
protoplast preparation were transferred to a 12 ml polypropylene
tube. Five micrograms of pShTh104 and 250 .mu.l of polyethylene
glycol (PEG) were added and gently mix by rotating the tube. The
reaction was incubated at 37.degree. C. for 30 minutes.
Transformations were diluted with 3 ml of STC buffer and the entire
amounts were plated onto COVE plates. The plates were incubated at
30.degree. C. for 7-10 days. Thirty transformants of the resulting
transformants were transferred to individual COVE plates and
incubated at 34.degree. C. for 5 days. Spore stocks were prepared
by collecting the spores in 0.1% TWEEN.RTM. 80. Cultures were
stored by preparing a glycerol stock of each (800 .mu.l spore
stock, 200 .mu.l 0.1% TWEEN.RTM. 80) and frozen at -80.degree.
C.
Example 4
Production of Malic Acid in Shake Flask Cultures
[0296] Spores from each transformant described in Example 3 and
Aspergillus oryzae NRRL 3488 as a control were plated onto
individual COVE plates and allowed to sporulate at 34.degree. C.
for 5 to 7 days. Spores were collected in 0.1% TWEEN.RTM. 80 and
counted using a hemacytometer. Seed cultures were prepared in 250
ml flasks containing 100 ml of seed medium B and inoculated with
2.times.10.sup.8 total spores. Seed cultures were grown for
approximately 17 hours at 30.degree. C. with shaking at 200 rpm.
Acid production cultures were prepared in 250 ml unbaffled flasks
containing 50 ml of acid production medium C and 3 ml of the 17
hour seed cultures. Cultures were incubated at 30.degree. C. with
shaking at 200 rpm for 2-10 days.
Example 5
HPLC Quantitation of Malic Acid of Shake Flask Cultures
[0297] Quantitation of malic acid for the shake flask cultures of
Example 4 was performed by Reverse Phase High Pressure Liquid
Chromatography (RP-HPLC) using an 1200 Series Binary LC System and
1200 Series Diode Array Detector (DAD) (Agilent Technologies, Santa
Clara, Calif. USA). Reverse phase separation was performed using an
Aqua 5.mu. C18 125 .ANG. 205.times.4.6 mm ID column and AQ C18
4.times.3.0 mm Security Guard Cartridge (Phenomenex, Inc.,
Torrance, Calif., USA). The mobile phase consisted of 10% methanol
(HPLC grade) and 90% 145 mM phosphate pH 1.5 buffer.
[0298] Whole culture samples were removed and diluted 1:10 in HPLC
Running Buffer composed of 850 ml of 64 mM phosphate buffer and 150
ml of methanol pH 1.65. The samples were then filtered through a 25
mm 0.45 micron polyethersulfone membrane (Whatman, Florham Park,
N.J., USA) and 1.5 ml of the filtrates were placed into HPLC vials
for acid analysis. The remaining amount of the shake flask cultures
were filtered through 3 layers of cheese cloth and rinsed three
times with 10 volumes of double distilled sterile water to remove
insoluble CaCO.sub.3. Cell pellets were harvested from the cheese
cloth, placed into a 15 ml culture tube and stored at -20.degree.
C.
[0299] RP-HPLC was performed using an injection volume of 10 .mu.l
at a flow rate of 0.7 ml/minute (isocratic) with a column
temperature of 25.degree. C. and run time of 11 minutes. Detection
was set at 210 nm, 8 nm bandwidth, with the reference at 360 nm, 40
nm bandwidth. The void time was determined to be 3.8 minutes. The
quantitative capabilities of the reverse phase method were
determined for malic acid by performing replicate injections of
serially diluted malic acid standards with concentrations ranging
from 49.2-3.93 mM. The relative standard deviation (RSD) for
replicate injections was .ltoreq.5%. Malic acid showed
R.sup.2.gtoreq.0.9999
[0300] Table 1 shows the relative increase in malic acid titer of
transformants Aspergillus oryzae ShTh1040-8 and Aspergillus oryzae
ShTh1040-28 compared to malic acid production of Aspergillus oryzae
NRRL 3488 as a control after 5 days of shake flask growth.
Aspergillus oryzae ShTh1040-8 and Aspergillus oryzae ShTh1040-28,
each containing the heterologous mae3 C4 dicarboxylic acid
transporter gene, produced a 2.1-fold and 2.2-fold increase in
malic acid titer, respectively, compared to Aspergillus oryzae NRRL
3488.
TABLE-US-00004 TABLE 1 Relative titer Strain of malic acid % CV
NRRL 3488 1 2.70% ShTh1040-8 2.1 12.60% ShTh1040-28 2.2 6.10%
Example 6
Fermentation of Aspergillus oryzae ShTh1040 Strains
[0301] Aspergillus oryzae transformants designated ShTh1040-8,
ShTh1040-28, and Aspergillus oryzae NRRL 3488 (control) were grown
for approximately 7 days at 32.degree. C. on PDA plates. A 5-6 ml
volume of sterile 50 mM sodium phosphate buffer (pH 6.8) containing
0.1% TWEEN.RTM. 80 was added to each plate and spores were
suspended by scraping with an inoculating loop. Each suspension was
transferred by pipette to a 50 ml conical tube. For each tube, 25
ml of sterile sodium phosphate buffer was added to a 500 ml
unbaffled flask containing 75 ml of seed medium, which was then
inoculated with 2 ml of spore suspension. The seed medium was
composed of 40 g glucose, 4.0 g (NH.sub.4).sub.2SO.sub.4, 0.75 g
KH.sub.2PO.sub.4, 0.75 g K.sub.2HPO.sub.4, 0.1 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, and deionized water to 1 liter.
The flasks were then incubated at 32.degree. C. and 180 rpm for
about 24 hours. Three seed flasks were combined to supply the 144
ml inoculum required per tank.
[0302] Three-liter fermentors containing 1.8 liters of medium were
individually inoculated by introducing 144 ml (8%) of the seed
culture broth from three combined seed flasks of either Aspergillus
oryzae transformant ShTh1040-8, Aspergillus oryzae transformant
ShTh1040-28, or Aspergillus oryzae NRRL 3488. The medium was
composed of 120 g glucose, 90 g CaCO.sub.3, 6 g Bacto peptone,
0.150 g KH.sub.2PO.sub.4, 0.150 g K.sub.2HPO.sub.4, 0.10 g
MgSO.7H.sub.2O, 0.10 g CaCl.sub.2-2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, and deionized water to 1
liter.
[0303] The fermentors were equilibrated at 32.+-.0.1.degree. C. and
stirred at 500 rpm. Inlet air flow was maintained at 1 v/v/m. No
acid or base additions were used for pH control.
[0304] Samples were withdrawn daily and analyzed for malic acid
production. Fermentations were completed after 7 days.
Example 7
HPLC Quantitation of Malic Acid of Fermentations
[0305] Quantitation of malic acid for the fermentations of Example
6 was performed as described in Example 5.
[0306] Table 2 shows the relative increase in malic acid titer of
transformants Aspergillus oryzae ShTh1040-8 and Aspergillus oryzae
ShTh1040-28 compared to malic acid production of Aspergillus oryzae
NRRL 3488 as a control. Aspergillus oryzae ShTh1040-8 and
Aspergillus oryzae ShTh1040-28, each containing the heterologous
mae3 C4 dicarboxylic acid transporter gene, produced a 1.98-fold
and 2.18-fold increase in malic acid titer, respectively, compared
to Aspergillus oryzae NRRL 3488.
TABLE-US-00005 TABLE 2 Relative Malic Transformant Acid Titer NRRL
3488 1.00 ShTh1040-28 1.98 ShTh1040-8 2.18
Example 8
Cloning of the Aspergillus oryzae NRRL 3488 Malate Dehydrogenase
Genes and Construction of Expression Vectors pShTh71 and
pShTh73
[0307] Malate dehydrogenase genes mdh1 and mdh3 were cloned from
Aspergillus oryzae NRRL 3488 genomic DNA by PCR amplification using
primers homologous to the mdh1 and mdh3 gene models
(AO090005000438, AO090701000013, respectively) found in the
published Aspergillus oryzae ATCC 42149 genome sequence (Galagan et
al., 2005, Nature 438: 1105-1115).
[0308] The Aspergillus oryzae mdh1 gene was amplified using primers
062390 and 062391, and the Aspergillus oryzae mdh3 gene was
amplified using primers 062388 and 062389 shown below. Aspergillus
oryzae NRRL 3488 was grown by inoculating 100 ml of YEG medium in a
shake flask with 2.times.10.sup.6 spores and incubating the flask
at 37.degree. C. overnight with shaking at 200 rpm. The mycelia
were harvested using a MIRACLOTH.RTM. lined funnel and
approximately 2 g of tissue was recovered and frozen in liquid
nitrogen. The mycelia were disrupted by grinding in a cold mortar
and pestle. Genomic DNA was isolated from the powdered mycelia
using a DNeasy.RTM. Plant Maxi Kit according to the manufacturer's
instructions.
TABLE-US-00006 Primer 062390: (SEQ ID NO: 9)
5'-ACACAACTGGCCATGTTCGCTGCTCGCCAGTCTTTCAACCTCCTCCA GA-3' Primer
062391: (SEQ ID NO: 10)
5'-AGTCACCTCTAGTTAATTAATTATTAAGGGTTGGCCTTGACGAAGTC AATACCCTTCTGA-3'
Primer 062388: (SEQ ID NO: 11)
5'-ACACAACTGGCCATGGTCAAAGCTGGTGAGTTAGCAATCCTTAACAG AT-3' Primer
062389: (SEQ ID NO: 12)
5'-AGTCACCTCTAGTTAATTAATTATTACTTTGGTGGTGGGTTCTTAAC
GAAGTCGATGCCT-3'
[0309] The amplification reactions were performed using an
EXPAND.RTM. High Fidelity PCR System according to the
manufacturer's instructions. Each PCR reaction contained 47 ng of
Aspergillus oryzae NRRL 3488 genomic DNA, 200 .mu.M dNTPs, 50 pM
forward primer, 50 pM reverse primer, 1.times. EXPAND.RTM. reaction
buffer, and 2.6 units of EXPAND.RTM. High Fidelity enzyme mix in a
final volume of 50 .mu.l. The amplification reactions were
incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. programmed for 1
cycle at 94.degree. C. for 2 minutes; 10 cycles each at 94.degree.
C. for 15 seconds, 62.2.degree. C. for 30 seconds, and 72.degree.
C. for 1 minute; 20 cycles each at 94.degree. C. for 15 seconds,
62.2.degree. C. for 30 seconds, and 72.degree. C. for 1 minute plus
0.5 second for each successive cycle; and 1 cycle at 72.degree. C.
for 7 minutes.
[0310] PCR products from each of the amplification reactions were
purified by 1% agarose gel electrophoresis in TAE buffer (50 mM
Tris base-50 mM acetate-0.1 mM disodium EDTA). A fragment of
approximately 1.3 kb from each amplification reaction was excised
from the gels and agarose extracted using a QIAQUICK.RTM. Gel
Extraction Kit. DNA sequence analysis employing an ABI3130XL DNA
Analyzer was used to confirm the integrity of the mdh1 and mdh3
coding sequences using primers 62399, 62400, 62396, 62393 shown
below.
TABLE-US-00007 Primer 62399: (SEQ ID NO: 13)
5'-CTTTGGTGTCACCACACTGG-3' Primer 62400: (SEQ ID NO: 14)
5'-GGGATTTGAACAGCAGAAGG-3' Primer 62396: (SEQ ID NO: 15)
5'-CTTAGCAAGGTCGCGGACAATGG-3' Primer 62393: (SEQ ID NO: 16)
5'-GGCACTGGGAATTGAATAC-3'
[0311] Each 1.3 kb fragment was then cloned into Nco I and Pac I
digested pBM120a (WO 2008/008950) using an In-Fusion.TM. Cloning
Kit according to the manufacturer's instructions resulting in
plasmids pShTh73 (FIG. 4) and pShTh71 (FIG. 6) for mdh1 and mdh3,
respectively. Plasmid pBM120a is an expression vector comprising
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus nidulans
triose phosphate isomerase) and the Aspergillus niger glucoamylase
terminator. Plasmids pShTh71 and pShTh73 were isolated using a
QIAfilter Maxi Plasmid Isolation Kit.
Example 9
Characterization of Aspergillus oryzae NRRL 3488 Malate
Dehydrogenase Genes
[0312] DNA sequencing of the Aspergillus oryzae NRRL 3488 malate
dehydrogenase mdh1 and mdh3 genes was performed with an ABI3130XL
DNA Analyzer using the primer walking technique with dye-terminator
chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60).
[0313] The nucleotide sequence (SEQ ID NO: 17) and deduced amino
acid sequence (SEQ ID NO: 18) of the Aspergillus oryzae NRRL 3488
malate dehydrogenase mdh1 gene are shown in FIG. 5. The genomic
coding sequence of 1294 bp (including stop codon) encodes a
polypeptide of 340 amino acids with a predicted mass of 36 kDa. The
coding sequence is interrupted by 4 introns of 85 bp (67-151 bp),
73 bp (270-342 bp), 60 bp (493-552 bp), and 53 bp (648-700 bp). The
G+C content of the mdh1 gene is 56.5% and 60.4% for the coding
region.
[0314] A comparative pairwise global alignment of amino acid
sequences in public databases was determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of EMBOSS
with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix. The alignment showed that the deduced amino acid
sequence of the Aspergillus oryzae NRRL 3488 malate dehydrogenase
mdh1 gene shares 61.9% sequence identity (excluding gaps) to the
deduced amino acid sequence of the Saccharomyces cerevisiae malate
dehydrogenase gene (MDH1; accession number YK1085W).
[0315] The nucleotide sequence (SEQ ID NO: 19) and deduced amino
acid sequence (SEQ ID NO: 20) of the Aspergillus oryzae NRRL 3488
malate dehydrogenase mdh3 gene are shown in FIG. 7. The genomic
coding sequence of 1430 bp (including stop codon) encodes a
polypeptide of 330 amino acids with a predicted mass of 35 kDa. The
coding sequence is interrupted by 7 introns of 57 bp (14-70 bp), 70
bp (103-172 bp), 74 bp (284-357 bp), 68 bp (446-513 bp), 58 bp
(892-949 bp), 48 bp (1035-1082 bp), and 62 bp (1228-1289 bp). The
G+C content of the coding region of the mdh3 gene is 50.3%.
[0316] A comparative pairwise global alignment of amino acid
sequences in public databases was determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of EMBOSS with gap open penalty
of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix. The
alignment showed that the deduced amino acid sequence of the
Aspergillus oryzae NRRL 3488 malate dehydrogenase mdh3 gene shares
47.8% sequence identity (excluding gaps) to the deduced amino acid
sequence of the Saccharomyces cerevisiae malate dehydrogenase gene
(MDH3; accession number YD1078C).
Example 10
Construction of Plasmids pShTh74 and pShTh75
[0317] Plasmid pShTh74 was constructed to contain a deletion of a
putative mitochondrial targeting sequence encoding amino acids 2 to
17 of Aspergillus oryzae NRRL 3488 MDH1. Plasmid pShTh75 was
constructed to contain a deletion of the predicted mitochondrial
targeting sequence encoding amino acids 2 to 17 and a substitution
of R48Y of MDH1. Both mutations are intended to prevent mdh1 gene
product from being targeted to and imported into the mitochondria,
so the malate dehydrogenase variant is localized to the
cytoplasm.
[0318] Plasmid pShTh74 was constructed by PCR amplifying the mdh1
gene from Aspergillus oryzae NRRL 3488 genomic DNA using
oligonucleotide primers 063183 shown below and primer 062391
(Example 8).
TABLE-US-00008 Primer 063183: (SEQ ID NO: 21)
5'-ACACAACTGGCCATGGCCTCTGCCAGCCAGGTGTG-3'
[0319] The amplification reaction was composed of 47 ng Aspergillus
oryzae NRRL 3488 genomic DNA, 200 .mu.M dNTPs, 50 pM forward
primer, 50 pM reverse primer, 1.times. EXPAND.RTM. reaction buffer,
and 2.6 units EXPAND.RTM. High Fidelity enzyme mix. The
amplification reaction was incubated in an EPPENDORF.RTM.
MASTERCYCLER.RTM. programmed for 1 cycle at 94.degree. C. for 3
minutes; 29 cycles each at 94.degree. C. for 15 seconds,
62.2.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and
1 cycle at 72.degree. C. for 7 minutes.
[0320] PCR products were purified by 1% agarose gel electrophoresis
in TAE buffer. A 1.2 kb fragment was excised from the gel and
agarose extracted using a QIAQUICK.RTM. Gel Extraction Kit, and
then cloned into Nco I and Pac I digested pBM120a (WO 2008/008950)
using an In-Fusion.TM. Cloning Kit resulting in plasmid pShTh74.
DNA sequence analysis employing an ABI3130XL DNA Analyzer was used
to confirm the integrity of the mdh1 DNA fragment.
[0321] Plasmid pShTh74 was mutagenized to pShTh75 using a
QUIKCHANGE.RTM. II SL Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, Calif., USA) to contain amino acid mutation R48Y using
primers 063184 and 063186 shown below. Mutation of the arginine
residue to tyrosine results in disruption of an amphipathic helix
that assists in targeting and transporting proteins across the
mitochondrial membrane.
TABLE-US-00009 Primer 063184: (SEQ ID NO: 22)
5'-CCTCAAGCTCAACCCCTACGTTTCTGAGCTTGCCCTCTAC-3' Primer 063186: (SEQ
ID NO: 23) 5'-GTAGAGGGCAAGCTCAGAAACGTAGGGGTTGAGCTTGAGG-3'
[0322] Plasmids pShTh74 and pShTh75 were isolated using a QIAfilter
Maxi Plasmid Isolation Kit.
Example 11
Construction of Expression Vector pSaMF21
[0323] Plasmid pSaMF21 was designed to contain the NAD-dependent
malate dehydrogenase (mdh3) gene sequence (DOGAN: AO090701000013),
a 1430 bp fragment from Aspergillus oryzae. The plasmid was
constructed by linearizing pShTh60 (FIG. 1) by restriction
digestion with Sex AI and Pac I. The digested vector was separated
by 0.8% agarose gel electrophoresis in TBE buffer and purified
using a QIAQUICK.RTM. Gel Extraction Kit. The mdh3 gene was
amplified from pShTh71 using primers 067522 and 067525.
TABLE-US-00010 Primer 067522: (SEQ ID NO: 28)
5'-AGAACATCGTCCATAATGGTCAAAGCTGGTGAGTTA-3' Primer 067525: (SEQ ID
NO: 29) 5'-GTGTCAGTCACCTCTAGTTATTACTTTGGTGGTGGGTTCT-3'
[0324] The PCR reaction was composed of 5 .mu.l 10.times. reaction
buffer, 1 .mu.l pShTh71 template (87 ng/.mu.l), 1 .mu.l primer
067522 (100 ng/.mu.l), 1 .mu.l primer 067525 (100 ng/.mu.l), 1
.mu.l dNTP mixture (10 mM), 45.5 .mu.l deionized water, and 0.5
.mu.l Herculase.RTM. HotStart DNA polymerase (Stratagene, La Jolla,
Calif., USA). The amplification reaction was incubated in an
EPPENDORF.RTM. MASTERCYCLER.RTM. programmed for 1 cycle at
95.degree. C. for 2 minutes; 10 cycles each at 95.degree. C. for 10
seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1.5
minutes; 20 cycles each at 95.degree. C. for 10 seconds, 50.degree.
C. for 30 seconds, and 72.degree. C. for 1.5 minutes plus 10
seconds per cycle. The PCR reaction was subjected to a restriction
digest with Dpn I for 1 hour to degrade any plasmid DNA template.
The PCR product was then purified using the MinElute.RTM. PCR
Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purified
PCR product was inserted into the vector using an In-Fusion.TM.
Advantage reaction composed of 2 .mu.l 5.times. buffer, 0.5 .mu.l
purified PCR product (110 ng/.mu.l), 1.7 .mu.l gel-purified Sex AI
and Pac I restriction digested pShTh60 (FIG. 1; 78 ng/.mu.l), 1
.mu.l In-Fusion.TM. enzyme and 4.8 .mu.l deinonized water. The
reaction was incubated at 37.degree. C. for 15 minutes followed by
50.degree. C. for 15 minutes after which it was placed on ice for 5
minutes and diluted with 40 .mu.l TE buffer resulting in pSaMF21
(FIG. 8). A 2 .mu.l aliquot of the ligation reaction was
transformed into ONE SHOT.RTM. TOP10 chemically competent E. coli
cells (Invitrogen, San Diego, Calif., USA) according to the
manufacturer's instructions. Transformants were plated onto
2XYT+amp plates and incubated at 37.degree. C. overnight. The
resulting transformants were picked and subjected to DNA sequencing
to confirm that the mdh3 gene was successfully integrated into the
vector.
Example 12
Cloning of the Aspergillus oryzae NRRL 3488 and Aspergillus oryzae
ATCC 56747 Pyruvate Carboxylase Genes
[0325] Pyruvate carboxylase genes (pyc) were cloned from
Aspergillus oryzae NRRL 3488 and Aspergillus oryzae ATCC 56747
genomic DNA by PCR amplification using primers homologous to the
putative pyruvate carboxylase gene model number AO090023000801
found in the published Aspergillus oryzae ATCC 42149 genome
sequence (Galagan et al., 2005, supra; DDBJ accession numbers
AP007150-AP007177) (uniprot accession number Q2UGL1).
[0326] The Aspergillus oryzae pyc genes were amplified using
primers 061929 and 061930 shown below. Aspergillus oryzae (NRRL
3488 and ATCC 56747) genomic DNA was isolated as described in
Example 1.
TABLE-US-00011 Primer 061929: (SEQ ID NO: 24)
5'-ACACAACTGGCCATGGCGGCTCCGTTTCGTCA-3' Primer 061930 (SEQ ID NO:
25) 5'-AGTCACCTCTAGTTAATTAATTATTACGCTTTGAC GATCTTGCAG-3'
[0327] The amplification reactions were performed using an
EXPAND.RTM. High Fidelity PCR System according to manufacturer's
instructions. The amplification reactions were composed of 47 ng
Aspergillus oryzae genomic DNA, 200 .mu.M dNTPs, 50 pM forward
primer, 50 pM reverse primer, 1.times. EXPAND.RTM. reaction buffer,
and 2.6 units EXPAND.RTM. High Fidelity enzyme mix in a final
volume of 50 .mu.l. The amplification reactions were incubated in
an EPPENDORF.RTM. MASTERCYCLER.RTM. programmed for 1 cycle at
94.degree. C. for 2 minutes; 10 cycles each at 94.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; 15 cycles each at 94.degree. C. for 15 seconds, 60.degree.
C. for 30 seconds, and 72.degree. C. for 1 minute plus 0.5 second
each successive cycle; and 1 cycle at 72.degree. C. for 7
minutes.
[0328] PCR products from the amplification reactions were purified
by 1% agarose gel electrophoresis in TAE buffer. Each 3.5 kb
fragment was excised from the gels and agarose extracted using a
QIAQUICK.RTM. Gel Extraction Kit.
Example 13
Characterization of the Aspergillus oryzae NRRL 3488 and ATCC 56747
Pyruvate Carboxylase Genes
[0329] DNA sequencing of the Aspergillus oryzae NRRL 3488 and ATCC
56747 pyruvate carboxylase genes (pyc) was performed with an
ABI3130XL DNA Analyzer using the primer walking technique with
dye-terminator chemistry (Giesecke et al., 1992, supra).
[0330] The nucleotide sequence (SEQ ID NO: 26) and deduced amino
acid sequence (SEQ ID NO: 27) of the Aspergillus oryzae pyruvate
carboxylase genes are shown in FIGS. 9A and 9B. Both the
Aspergillus oryzae NRRL 3488 and ATCC 56747 pyruvate carboxylase
genes have the same nucleotide sequence. The genomic coding
sequence of 3643 bp (including one stop codon) encodes a
polypeptide of 1193 amino acids with a predicted mass of 131 kDa.
The coding sequence is interrupted by 1 intron of 61 bp (3475-3535
bp). The G+C content of the coding region of the gene is 57.1%.
[0331] A comparative pairwise global alignment of amino acid
sequences in public databases was determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of EMBOSS with gap open penalty
of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix. The
alignment showed that the deduced amino acid sequence of the
Aspergillus oryzae NRRL 3488 or ATCC 56747 pyruvate carboxylase pyc
gene shares 68.4% sequence identity to the deduced amino acid
sequence of the Saccharomyces cerevisiae pyruvate carboxylase gene
(PYC1; accession number YG1062W).
Example 14
Construction of Expression Vector pRyan1
[0332] Plasmid pRyan1 was constructed to contain the pyruvate
carboxylase (pyc) gene sequence (DOGAN: AO090023000801), a 3646 bp
fragment from Aspergillus oryzae (including two stop codons). This
plasmid was constructed by linearizing pShTh60 (FIG. 1) by
restriction digestion with Sex AI and Pac I. The digested vector
was separated by 0.8% agarose gel electrophoresis in TBE buffer and
purified using a QIAQUICK.RTM. Gel Extraction Kit. The pyc gene was
amplified from Aspergillus oryzae NRRL 3488 genomic DNA using
primers 066549 and 067388 shown below.
TABLE-US-00012 Primer 066549: (SEQ ID NO: 30)
5'-TAGAACATCGTCCATAATGGCGGCTCCGTTTCGTCA-3' Primer 067388: (SEQ ID
NO: 31) 5'-GTGTCAGTCACCTCTAGTTATTATTACGCTTTGACGATCT-3'
[0333] The PCR reaction was composed of 5 .mu.l 10.times. reaction
buffer, 1 .mu.l Aspergillus oryzae NRRL3488 genomic DNA (110
ng/.mu.l), 1 .mu.l primer 066549 (100 ng/.mu.l), 1 .mu.l primer
067388 (100 ng/.mu.l), 1 .mu.l dNTP mixture (10 mM), 45.5 .mu.l
deionized water, and 0.5 .mu.l Herculase.RTM. HotStart DNA
polymerase. The amplification reaction was incubated in an
EPPENDORF.RTM. MASTERCYCLER.RTM. programmed for 1 cycle at
95.degree. C. for 2 minutes; 10 cycles each at 95.degree. C. for 10
seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 3.5
minutes; 20 cycles each at 95.degree. C. for 10 seconds, 58.degree.
C. for 30 seconds, and 72.degree. C. for 3.5 minutes plus 10
seconds per cycle. The PCR product was then purified using a
MinElute.RTM. PCR Purification Kit.
[0334] The purified PCR product was inserted into the vector using
an In-Fusion.TM. Advantage reaction composed of 2 .mu.l 5.times.
buffer, 1 .mu.l purified PCR product (144 ng/.mu.l), 2 .mu.l gel
purified Sex AI and Pac I restriction digested pShTh60 (FIG. 1; 78
ng/.mu.l), 1 .mu.l In-Fusion.TM. enzyme and 4 .mu.l deionized
water. The reaction was incubated at 37.degree. C. for 15 minutes
followed by 50.degree. C. for 15 minutes after which it was placed
on ice for 5 minutes and diluted with 40 .mu.l TE buffer resulting
in pRYAN1 (FIG. 10). A 2 .mu.l aliquot of the ligation reaction was
transformed into ONE SHOT.RTM. TOP10 chemically competent E. coli
cells according to the manufacturer's instructions. Transformants
were plated onto 2XYT+amp plates and incubated at 37.degree. C.
overnight. The resulting transformants were picked and subjected to
DNA sequencing to confirm that the pyc gene was successfully
integrated into the vector. Nucleotide 1308 was changed from C to
T, but did not affect the protein sequence.
Example 15
Transformation of pShTh104, pSaMF21 and pRyan1 into Aspergillus
oryzae NRRL 3488 (SaMF2103)
[0335] Plasmids pShTh104, pSaMF21 and pRyan1 were transformed into
Aspergillus oryzae NRRL 3488 to assay C4 dicarboxylic acid
production for strains containing the mae3 C4 dicarboxylic acid
transporter, the mdh3 NBD-dependent malate dehydrogenase, and the
pyc pyruvate carboxylase genes described above.
[0336] 100 .mu.l of protoplast preparation (prepared as described
in Example 3) was transferred to a 12 ml polypropylene tube. To
this was added 2.6 .mu.g pSaMF21, 5.5 .mu.g pShTh104, 4.73 .mu.g
pRyan1 and 250 .mu.l PEG solution (60% w/v polyethylene glycol
(PEG), 10 mM Tris 6.5, 10 mM CaCl) followed by gentle mixing and
incubation at 37.degree. C. for 30 minutes. Each transformation was
diluted with 9 ml of STC buffer, followed by plating three separate
3 ml aliquots onto COVE plates. Each plate was then incubated at
34.degree. C. for 7-10 days. Sixty SaMF2103 transformants were
transferred to individual COVE plates and incubated at 34.degree.
C. for 5 days. Spore stocks were prepared by collecting the spores
in 0.1% TWEEN.RTM. 80. Cultures were stored by preparing a glycerol
stock of each (800 .mu.l spore stock, 200 .mu.l 0.1% TWEEN.RTM. 80)
and frozen at -80.degree. C.
[0337] Extraction of DNA from the top five malic acid producing
strains (SaMF2103-14, 29, 37, 39, 53) were performed by inoculating
approximately 2.times.10.sup.7 spores into 100 ml YEG medium and
incubating the flask at 34.degree. C. for 16-18 hours at 160 rpm.
Mycelia were collected by pouring the culture through a sterile
vacuum filtration unit. The biomass was incubated in liquid
nitrogen for about 10 seconds, then placed onto 2 layers of
cheesecloth lined with MIRACLOTH.RTM.. The cloth was folded into a
pouch and smashed with a hammer 12-15 times. The crushed biomass
was transferred to a sterile 50 ml conical tube, to which was added
10 ml of 1.times. Lysis Buffer (100 mM EDTA, 10 mM Tris pH 8.0, 1%
Triton X-100, 0.5 M guanidine-HCl, 200 mM NaCl) and 3 .mu.l 100
mg/ml RNase A. Following 5 minutes of incubation at room
temperature, 150 .mu.l 20 mg/ml Proteinase K (QIAGEN Inc.,
Valencia, Calif., USA) was added, mixed by inversion and incubated
at 50.degree. C. for 1 hour. The tubes were centrifuged for 20
minutes at 7240.times.g. The supernatant was poured into a Midi-Tip
(QIAGEN Inc., Valencia, Calif., USA) pre-equilibrated with 4 ml QBT
buffer and allowed to flow by gravity. The tips were washed with 15
ml QC buffer (QIAGEN Inc., Valencia, Calif., USA) then eluted with
5 ml of QF buffer (QIAGEN Inc., Valencia, Calif., USA). To these
3.5 ml of isopropanol was added and mixed, then centrifuged for 20
minutes at 4.degree. C. at 12,380.times.g. The supernatants were
discarded and the pellets were washed with 2 ml cold 70% ethanol
before being centrifuged for 10 minutes at 4.degree. C. at
12,380.times.g. The pellets were air dried, then resuspended in 100
.mu.l EB buffer (QIAGEN Inc., Valencia, Calif., USA). The DNA
concentrations were 71 ng/.mu.l SaMF2103-14, 220 ng/.mu.l
SaMF2103-29, 210 ng/.mu.l SaMF2103-37, 230 ng/.mu.l SaMF2103-39,
233 ng/.mu.l SaMF2103-53.
TABLE-US-00013 Primer 062012: (SEQ ID NO: 32)
5'-GGAAACGTCAAGCGGCTTGC-3'
[0338] PCR reactions to test for the presence of the pShTh104
expression cassette were composed of 2.5 .mu.l 10.times. reaction
buffer, 1 .mu.l template (80-300 ng/.mu.l), 0.5 .mu.l primer 065067
(Example 1; 50 pM), 0.5 .mu.l primer 065130 (Example 1; 50 pM), 0.5
.mu.l dNTP mixture (10 mM), 19.75 .mu.l deionized water, and 0.25
.mu.l Herculase.RTM. HotStart DNA polymerase. PCR reactions to test
for the presence of the pSaMF21 expression cassette were composed
of 2.5 .mu.l 10.times. reaction buffer, 1 .mu.l template (80-300
ng/.mu.l), 0.5 .mu.l primer 065067 (Example 1; 50 pM), 0.5 .mu.l
primer 062400 (Example 8; 50 pM), 0.5 .mu.l dNTP mixture (10 mM),
19.75 .mu.l deionized water, and 0.25 .mu.l of Herculase.RTM.
HotStart DNA polymerase. PCR reactions to test for the presence of
the pRyan1 expression cassettes were composed of 2.5 .mu.l
10.times. reaction buffer, 1 .mu.l template (80-300 ng/.mu.l), 0.5
.mu.l primer 065067 (Example 1; 50 pM), 0.5 .mu.l primer 062012
(see above; 50 pM), 0.5 .mu.l dNTP mixture (10 mM), 19.75 .mu.l
deionized water, and 0.25 .mu.l Herculase.RTM. HotStart DNA
polymerase. Aspergillus oryzae NRRL 3488 genomic DNA (110 ng/.mu.l)
was used as a negative control template for all three expression
cassettes and the plasmids (diluted to 20 ng/.mu.l) were used as
positive control templates. The amplification reactions were
incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. programmed for 1
cycle at 95.degree. C. for 2 minutes; 10 cycles each at 95.degree.
C. for 10 seconds, 55.degree. C. for 30 seconds, and 72.degree. C.
for 1.5 minutes; 20 cycles each at 95.degree. C. for 10 seconds,
50.degree. C. for 30 seconds, and 72.degree. C. for 1.5 minute plus
10 seconds per cycle. The PCR products (5 .mu.l) from each sample
(in this order pShTh104, pSaMF21, pRyan1) were analyzed by 0.8%
agarose gel electrophoresis. The five samples tested (and positive
controls) had expected band sizes while the Aspergillus oryzae NRRL
3488 control samples did not.
Example 16
Production of Malic Acid in Shake Flask Cultures
[0339] Spores from each transformant described in Example 15 and
Aspergillus oryzae NRRL 3488 as a control were plated onto
individual PDA plates and allowed to sporulate at 34.degree. C. for
5 to 7 days. Spores were collected in 0.05% TWEEN.RTM. 80. Seed
cultures were prepared in 250 ml flasks containing 100 ml of seed
medium B and inoculated with 300 .mu.l spore stock. Seed cultures
were grown for approximately 22 hours at 30.degree. C. with shaking
at 200 rpm. Acid production cultures were prepared in 250 ml
unbaffled flasks containing 50 ml of acid production medium C and 3
ml of the 17 hour seed cultures. Cultures were incubated at
30.degree. C. with shaking at 200 rpm for 3 days.
[0340] Aspergillus oryzae SaMF2103 transformants which contain the
heterologous mae3 C4 dicarboxylic acid transporter gene, the
heterologous mdh3 NBD-dependent malate dehydrogenase gene, and the
heterologous pyc pyruvate carboxylase gene (Example 15), produced
malic acid increases in titer of up to 2.6-fold compared to
Aspergillus oryzae NRRL 3488 control.
Example 17
Fermentation of Aspergillus oryzae ShTh1040 and SaMF2103
Strains
[0341] Aspergillus oryzae strains ShTh1040-31 and ShTh1040-44 (each
containing the mae3 C4 dicarboxylic acid transporter gene; Example
3), and SaMF2103-37 and SaMF2103-39 (each containing the mae3 C4
dicarboxylic acid transporter gene, the mdh3 NBD-dependent malate
dehydrogenase gene, and the pyc pyruvate carboxylase gene; Example
15) were grown for approximately 7 days at 34.degree. C. on PDA
plates. A 5-6 ml volume of sterile 50 mM sodium phosphate pH 6.8
buffer containing 0.2% TWEEN.RTM. 80 was added to each of the
plates and spores were suspended by scraping with an inoculating
loop. Each of the suspended spores was transferred from the plate
by pipette to a 50 ml conical tube. For each tube, 25 ml of the
sterile 50 mM sodium phosphate pH 6.8 buffer containing 0.2%
TWEEN.RTM. 80 was added to a 500 ml unbaffled flask containing 75
ml of seed medium, which was then inoculated with 2 ml of spore
suspension. The seed medium was composed of 40 g glucose, 6 g Bacto
peptone, 0.75 g KH.sub.2PO.sub.4, 0.75 g K.sub.2HPO.sub.4, 0.1 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, and deionized water to 1 liter.
The flasks were then incubated at 34.degree. C. and 180 rpm for
approximately 24 hours. Three seed flasks were combined to supply
144 ml inoculum per tank. Three-liter fermentors containing 1.8
liters of medium were individually inoculated with the strain of
choice by introducing 144 ml (8%) of the seed culture broth from
three combined seed flasks of either Aspergillus oryzae
transformant ShTh1040-31, Aspergillus oryzae transformant
ShTh1040-44, Aspergillus oryzae transformant SaMF2103-37 or
Aspergillus oryzae transformant SaMF2103-39. The medium was
composed of 120 g glucose, 120 g CaCO.sub.3, 9 g Bacto peptone,
0.150 g KH.sub.2PO.sub.4, 0.150 g K.sub.2HPO.sub.4, 0.10 g
MgSO.7H.sub.2O, 0.10 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, 1.22 mg biotin, and deionized
water to 1 liter.
[0342] The fermentors were equilibrated at 34.+-.0.1.degree. C. and
stirred at 500 rpm. Inlet air flow was maintained at 1 v/v/m. No
acid or base additions were used for pH control. Feeds consisting
of 20% glucose were administered to each tank at rate of
approximately 7.3 g/hr beginning at about 43 hours into the
fermentations. An additional 100 g of CaCO.sub.3 was introduced to
each tank on day 5 of the fermentations. Samples were withdrawn
daily and analyzed for malic acid production. Fermentations were
completed after 8 days.
Example 18
HPLC Quantitation of Malic Acid of Fermentations
[0343] Quantitation of malic acid for the fermentations of Example
17 was performed as described in Example 5.
[0344] Table 3 shows the relative malic acid titer of Aspergillus
oryzae transformants ShTh1040-44, SaMF2103-37, SaMF2103-39, and
ShTh1040-31. The relative ranking of each type of transformant is
consistent with the shake flask results. Both SaMF2103
transformants which contain the heterologous mae3 C4 dicarboxylic
acid transporter gene, the heterologous mdh3 NBD-dependent malate
dehydrogenase gene, and the heterologous pyc pyruvate carboxylase
gene (Example 15) yielded higher malic acid titers than either
ShTh1040 transformant which contain the heterologous mae3 C4
dicarboxylic acid transporter gene, but not the heterologous mdh3
NBD-dependent malate dehydrogenase gene or the heterologous pyc
pyruvate carboxylase gene (Example 3).
TABLE-US-00014 TABLE 3 Relative Malic Transformant Acid Titer
ShTh1040-31 1.00 ShTh1040-44 1.21 SaMF2103-37 1.40 SaMF2103-39
1.50
Example 19
Cloning of a Aspergillus terreus C4 Dicarboxylic Acid Transporter
Gene and Construction of Expression Vector pShTh122AtC4T
[0345] The 1182 bp Aspergillus terreus C4 dicarboxylic acid
transport protein gene atc4t (ATEG.sub.--00085) was synthetically
constructed into pAtC4T (FIG. 11; DNA2.0, Menlo Park, Calif., USA).
The atc4t gene was amplified from pAtC4T using primers 069739 and
069740 shown below.
TABLE-US-00015 Primer 069739: (SEQ ID NO: 38)
5'-GTGTGATAGAACATCGTCCATAATGTTTGAGAACACTGCCCC-3' Primer 069740:
(SEQ ID NO: 39)
5'-GTCAGTCACCTCTAGTTAATTAATTACTCCACCACATCCTCGTC-3'
[0346] The PCR reaction mixture was composed of 50 ng of pAtC4T
template, 200 .mu.M dNTP mixture, 50 pM of primer 069739, 50 pM
primer 069740, 1.times. Pol1 reaction buffer (New England Biolabs,
MA, USA), 3% DMSO, 1 unit of Vent DNA Polymerase (New England
Biolabs), and deionized water 50 .mu.l. The PCR reaction was
incubated in an EPPENDORF MASTERCYCLER.RTM. programmed for 1 cycle
at 94.degree. C. for 3 minutes; 35 cycles each at 94.degree. C. for
15 seconds, 59.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; and 1 cycle at 72.degree. C. for 5 minutes. The PCR product
was purified by 1% agarose gel electrophoresis in TAE buffer and
purified using a QIAQUICK.RTM. Gel Extraction Kit.
[0347] Plasmid pShTh60 (FIG. 1) was digested with Sex AI and Pac I
then separated by 0.8% agarose gel electrophoresis in TBE buffer
(10.8 g/L Tris Base, 5.5 g/L boric acid, 2 mM EDTA, pH 8.0) and
purified using a QIAQUICK.RTM. Gel Extraction Kit. The purified PCR
product above was then inserted into the digested vector using an
InFusion.TM. Cloning Kit according to the manufacturer's
instructions resulting in plasmid pShTh122AtC4T (FIG. 12). Plasmid
pShTh122AtC4T was isolated using a QIAfilter Maxi Plasmid Isolation
Kit. DNA sequence analysis was used to confirm the integrity of the
atc4t coding sequence using primers 996270 and 065067 described in
the Examples above.
[0348] The nucleotide sequence (SEQ ID NO: 33) and deduced amino
acid sequence (SEQ ID NO: 34) of the atc4t gene are shown in FIG.
13. The genomic coding sequence of 1182 bp (including stop codon)
encodes a polypeptide of 393 amino acids with a predicted mass of
43.2 kDa and an isoelectric pH of 6.54. The gene contains no
introns.
Example 20
Transformation of pShTh12AtC4T into Aspergillus oryzae NRRL3488
[0349] The pShTh12AtC4T vector in Example 19 was prepared for
transformation by restriction digestion with Pme I. The
approximately 5 kb expression cassette was separated from the
vector sequences by 0.8% agarose gel electrophoresis in TBE buffer
and purified using a QIAQUICK.RTM. Gel Extraction Kit.
[0350] Four transformation reactions were prepared. For each
reaction, a 100 .mu.l solution of protoplast preparation (prepared
as described in Example 3) was transferred to a 12 ml polypropylene
tube, to which was added 2-5 .mu.g of digested plasmid vector above
and 250 .mu.l of polyethylene glycol solution (60% w/v polyethylene
glycol (PEG), 10 mM Tris 6.5, 10 mM CaCl), followed by gentle
mixing and incubation at 37.degree. C. for 30 minutes. Each
transformation reaction was diluted with 6 ml STC, followed by
three separate aliquots onto COVE plates. Each plate was then
incubated at 34.degree. C. for 7-10 days. The resulting
transformants were transferred to individual COVE plates and
incubated at 34.degree. C. for 5 days. Spore stocks were prepared
by collecting the spores in 0.1% TWEEN.RTM. 80. Cultures were
stored by preparing a glycerol stock of each (800 .mu.l spore
stock, 200 .mu.l 0.1% TWEEN.RTM. 80) and frozen at -80.degree.
C.
Example 21
Production of Malic Acid in Shake Flask Cultures
[0351] Spores from each transformant described in Example 20
(ShTh1220) and Aspergillus oryzae NRRL 3488 as a control were
plated onto individual PDA plates and allowed to sporulate at
34.degree. C. for 5 to 7 days. Spores were collected in 0.05%
TWEEN.RTM. 80. Seed cultures were prepared in 250 ml flasks
containing 100 ml of seed medium B and inoculated with 300 .mu.l
spore stock. Seed cultures were grown for approximately 22 hours at
30.degree. C. with shaking at 200 rpm. Acid production cultures
were prepared in 250 ml unbaffled flasks containing 50 ml of acid
production medium C and 3 ml of the 17 hour seed cultures. Cultures
were incubated at 30.degree. C. with shaking at 200 rpm for 3
days.
[0352] Aspergillus oryzae ShTh1220 transformants, which contain the
heterologous Aspergillus terreus C4 dicarboxylic acid transport
protein gene atc4t, produced malic acid increases in titer of up to
1.9-fold compared to Aspergillus oryzae NRRL 3488 control.
Example 22
Fermentation of Aspergillus oryzae ShTh1220 Strains
[0353] Aspergillus oryzae ShTh1220-11, ShTh1220-22 and ShTh1220-25
were grown for approximately 7 days at 34.degree. C. on PDA plates.
A 5-6 ml volume of sterile 50 mM sodium phosphate pH 6.8 buffer
containing 0.2% TWEEN.RTM. 80 was added to each of the plates and
spores were suspended by scraping with an inoculating loop. Each of
the suspended spores was transferred from the plate by pipette to a
50 ml conical tube. For each tube, 25 ml of the sterile 50 mM
sodium phosphate pH 6.8 buffer containing 0.2% TWEEN.RTM. 80 was
added to a 500 ml unbaffled flask containing 75 ml of seed medium,
which was then inoculated with 2 ml of spore suspension. The seed
medium was composed of 40 g glucose, 6 g Bacto-peptone, 0.75 g
KH.sub.2PO.sub.4, 0.75 g K.sub.2HPO.sub.4, 0.1 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, and deionized water to 1 liter.
The flasks were then incubated at 34.degree. C. and 180 rpm for
approximately 24 hours. Three seed flasks were combined to supply
144 ml inoculum per tank. Three-liter fermentors containing 1.8
liters of medium were individually inoculated with the strain of
choice by introducing 144 ml (8%) of the seed culture broth from
three combined seed flasks of either Aspergillus oryzae
transformant ShTh1220-11, Aspergillus oryzae transformant
ShTh1220-22 or Aspergillus oryzae transformant ShTh1220-25. The
medium was composed of 60 g glucose, 120 g CaCO.sub.3, 9 g
Bacto-peptone, 0.150 g KH.sub.2PO.sub.4, 0.150 g K.sub.2HPO.sub.4,
0.10 g MgSO.7H.sub.2O, 0.10 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, 1.22 mg biotin, and deionized
water to 1 liter.
[0354] The fermentors were equilibrated at 34.+-.0.1.degree. C. and
stirred at 500 rpm. Inlet air flow was maintained at 1 v/v/m. No
acid or base additions were used for pH control. Feeds consisting
of 25% glucose were administered to each tank at the rate of
approximately 7.3 g/hr beginning at about 43 hours into the
fermentations. After about 92 hours of fermentation the feed rates
were increased to approximately 9.3 g/hr, and then returned to
approximately 7.3 g/hr at about 164 hours. An additional 100 g of
CaCO.sub.3 was introduced to each tank on day 5 of the
fermentations. Samples were withdrawn daily and analyzed for malic
acid production. Fermentations were completed after 8 days.
Example 23
HPLC Quantitation of Malic Acid of Fermentations
[0355] Quantitation of malic acid for the fermentations of Example
22 was performed as described in Example 5.
[0356] Table 4 shows the relative malic acid titer of Aspergillus
oryzae transformants ShTh1220-11, ShTh1220-22, and ShTh1220-25
compared to the malic acid production of ShTh1040-22. The relative
ranking of each transformant is consistent with the shake flask
results by demonstrating that ShTh1220 transformants (which contain
the heterologous Aspergillus terreus C4 dicarboxylic acid transport
protein gene atc4t) yielded similar malic acid titer to the
ShTh1040 transformant (which contains the heterologous mae3 C4
dicarboxylic acid transporter gene and is shown in Example 7 to
have increased titer over the Aspergillus oryzae NRRL 3488 control
lacking a heterologous C4 dicarboxylic acid transporter).
TABLE-US-00016 TABLE 4 Relative Malic Transformant Acid Titer
ShTh1040-22 1.00 ShTh1220-11 0.97 ShTh1220-22 0.96 ShTh1220-25
0.96
Example 24
Cloning of a Schizosaccharomyces pombe C4 Dicarboxylic Acid
Transporter Gene and Construction of Expression Vector pSaMF27
[0357] The putative Schizosaccharomyces pombe C4 dicarboxylic acid
transport protein gene mae1 (GenBank: U21002.1) was codon optimized
for expression in Aspergillus oryzae and synthetically constructed
into p0941304_sspMAE1_pMK
[0358] (FIG. 14; GENEART, Burlingame, Calif., USA). The mae1 gene
was amplified from p0941304_sspMAE1_pMK using primers 068320 and
068808 shown below.
TABLE-US-00017 Primer 068320: (SEQ ID NO: 40)
5'-GAACATCGTCCATAATGGGAGAATTGAAGGAAATTC-3' Primer 068808: (SEQ ID
NO: 41) 5'-GGTGTCAGTCACCTCTAGTTATTATTAGACCGACTCGTGT-3'
[0359] The PCR reaction was composed of 5 .mu.l 10.times. reaction
buffer, 1 .mu.l 0941304_sspMAE1_pMK template (50 ng/.mu.l), 1 .mu.l
primer 068320 (100 ng/.mu.l), 1 .mu.l primer 068808 (100 ng/.mu.l),
1 .mu.l dNTP mixture (10 mM), 45.5 .mu.l deionized water, and 0.5
.mu.l Herculase.RTM. HotStart DNA polymerase. The amplification
reaction was incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM.
programmed for 1 cycle at 95.degree. C. for 2 minutes; 10 cycles
each at 95.degree. C. for 10 seconds, 55.degree. C. for 30 seconds,
and 72.degree. C. for 1.5 minutes; 20 cycles each at 95.degree. C.
for 10 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for
1.5 minutes plus 10 seconds per cycle. The PCR reaction was
subjected to restriction digestion with Dpn I for 1 hour to degrade
any plasmid DNA template then purified using the Qiagen
MinElute.RTM. PCR purification kit.
[0360] Plasmid pShTh60 (FIG. 1) was digested with Sex AI and Pac I
then separated by 0.8% agarose gel electrophoresis in TBE buffer
and purified using a QIAQUICK.RTM. Gel Extraction Kit. The purified
PCR product above was then inserted into the digested vector using
an In-Fusion Advantage.TM. Reaction Kit in a reaction composed of 2
.mu.l 5.times. buffer, 0.5 .mu.l purified PCR product (148
ng/.mu.l), 2.5 .mu.l digested and gel purified pShTh60 (78
ng/.mu.l), 1 .mu.l InFusion.TM. enzyme and 4 .mu.l deionized water.
The reaction was incubated at 37.degree. C. for 15 minutes then
50.degree. C. for 15 minutes after which it was placed on ice for 5
minutes and diluted with 40 .mu.l TE buffer resulting in pSaMF27
(FIG. 15).
[0361] A 2 .mu.l aliquot of the ligation reaction above was
transformed into ONE SHOT.RTM. TOP10 chemically competent E. coli
cells according to the manufacturer's instructions. Transformants
were plated onto 2XYT+amp plates and incubated at 37.degree. C.
overnight. The resulting transformants were picked and subjected to
DNA sequencing using primers 065067 and 996270 (see Examples above)
to confirm that the mae1 gene was successfully integrated into the
vector.
[0362] The codon-optimized nucleotide sequence (CO), deduced amino
acid sequence, and wild-type nucleotide sequence (WT) of the
Schizosaccharomyces pombe mae1 gene are shown in FIGS. 16A and 16B
(SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO:37, respectively). The
coding sequence is 1317 bp including the stop codon. The encoded
predicted protein is 439 amino acids with a predicted mass of 49.4
kDa and an isoelectric pH of 8.24. The gene contains no introns.
Using the SignalP program (Nielsen et al., 1997, Protein
Engineering 10:1-6), a signal peptide of 49 residues was predicted.
Based on this program, the predicted mature protein contains 389
amino acids with a predicted molecular mass of 43.7 kDa and an
isoelectric pH of 7.67.
Example 25
Transformation of pSaMF27 into Aspergillus oryzae NRRL3488
[0363] The pSaMF27 vector in Example 24 was prepared for
transformation by restriction digestion with Pme I for 1 hour at
37.degree. C. The 5103 bp expression cassette was purified by 0.8%
agarose gel electrophoresis in TBE buffer and a QIAQUICK.RTM. Gel
Extraction Kit according to manufacturer's instructions.
[0364] Three transformation reactions were prepared. For each
reaction, 100 .mu.l of protoplast preparation (prepared as
described in Example 3) was transferred to a 12 ml polypropylene
tube. To this was added 5 .mu.g of ampicillin marker-free,
linearized pSaMF27 vector and 250 .mu.l of polyethylene glycol
(PEG) followed by gentle mixing and incubation at 37.degree. C. for
30 minutes. Each transformation was diluted with 3 ml of STC buffer
followed by plating onto COVE plates. Each plate was then incubated
at 34.degree. C. for 7-10 days. The resulting transformants were
transferred to individual COVE plates and incubated at 34.degree.
C. for 5 days. Spore stocks were prepared by collecting the spores
in 0.1% TWEEN.RTM. 80. Cultures were stored by preparing a glycerol
stock of each (800 .mu.l spore stock, 200 .mu.l 0.1% TWEEN.RTM. 80)
and frozen at -80.degree. C.
Example 26
Production of Malic Acid in Shake Flask Cultures
[0365] Spores from each transformant described in Example 25 and
Aspergillus oryzae NRRL 3488 as a control were plated onto
individual PDA plates and allowed to sporulate at 34.degree. C. for
5 to 7 days. Spores were collected in 0.05% TWEEN.RTM. 80. Seed
cultures were prepared in 250 ml flasks containing 100 ml of seed
medium B and inoculated with 300 .mu.l spore stock. Seed cultures
were grown for approximately 22 hours at 30.degree. C. with shaking
at 200 rpm. Acid production cultures were prepared in 250 ml
unbaffled flasks containing 50 ml of acid production medium C and 3
ml of the 17 hour seed cultures. Cultures were incubated at
30.degree. C. with shaking at 200 rpm for 3 days.
[0366] Aspergillus oryzae SaMF27 transformants, which contain the
heterologous S. pombe C4 dicarboxylic acid transport protein gene
mae1, produced malic acid increases in titer of up to 1.9-fold
compared to Aspergillus oryzae NRRL 3488 control.
Example 27
Fermentation of Aspergillus oryzae SaMF27 Strains
[0367] Aspergillus oryzae ShTh1040-44, SaMF27-2, SaMF27-4 and
SaMF27-7 were grown for approximately 7 days at 34.degree. C. on
PDA plates. A 5-6 ml volume of sterile 50 mM sodium phosphate pH
6.8 buffer containing 0.2% TWEEN.RTM. 80 were added to each of the
plates and spores were suspended by scraping with an inoculating
loop. Each of the suspended spores was transferred from the plate
by pipette to a 50 ml conical tube. For each tube, 25 ml of the
sterile 50 mM sodium phosphate pH 6.8 buffer containing 0.2%
TWEEN.RTM. 80 was added to a 500 ml unbaffled flask containing 75
ml of seed medium, which was then inoculated with 2 ml of spore
suspension. The seed medium was composed of 40 g glucose, 6 g Bacto
peptone, 0.75 g KH.sub.2PO.sub.4, 0.75 g K.sub.2HPO.sub.4, 0.1 g
MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.2H.sub.2O, 0.005 g
FeSO.sub.4.7H.sub.2O, 0.005 g NaCl, and deionized water to 1 liter.
The flasks were then incubated at 34.degree. C. and 180 rpm for
approximately 24 hours. Three seed flasks were combined to supply
144 ml inoculum per tank. Three-liter fermentors containing 1.8
liters of medium were individually inoculated with the strain of
choice by introducing 144 ml (8%) of the seed culture broth from
three combined seed flasks of either Aspergillus oryzae
transformant ShTh1040-44, Aspergillus oryzae transformant SaMF27-2,
Aspergillus oryzae transformant SaMF27-4 or Aspergillus oryzae
transformant SaMF27-7. The medium was composed of 120 g glucose,
120 g CaCO.sub.3, 9 g Bacto peptone, 0.150 g KH.sub.2PO.sub.4,
0.150 g K.sub.2HPO.sub.4, 0.10 g MgSO.7H.sub.2O, 0.10 g
CaCl.sub.2.2H.sub.2O, 0.005 g FeSO.sub.4.7H.sub.2O, 0.005 g NaCl,
1.22 mg biotin, and deionized water to 1 liter.
[0368] The fermentors were equilibrated at 34.+-.0.1.degree. C. and
stirred at 500 rpm. Inlet air flow was maintained at 1 v/v/m. No
acid or base additions were used for pH control. Feeds consisting
of 20% glucose were administered to each tank at rate of
approximately 7.3 g/hr beginning at about 43 hours into the
fermentations. An additional 100 g of CaCO.sub.3 was introduced to
each tank on day 5 of the fermentations. Samples were withdrawn
daily and analyzed for malic acid production. Fermentations were
completed after 8 days.
Example 28
HPLC Quantitation of Malic Acid of Fermentations
[0369] Quantitation of malic acid for the fermentations of Example
27 was performed as described in Example 5.
[0370] Table 5 shows the relative malic acid titer of Aspergillus
oryzae transformants SaMF27-2, SaMF27-4, and SaMF27-7 compared to
the malic acid production of ShTh1040-44. The relative ranking of
each transformant is consistent with the shake flask results by
demonstrating that SaMF27 transformants (which contain the
heterologous S. pombe C4 dicarboxylic acid transport protein gene
mae1) gave similar malic acid yields to the ShTh1040 transformant
(which contains the heterologous mae3 C4 dicarboxylic acid
transporter gene and is shown in Example 7 to have increased titer
over the Aspergillus oryzae NRRL 3488 control lacking a
heterologous C4 dicarboxylic acid transporter).
TABLE-US-00018 TABLE 5 Relative Malic Transformant Acid Titer
ShTh1040-44 1.00 SaMF27-2 0.89 SaMF27-4 0.95 SaMF27-7 0.84
[0371] The present invention may be further described by the
following numbered paragraphs:
[0372] [1] A method of producing a C4 dicarboxylic acid (e.g.,
malic acid), comprising: (a) cultivating in a medium a filamentous
fungal host cell comprising one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase; wherein the filamentous fungal host cell secretes (or
is capable of secreting) increased levels of the C4 dicarboxylic
acid compared to the filamentous fungal host cell without the one
or more (several) heterologous polynucleotides encoding the C4
dicarboxylic acid transporter, the malate dehydrogenase, and the
pyruvate carboxylase when cultivated under the same conditions; and
(b) recovering the C4 dicarboxylic acid.
[0373] [2] The method of paragraph [1], wherein the C4 dicarboxylic
acid transporter is selected from the group consisting of (a) a C4
dicarboxylic acid transporter comprising an amino acid sequence
having at least 60% sequence identity, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity with SEQ ID NO: 8, SEQ ID NO: 34, or SEQ
ID NO: 36; (b) a C4 dicarboxylic acid transporter encoded by a
polynucleotide that hybridizes under at least low stringency
conditions, e.g., medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO:
35, or SEQ ID NO: 37; or a full-length complementary strand
thereof; (c) a C4 dicarboxylic acid transporter encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID
NO: 37; (d) a C4 dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (several)
amino acids of the mature polypeptide of SEQ ID NO: 8, SEQ ID NO:
34, or SEQ ID NO: 36; and (e) a fragment of the C4 dicarboxylic
acid transporter of (a), (b), (c), or (d) that has C4 dicarboxylic
acid transporter activity.
[0374] [3] The method of paragraph 1 or 2, wherein the C4
dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 8.
[0375] [4] The method of any one of paragraphs 1-3, wherein the C4
dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 34.
[0376] [5] The method of any one of paragraphs 1-4, wherein the C4
dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 36.
[0377] [6] The method of any one of paragraphs 1-5, wherein the C4
dicarboxylic acid transporter is encoded by a polynucleotide that
hybridizes under very low stringency conditions, low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with SEQ ID NO: 7 or the full-length complementary
strand thereof.
[0378] [7] The method of any one of paragraphs 1-6, wherein the C4
dicarboxylic acid transporter is encoded by a polynucleotide that
hybridizes under very low stringency conditions, low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with SEQ ID NO: 33 or the full-length complementary
strand thereof.
[0379] [8] The method of any one of paragraphs 1-7, wherein the C4
dicarboxylic acid transporter is encoded by a polynucleotide that
hybridizes under very low stringency conditions, low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with SEQ ID NO: 35 or SEQ ID NO: 37; or a full-length
complementary strand thereof.
[0380] [9] The method of any one of paragraphs 1-8, wherein the C4
dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 7.
[0381] [10] The method of any one of paragraphs 1-9, wherein the C4
dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 33.
[0382] [11] The method of any one of paragraphs 1-10, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 35 or SEQ ID NO:
37.
[0383] [12] The method of any one of paragraphs 1-11, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 8.
[0384] [13] The method of any one of paragraphs 1-11, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 34.
[0385] [14] The method of any one of paragraphs 1-11, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 36.
[0386] [15] The method of any of paragraphs 1-14, wherein the
heterologous first polynucleotide encoding the C4 dicarboxylic acid
transporter is operably linked to a promoter foreign to the first
polynucleotide.
[0387] [16] The method of any of paragraphs 1-15, wherein the
malate dehydrogenase is selected from the group consisting of (a) a
malate dehydrogenase comprising an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or 100% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20; (b)
a malate dehydrogenase encoded by a polynucleotide that hybridizes
under at least low stringency conditions, e.g., medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
17 or SEQ ID NO: 19, (ii) the cDNA sequence contained in SEQ ID NO:
17 or SEQ ID NO: 19, or (iii) a full-length complementary strand of
(i) or (ii); (c) a malate dehydrogenase encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity
with SEQ ID NO: 17 or SEQ ID NO: 19; (d) a malate dehydrogenase
variant comprising a substitution, deletion, and/or insertion of
one or more (several) amino acids of the mature polypeptide of SEQ
ID NO: 18 or SEQ ID NO: 20; and (e) a fragment of the malate
dehydrogenase of (a), (b), (c), or (d) that has malate
dehydrogenase activity.
[0388] [17] The method of any of paragraphs 1-16, wherein the
malate dehydrogenase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity with SEQ ID NO: 18.
[0389] [18] The method of any of paragraphs 1-17, wherein the
malate dehydrogenase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity with SEQ ID NO: 20.
[0390] [19] The method of any of paragraphs 1-18, wherein the
malate dehydrogenase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, or high stringency conditions
with (i) SEQ ID NO: 17, (ii) the cDNA sequence contained in SEQ ID
NO: 17, or (iii) the full-length complementary strand of (i) or
(ii).
[0391] [20] The method of any of paragraphs 1-19, wherein the
malate dehydrogenase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, or high stringency conditions
with (i) SEQ ID NO: 19, (ii) the cDNA sequence contained in SEQ ID
NO: 19, or (iii) the full-length complementary strand of (i) or
(ii).
[0392] [21] The method of any of paragraphs 1-20, wherein the
malate dehydrogenase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
17.
[0393] [22] The method of any of paragraphs 1-21, wherein the
malate dehydrogenase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
19.
[0394] [23] The method of any of paragraphs 1-22, wherein the
malate dehydrogenase comprises or consists of SEQ ID NO: 18.
[0395] [24] The method of any of paragraphs 1-22, wherein the
malate dehydrogenase comprises or consists of SEQ ID NO: 20.
[0396] [25] The method of any of paragraphs 1-15, wherein the
malate dehydrogenase is a variant of a parent malate dehydrogenase
comprising (i) a deletion at positions equivalent to or
corresponding to amino acids positions 2 to 17 or a portion thereof
of SEQ ID NO: 18, and (ii) a substitution at a position equivalent
to amino acid 48 of SEQ ID NO: 18; wherein the deletion and the
substitution reduce mitochondrial import in vivo of the malate
dehydrogenase variant thereby increasing the level of the malate
dehydrogenase variant in the cytosol, and wherein the filamentous
fungal host cell secretes (or is capable of secreting) increased
levels of the C4 dicarboxylic acid (e.g., malic acid) compared to
the filamentous fungal host cell without the polynucleotide
encoding the malate dehydrogenase variant when cultivated under the
same conditions.
[0397] [26] The method of paragraph 25, wherein the parent malate
dehydrogenase is selected from the group consisting of (a) a malate
dehydrogenase comprising an amino acid sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to SEQ ID NO: 18; (b) a malate dehydrogenase encoded by a
polynucleotide that hybridizes under at least low stringency
conditions, e.g., medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 17, or its full-length
complementary strand; and (c) a malate dehydrogenase encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to SEQ ID NO: 17.
[0398] [27] The method of paragraph 26, wherein the parent malate
dehydrogenase comprises or consists of SEQ ID NO: 18.
[0399] [28] The method of any of paragraphs 25-27, wherein the
malate dehydrogenase variant comprises an amino acid sequence
having at least 60% sequence identity, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18.
[0400] [29] The method of any of paragraphs 25-28, wherein the
variant comprises the deletions
Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
[0401] [30] The method of any of paragraphs 1-29, wherein the
heterologous second polynucleotide encoding the malate
dehydrogenase is operably linked to a promoter foreign to the
second polynucleotide.
[0402] [31] The method of any of paragraphs 1-30, wherein the
pyruvate carboxylase is selected from the group consisting of (a) a
pyruvate carboxylase comprising an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least at least 98%, at least 99%, or 100% with SEQ ID NO: 27; (b) a
pyruvate carboxylase encoded by a polynucleotide that hybridizes
under at least low stringency conditions, e.g., medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
26, (ii) the cDNA sequence contained in SEQ ID NO: 26, or (iii) the
full-length complementary strand of (i) or (ii); (c) a pyruvate
carboxylase encoded by a polynucleotide comprising a nucleotide
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 26; (d) a
pyruvate carboxylase variant comprising a substitution, deletion,
and/or insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 26; and (e) a fragment of the pyruvate
carboxylase of (a), (b), (c), or (d) that has pyruvate carboxylase
activity.
[0403] [32] The method of any of paragraphs 1-31, wherein the
pyruvate carboxylase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
with SEQ ID NO: 27.
[0404] [33] The method of any of paragraphs 1-32, wherein the
pyruvate carboxylase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, or high stringency conditions
with (i) SEQ ID NO: 26, (ii) the cDNA sequence contained in SEQ ID
NO: 26, or (iii) the full-length complementary strand of (i) or
(ii).
[0405] [34] The method of any of paragraphs 1-33, wherein the
pyruvate carboxylase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
26.
[0406] [35] The method of any of paragraphs 1-34, wherein the
pyruvate carboxylase comprises or consists of SEQ ID NO: 27.
[0407] [36] The method of any of paragraphs 1-35, wherein the
pyruvate carboxylase is a variant of a mitochondrial pyruvate
carboxylase.
[0408] [37] The method of any of paragraphs 1-36, wherein the
heterologous third polynucleotide encoding the pyruvate carboxylase
is operably linked to a promoter foreign to the third
polynucleotide.
[0409] [38] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter.
[0410] [39] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous second
polynucleotide encoding a malate dehydrogenase.
[0411] [40] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous third
polynucleotide encoding a pyruvate carboxylase.
[0412] [41] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter and a
heterologous second polynucleotide encoding a malate
dehydrogenase.
[0413] [42] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter and a
heterologous third polynucleotide encoding a pyruvate
carboxylase.
[0414] [43] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous second
polynucleotide encoding a malate dehydrogenase and a heterologous
third polynucleotide encoding a pyruvate carboxylase.
[0415] [44] The method of any of paragraphs 1-37, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase.
[0416] [45] The method of any of paragraphs 1-44, wherein the
filamentous fungal host cell is selected from the group consisting
of an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,
Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trametes, and Trichoderma cell.
[0417] [46] The method of paragraph 45, wherein the host cell is an
Aspergillus host cell, such as Aspergillus oryzae.
[0418] [47] The method of any of paragraphs 1-46, wherein the level
of the C4 dicarboxylic acid (e.g., malic acid) is increased by at
least 25%, e.g., by at least 50%, at least 100%, at least 200%, or
at 500% compared to the filamentous fungal host cell without the
one or more (several) heterologous polynucleotides encoding the C4
dicarboxylic acid transporter, the malate dehydrogenase, and the
pyruvate carboxylase when cultivated under the same conditions.
[0419] [48] A method for increasing C4 dicarboxylic acid (e.g.,
malic acid) production, comprising: (a) transforming into a
filamentous fungal host cell one or more (several) polynucleotides
selected from the group consisting of a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase, wherein the filamentous fungal host cell secretes (or
is capable of secreting) increased levels of the C4 dicarboxylic
acid compared to the filamentous fungal host cell without the one
or more (several) heterologous polynucleotides encoding the C4
dicarboxylic acid transporter, the malate dehydrogenase, and the
pyruvate carboxylase when cultivated under the same conditions; (b)
cultivating the transformed filamentous fungal host cell in a
medium; and (c) recovering the C4 dicarboxylic acid.
[0420] [49] The method of paragraph 48, wherein the C4 dicarboxylic
acid transporter is selected from the group consisting of (a) a C4
dicarboxylic acid transporter comprising an amino acid sequence
having at least 60% sequence identity, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity with SEQ ID NO: 8, SEQ ID NO: 34, or SEQ
ID NO: 36; (b) a C4 dicarboxylic acid transporter encoded by a
polynucleotide that hybridizes under at least low stringency
conditions, e.g., medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO:
35 or SEQ ID NO: 37, or a full-length complementary strand thereof;
(c) a C4 dicarboxylic acid transporter encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or 100% sequence
identity with SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID
NO: 37; (d) a C4 dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (several)
amino acids of the mature polypeptide of SEQ ID NO: 8, SEQ ID NO:
34, or SEQ ID NO: 36; and (e) a fragment of the C4 dicarboxylic
acid transporter of (a), (b), (c), or (d) that has C4 dicarboxylic
acid transporter activity.
[0421] [50] The method of paragraph 48 or 49, wherein the C4
dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 8.
[0422] [51] The method of any one of paragraphs 48-50, wherein the
C4 dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 34.
[0423] [52] The method of any one of paragraphs 48-51, wherein the
C4 dicarboxylic acid transporter comprises an amino acid sequence
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, 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
SEQ ID NO: 36.
[0424] [53] The method of any one of paragraphs 48-52, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
that hybridizes under very low stringency conditions, low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 7 or the full-length
complementary strand thereof.
[0425] [54] The method of any one of paragraphs 48-53, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
that hybridizes under very low stringency conditions, low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 33, or the full-length
complementary strand thereof.
[0426] [55] The method of any one of paragraphs 48-54, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
that hybridizes under very low stringency conditions, low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 35 or SEQ ID NO: 37, or a
full-length complementary strand thereof.
[0427] [56] The method of any one of paragraphs 48-55, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 7.
[0428] [57] The method of any one of paragraphs 48-56, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 33.
[0429] [58] The method of any one of paragraphs 48-57, wherein the
C4 dicarboxylic acid transporter is encoded by a polynucleotide
comprising a nucleotide sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 35 or SEQ ID NO:
37.
[0430] [59] The method of any one of paragraphs 48-58, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 8.
[0431] [60] The method of any one of paragraphs 48-58, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 34.
[0432] [61] The method of any one of paragraphs 48-58, wherein the
C4 dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 36.
[0433] [62] The method of any of paragraphs 48-61, wherein the
heterologous first polynucleotide encoding the C4 dicarboxylic acid
transporter is operably linked to a promoter foreign to the first
polynucleotide.
[0434] [63] The method of any of paragraphs 48-62, wherein the
malate dehydrogenase is selected from the group consisting of (a) a
malate dehydrogenase comprising an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or 100% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20; (b)
a malate dehydrogenase encoded by a polynucleotide that hybridizes
under at least low stringency conditions, e.g., medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
17 or SEQ ID NO: 19, (ii) the cDNA sequence contained in SEQ ID NO:
17 or SEQ ID NO: 19, or (iii) a full-length complementary strand of
(i) or (ii); (c) a malate dehydrogenase encoded by a polynucleotide
comprising a nucleotide sequence having at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or 100% sequence
identity with SEQ ID NO: 17 or SEQ ID NO: 19; (d) a malate
dehydrogenase variant comprising a substitution, deletion, and/or
insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 18 or SEQ ID NO: 20; and (e) a fragment
of the malate dehydrogenase of (a), (b), (c), or (d) that has
malate dehydrogenase activity.
[0435] [64] The method of any of paragraphs 48-63, wherein the
malate dehydrogenase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity with SEQ ID NO: 18.
[0436] [65] The method of any of paragraphs 48-64, wherein the
malate dehydrogenase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity with SEQ ID NO: 20.
[0437] [66] The method of any of paragraphs 48-65, wherein the
malate dehydrogenase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with (i) SEQ ID NO: 17, (ii) the
cDNA sequence contained in SEQ ID NO: 17, or (iii) the full-length
complementary strand of (i) or (ii).
[0438] [67] The method of any of paragraphs 48-66, wherein the
malate dehydrogenase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with (i) SEQ ID NO: 19, (ii) the
cDNA sequence contained in SEQ ID NO: 19, or (iii) the full-length
complementary strand of (i) or (ii).
[0439] [68] The method of any of paragraphs 48-67, wherein the
malate dehydrogenase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
17.
[0440] [69] The method of any of paragraphs 48-68, wherein the
malate dehydrogenase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
19.
[0441] [70] The method of any of paragraphs 48-69, wherein the
malate dehydrogenase comprises or consists of SEQ ID NO: 18.
[0442] [71] The method of any of paragraphs 48-69, wherein the
malate dehydrogenase comprises or consists of SEQ ID NO: 20.
[0443] [72] The method of any of paragraphs 48-62, wherein the
malate dehydrogenase is a variant of a parent malate dehydrogenase
comprising (i) a deletion at positions equivalent to amino acids
positions 2 to 17 or a portion thereof of SEQ ID NO: 18, and (ii) a
substitution at a position equivalent to amino acid 48 of SEQ ID
NO: 18; wherein the deletion and the substitution reduce
mitochondrial import in vivo of the malate dehydrogenase variant
thereby increasing the level of the malate dehydrogenase variant in
the cytosol, and wherein the filamentous fungal host cell secretes
(or is capable of secreting) increased levels of the C4
dicarboxylic acid (e.g., malic acid) compared to the filamentous
fungal host cell without the polynucleotide encoding the malate
dehydrogenase variant when cultivated under the same
conditions.
[0444] [73] The method of paragraph 72, wherein the parent malate
dehydrogenase is selected from the group consisting of (a) a malate
dehydrogenase comprising an amino acid sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to SEQ ID NO: 18; (b) a malate dehydrogenase encoded by a
polynucleotide that hybridizes under at least low stringency
conditions, e.g., medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with SEQ ID NO: 17 or its full-length
complementary strand; and (c) a malate dehydrogenase encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to SEQ
ID NO: 17.
[0445] [74] The method of paragraph 73, wherein the parent malate
dehydrogenase comprises or consists of SEQ ID NO: 18.
[0446] [75] The method of any of paragraphs 72-74, wherein the
malate dehydrogenase variant comprises an amino acid sequence
having at least 60% sequence identity, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18.
[0447] [76] The method of any of paragraphs 72-75, wherein the
variant comprises the deletions
Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
[0448] [77] The method of any of paragraphs 48-76, wherein the
heterologous second polynucleotide encoding the malate
dehydrogenase is operably linked to a promoter foreign to the
second polynucleotide.
[0449] [78] The method of any of paragraphs 48-77, wherein the
pyruvate carboxylase is selected from the group consisting of (a) a
pyruvate carboxylase comprising an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity with SEQ ID NO: 27; (b) a pyruvate carboxylase
encoded by a polynucleotide that hybridizes under at least low
stringency conditions, e.g., medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with (i) SEQ ID NO: 26, (ii) the
cDNA sequence contained in SEQ ID NO: 26, or (iii) the full-length
complementary strand of (i) or (ii); (c) a pyruvate carboxylase
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% sequence identity with SEQ ID NO: 26; (d) a pyruvate
carboxylase variant comprising a substitution, deletion, and/or
insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 27; and (e) a fragment of the pyruvate
carboxylase of (a), (b), (c), or (d) that has pyruvate carboxylase
activity.
[0450] [79] The method of any of paragraphs 48-78, wherein the
pyruvate carboxylase comprises an amino acid sequence having at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
with SEQ ID NO: 27.
[0451] [80] The method of any of paragraphs 48-79, wherein the
pyruvate carboxylase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with (i) SEQ ID NO: 26, (ii) the
cDNA sequence contained in SEQ ID NO: 26, or (iii) the full-length
complementary strand of (i) or (ii).
[0452] [81] The method of any of paragraphs 48-80, wherein the
pyruvate carboxylase is encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO:
26.
[0453] [82] The method of any of paragraphs 48-81, wherein the
pyruvate carboxylase comprises or consists of SEQ ID NO: 27.
[0454] [83] The method of any of paragraphs 48-82, wherein the
pyruvate carboxylase is a variant of a mitochondrial pyruvate
carboxylase.
[0455] [84] The method of any of paragraphs 48-83, wherein the
heterologous third polynucleotide encoding the pyruvate carboxylase
is operably linked to a promoter foreign to the third
polynucleotide.
[0456] [85] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter.
[0457] [86] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous second
polynucleotide encoding a malate dehydrogenase.
[0458] [87] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous third
polynucleotide encoding a pyruvate carboxylase.
[0459] [88] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter and a
heterologous second polynucleotide encoding a malate
dehydrogenase.
[0460] [89] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter and a
heterologous third polynucleotide encoding a pyruvate
carboxylase.
[0461] [90] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous second
polynucleotide encoding a malate dehydrogenase and a heterologous
third polynucleotide encoding a pyruvate carboxylase.
[0462] [91] The method of any of paragraphs 48-84, wherein the
filamentous fungal host cell comprises a heterologous first
polynucleotide encoding a C4 dicarboxylic acid transporter, a
heterologous second polynucleotide encoding a malate dehydrogenase,
and a heterologous third polynucleotide encoding a pyruvate
carboxylase.
[0463] [92] The method of any of paragraphs 48-91, wherein the
filamentous fungal host cell is selected from the group consisting
of an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,
Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,
Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trametes, and Trichoderma cell.
[0464] [93] The method of paragraph 92, wherein the host cell is an
Aspergillus host cell, such as Aspergillus oryzae.
[0465] [94] The method of any of paragraphs 48-93, wherein the
level of the C4 dicarboxylic acid (e.g., malic acid) is increased
by at least 25%, e.g., by at least 50%, at least 100%, at least
200%, or at 500% compared to the filamentous fungal host cell
without the one or more (several) heterologous polynucleotides
encoding the C4 dicarboxylic acid transporter, the malate
dehydrogenase, and the pyruvate carboxylase when cultivated under
the same conditions.
[0466] [95] A filamentous fungal host cell comprising one or more
(several) polynucleotides selected from the group consisting of a
heterologous first polynucleotide encoding a C4 dicarboxylic acid
transporter, a heterologous second polynucleotide encoding a malate
dehydrogenase, and a heterologous third polynucleotide encoding a
pyruvate carboxylase; wherein the filamentous fungal host cell
secretes (or is capable of secreting) increased levels of the C4
dicarboxylic acid (e.g., malic acid) compared to the filamentous
fungal host cell without the one or more (several) heterologous
polynucleotides encoding the C4 dicarboxylic acid transporter, the
malate dehydrogenase, and the pyruvate carboxylase when cultivated
under the same conditions.
[0467] [96] The filamentous fungal host cell of paragraph 95,
wherein the C4 dicarboxylic acid transporter is selected from the
group consisting of (a) a C4 dicarboxylic acid transporter
comprising an amino acid sequence having at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity
with SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO: 36; (b) a C4
dicarboxylic acid transporter encoded by a polynucleotide that
hybridizes under at least low stringency conditions, e.g., medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with SEQ
ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, or a
full-length complementary strand thereof; (c) a C4 dicarboxylic
acid transporter encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7, SEQ
ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37; (d) a C4 dicarboxylic
acid transporter variant comprising a substitution, deletion,
and/or insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 8, SEQ ID NO: 34, or SEQ ID NO: 36; and
(e) a fragment of the C4 dicarboxylic acid transporter of (a), (b),
(c), or (d) that has C4 dicarboxylic acid transporter activity.
[0468] [97] The filamentous fungal host cell of paragraph 95 or 96,
wherein the C4 dicarboxylic acid transporter comprises an amino
acid sequence having at least 60%, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 91%, 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 SEQ ID NO: 8.
[0469] [98] The filamentous fungal host cell of any one of
paragraphs 95-97, wherein the C4 dicarboxylic acid transporter
comprises an amino acid sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 34.
[0470] [99] The filamentous fungal host cell of any one of
paragraphs 95-98, wherein the C4 dicarboxylic acid transporter
comprises an amino acid sequence having at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, 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 SEQ ID NO: 36.
[0471] [100] The filamentous fungal host cell of any one of
paragraphs 95-99, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under very low
stringency conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 7 or
the full-length complementary strand thereof.
[0472] [101] The filamentous fungal host cell of any one of
paragraphs 95-100, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under very low
stringency conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 33
or the full-length complementary strand thereof.
[0473] [102] The filamentous fungal host cell of any one of
paragraphs 95-101, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide that hybridizes under very low
stringency conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 35
or SEQ ID NO: 37, or a full-length complementary strand
thereof.
[0474] [103] The filamentous fungal host cell of any one of
paragraphs 95-102, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60%, e.g., at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, 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 SEQ ID
NO: 7.
[0475] [104] The filamentous fungal host cell of any one of
paragraphs 95-103, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60%, e.g., at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, 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 SEQ ID
NO: 33.
[0476] [105] The filamentous fungal host cell of any one of
paragraphs 95-104, wherein the C4 dicarboxylic acid transporter is
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60%, e.g., at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, 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 SEQ ID
NO: 35 or SEQ ID NO: 37.
[0477] [106] The filamentous fungal host cell of any one of
paragraphs 95-105, wherein the C4 dicarboxylic acid transporter
comprises or consists of SEQ ID NO: 8.
[0478] [107] The filamentous fungal host cell of any one of
paragraphs 95-105, wherein the C4 dicarboxylic acid transporter
comprises or consists of SEQ ID NO: 34.
[0479] [108] The filamentous fungal host cell of any one of
paragraphs 95-105, wherein the C4 dicarboxylic acid transporter
comprises or consists of SEQ ID NO: 36.
[0480] [109] The filamentous fungal host cell of any of paragraphs
95-108, wherein the heterologous first polynucleotide encoding the
C4 dicarboxylic acid transporter is operably linked to a promoter
foreign to the third polynucleotide.
[0481] [110] The filamentous fungal host cell of any of paragraphs
95-109, wherein the malate dehydrogenase is selected from the group
consisting of (a) a malate dehydrogenase comprising an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 18 or SEQ ID
NO: 20; (b) a malate dehydrogenase encoded by a polynucleotide that
hybridizes under at least low stringency conditions, e.g., medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with (i)
SEQ ID NO: 17 or SEQ ID NO: 19, (ii) the cDNA sequence contained in
SEQ ID NO: 17 or SEQ ID NO: 19, or (iii) a full-length
complementary strand of (i) or (ii); (c) a malate dehydrogenase
encoded by a polynucleotide comprising a nucleotide sequence having
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% sequence identity with SEQ ID NO: 17 or SEQ ID NO: 19; (d) a
malate dehydrogenase variant comprising a substitution, deletion,
and/or insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 18 or SEQ ID NO: 20; and (e) a fragment
of the malate dehydrogenase of (a), (b), (c), or (d) that has
malate dehydrogenase activity.
[0482] [111] The filamentous fungal host cell of any of paragraphs
95-110, wherein the malate dehydrogenase comprises an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 18.
[0483] [112] The filamentous fungal host cell of any of paragraphs
95-111, wherein the malate dehydrogenase comprises an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 20.
[0484] [113] The filamentous fungal host cell of any of paragraphs
95-112, wherein the malate dehydrogenase is encoded by a
polynucleotide that hybridizes under low stringency conditions,
medium stringency conditions, medium-high stringency conditions,
high stringency conditions, or very high stringency conditions with
(i) SEQ ID NO: 17, (ii) the cDNA sequence contained in SEQ ID NO:
17, or (iii) the full-length complementary strand of (i) or
(ii).
[0485] [114] The filamentous fungal host cell of any of paragraphs
95-113, wherein the malate dehydrogenase is encoded by a
polynucleotide that hybridizes under low stringency conditions,
medium stringency conditions, medium-high stringency conditions,
high stringency conditions, or very high stringency conditions with
(i) SEQ ID NO: 19, (ii) the cDNA sequence contained in SEQ ID NO:
19, or (iii) the full-length complementary strand of (i) or
(ii).
[0486] [115] The filamentous fungal host cell of any of paragraphs
95-114, wherein the malate dehydrogenase is encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity with SEQ ID NO: 17.
[0487] [116] The filamentous fungal host cell of any of paragraphs
95-115, wherein the malate dehydrogenase is encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity with SEQ ID NO: 19.
[0488] [117] The filamentous fungal host cell of any of paragraphs
95-116, wherein the malate dehydrogenase comprises or consists of
SEQ ID NO: 18.
[0489] [118] The filamentous fungal host cell of any of paragraphs
95-116, wherein the malate dehydrogenase comprises or consists of
SEQ ID NO: 20.
[0490] [119] The filamentous fungal host cell of any of paragraphs
95-109, wherein the malate dehydrogenase is a variant of a parent
malate dehydrogenase comprising (i) a deletion at positions
equivalent to amino acids positions 2 to 17 or a portion thereof of
SEQ ID NO: 18, and (ii) a substitution at a position equivalent to
amino acid 48 of SEQ ID NO: 18; wherein the deletion and the
substitution reduce mitochondrial import in vivo of the malate
dehydrogenase variant thereby increasing the level of the malate
dehydrogenase variant in the cytosol, and wherein the filamentous
fungal host cell secretes (or is capable of secreting) increased
levels of the C4 dicarboxylic acid (e.g., malic acid) compared to
the filamentous fungal host cell without the polynucleotide
encoding the malate dehydrogenase variant when cultivated under the
same conditions.
[0491] [120] The filamentous fungal host cell of paragraph 119,
wherein the parent malate dehydrogenase is selected from the group
consisting of (a) a malate dehydrogenase comprising an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to SEQ ID NO: 18; (b) a malate
dehydrogenase encoded by a polynucleotide that hybridizes under at
least low stringency conditions, e.g., medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 17,
or its full-length complementary strand; and (c) a malate
dehydrogenase encoded by a polynucleotide comprising a nucleotide
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to SEQ ID NO: 17.
[0492] [121] The filamentous fungal host cell of paragraph 120,
wherein the parent malate dehydrogenase comprises or consists of
SEQ ID NO: 18.
[0493] [122] The filamentous fungal host cell of any of paragraphs
119-121, wherein the malate dehydrogenase variant comprises an
amino acid sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18.
[0494] [123] The filamentous fungal host cell of any of paragraphs
119-122, wherein the variant comprises the deletions
Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
[0495] [124] The filamentous fungal host cell of any of paragraphs
119-123, wherein the heterologous second polynucleotide encoding
the malate dehydrogenase is operably linked to a promoter foreign
to the first polynucleotide.
[0496] [125] The filamentous fungal host cell of any of paragraphs
95-124, wherein the pyruvate carboxylase is selected from the group
consisting of (a) a pyruvate carboxylase comprising an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 27; (b) a
pyruvate carboxylase encoded by a polynucleotide that hybridizes
under at least low stringency conditions, e.g., medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) SEQ ID NO:
26, (ii) the cDNA sequence contained in SEQ ID NO: 26, or (iii) the
full-length complementary strand of (i) or (ii); (c) a pyruvate
carboxylase encoded by a polynucleotide comprising a nucleotide
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity with SEQ ID NO: 26; (d) a
pyruvate carboxylase variant comprising a substitution, deletion,
and/or insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 27; and (e) a fragment of the pyruvate
carboxylase of (a), (b), (c), or (d) that has pyruvate carboxylase
activity.
[0497] [126] The filamentous fungal host cell of any of paragraphs
95-125, wherein the pyruvate carboxylase comprises an amino acid
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% with SEQ ID NO: 27.
[0498] [127] The filamentous fungal host cell of any of paragraphs
95-126, wherein the pyruvate carboxylase is encoded by a
polynucleotide that hybridizes under low stringency conditions,
medium stringency conditions, medium-high stringency conditions,
high stringency conditions, or very high stringency conditions with
(i) SEQ ID NO: 26, (ii) the cDNA sequence contained in SEQ ID NO:
26, or (iii) the full-length complementary strand of (i) or
(ii).
[0499] [128] The filamentous fungal host cell of any of paragraphs
95-127, wherein the pyruvate carboxylase is encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity with SEQ ID NO: 26.
[0500] [129] The filamentous fungal host cell of any of paragraphs
95-128, wherein the pyruvate carboxylase comprises or consists of
SEQ ID NO: 27.
[0501] [130] The filamentous fungal host cell of any of paragraphs
95-129, wherein the pyruvate carboxylase is a variant of a
mitochondrial pyruvate carboxylase.
[0502] [131] The filamentous fungal host cell of any of paragraphs
95-130, wherein the heterologous third polynucleotide encoding the
pyruvate carboxylase is operably linked to a promoter foreign to
the second polynucleotide.
[0503] [132] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous first polynucleotide
encoding a C4 dicarboxylic acid transporter.
[0504] [133] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous second polynucleotide
encoding a malate dehydrogenase.
[0505] [134] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous third polynucleotide
encoding a pyruvate carboxylase.
[0506] [135] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous first polynucleotide
encoding a C4 dicarboxylic acid transporter and a heterologous
second polynucleotide encoding a malate dehydrogenase.
[0507] [136] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous first polynucleotide
encoding a C4 dicarboxylic acid transporter and a heterologous
third polynucleotide encoding a pyruvate carboxylase.
[0508] [137] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous second polynucleotide
encoding a malate dehydrogenase and a heterologous third
polynucleotide encoding a pyruvate carboxylase.
[0509] [138] The filamentous fungal host cell of any of paragraphs
95-131, which comprises a heterologous first polynucleotide
encoding a C4 dicarboxylic acid transporter, a heterologous second
polynucleotide encoding a malate dehydrogenase, and a heterologous
third polynucleotide encoding a pyruvate carboxylase.
[0510] [139] The filamentous fungal host cell of any of paragraphs
95-138, which is selected from the group consisting of an
Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phiebia, Piromyces, Pleurotus, Rhizopus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, and Trichoderma cell.
[0511] [140] The filamentous fungal host cell of paragraph 139,
wherein the host cell is an Aspergillus host cell, such as
Aspergillus oryzae.
[0512] [141] The filamentous fungal host cell of any of paragraphs
95-140, wherein the level of the C4 dicarboxylic acid (e.g., malic
acid) is increased by at least 25%, e.g., by at least 50%, at least
100%, at least 200%, or at 500% compared to the filamentous fungal
host cell without the one or more (several) heterologous
polynucleotides encoding the C4 dicarboxylic acid transporter, the
malate dehydrogenase, and the pyruvate carboxylase when cultivated
under the same conditions.
[0513] [142] An isolated variant of a parent malate dehydrogenase,
comprising (i) a deletion at positions equivalent to amino acids
positions 2 to 17 or a portion thereof of SEQ ID NO: 18, and (ii) a
substitution at a position equivalent to amino acid 48 of SEQ ID
NO: 18.
[0514] [143] The variant of paragraph 142, wherein the parent
malate dehydrogenase is (a) a malate dehydrogenase comprising an
amino acid sequence having at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18; or
(b) a malate dehydrogenase encoded by a polynucleotide that
hybridizes under at least low stringency conditions, e.g., medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with SEQ
ID NO: 17 or its full-length complementary strand; or (c) a malate
dehydrogenase encoded by a polynucleotide comprising a nucleotide
sequence having at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to SEQ ID NO: 17.
[0515] [144] The variant of paragraph 142, wherein the parent
malate dehydrogenase comprises or consists of SEQ ID NO: 18.
[0516] [145] The variant of any of paragraphs 142-144, which
comprises an amino acid sequence having at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to
the amino acid sequence of the parent malate dehydrogenase.
[0517] [145] The variant of any of paragraphs 142-145, which
comprises the deletions Phe2*+Ala3*+Ala4*+Arg5*+Gln6*+Ser7*+Phe8*.
Asn9*+Leu10*+Leu11*+Gln12*+Lys13*+Arg14*+Ala15*+Phe16*+Ser17* of
SEQ ID NO: 18 and the substitution Arg48Tyr of SEQ ID NO: 18.
[0518] [146] An isolated polynucleotide encoding the variant of any
of paragraphs 142-146.
[0519] [147] A nucleic acid construct comprising the polynucleotide
of paragraph 147.
[0520] [148] A recombinant expression vector comprising the
polynucleotide of paragraph 147.
[0521] [149] A recombinant host cell comprising the polynucleotide
of paragraph 147.
[0522] [150] The method of any one of paragraphs 1-94, wherein the
C4 dicarboxylic acid is malic acid.
[0523] [151] The filamentous fungal host cell of any of paragraphs
95-140, wherein the C4 dicarboxylic acid is malic acid.
[0524] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
41140DNAAspergillus oryzae 1gtgatagaac atcgtccata atgctgacac
ctcccaagtt 40243DNAAspergillus oryzae 2agtcacctct agttaattaa
ttactaatca gatacatcct cat 43325DNAAspergillus oryzae 3ctatagcgaa
atggattgat tgtct 25420DNAAspergillus oryzae 4tgaccttcca cgctgaccac
20519DNAAspergillus oryzae 5ctaatcagat acatcctca
19619DNAAspergillus oryzae 6atgctgacac ctcccaagt
1971143DNAAspergillus oryzae 7atgctgacac ctcccaagtt tgaggatgag
aagcagctgg gccccgtggg tatccgggag 60aggcttcgcc atttcacttg ggcctggtac
acattaacga tgagtggagg agggctggcc 120gtcctcatca tcagccagcc
ctttgggttc cgcggattga gagagatcgg catcgctgtc 180tatatcctca
acctgatcct cttcgccctt gtctgctcta ccatggctat aaggttcatc
240ctgcacggca accttctgga gtccctccgt catgaccgcg agggtctctt
cttcccgacc 300ttctggctct ccgtcgcaac catcatctgc ggcttgtctc
gctacttcgg tgaagaatcg 360aatgagtcct tccaactagc cctcgaagcc
ctcttctgga tctactgcgt ctgcacctta 420ctcgtcgcaa tcatccaata
ctcgttcgtc ttctcatccc acaagtacgg ccttcaaacc 480atgatgcctt
catggatcct tccagccttc cccatcatgc tcagcggcac catcgcctcc
540gtcatcggtg aacaacaacc cgctcgcgca gccctcccca tcatcggcgc
cggcgtcacc 600ttccagggcc tcggcttctc catcagcttc atgatgtacg
cccactacat cggccgactg 660atggagtccg gcctccccca cagcgaccac
agaccaggca tgttcatctg cgtcggaccc 720cccgccttca cagccctcgc
cctcgtcggc atgagcaaag gcctccccga agacttcaag 780ctgctccacg
acgcccacgc cctggaagat ggccgcatca tcgagctgct ggccatctcc
840gccggcgtct tcctctgggc cctgagtctc tggttcttct gcatcgccat
tgtcgccgtc 900atccgctcgc cccccgaggc cttccacctc aactggtggg
ccatggtctt ccccaacacc 960ggcttcaccc tggccaccat caccctgggc
aaggctctca acagtaacgg tgtgaagggc 1020gtcggttccg ccatgtctat
ctgcatcgtg tgcatgtata tcttcgtctt cgtcaacaat 1080gtccgcgccg
ttatccggaa ggatatcatg tacccgggta aagatgagga tgtatctgat 1140tag
11438380PRTAspergillus oryzae 8Met Leu Thr Pro Pro Lys Phe Glu Asp
Glu Lys Gln Leu Gly Pro Val 1 5 10 15 Gly Ile Arg Glu Arg Leu Arg
His Phe Thr Trp Ala Trp Tyr Thr Leu 20 25 30 Thr Met Ser Gly Gly
Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe 35 40 45 Gly Phe Arg
Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn 50 55 60 Leu
Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile 65 70
75 80 Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly
Leu 85 90 95 Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile
Cys Gly Leu 100 105 110 Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser
Phe Gln Leu Ala Leu 115 120 125 Glu Ala Leu Phe Trp Ile Tyr Cys Val
Cys Thr Leu Leu Val Ala Ile 130 135 140 Ile Gln Tyr Ser Phe Val Phe
Ser Ser His Lys Tyr Gly Leu Gln Thr 145 150 155 160 Met Met Pro Ser
Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly 165 170 175 Thr Ile
Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu 180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile 195
200 205 Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser
Gly 210 215 220 Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys
Val Gly Pro 225 230 235 240 Pro Ala Phe Thr Ala Leu Ala Leu Val Gly
Met Ser Lys Gly Leu Pro 245 250 255 Glu Asp Phe Lys Leu Leu His Asp
Ala His Ala Leu Glu Asp Gly Arg 260 265 270 Ile Ile Glu Leu Leu Ala
Ile Ser Ala Gly Val Phe Leu Trp Ala Leu 275 280 285 Ser Leu Trp Phe
Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro 290 295 300 Pro Glu
Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr 305 310 315
320 Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335 Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val
Cys Met 340 345 350 Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val
Ile Arg Lys Asp 355 360 365 Ile Met Tyr Pro Gly Lys Asp Glu Asp Val
Ser Asp 370 375 380 949DNAAspergillus oryzae 9acacaactgg ccatgttcgc
tgctcgccag tctttcaacc tcctccaga 491060DNAAspergillus oryzae
10agtcacctct agttaattaa ttattaaggg ttggccttga cgaagtcaat acccttctga
601149DNAAspergillus oryzae 11acacaactgg ccatggtcaa agctggtgag
ttagcaatcc ttaacagat 491260DNAAspergillus oryzae 12agtcacctct
agttaattaa ttattacttt ggtggtgggt tcttaacgaa gtcgatgcct
601320DNAAspergillus oryzae 13ctttggtgtc accacactgg
201420DNAAspergillus oryzae 14gggatttgaa cagcagaagg
201523DNAAspergillus oryzae 15cttagcaagg tcgcggacaa tgg
231619DNAAspergillus oryzae 16ggcactggga attgaatac
19171294DNAAspergillus oryzae 17atgttcgctg ctcgccagtc tttcaacctc
ctccagaagc gcgccttctc cgcctctgcc 60agccaggtgt gtgattgaat ggatccattg
gacctcggag ctagctctgc aacatcaaca 120aaactaacat actaacttat
cttcttcata ggcttccaag gttgccgttc ttggtgccgc 180tggtggcatt
ggccagcctc tctcccttct cctcaagctc aacccccgtg tttctgagct
240tgccctctac gatatccgcg gtggccctgg tatgtttttg cacagcttgc
aacatctccg 300acttcggtga ttcaagacag ggctaacata aggatacaat
aggtgttgcc gctgacctga 360gccacatcaa caccaacagc accgtctctg
gctacgaggc taccccctct ggcctccgtg 420atgctctcaa gggctccgag
atcgtcctca tccctgccgg tgttcctcgc aagcccggca 480tgacccgtga
cggtatgaac cgttaacttg tcaatggcac tgggaattga atactaatta
540taatatcgcc agacctgttc aacaccaacg cctccattgt ccgcgacctt
gctaaggccg 600ccgccgaggc ttcccccgag gccaacatcc tcgtcatctc
caaccctgta tgacgctttc 660cacccactgc taccagttat ctcgcgctaa
ttgcaatcag gtcaactcca ccgtccccat 720cgtctctgag gtcttcaagt
ccaagggtgt ctacaacccc aagcgtctct tcggtgtcac 780tacccttgac
gttgtccgtg cctctcgctt catctcccag gtccagaaga ccgacccctc
840caacgaggcc gtcactgtcg tcggtggtca ctccggtgtg accattgtcc
ctcttctctc 900ccagtccagc caccccagca ttgagggtaa gacccgcgat
gagctcgtca accgcatcca 960gttcggtggt gatgaggttg tcaaggccaa
ggatggtgct ggctctgcca ccctctccat 1020ggccatggct ggtgctcgca
tggctgagtc cctcctgaag gccgcccagg gtgagaaggg 1080tgtcgttgag
cccactttcg tcgacagccc tctctacaag gaccagggtg ttgacttctt
1140cgcctccaag gtcgagctcg gccccaacgg tgttgagaag atcctccccg
ttggccaggt 1200caacgcctac gaggagaagc tcctcgaggc ctgccttggt
gacctcaaga agaacatcca 1260gaagggtatt gacttcgtca aggccaaccc ttaa
129418340PRTAspergillus oryzae 18Met Phe Ala Ala Arg Gln Ser Phe
Asn Leu Leu Gln Lys Arg Ala Phe 1 5 10 15 Ser Ala Ser Ala Ser Gln
Ala Ser Lys Val Ala Val Leu Gly Ala Ala 20 25 30 Gly Gly Ile Gly
Gln Pro Leu Ser Leu Leu Leu Lys Leu Asn Pro Arg 35 40 45 Val Ser
Glu Leu Ala Leu Tyr Asp Ile Arg Gly Gly Pro Gly Val Ala 50 55 60
Ala Asp Leu Ser His Ile Asn Thr Asn Ser Thr Val Ser Gly Tyr Glu 65
70 75 80 Ala Thr Pro Ser Gly Leu Arg Asp Ala Leu Lys Gly Ser Glu
Ile Val 85 90 95 Leu Ile Pro Ala Gly Val Pro Arg Lys Pro Gly Met
Thr Arg Asp Asp 100 105 110 Leu Phe Asn Thr Asn Ala Ser Ile Val Arg
Asp Leu Ala Lys Ala Ala 115 120 125 Ala Glu Ala Ser Pro Glu Ala Asn
Ile Leu Val Ile Ser Asn Pro Val 130 135 140 Asn Ser Thr Val Pro Ile
Val Ser Glu Val Phe Lys Ser Lys Gly Val 145 150 155 160 Tyr Asn Pro
Lys Arg Leu Phe Gly Val Thr Thr Leu Asp Val Val Arg 165 170 175 Ala
Ser Arg Phe Ile Ser Gln Val Gln Lys Thr Asp Pro Ser Asn Glu 180 185
190 Ala Val Thr Val Val Gly Gly His Ser Gly Val Thr Ile Val Pro Leu
195 200 205 Leu Ser Gln Ser Ser His Pro Ser Ile Glu Gly Lys Thr Arg
Asp Glu 210 215 220 Leu Val Asn Arg Ile Gln Phe Gly Gly Asp Glu Val
Val Lys Ala Lys 225 230 235 240 Asp Gly Ala Gly Ser Ala Thr Leu Ser
Met Ala Met Ala Gly Ala Arg 245 250 255 Met Ala Glu Ser Leu Leu Lys
Ala Ala Gln Gly Glu Lys Gly Val Val 260 265 270 Glu Pro Thr Phe Val
Asp Ser Pro Leu Tyr Lys Asp Gln Gly Val Asp 275 280 285 Phe Phe Ala
Ser Lys Val Glu Leu Gly Pro Asn Gly Val Glu Lys Ile 290 295 300 Leu
Pro Val Gly Gln Val Asn Ala Tyr Glu Glu Lys Leu Leu Glu Ala 305 310
315 320 Cys Leu Gly Asp Leu Lys Lys Asn Ile Gln Lys Gly Ile Asp Phe
Val 325 330 335 Lys Ala Asn Pro 340 191430DNAAspergillus oryzae
19atggtcaaag ctggtgagtt agcaatcctt aacagatgac actctcatag gtactaactc
60gaaacgttag cggtacttgg agcttctggt ggcattggcc aggtatggat atccccacgc
120cttacaaccc tggtcacaat atgaccttgt tcgatactga ctatctccca
agccactgtc 180tctcctgttg aagacctgtc ccttagttga agagcttgct
ctctacgatg ttgtgaacac 240ccctggtgtt gctgctgatc tatcccacat
ctcgtctatc gctgtacgtt actgccacaa 300tgcgaattgc ccgatggaag
aggcgaaaaa tggtatcttg cttacctggg cgattagaaa 360atctctggtt
ttctgcccaa agatgatggg ctgaagcagg cccttactgg tgctaatatt
420gttgtcatcc cggctggtat tccccgtaag tccctaccct ttcgcattgc
tcctcgtatg 480ttcgctggtg gccagttttc tgatagttga taggcaagcc
tggtatgacc cgtgacgacc 540tcttcaagat caacgccggc atagtgcgag
acttggtcaa gggtatcgcc gagttctgcc 600ccaaggcctt tgttctggtt
atctcaaacc ccgttaattc tactgttcct attgctgcag 660aggtgctcaa
agccgctggc gtctttgacc cgaagcgcct ctttggtgtc accacactgg
720acgtcgttcg tgcagagact ttcacccaag agttctcggg ccagaaggat
ccttctgctg 780ttcaaatccc agttgttggt ggccactctg gagagaccat
tgtccccctc ttcagcaaga 840ctacccccgc aattcagata cccgaggaga
agtatgacgc actgatccac cgtaggttgt 900cccaaagaat ctcatgaata
tcttgctgta agcactaact atgcttcagg cgtccaattt 960ggtggagatg
aggtggtcca agctaaggac ggtgctggtt ccgccacctt gtctatggcc
1020tatgccggtt acaggtaggg atgctgcgta ccgtgagagc actcgcggct
aacatgccat 1080aggttcgctg agagtgtaat caaagcttca aagggtcaaa
cgggtattgt cgagcctacc 1140ttcgtctacc tgcctggaat tcccggcggt
gatgagatcg ttaaggcaac tggcgtggaa 1200ttcttctcta ctcttgtaac
cttaggagta agattcatct cctcacagaa tcttcgttca 1260tatcacgcca
ggctaacgct attaaacaga ctaatggcgc agagaaggct agcaacgttc
1320ttgagggcgt gaccgagaag gaaaagaagc ttctcgaggc ttgcacgaaa
ggccttaagg 1380gtaatatcga gaaaggcatc gacttcgtta agaacccacc
accaaagtaa 143020330PRTAspergillus oryzae 20Met Val Lys Ala Ala Val
Leu Gly Ala Ser Gly Gly Ile Gly Gln Pro 1 5 10 15 Leu Ser Leu Leu
Leu Lys Thr Cys Pro Leu Val Glu Glu Leu Ala Leu 20 25 30 Tyr Asp
Val Val Asn Thr Pro Gly Val Ala Ala Asp Leu Ser His Ile 35 40 45
Ser Ser Ile Ala Lys Ile Ser Gly Phe Leu Pro Lys Asp Asp Gly Leu 50
55 60 Lys Gln Ala Leu Thr Gly Ala Asn Ile Val Val Ile Pro Ala Gly
Ile 65 70 75 80 Pro Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Lys
Ile Asn Ala 85 90 95 Gly Ile Val Arg Asp Leu Val Lys Gly Ile Ala
Glu Phe Cys Pro Lys 100 105 110 Ala Phe Val Leu Val Ile Ser Asn Pro
Val Asn Ser Thr Val Pro Ile 115 120 125 Ala Ala Glu Val Leu Lys Ala
Ala Gly Val Phe Asp Pro Lys Arg Leu 130 135 140 Phe Gly Val Thr Thr
Leu Asp Val Val Arg Ala Glu Thr Phe Thr Gln 145 150 155 160 Glu Phe
Ser Gly Gln Lys Asp Pro Ser Ala Val Gln Ile Pro Val Val 165 170 175
Gly Gly His Ser Gly Glu Thr Ile Val Pro Leu Phe Ser Lys Thr Thr 180
185 190 Pro Ala Ile Gln Ile Pro Glu Glu Lys Tyr Asp Ala Leu Ile His
Arg 195 200 205 Val Gln Phe Gly Gly Asp Glu Val Val Gln Ala Lys Asp
Gly Ala Gly 210 215 220 Ser Ala Thr Leu Ser Met Ala Tyr Ala Gly Tyr
Arg Phe Ala Glu Ser 225 230 235 240 Val Ile Lys Ala Ser Lys Gly Gln
Thr Gly Ile Val Glu Pro Thr Phe 245 250 255 Val Tyr Leu Pro Gly Ile
Pro Gly Gly Asp Glu Ile Val Lys Ala Thr 260 265 270 Gly Val Glu Phe
Phe Ser Thr Leu Val Thr Leu Gly Thr Asn Gly Ala 275 280 285 Glu Lys
Ala Ser Asn Val Leu Glu Gly Val Thr Glu Lys Glu Lys Lys 290 295 300
Leu Leu Glu Ala Cys Thr Lys Gly Leu Lys Gly Asn Ile Glu Lys Gly 305
310 315 320 Ile Asp Phe Val Lys Asn Pro Pro Pro Lys 325 330
2135DNAAspergillus oryzae 21acacaactgg ccatggcctc tgccagccag gtgtg
352240DNAAspergillus oryzae 22cctcaagctc aacccctacg tttctgagct
tgccctctac 402340DNAAspergillus oryzae 23gtagagggca agctcagaaa
cgtaggggtt gagcttgagg 402432DNAAspergillus oryzae 24acacaactgg
ccatggcggc tccgtttcgt ca 322545DNAAspergillus oryzae 25agtcacctct
agttaattaa ttattacgct ttgacgatct tgcag 45263643DNAAspergillus
oryzae 26atggcggctc cgtttcgtca gcctgaggag gcggtcgatg acaccgagtt
catcgatgac 60caccatgaac acctccgtga taccgtgcac catcggttgc gcgccaattc
ctccattatg 120cacttccaga agatcctcgt cgccaaccgt ggtgagatcc
ccattcgtat cttcagaacg 180gcccacgagc tgtccttgca gacggttgct
atctactctc atgaggatcg actgtcaatg 240caccgtcaaa aggccgatga
ggcctacatg attggccacc gcggtcagta cacccctgtc 300ggtgcgtacc
tggcgggcga tgagatcatc aagatcgccc tggagcacgg tgtccagctg
360atccacccgg gctacggttt cttgtccgag aacgccgact tcgcccgcaa
ggttgagaac 420gccggcattg tctttgtggg acccactccc gataccattg
acagcttggg tgacaaggtg 480tcggcccgtc ggctggccat taagtgcgag
gtccctgtcg ttccgggtac ggagggcccc 540gtcgagcgct atgaggaggt
caaggcgttc acagacacct atggcttccc catcatcatc 600aaggctgcct
ttggcggtgg tggccgtggt atgcgtgtgg tccgtgacca ggccgagctg
660cgtgactcgt tcgagcgagc cacctctgag gcccgctccg ccttcggcaa
tggtaccgtc 720ttcgtcgagc gcttcctcga caaacccaag cacattgaag
tccagcttct gggtgacagc 780cacggcaacg ttgtccatct gtttgagcgt
gactgctccg tgcagcgtcg tcaccagaag 840gtcgttgagg ttgctccggc
taaggacctg ccagccgatg tccgggaccg catcctggcc 900gatgctgtga
agctggccaa gtccgtcaac taccgtaacg ccggtacagc tgagttcctg
960gtggaccagc agaaccgcca ctacttcatt gaaatcaatc ctcgtatcca
agtcgagcac 1020accatcaccg aagagattac tggtatcgat atcgtggctg
cacagatcca gattgctgct 1080ggtgcaagcc tcgagcaact gggcctgact
caggaccgca tctccgcccg cggatttgcc 1140attcaatgtc gtatcaccac
ggaagatccc gccaaggggt tctctccgga tactggtaag 1200attgaggttt
atcgttccgc tggtggtaac ggtgtccgtc tggatggtgg taacggtttc
1260gctggtgcta tcatcacccc tcactacgac tccatgctgg tcaagtgcac
ctgccgtggt 1320tcgacctatg aaatcgctcg tcgcaaggtt gtgcgtgcct
tggtcgagtt ccgtattcgt 1380ggtgtgaaga ccaacattcc cttcctgact
tcgcttctga gccacccgac cttcgtcgat 1440ggaaactgct ggaccacttt
catcgacgac acccctgaat tgttctctct tgtcggcagt 1500cagaaccgtg
cccagaagct gctcgcatac ctcggcgatg tagctgtcaa cggtagtagc
1560atcaagggcc aaattggcga gcccaagctc aagggtgatg tcatcaagcc
gaagcttttc 1620gatgccgagg gcaagccgct tgacgtttcc gccccctgca
ccaagggttg gaagcagatt 1680ctggaccggg agggtccggc tgcctttgcg
aaggccgtgc gtgccaacaa gggttgcttg 1740atcatggata ctacctggcg
tgacgcccac cagtctttgc tggccacccg tgtgcgtacc 1800atcgacttgt
tgaacatcgc ccatgagacc agctacgcct actccaatgc gtacagtttg
1860gaatgctggg gtggtgctac cttcgatgtg gccatgcgtt tcctctatga
ggacccctgg 1920gaccgcctgc gcaagatgcg taaggctgtt cctaacatcc
cattccagat gttgctccgt 1980ggtgccaacg gtgtcgccta ctcttccctc
ccagacaacg ccatctacca cttctgtaag 2040caggctaaga agtgcggtgt
cgacattttc cgtgttttcg acgccctcaa cgatgtcgat 2100cagctcgagg
tcggtatcaa ggctgttcat gctgccgagg gtgttgtcga ggccaccatg
2160tgctacagcg gtgacatgct gaacccccac aagaagtaca acctggagta
ctacatggcc 2220ttggtggata agattgtagc catgaagcct cacatccttg
gtatcaagga tatggccggt 2280gtgctgaagc cccaggccgc tcgcctgttg
gtgggctcca tccgtcagcg ctaccctgac 2340cttcccatcc acgtccacac
ccacgactcc
gctggtactg gtgtagcttc catgattgcc 2400tgtgcccagg cgggtgccga
cgccgtggac gccgcgaccg acagcatgtc cggtatgacc 2460tcccagccta
gcattggtgc cattctggcc tctcttgagg gcactgagca agaccccggt
2520ctcaacctcg cccacgtgcg cgctattgat agctactggg cacagctgcg
cttgctctac 2580tctcctttcg aggcgggtct cactggcccc gaccctgagg
tctacgagca cgagatccct 2640ggtggtcagt tgaccaacct tatcttccag
gccagtcagc tcggcttggg ccagcagtgg 2700gccgaaacca agaaggccta
tgaggcggct aatgatttac tcggcgacat tgtaaaggtc 2760actcccacct
ccaaggtggt cggtgacttg gctcagttca tggtctcgaa caaactgact
2820ccagaggatg ttgttgagcg tgctggtgag ctggacttcc ctggttctgt
gctcgaattc 2880ctcgaaggtc tcatgggaca gcccttcggt ggattccccg
agccattgcg ctcccgcgcc 2940ctgcgcgatc gccgcaagct cgagaagcgt
ccaggtctct acctcgagcc tttggatttg 3000gctaagatca agagccagat
ccgtgagaag ttcggtgctg ctactgagta tgacgtggcc 3060agctatgcca
tgtatcccaa ggtcttcgag gactacaaga agttcgtcca gaagttcggt
3120gatctctccg tcttgcccac acggtacttc ttggccaagc ctgagattgg
cgaggagttc 3180cacgttgagc tggagaaggg taaggtgctc atcctgaagt
tgttggccat cggccctctt 3240tcagagcaga ctggtcagcg tgaggtcttc
tacgaagtca acggtgaggt gcgccaggtc 3300gctgttgatg acaacaaggc
ttccgtggac aacacttcac gccctaaggc cgatgtgggt 3360gacagcagcc
aggtcggtgc tcctatgagc ggtgtggttg ttgaaatccg tgtccacgat
3420ggtctggagg ttaagaaggg tgacccactt gccgtcctga gtgccatgaa
gatggtaagt 3480tcattccgaa tcatttttct cactggtcaa ctacagatgc
taacagctta tccaggaaat 3540ggttatctct gctcctcaca gtggaaaggt
ctccagcttg ctggtcaagg agggcgattc 3600tgtggatggc caggatctcg
tctgcaagat cgtcaaagcg taa 3643271193PRTAspergillus oryzae 27Met Ala
Ala Pro Phe Arg Gln Pro Glu Glu Ala Val Asp Asp Thr Glu 1 5 10 15
Phe Ile Asp Asp His His Glu His Leu Arg Asp Thr Val His His Arg 20
25 30 Leu Arg Ala Asn Ser Ser Ile Met His Phe Gln Lys Ile Leu Val
Ala 35 40 45 Asn Arg Gly Glu Ile Pro Ile Arg Ile Phe Arg Thr Ala
His Glu Leu 50 55 60 Ser Leu Gln Thr Val Ala Ile Tyr Ser His Glu
Asp Arg Leu Ser Met 65 70 75 80 His Arg Gln Lys Ala Asp Glu Ala Tyr
Met Ile Gly His Arg Gly Gln 85 90 95 Tyr Thr Pro Val Gly Ala Tyr
Leu Ala Gly Asp Glu Ile Ile Lys Ile 100 105 110 Ala Leu Glu His Gly
Val Gln Leu Ile His Pro Gly Tyr Gly Phe Leu 115 120 125 Ser Glu Asn
Ala Asp Phe Ala Arg Lys Val Glu Asn Ala Gly Ile Val 130 135 140 Phe
Val Gly Pro Thr Pro Asp Thr Ile Asp Ser Leu Gly Asp Lys Val 145 150
155 160 Ser Ala Arg Arg Leu Ala Ile Lys Cys Glu Val Pro Val Val Pro
Gly 165 170 175 Thr Glu Gly Pro Val Glu Arg Tyr Glu Glu Val Lys Ala
Phe Thr Asp 180 185 190 Thr Tyr Gly Phe Pro Ile Ile Ile Lys Ala Ala
Phe Gly Gly Gly Gly 195 200 205 Arg Gly Met Arg Val Val Arg Asp Gln
Ala Glu Leu Arg Asp Ser Phe 210 215 220 Glu Arg Ala Thr Ser Glu Ala
Arg Ser Ala Phe Gly Asn Gly Thr Val 225 230 235 240 Phe Val Glu Arg
Phe Leu Asp Lys Pro Lys His Ile Glu Val Gln Leu 245 250 255 Leu Gly
Asp Ser His Gly Asn Val Val His Leu Phe Glu Arg Asp Cys 260 265 270
Ser Val Gln Arg Arg His Gln Lys Val Val Glu Val Ala Pro Ala Lys 275
280 285 Asp Leu Pro Ala Asp Val Arg Asp Arg Ile Leu Ala Asp Ala Val
Lys 290 295 300 Leu Ala Lys Ser Val Asn Tyr Arg Asn Ala Gly Thr Ala
Glu Phe Leu 305 310 315 320 Val Asp Gln Gln Asn Arg His Tyr Phe Ile
Glu Ile Asn Pro Arg Ile 325 330 335 Gln Val Glu His Thr Ile Thr Glu
Glu Ile Thr Gly Ile Asp Ile Val 340 345 350 Ala Ala Gln Ile Gln Ile
Ala Ala Gly Ala Ser Leu Glu Gln Leu Gly 355 360 365 Leu Thr Gln Asp
Arg Ile Ser Ala Arg Gly Phe Ala Ile Gln Cys Arg 370 375 380 Ile Thr
Thr Glu Asp Pro Ala Lys Gly Phe Ser Pro Asp Thr Gly Lys 385 390 395
400 Ile Glu Val Tyr Arg Ser Ala Gly Gly Asn Gly Val Arg Leu Asp Gly
405 410 415 Gly Asn Gly Phe Ala Gly Ala Ile Ile Thr Pro His Tyr Asp
Ser Met 420 425 430 Leu Val Lys Cys Thr Cys Arg Gly Ser Thr Tyr Glu
Ile Ala Arg Arg 435 440 445 Lys Val Val Arg Ala Leu Val Glu Phe Arg
Ile Arg Gly Val Lys Thr 450 455 460 Asn Ile Pro Phe Leu Thr Ser Leu
Leu Ser His Pro Thr Phe Val Asp 465 470 475 480 Gly Asn Cys Trp Thr
Thr Phe Ile Asp Asp Thr Pro Glu Leu Phe Ser 485 490 495 Leu Val Gly
Ser Gln Asn Arg Ala Gln Lys Leu Leu Ala Tyr Leu Gly 500 505 510 Asp
Val Ala Val Asn Gly Ser Ser Ile Lys Gly Gln Ile Gly Glu Pro 515 520
525 Lys Leu Lys Gly Asp Val Ile Lys Pro Lys Leu Phe Asp Ala Glu Gly
530 535 540 Lys Pro Leu Asp Val Ser Ala Pro Cys Thr Lys Gly Trp Lys
Gln Ile 545 550 555 560 Leu Asp Arg Glu Gly Pro Ala Ala Phe Ala Lys
Ala Val Arg Ala Asn 565 570 575 Lys Gly Cys Leu Ile Met Asp Thr Thr
Trp Arg Asp Ala His Gln Ser 580 585 590 Leu Leu Ala Thr Arg Val Arg
Thr Ile Asp Leu Leu Asn Ile Ala His 595 600 605 Glu Thr Ser Tyr Ala
Tyr Ser Asn Ala Tyr Ser Leu Glu Cys Trp Gly 610 615 620 Gly Ala Thr
Phe Asp Val Ala Met Arg Phe Leu Tyr Glu Asp Pro Trp 625 630 635 640
Asp Arg Leu Arg Lys Met Arg Lys Ala Val Pro Asn Ile Pro Phe Gln 645
650 655 Met Leu Leu Arg Gly Ala Asn Gly Val Ala Tyr Ser Ser Leu Pro
Asp 660 665 670 Asn Ala Ile Tyr His Phe Cys Lys Gln Ala Lys Lys Cys
Gly Val Asp 675 680 685 Ile Phe Arg Val Phe Asp Ala Leu Asn Asp Val
Asp Gln Leu Glu Val 690 695 700 Gly Ile Lys Ala Val His Ala Ala Glu
Gly Val Val Glu Ala Thr Met 705 710 715 720 Cys Tyr Ser Gly Asp Met
Leu Asn Pro His Lys Lys Tyr Asn Leu Glu 725 730 735 Tyr Tyr Met Ala
Leu Val Asp Lys Ile Val Ala Met Lys Pro His Ile 740 745 750 Leu Gly
Ile Lys Asp Met Ala Gly Val Leu Lys Pro Gln Ala Ala Arg 755 760 765
Leu Leu Val Gly Ser Ile Arg Gln Arg Tyr Pro Asp Leu Pro Ile His 770
775 780 Val His Thr His Asp Ser Ala Gly Thr Gly Val Ala Ser Met Ile
Ala 785 790 795 800 Cys Ala Gln Ala Gly Ala Asp Ala Val Asp Ala Ala
Thr Asp Ser Met 805 810 815 Ser Gly Met Thr Ser Gln Pro Ser Ile Gly
Ala Ile Leu Ala Ser Leu 820 825 830 Glu Gly Thr Glu Gln Asp Pro Gly
Leu Asn Leu Ala His Val Arg Ala 835 840 845 Ile Asp Ser Tyr Trp Ala
Gln Leu Arg Leu Leu Tyr Ser Pro Phe Glu 850 855 860 Ala Gly Leu Thr
Gly Pro Asp Pro Glu Val Tyr Glu His Glu Ile Pro 865 870 875 880 Gly
Gly Gln Leu Thr Asn Leu Ile Phe Gln Ala Ser Gln Leu Gly Leu 885 890
895 Gly Gln Gln Trp Ala Glu Thr Lys Lys Ala Tyr Glu Ala Ala Asn Asp
900 905 910 Leu Leu Gly Asp Ile Val Lys Val Thr Pro Thr Ser Lys Val
Val Gly 915 920 925 Asp Leu Ala Gln Phe Met Val Ser Asn Lys Leu Thr
Pro Glu Asp Val 930 935 940 Val Glu Arg Ala Gly Glu Leu Asp Phe Pro
Gly Ser Val Leu Glu Phe 945 950 955 960 Leu Glu Gly Leu Met Gly Gln
Pro Phe Gly Gly Phe Pro Glu Pro Leu 965 970 975 Arg Ser Arg Ala Leu
Arg Asp Arg Arg Lys Leu Glu Lys Arg Pro Gly 980 985 990 Leu Tyr Leu
Glu Pro Leu Asp Leu Ala Lys Ile Lys Ser Gln Ile Arg 995 1000 1005
Glu Lys Phe Gly Ala Ala Thr Glu Tyr Asp Val Ala Ser Tyr Ala 1010
1015 1020 Met Tyr Pro Lys Val Phe Glu Asp Tyr Lys Lys Phe Val Gln
Lys 1025 1030 1035 Phe Gly Asp Leu Ser Val Leu Pro Thr Arg Tyr Phe
Leu Ala Lys 1040 1045 1050 Pro Glu Ile Gly Glu Glu Phe His Val Glu
Leu Glu Lys Gly Lys 1055 1060 1065 Val Leu Ile Leu Lys Leu Leu Ala
Ile Gly Pro Leu Ser Glu Gln 1070 1075 1080 Thr Gly Gln Arg Glu Val
Phe Tyr Glu Val Asn Gly Glu Val Arg 1085 1090 1095 Gln Val Ala Val
Asp Asp Asn Lys Ala Ser Val Asp Asn Thr Ser 1100 1105 1110 Arg Pro
Lys Ala Asp Val Gly Asp Ser Ser Gln Val Gly Ala Pro 1115 1120 1125
Met Ser Gly Val Val Val Glu Ile Arg Val His Asp Gly Leu Glu 1130
1135 1140 Val Lys Lys Gly Asp Pro Leu Ala Val Leu Ser Ala Met Lys
Met 1145 1150 1155 Glu Met Val Ile Ser Ala Pro His Ser Gly Lys Val
Ser Ser Leu 1160 1165 1170 Leu Val Lys Glu Gly Asp Ser Val Asp Gly
Gln Asp Leu Val Cys 1175 1180 1185 Lys Ile Val Lys Ala 1190
2836DNAAspergillus oryzae 28agaacatcgt ccataatggt caaagctggt gagtta
362940DNAAspergillus oryzae 29gtgtcagtca cctctagtta ttactttggt
ggtgggttct 403036DNAAspergillus oryzae 30tagaacatcg tccataatgg
cggctccgtt tcgtca 363140DNAAspergillus oryzae 31gtgtcagtca
cctctagtta ttattacgct ttgacgatct 403220DNAAspergillus oryzae
32ggaaacgtca agcggcttgc 20331182DNAAspergillus terreus 33atgtttgaga
acactgcccc tccagggagc tcccgctccg actctggcat cctggaccat 60gaattcgaga
agcagccggg ttccgtgggc atgcgtgaac gcatccgcca ttttacctgg
120gcctggtata ctctcacaat gagtgctggt ggcttggccc tcctccttgg
gagccagcca 180aacaccttca ccggcctgag ggagattgga ctcgccgtgt
acctgctcaa cctgctcttc 240tttgccctgg tctgctcgac catggccggc
cggttcatcc tgcacggagg gctggtcgac 300tctctccggc acgaacgcga
gggcatcttc ttcccaacct tctggctctc gatcgccacc 360atcatcacag
gcctgtaccg ctacttcggc gaagacgccg gacgcccctt cgtgctcgcc
420ctcgaagccc tcttctggat ctactgcgct tgcaccctcc tcgtcgccgt
catccaatac 480tcctggctct tctccggccc caaataccgc ctccaaaccg
ccatgcccgg ctggatcctc 540cccgccttcc ctgtcatgct ctctggcacc
atcgcctccg tcatcgccga gcagcagccg 600gcccgcgccg ccatccccat
catcgtcgcc ggcaccacct tccagggcct gggcttctcc 660atcagcatga
tcatgtacgc ccactacgtc ggccgcctca tggagtccgg cctgccgtgc
720cgcgagcacc gcccgggcat gttcatcgcc gtcggcccgc cggctttcac
ggcgctggcc 780ctcgtcggca tgaccaaggg gctcccgcac gacttccagc
tcatcggcga tgacttcgcc 840ttcgaggatg cccgcatcct gcagctgctg
gcgatcgccg tcggcgtgtt tctctgggcg 900ctgagtctgt ggttcttttg
cattgcggcc attgccgtcg tgcgctcccc gccaacggcc 960ttccacctga
gctggtgggc catggtcttc cccaacacgg gcttcaccct cgccacgatc
1020aacctgggta cggccctcaa gagcgagggt atccagggtg tggggacggc
catgtcgatt 1080ggaattgtgt ctattttctt gtttgtgttt atcagccatg
tgcgggctgt catcaggaaa 1140gacattatgt atcctgggaa agacgaggat
gtggtggagt aa 118234393PRTAspergillus terreus 34Met Phe Glu Asn Thr
Ala Pro Pro Gly Ser Ser Arg Ser Asp Ser Gly 1 5 10 15 Ile Leu Asp
His Glu Phe Glu Lys Gln Pro Gly Ser Val Gly Met Arg 20 25 30 Glu
Arg Ile Arg His Phe Thr Trp Ala Trp Tyr Thr Leu Thr Met Ser 35 40
45 Ala Gly Gly Leu Ala Leu Leu Leu Gly Ser Gln Pro Asn Thr Phe Thr
50 55 60 Gly Leu Arg Glu Ile Gly Leu Ala Val Tyr Leu Leu Asn Leu
Leu Phe 65 70 75 80 Phe Ala Leu Val Cys Ser Thr Met Ala Gly Arg Phe
Ile Leu His Gly 85 90 95 Gly Leu Val Asp Ser Leu Arg His Glu Arg
Glu Gly Ile Phe Phe Pro 100 105 110 Thr Phe Trp Leu Ser Ile Ala Thr
Ile Ile Thr Gly Leu Tyr Arg Tyr 115 120 125 Phe Gly Glu Asp Ala Gly
Arg Pro Phe Val Leu Ala Leu Glu Ala Leu 130 135 140 Phe Trp Ile Tyr
Cys Ala Cys Thr Leu Leu Val Ala Val Ile Gln Tyr 145 150 155 160 Ser
Trp Leu Phe Ser Gly Pro Lys Tyr Arg Leu Gln Thr Ala Met Pro 165 170
175 Gly Trp Ile Leu Pro Ala Phe Pro Val Met Leu Ser Gly Thr Ile Ala
180 185 190 Ser Val Ile Ala Glu Gln Gln Pro Ala Arg Ala Ala Ile Pro
Ile Ile 195 200 205 Val Ala Gly Thr Thr Phe Gln Gly Leu Gly Phe Ser
Ile Ser Met Ile 210 215 220 Met Tyr Ala His Tyr Val Gly Arg Leu Met
Glu Ser Gly Leu Pro Cys 225 230 235 240 Arg Glu His Arg Pro Gly Met
Phe Ile Ala Val Gly Pro Pro Ala Phe 245 250 255 Thr Ala Leu Ala Leu
Val Gly Met Thr Lys Gly Leu Pro His Asp Phe 260 265 270 Gln Leu Ile
Gly Asp Asp Phe Ala Phe Glu Asp Ala Arg Ile Leu Gln 275 280 285 Leu
Leu Ala Ile Ala Val Gly Val Phe Leu Trp Ala Leu Ser Leu Trp 290 295
300 Phe Phe Cys Ile Ala Ala Ile Ala Val Val Arg Ser Pro Pro Thr Ala
305 310 315 320 Phe His Leu Ser Trp Trp Ala Met Val Phe Pro Asn Thr
Gly Phe Thr 325 330 335 Leu Ala Thr Ile Asn Leu Gly Thr Ala Leu Lys
Ser Glu Gly Ile Gln 340 345 350 Gly Val Gly Thr Ala Met Ser Ile Gly
Ile Val Ser Ile Phe Leu Phe 355 360 365 Val Phe Ile Ser His Val Arg
Ala Val Ile Arg Lys Asp Ile Met Tyr 370 375 380 Pro Gly Lys Asp Glu
Asp Val Val Glu 385 390 351317DNASchizosaccharomyces pombe
35atgggagaat tgaaggaaat tctcaagcag cgctaccatg aattgctcga ctggaacgtc
60aaagcacccc acgtccctct ctcgcagagg ttgaagcatt tcacatggtc gtggttcgcg
120tgtacgatgg caaccggtgg cgtcggactc atcatcggat ccttcccttt
ccgattctac 180ggactcaaca cgatcggcaa gattgtgtac atcctccaga
ttttcctctt ctccttgttc 240ggctcgtgta tgctcttcag gttcatcaag
tatccgtcca caatcaagga ctcctggaac 300catcatctcg agaaactctt
cattgcgact tgtctcctct cgatttcgac attcatcgat 360atgttggcga
tctacgccta ccccgacaca ggcgagtgga tggtgtgggt catccgaatc
420ctctactaca tctacgtcgc ggtctccttc atttactgtg tgatggcgtt
cttcacgatc 480ttcaacaacc acgtctatac cattgaaacc gcctcgcctg
catggatcct ccctatcttc 540cctccgatga tctgtggtgt cattgccggt
gcggtgaact ccacccagcc tgcgcaccag 600ctcaaaaaca tggtgatttt
cggaatcctc ttccagggat tgggtttctg ggtctacttg 660ctcttgttcg
cagtcaacgt gctccggttc ttcacggtcg gcttggcaaa gccccaggac
720cgacctggca tgttcatgtt cgtgggacct cctgcgttct ccggcttggc
actcatcaac 780atcgcgaggg gtgccatggg ctcgaggccg tacatcttcg
tgggagcaaa ctcctcggaa 840tacttgggtt tcgtgtcgac gttcatggcg
attttcatct ggggcttggc agcatggtgt 900tattgtctcg ccatggtgtc
cttcctcgca ggcttcttca cacgcgcacc tttgaagttc 960gcgtgtggtt
ggttcgcatt catcttcccc aacgtgggct tcgtgaactg tacgattgag
1020atcggcaaga tgatcgactc caaagccttc cagatgttcg gccacattat
cggtgtcatc 1080ctctgtatcc agtggatttt gctcatgtat ttgatggtgc
gtgcgttctt ggtcaacgac 1140ttgtgttatc ccggtaaaga cgaggacgcc
catccgcctc ccaaacccaa cacaggcgtc 1200ctcaacccca ccttccctcc
cgaaaaagca cctgcctccc tcgaaaaagt cgatacacat 1260gtcacttcca
ctggcggaga gtcggatcct ccgtcctccg aacacgagtc ggtctaa
131736438PRTSchizosaccharomyces pombe 36Met Gly Glu Leu Lys Glu Ile
Leu Lys Gln Arg Tyr His Glu Leu Leu 1 5 10 15 Asp Trp Asn Val Lys
Ala Pro His Val Pro Leu Ser Gln Arg Leu Lys 20 25 30
His Phe Thr Trp Ser Trp Phe Ala Cys Thr Met Ala Thr Gly Gly Val 35
40 45 Gly Leu Ile Ile Gly Ser Phe Pro Phe Arg Phe Tyr Gly Leu Asn
Thr 50 55 60 Ile Gly Lys Ile Val Tyr Ile Leu Gln Ile Phe Leu Phe
Ser Leu Phe 65 70 75 80 Gly Ser Cys Met Leu Phe Arg Phe Ile Lys Tyr
Pro Ser Thr Ile Lys 85 90 95 Asp Ser Trp Asn His His Leu Glu Lys
Leu Phe Ile Ala Thr Cys Leu 100 105 110 Leu Ser Ile Ser Thr Phe Ile
Asp Met Leu Ala Ile Tyr Ala Tyr Pro 115 120 125 Asp Thr Gly Glu Trp
Met Val Trp Val Ile Arg Ile Leu Tyr Tyr Ile 130 135 140 Tyr Val Ala
Val Ser Phe Ile Tyr Cys Val Met Ala Phe Phe Thr Ile 145 150 155 160
Phe Asn Asn His Val Tyr Thr Ile Glu Thr Ala Ser Pro Ala Trp Ile 165
170 175 Leu Pro Ile Phe Pro Pro Met Ile Cys Gly Val Ile Ala Gly Ala
Val 180 185 190 Asn Ser Thr Gln Pro Ala His Gln Leu Lys Asn Met Val
Ile Phe Gly 195 200 205 Ile Leu Phe Gln Gly Leu Gly Phe Trp Val Tyr
Leu Leu Leu Phe Ala 210 215 220 Val Asn Val Leu Arg Phe Phe Thr Val
Gly Leu Ala Lys Pro Gln Asp 225 230 235 240 Arg Pro Gly Met Phe Met
Phe Val Gly Pro Pro Ala Phe Ser Gly Leu 245 250 255 Ala Leu Ile Asn
Ile Ala Arg Gly Ala Met Gly Ser Arg Pro Tyr Ile 260 265 270 Phe Val
Gly Ala Asn Ser Ser Glu Tyr Leu Gly Phe Val Ser Thr Phe 275 280 285
Met Ala Ile Phe Ile Trp Gly Leu Ala Ala Trp Cys Tyr Cys Leu Ala 290
295 300 Met Val Ser Phe Leu Ala Gly Phe Phe Thr Arg Ala Pro Leu Lys
Phe 305 310 315 320 Ala Cys Gly Trp Phe Ala Phe Ile Phe Pro Asn Val
Gly Phe Val Asn 325 330 335 Cys Thr Ile Glu Ile Gly Lys Met Ile Asp
Ser Lys Ala Phe Gln Met 340 345 350 Phe Gly His Ile Ile Gly Val Ile
Leu Cys Ile Gln Trp Ile Leu Leu 355 360 365 Met Tyr Leu Met Val Arg
Ala Phe Leu Val Asn Asp Leu Cys Tyr Pro 370 375 380 Gly Lys Asp Glu
Asp Ala His Pro Pro Pro Lys Pro Asn Thr Gly Val 385 390 395 400 Leu
Asn Pro Thr Phe Pro Pro Glu Lys Ala Pro Ala Ser Leu Glu Lys 405 410
415 Val Asp Thr His Val Thr Ser Thr Gly Gly Glu Ser Asp Pro Pro Ser
420 425 430 Ser Glu His Glu Ser Val 435
371317DNASchizosaccharomyces pombe 37atgggtgaac tcaaggaaat
cttgaaacag aggtatcatg agttgcttga ctggaatgtc 60aaagcccctc atgtccctct
cagtcaacga ctgaagcatt ttacatggtc ttggtttgca 120tgtactatgg
caactggtgg tgttggtttg attattggtt ctttcccctt tcgattttat
180ggtcttaata caattggcaa aattgtttat attcttcaaa tctttttgtt
ttctctcttt 240ggatcatgca tgctttttcg ctttattaaa tatccttcaa
ctatcaagga ttcctggaac 300catcatttgg aaaagctttt cattgctact
tgtcttcttt caatatccac gttcatcgac 360atgcttgcca tatacgccta
tcctgatacc ggcgagtgga tggtgtgggt cattcgaatc 420ctttattaca
tttacgttgc agtatccttt atatactgcg taatggcttt ttttacaatt
480ttcaacaacc atgtatatac cattgaaacc gcatctcctg cttggattct
tcctattttc 540cctcctatga tttgtggtgt cattgctggc gccgtcaatt
ctacacaacc cgctcatcaa 600ttaaaaaata tggttatctt tggtatcctc
tttcaaggac ttggtttttg ggtttatctt 660ttactgtttg ccgtcaatgt
cttacggttt tttactgtag gcctggcaaa accccaagat 720cgacctggta
tgtttatgtt tgtcggtcca ccagctttct caggtttggc cttaattaat
780attgcgcgtg gtgctatggg cagtcgccct tatatttttg ttggcgccaa
ctcatccgag 840tatcttggtt ttgtttctac ctttatggct atttttattt
ggggtcttgc tgcttggtgt 900tactgtctcg ccatggttag ctttttagcg
ggctttttca ctcgagcccc tctcaagttt 960gcttgtggat ggtttgcatt
cattttcccc aacgtgggtt ttgttaattg taccattgag 1020ataggtaaaa
tgatagattc caaagctttc caaatgtttg gacatatcat tggggtcatt
1080ctttgtattc agtggatcct cctaatgtat ttaatggtcc gtgcgtttct
cgtcaatgat 1140ctttgctatc ctggcaaaga cgaagatgcc catcctccac
caaaaccaaa tacaggtgtc 1200cttaacccta ccttcccacc tgaaaaagca
cctgcatctt tggaaaaagt cgatacacat 1260gtcacatcta ctggtggtga
atcggatcct cctagtagtg aacatgaaag cgtttaa 13173842DNAAspergillus
terreus 38gtgtgataga acatcgtcca taatgtttga gaacactgcc cc
423944DNAAspergillus terreus 39gtcagtcacc tctagttaat taattactcc
accacatcct cgtc 444036DNASchizosaccharomyces pombe 40gaacatcgtc
cataatggga gaattgaagg aaattc 364140DNASchizosaccharomyces pombe
41ggtgtcagtc acctctagtt attattagac cgactcgtgt 40
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