U.S. patent application number 13/926421 was filed with the patent office on 2013-10-31 for methods for improved c4-dicarboxylic acid production in filamentous fungi.
The applicant listed for this patent is Novozymes, Inc.. Invention is credited to Sheryl Luttringer, Debbie Yaver.
Application Number | 20130288321 13/926421 |
Document ID | / |
Family ID | 44504321 |
Filed Date | 2013-10-31 |
United States Patent
Application |
20130288321 |
Kind Code |
A1 |
Luttringer; Sheryl ; et
al. |
October 31, 2013 |
Methods for Improved C4-Dicarboxylic Acid Production in Filamentous
Fungi
Abstract
The present invention relates to methods of producing
C4-dicarboxylic acids, such as malic acid, comprising: (a)
cultivating a host cell comprising a polynucleotide encoding a
C4-dicarboxylic acid transporter; and (b) recovering the
C4-dicarboxylic acid. The present invention also relates to methods
for increasing C4-dicarboxylic acid production, as well as host
cells comprising the polynucleotides.
Inventors: |
Luttringer; Sheryl; (Loomis,
CA) ; Yaver; Debbie; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes, Inc. |
Davis |
CA |
US |
|
|
Family ID: |
44504321 |
Appl. No.: |
13/926421 |
Filed: |
June 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13165719 |
Jun 21, 2011 |
8497103 |
|
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13926421 |
|
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61357007 |
Jun 21, 2010 |
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Current U.S.
Class: |
435/145 ;
435/254.3 |
Current CPC
Class: |
C07K 14/38 20130101;
C12P 7/46 20130101 |
Class at
Publication: |
435/145 ;
435/254.3 |
International
Class: |
C12P 7/46 20060101
C12P007/46 |
Claims
1: A host cell comprising a heterologous polynucleotide encoding a
C4-dicarboxylic acid transporter, wherein the heterologous
polynucleotide: (a) encodes a C4-dicarboxylic acid transporter
having at least 95% sequence identity to SEQ ID NO: 4; (b)
hybridizes under high stringency conditions with the full-length
complementary strand of SEQ ID NO: 3; or (c) has at least 95%
sequence identity to SEQ ID NO: 3; wherein the host cell is capable
of secreting increased levels of C4-dicarboxylic acid compared to
the host cell without the heterologous polynucleotide encoding the
C4-dicarboxylic acid transporter when cultivated under the same
conditions.
2: The host cell of claim 1, wherein the heterologous
polynucleotide encodes a C4-dicarboxylic acid transporter having at
least 95% sequence identity to SEQ ID NO: 4.
3: The host cell of claim 1, wherein the heterologous
polynucleotide encodes a C4-dicarboxylic acid transporter having at
least 97% sequence identity to SEQ ID NO: 4.
4: The host cell of claim 1, wherein the heterologous
polynucleotide encodes a C4-dicarboxylic acid transporter having at
least 99% sequence identity to SEQ ID NO: 4.
5: The host cell of claim 1, wherein the heterologous
polynucleotide encodes a C4-dicarboxylic acid transporter that
comprises or consists of SEQ ID NO: 4.
6: The host cell of claim 1, wherein the heterologous
polynucleotide hybridizes under high stringency conditions with the
full-length complementary strand of SEQ ID NO: 3.
7: The host cell of claim 1, wherein the heterologous
polynucleotide hybridizes under very high stringency conditions
with the full-length complementary strand of SEQ ID NO: 3.
8: The host cell of claim 1, wherein the heterologous
polynucleotide has at least 95% sequence identity to SEQ ID NO:
3.
9: The host cell of claim 1, wherein the heterologous
polynucleotide has at least 97% sequence identity to SEQ ID NO:
3.
10: The host cell of claim 1, wherein the heterologous
polynucleotide has at least 99% sequence identity to SEQ ID NO:
3.
11: The host cell of claim 1, wherein the heterologous
polynucleotide comprises or consists of SEQ ID NO: 3.
12: The host cell of claim 1, wherein the heterologous
polynucleotide encoding the C4-dicarboxylic acid transporter is
operably linked to a promoter foreign to the polynucleotide.
13: The host cell of claim 1, wherein the host cell is a
filamentous fungal host cell.
14: The host cell of claim 13, wherein the host cell is an
Aspergillus host cell.
15: The host cell of claim 14, wherein the Aspergillus host cell is
an Aspergillus oryzae host cell.
16: The host cell of claim 1, wherein the host cell is capable of
secreting at least 50% more C4-dicarboxylic acid compared to the
host cell without the heterologous polynucleotide encoding the
C4-dicarboxylic acid transporter when cultivated under the same
conditions.
17: The host cell of claim 1, further comprising a heterologous
polynucleotide encoding a malate dehydrogenase.
18: The host cell of claim 1, further comprising a heterologous
polynucleotide encoding a pyruvate carboxylase.
19: The host cell of claim 1, wherein the C4-dicarboxylic acid is
malic acid.
20: A method of producing a C4-dicarboxylic acid, comprising: (a)
cultivating the host cell of claim 1 in a medium; and (b)
recovering the C4-dicarboxylic acid.
21: The method of claim 20, wherein the heterologous polynucleotide
encodes a C4-dicarboxylic acid transporter having at least 95%
sequence identity to SEQ ID NO: 4.
22: The method of claim 20, wherein the C4 dicarboxylic acid is
malic acid.
23: The method of claim 20, wherein the host cell is an Aspergillus
host cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/165,719 filed Jun. 21, 2011, which claims priority benefit
of U.S. provisional application No. 61/357,007, filed Jun. 21,
2010. The content of these applications is incorporated herein by
reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods for improving the
production of C4-dicarboxylic acids (e.g., malic acid) 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). 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.
[0008] 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.
[0009] 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).
PCT Application No. PCT/US10/47002, entitled "Methods for Improving
Malic Acid Production in Filamentous Fungi" filed Aug. 27, 2010,
the content of which is hereby incorporated by reference in its
entirety, describes malic acid production in filamentous fungi.
[0010] It would be advantageous in the art to improve
C4-dicarboxylic acid production, such as malic acid production, in
Aspergillus as a result of genetic engineering using recombinant
DNA techniques. The present invention provides, inter alia, methods
for improving C4-dicarboxylic acid production (e.g., malic acid
production).
SUMMARY OF THE INVENTION
[0011] The present invention relates to methods of producing
C4-dicarboxylic acids (e.g., malic acid). In one aspect, the method
comprises (a) cultivating a host cell (e.g., a filamentous fungal
host cell) comprising a heterologous polynucleotide encoding a
C4-dicarboxylic acid transporter described herein; and (b)
recovering the C4-dicarboxylic acid (e.g., malic acid). In another
aspect, the method comprises (a) transforming into host cell (e.g.,
a filamentous fungal host cell) a heterologous polynucleotide
encoding a C4-dicarboxylic acid transporter described herein; (b)
cultivating the transformed organism in a medium; and (c)
recovering the C4-dicarboxylic acid (e.g., malic acid). In some
aspects of the methods, the host cell further comprises a
heterologous polynucleotide encoding a malate dehydrogenase and/or
a pyruvate carboxylase.
[0012] The present invention also relates to a host cell (e.g., a
filamentous fungal host cell, such as Aspergillus oryzae)
comprising a polynucleotide described herein wherein the host cell
secretes and/or is capable of secreting increased levels of a
C4-dicarboxylic acid (e.g., malic acid). In some aspects, the host
cell further comprises a heterologous polynucleotide encoding a
malate dehydrogenase and/or a pyruvate carboxylase.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a restriction map of pAcC4T.
[0014] FIG. 2 shows a restriction map of pShTh60.
[0015] FIG. 3 shows a restriction map of pShTh120AcC4T.
[0016] FIG. 4 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus clavatus C4-dicarboxylic acid
transporter gene (SEQ ID NOs: 1 and 2, respectively).
[0017] FIG. 5 shows a restriction map of pAfC4T.
[0018] FIG. 6 shows a restriction map of pShTh121AfC4T.
[0019] FIG. 7 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus fumigates C4-dicarboxylic acid
transporter gene (SEQ ID NOs: 3 and 4, respectively).
[0020] FIG. 8 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus oryzae NRRL 3488 malate
dehydrogenase gene (mdh3) (SEQ ID NOs: 11 and 12,
respectively).
[0021] FIGS. 9A and 9B together show the genomic DNA sequence and
the deduced amino acid sequence of an Aspergillus oryzae NRRL 3488
pyruvate carboxylase gene (pyc) (SEQ ID NOs: 13 and 14,
respectively).
Definitions
[0022] 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.
[0023] In some aspects, 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 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%, at least 99%, or at least 100% of
the C4-dicarboxylic acid transporter activity (e.g., malic acid
transporter activity) of the mature polypeptide sequence of SEQ ID
NO: 2 or the mature polypeptide sequence of SEQ ID NO: 4.
[0024] 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.
[0025] In some aspects, 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 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%, at least 99%, or at least 100% of the
malate dehydrogenase activity of the mature polypeptide sequence of
SEQ ID NO: 12.
[0026] 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.
[0027] In some aspects, 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 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%, at least 99%, or at least 100% of the
pyruvate carboxylase activity of the mature polypeptide sequence of
SEQ ID NO: 14.
[0028] 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 (e.g., two, several) extra copies of
the polynucleotide into the host cell.
[0029] Isolated/purified: The terms "isolated" and "purified" mean
a polypeptide or polynucleotide that is removed from at least one
component with which it is naturally associated. For example, a
polypeptide may be 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 93% pure, at
least 95% pure, at least 97%, at least 98% pure, or at least 99%
pure, as determined by SDS-PAGE and a polynucleotide may be 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%, at least 93% pure, at least 95% pure, at least 97%,
at least 98% pure, or at least 99% pure, as determined by agarose
electrophoresis.
[0030] Coding sequence: The term "coding sequence" means a
polynucleotide sequence, which specifies the amino acid sequence of
a polypeptide. 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 sequence of genomic DNA, cDNA, a synthetic
polynucleotide, and/or a recombinant polynucleotide.
[0031] cDNA sequence: The term "cDNA sequence" means a sequence of
DNA following reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell.
[0032] The initial, primary RNA transcript from genomic DNA is a
precursor to mRNA that is processed through a series of steps,
including splicing, before appearing as mature spliced mRNA. A cDNA
sequence lacks intervening intron sequences that may be present in
the corresponding genomic DNA sequence. Accordingly, the phrase
"the cDNA sequence of SEQ ID NO: X" intends the resulting sequence
after the intervening intron sequences of SEQ ID NO: X, if present,
are removed. In some instances--when a referenced genomic DNA
sequence lacks intervening intron sequences--a cDNA sequence may be
identical to its corresponding genomic DNA sequence.
[0033] Genomic DNA sequence: The term "genomic DNA sequence" means
a DNA sequence found in the genome of a source organism (e.g., a
eukaryotic or prokaryotic genome). In some instances, a genomic DNA
sequence from a eukaryotic genome contains one or more intervening
intron sequences that are removed from the primary RNA transcript
as a result of RNA splicing. Accordingly, the phrase "the genomic
DNA sequence of SEQ ID NO: Y" intends the corresponding DNA
sequence from the source organism which includes intervening intron
sequences, if any, that are present before RNA splicing.
[0034] Mature polypeptide sequence: The term "mature polypeptide
sequence" means the portion of the referenced polypeptide sequence
after any post-translational sequence modifications (such as
N-terminal processing and/or C-terminal truncation). In some
instances, the mature polypeptide sequence may be identical to the
entire referenced polypeptide sequence. In one aspect, the mature
polypeptide sequence is amino acids 53 to 392 of SEQ ID NO: 2 based
on the Vector NIT.RTM. program (Invitrogen, CA, USA) that predicts
amino acids 1 to 52 of SEQ ID NO: 2 are a signal peptide. In
another aspect, the mature polypeptide sequence is amino acids 1 to
393 of SEQ ID NO: 4.
[0035] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means the portion of the referenced
polynucleotide sequence (e.g., genomic or cDNA sequence) that
encodes a mature polypeptide sequence. In some instances, the
mature polypeptide coding sequence may be identical to the entire
referenced polynucleotide sequence. In one aspect, the mature
polypeptide coding sequence is nucleotides 157 to 1179 of SEQ ID
NO: 1 based on the Vector NIT.RTM. program (Invitrogen, CA, USA)
that predicts nucleotides 1 to 156 of SEQ ID NO: 1 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 1 to 1182 of SEQ ID NO: 3.
[0036] Fragment: The term "fragment" means a polypeptide having one
or more (e.g., two, several) amino acids deleted from the amino
and/or carboxyl terminus of a referenced polypeptide sequence. In
one aspect, the fragment has C4-dicarboxylic acid transporter
activity. In another aspect, a fragment contains at least 332 amino
acid residues, e.g., at least 352 amino acid residues or at least
372 amino acid residues of SEQ ID NO: 2. In another aspect, a
fragment contains at least 332 amino acid residues, e.g., at least
352 amino acid residues or at least 372 amino acid residues of SEQ
ID NO: 4.
[0037] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g., two, several) nucleotides deleted from
the 5' and/or 3' end of the referenced nucleotide sequence. In one
aspect, the subsequence encodes a fragment having C4-dicarboxylic
acid transporter activity. In another aspect, a subsequence
contains at least 996 nucleotides, e.g., at least 1056 nucleotides
or at least 1116 nucleotides of SEQ ID NO: 1. In another aspect, a
subsequence contains at least 996 nucleotides, e.g., at least 1056
nucleotides or at least 1116 nucleotides of SEQ ID NO: 3.
[0038] Allelic variant: The term "allelic variant" means 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.
[0039] Sequence Identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0040] For purposes of the present invention, the degree of
sequence 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 Genet. 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)
[0041] For purposes of the present invention, the degree of
sequence 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)
[0042] 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.
[0043] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule--single-stranded or
double-stranded--which is isolated from a naturally occurring gene,
modified to contain segments of nucleic acids in a manner that
would not otherwise exist in nature, or synthetic, wherein the
nucleic acid molecule comprises one or more (e.g., two, several)
control sequences.
[0044] Control sequence: The term "control sequence" means a
nucleic acid sequence necessary for polypeptide expression. Control
sequences may be native or foreign to the polynucleotide encoding
the polypeptide, and native or foreign to each other. Such control
sequences include, but are not limited to, a leader sequence,
polyadenylation sequence, propeptide sequence, promoter sequence,
signal peptide sequence, and transcription terminator sequence. 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 polynucleotide
encoding a polypeptide.
[0045] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs the
expression of the coding sequence.
[0046] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a polypeptide and is operably linked to control sequences,
wherein the control sequences provide for expression of the
polynucleotide encoding the polypeptide. At a minimum, the
expression vector comprises a promoter sequence, and
transcriptional and translational stop signal sequences.
[0047] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, and the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention (e.g., a polynucleotide
encoding a C4-dicarboxylic acid transporter). 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.
[0048] Variant: The term "variant" means a polypeptide having
activity, e.g., C4-dicarboxylic acid transporter activity,
comprising an alteration, i.e., a substitution, insertion, and/or
deletion of one or more (e.g., two, several) amino acid residues at
one or more 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, e.g., 1-3 amino acids,
adjacent to an amino acid occupying a position.
[0049] Volumetric productivity: The term "volumetric productivity"
refers to the amount of referenced product produced (e.g., the
amount of a C4-dicarboxylic acid produced) per volume of the system
used (e.g., the total volume of media and contents therein) per
unit of time.
[0050] Fermentable medium: The term "fermentable medium" refers to
a medium comprising one or more (e.g., two, several) sugars, such
as glucose, fructose, sucrose, cellobiose, xylose, xylulose,
arabinose, mannose, galactose, and/or soluble oligosaccharides,
wherein the medium is capable, in part, of being converted
(fermented) by a host cell into a desired product, such as a
C4-dicarboxylic acid. In some instances, the fermentation medium is
derived from a natural source, such as sugar cane, starch, or
cellulose, and may be the result of pretreating the source by
enzymatic hydrolysis (saccharification).
[0051] 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".
[0052] 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.
[0053] 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
[0054] The present invention describes the overexpression of
specific genes in a host cell, such as a filamentous fungus (e.g.,
Aspergillus) to enhance the production of C4-dicarboxylic acids,
(e.g., malic acid) that encompasses transport of the
C4-dicarboxylic acid out of the cell via a C4-dicarboxylic acid
transporter. In the present invention, the C4-dicarboxylic acid
transporter can be any described 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
C4-dicarboxylic acid in high titers. The recombinant host cell may
further comprise a heterologous polynucleotide encoding a malate
dehydrogenase and/or a heterologous polynucleotide encoding a
pyruvate carboxylase.
C4-Dicarboxylic Acid Transporters and Polynucleotides Encoding
C4-Dicarboxylic Acid Transporters
[0055] In one aspect of the recombinant host cells and methods
described herein, the C4-dicarboxylic acid transporter is selected
from: (a) a C4-dicarboxylic acid transporter having at least 60%
sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide
sequence thereof; (b) a C4-dicarboxylic acid transporter encoded by
a polynucleotide that hybridizes under low stringency conditions
with SEQ ID NO: 1 or 3, the mature polypeptide coding sequence
thereof, or the full-length complementary strand of the foregoing;
(c) a C4-dicarboxylic acid transporter encoded by a polynucleotide
having at least 60% sequence identity to SEQ ID NO: 1 or 3, the
mature polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing; (d) a C4-dicarboxylic acid
transporter variant comprising a substitution, deletion, and/or
insertion of one or more (e.g., two, several) amino acids of SEQ ID
NO: 2 or 4, or the mature polypeptide sequence thereof; and (e) a
fragment of a polypeptide of (a), (b), (c), or (d) that has
C4-dicarboxylic acid transporter activity.
[0056] In one aspect, the C4-dicarboxylic acid transporter
comprises or consists of an amino acid sequence 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4,
or the mature polypeptide sequence thereof. In one aspect, the
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: 2 or 4, or the mature polypeptide sequence
thereof.
[0057] In one aspect, the C4-dicarboxylic acid transporter
comprises or consists of an amino acid sequence 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or the
mature polypeptide sequence thereof. In one aspect, the
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: 2 or the mature polypeptide sequence thereof.
In another aspect, the C4-dicarboxylic acid transporter comprises
an amino acid sequence 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%, at least 99%, or
100% sequence identity to SEQ ID NO: 4 or the mature polypeptide
sequence thereof. In one aspect, the 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: 4 or the mature polypeptide sequence thereof.
[0058] In one aspect, the C4-dicarboxylic acid transporter
comprises or consists of the amino acid sequence of SEQ ID NO: 2,
the mature polypeptide sequence of SEQ ID NO: 2, an allelic variant
thereof, or a fragment of the foregoing, having C4-dicarboxylic
acid transporter activity. In another aspect, the C4-dicarboxylic
acid transporter comprises or consists of the amino acid sequence
of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic acid
transporter comprises or consists of the mature polypeptide
sequence of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic
acid transporter comprises or consists of amino acids 1 to 392 of
SEQ ID NO: 2.
[0059] In one aspect, the C4-dicarboxylic acid transporter
comprises or consists of the amino acid sequence of SEQ ID NO: 4,
the mature polypeptide sequence of SEQ ID NO: 4, an allelic variant
thereof, or a fragment of the foregoing, having C4-dicarboxylic
acid transporter activity. In another aspect, the C4-dicarboxylic
acid transporter comprises or consists of the amino acid sequence
of SEQ ID NO: 4. In another aspect, the C4-dicarboxylic acid
transporter comprises or consists of the mature polypeptide
sequence of SEQ ID NO: 4. In another aspect, the C4-dicarboxylic
acid transporter comprises or consists of amino acids 1 to 393 of
SEQ ID NO: 4.
[0060] In one aspect, the C4-dicarboxylic acid transporter is
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: 1 or 3, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing (see, e.g., J. Sambrook, E.
F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory
Manual, 2d edition, Cold Spring Harbor, N.Y.).
[0061] In one aspect, the C4-dicarboxylic acid transporter is
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: 1, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing. In another aspect, the
C4-dicarboxylic acid transporter is 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: 3, the mature polypeptide coding sequence thereof, of
the full-length complementary strand of the foregoing.
[0062] In one aspect, the C4-dicarboxylic acid transporter is
encoded by a polynucleotide having at least 65%, e.g., at least
70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 1 or 3, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing.
[0063] In one aspect, the C4-dicarboxylic acid transporter is
encoded by a polynucleotide having at least 65%, e.g., at least
70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 1, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing.
[0064] In one aspect, the C4-dicarboxylic acid transporter is
encoded by a polynucleotide having at least 65%, e.g., at least
70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 3, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing.
[0065] In one aspect, the C4-dicarboxylic acid transporter is
encoded by SEQ ID NO: 1 or 3, or the mature polypeptide coding
sequence thereof. In one aspect, the C4-dicarboxylic acid
transporter is encoded by SEQ ID NO: 1 or the mature polypeptide
coding sequence thereof. In one aspect, the C4-dicarboxylic acid
transporter is encoded by SEQ ID NO: 1. In one aspect, the
C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 3 or the
mature polypeptide coding sequence thereof. In one aspect, the
C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 3. In one
aspect, the C4-dicarboxylic acid transporter is encoded by a
subsequence of SEQ ID NO: 1 or 3, wherein the subsequence encodes a
polypeptide having C4-dicarboxylic acid transporter activity. In
one aspect, the C4-dicarboxylic acid transporter is encoded by a
subsequence of SEQ ID NO: 1, wherein the subsequence encodes a
polypeptide having C4-dicarboxylic acid transporter activity. In
one aspect, the C4-dicarboxylic acid transporter is encoded by a
subsequence of SEQ ID NO: 3, wherein the subsequence encodes a
polypeptide having C4-dicarboxylic acid transporter activity.
[0066] In one aspect, the C4-dicarboxylic acid transporter is a
variant comprising a substitution, deletion, and/or insertion of
one or more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4,
or the mature polypeptide sequence thereof. In one aspect, the
C4-dicarboxylic acid transporter is a variant comprising a
substitution, deletion, and/or insertion of one or more amino acids
of SEQ ID NO: 2. In one aspect, the C4-dicarboxylic acid
transporter is a variant comprising a substitution, deletion,
and/or insertion of one or more amino acids of the mature
polypeptide sequence of SEQ ID NO: 2. In one aspect, the
C4-dicarboxylic acid transporter is a variant comprising a
substitution, deletion, and/or insertion of one or more amino acids
of SEQ ID NO: 4. In one aspect, the C4-dicarboxylic acid
transporter is a variant comprising a substitution, deletion,
and/or insertion of one or more amino acids of the mature
polypeptide sequence of SEQ ID NO: 4.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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
activity (e.g., 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 referenced parent
polypeptide.
[0071] 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).
[0072] 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.
[0073] In some aspects, the total number of amino acid
substitutions, deletions and/or insertions of SEQ ID NO: 2 or 4, or
the mature polypeptide sequence thereof, is not more than 10, e.g.,
not more than 1, 2, 3, 4, 5, 6, 7, 8 or 9.
[0074] In another aspect, the C4-dicarboxylic acid transporter is a
fragment of SEQ ID NO: 2 or 4, or the mature polypeptide sequence
thereof, wherein the fragment has C4-dicarboxylic acid transporter
activity. In one aspect, the C4-dicarboxylic acid transporter is a
fragment of SEQ ID NO: 2 or the mature polypeptide sequence
thereof, wherein the fragment has C4-dicarboxylic acid transporter
activity. In one aspect, the fragment contains at least 332 amino
acid residues, e.g., at least 352 amino acid residues, or at least
372 amino acid residues of SEQ ID NO: 2. In one aspect, the
fragment contains a C4-dicarboxylic acid transporter domain, e.g.,
the putative transporter domain of amino acids 39 to 337 of SEQ ID
NO: 2. In another aspect, the C4-dicarboxylic acid transporter is a
fragment of SEQ ID NO: 4 or the mature polypeptide sequence
thereof, wherein the fragment has C4-dicarboxylic acid transporter
activity. In one aspect, the fragment contains at least 332 amino
acid residues, e.g., at least 352 amino acid residues, or at least
372 amino acid residues of SEQ ID NO: 4. In one aspect, the
fragment contains a C4-dicarboxylic acid transporter domain, e.g.,
the putative transporter domain of amino acids 41 to 338 of SEQ ID
NO: 4.
[0075] 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).
[0076] 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.
[0077] Techniques used to isolate or clone a polynucleotide--such
as a polynucleotide encoding a C4-dicarboxylic acid transporter--as
well as any other polypeptide used in any of the aspects mentioned
herein, 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.
[0078] The polynucleotide of SEQ ID NO: 1 or 3, or a subsequence
thereof; as well as the amino acid sequence of SEQ ID NO: 2 or 4;
or a fragment thereof; may be used to design nucleic acid probes to
identify and clone DNA encoding a C4-dicarboxylic acid transporter
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, e.g., at
least 14 nucleotides, at least 25 nucleotides, at least 35
nucleotides, at least 70 nucleotides in lengths. The probes may be
longer, e.g., at least 100 nucleotides, at least 200 nucleotides,
at least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides in lengths. Even longer probes may be used, e.g., 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.
[0079] A genomic DNA or cDNA library prepared from such other
strains may be screened for DNA that hybridizes with the probes
described above and encodes a polypeptide having C4-dicarboxylic
acid transporter activity. Genomic or other DNA from such other
strains 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: 1 or 3, or a subsequence thereof, the carrier material is
preferably used in a Southern blot.
[0080] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe corresponding to SEQ ID NO: 1 or 3, the mature
polypeptide coding sequence of SEQ ID NO: 1 or 3, or the
full-length complementary strand thereof, or a subsequence of the
foregoing; under very low to very high stringency conditions.
Molecules to which the nucleic acid probe hybridizes under these
conditions can be detected using, for example, X-ray film.
[0081] In one aspect, the nucleic acid probe is SEQ ID NO: 1 or 3.
In another aspect, the nucleic acid probe is the mature polypeptide
coding sequence of SEQ ID NO: 1 or 3. In another aspect, the
nucleic acid probe is the mature polypeptide coding sequence of SEQ
ID NO: 1. In another aspect, the nucleic acid probe is SEQ ID NO:
1. In another aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 3. In another aspect, the
nucleic acid probe is SEQ ID NO: 3. In another aspect, the nucleic
acid probe is a polynucleotide that encodes the polypeptide of SEQ
ID NO: 2 or a fragment thereof. In another aspect, the nucleic acid
probe is a polynucleotide that encodes the polypeptide of SEQ ID
NO: 4 or a fragment thereof.
[0082] 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. 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), and at 70.degree. C. (very high
stringency).
[0083] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization and 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, Proc. Natl. Acad. Sci. 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. The carrier material is finally 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.
[0084] The C4-dicarboxylic acid transporter of the present
invention may be obtained from a microorganism of any genus. As
used herein, the term "obtained from" in connection with a given
source shall mean that the polypeptide encoded by a polynucleotide
is produced by the source or by a cell in which the polynucleotide
from the source has been inserted.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] In another aspect, the C4-dicarboxylic acid transporter is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasfi, Saccharomyces
kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis
C4-dicarboxylic acid transporter.
[0092] In another aspect, the C4-dicarboxylic acid transporter is
an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus
clavatus, 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.
[0093] In one aspect, the C4-dicarboxylic acid transporter is an
Aspergillus C4-dicarboxylic acid transporter, such as an
Aspergillus clavatus C4-dicarboxylic acid transporter or an
Aspergillus fumigatus C4-dicarboxylic acid transporter. In one
aspect, the C4-dicarboxylic acid transporter an Aspergillus
clavatus C4-dicarboxylic acid transporter of SEQ ID NO: 2. In
another aspect, the C4-dicarboxylic acid transporter an Aspergillus
fumigatus C4-dicarboxylic acid transporter of SEQ ID NO: 4.
[0094] 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.
[0095] 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).
[0096] 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.).
Malate Dehydrogenases and Polynucleotides Encoding Malate
Dehydrogenases
[0097] In some aspects of the recombinant host cells and methods of
use thereof, the host cells have malate dehydrogenase activity. In
some aspects, the host cells comprise a heterologous polynucleotide
encoding a malate dehydrogenase. The malate dehydrogenase can be
any malate dehydrogenase that is suitable for practicing the
invention. In one aspect, the malate dehydrogenase is an enzyme
that is present in the cytosol of the host cell.
[0098] In one aspect of the recombinant host cells and methods
described herein, the malate dehydrogenase is (a) a malate
dehydrogenase having at least 60% sequence identity to SEQ ID NO:
12 or the mature polypeptide sequence thereof; (b) a malate
dehydrogenase encoded by a polynucleotide that hybridizes under low
stringency conditions with (i) SEQ ID NO: 11 or the mature
polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ
ID NO: 11 or the mature polypeptide coding sequence thereof, or
(iii) the full-length complementary strand of (i) or (ii); (c) a
malate dehydrogenase encoded by a polynucleotide having at least
60% sequence identity to (iv) SEQ ID NO: 11 or the mature
polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ
ID NO: 11 or the mature polypeptide coding sequence thereof; or
(vi) the full-length complementary strand of (iv) or (v); (d) a
malate dehydrogenase variant comprising a substitution, deletion,
and/or insertion of one or more (e.g., two, several) amino acids of
SEQ ID NO: 12 or the mature polypeptide sequence thereof; and (e) a
fragment of a polypeptide of (a), (b), (c), or (d) that has malate
dehydrogenase activity.
[0099] In one aspect, the malate dehydrogenase comprises or
consists of 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 to SEQ ID NO: 12 or the mature
polypeptide sequence thereof. In one aspect, the 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: 12 or the mature polypeptide sequence thereof.
[0100] In one aspect, the malate dehydrogenase comprises or
consists of the amino acid sequence of SEQ ID NO: 12, the mature
polypeptide sequence of SEQ ID NO: 12, an allelic variant thereof,
or a fragment of the foregoing, having malate dehydrogenase
activity. In another aspect, the malate dehydrogenase comprises or
consists of the amino acid sequence of SEQ ID NO: 12. In another
aspect, the malate dehydrogenase comprises or consists of the
mature polypeptide sequence of SEQ ID NO: 12. In another aspect,
the malate dehydrogenase comprises or consists of amino acids 1 to
330 of SEQ ID NO: 12.
[0101] In one aspect, the malate dehydrogenase is 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: 11 or the mature
polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ
ID NO: 11 or the mature polypeptide coding sequence thereof, or
(iii) the full-length complementary strand of (i) or (ii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, supra).
[0102] In one aspect, the malate dehydrogenase is encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to (iv) SEQ ID NO: 11 or the mature polypeptide
coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 11 or
the mature polypeptide coding sequence thereof; or (vi) the
full-length complementary strand of (iv) or (v).
[0103] In one aspect, the malate dehydrogenase is encoded by SEQ ID
NO: 11, or the mature polypeptide coding sequence thereof. In one
aspect, the malate dehydrogenase is encoded by SEQ ID NO: 11. In
one aspect, the malate dehydrogenase is encoded by the mature
polypeptide coding sequence of SEQ ID NO: 11. In one aspect, the
malate dehydrogenase is encoded by a subsequence of SEQ ID NO: 11,
wherein the subsequence encodes a polypeptide having malate
dehydrogenase activity. In one aspect, the subsequence contains at
least 885 nucleotides, e.g., at least 930 nucleotides or at least
975 nucleotides of SEQ ID NO: 11.
[0104] In one aspect, the malate dehydrogenase is a variant
comprising a substitution, deletion, and/or insertion of one or
more (e.g., two, several) amino acids of SEQ ID NO: 12, or the
mature polypeptide sequence thereof, as described supra. In one
aspect, the malate dehydrogenase is a variant comprising a
substitution, deletion, and/or insertion of one or more amino acids
of SEQ ID NO: 12. In one aspect, the malate dehydrogenase is a
variant comprising a substitution, deletion, and/or insertion of
one or more amino acids of the mature polypeptide sequence of SEQ
ID NO: 12. In some aspects, the total number of amino acid
substitutions, deletions and/or insertions of the mature
polypeptide sequence of SEQ ID NO: 12 or the mature polypeptide
sequence thereof is not more than 10, e.g., not more than 1, 2, 3,
4, 5, 6, 7, 8 or 9.
[0105] In another aspect, the malate dehydrogenase is a fragment of
SEQ ID NO: 12, or the mature polypeptide sequence thereof, wherein
the fragment has malate dehydrogenase activity. In one aspect, the
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: 12.
[0106] The malate dehydrogenase may also be an allelic variant or
artificial variant of a malate dehydrogenase.
[0107] The malate dehydrogenase can also include fused polypeptides
or cleavable fusion polypeptides, as described supra.
[0108] Techniques used to isolate or clone a polynucleotide
encoding a malate dehydrogenase are described supra.
[0109] The polynucleotide of SEQ ID NO: 11; or a subsequence
thereof; as well as the amino acid sequence of SEQ ID NO: 12; 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. 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.
[0110] In one aspect, the nucleic acid probe is SEQ ID NO: 11. In
another aspect, the nucleic acid probe is the mature polypeptide
coding sequence of SEQ ID NO: 11. In another aspect, the nucleic
acid probe is a polynucleotide sequence that encodes SEQ ID NO: 12,
the mature polypeptide sequence thereof, or a fragment of the
foregoing.
[0111] For long probes of at least 100 nucleotides in length, very
low to very high stringency and washing conditions are defined as
described supra. For short probes of about 15 nucleotides to about
70 nucleotides in length, stringency and washing conditions are
defined as described supra.
[0112] The malate dehydrogenase may be obtained from microorganisms
of any genus. In one aspect, the malate dehydrogenase may be a
bacterial, a yeast, or a filamentous fungal malate dehydrogenase
obtained from the microorganisms described herein. In another
aspect, the malate dehydrogenase is an Aspergillus oryzae malate
dehydrogenase, e.g., the Aspergillus oryzae malate dehydrogenase of
SEQ ID NO: 12.
[0113] Other 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), the Aspergillus oryzae
malate dehydrogenase of SEQ ID NO: 16 (encoded by the
polynucleotide sequence of SEQ ID NO: 15; see U.S. application Ser.
No. 12/870,523, entitled "Methods for Improving Malic Acid
Production in Filamentous Fungi" filed Aug. 27, 2010), or any
aspect of the malate dehydrogenase described in the respective
reference therein.
[0114] The invention embraces any aspect of sequence identity,
hybridization, variants and fragments described herein as applied
to other malate dehydrogenase polypeptide sequences and
polynucleotide sequences described above. For example, in one
aspect, the malate dehydrogenase is (a) a malate dehydrogenase
having at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 16, or the mature polypeptide
sequence thereof; (b) a malate dehydrogenase encoded by a
polynucleotide that hybridizes under 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: 15 or the mature polypeptide coding
sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 15 or the
mature polypeptide coding sequence thereof, or (iii) the
full-length complementary strand of the (i) or (ii); (c) a malate
dehydrogenase encoded by a polynucleotide having at least 60%,
e.g., at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, 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%, at least 99%, or 100% sequence identity to (iv)
SEQ ID NO: 15 or the mature polypeptide coding sequence thereof,
(v) the cDNA sequence of SEQ ID NO: 15 or the mature polypeptide
coding sequence thereof, or (vi) the full-length complementary
strand of the (iv) or (v); (d) a malate dehydrogenase variant
comprising a substitution, deletion, and/or insertion of one or
more (e.g., two, several) amino acids of SEQ ID NO: 16 or the
mature polypeptide sequence thereof; or (e) a fragment of a
polypeptide of (a), (b), (c), or (d) that has malate dehydrogenase
activity.
[0115] 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.
Pyruvate Carboxylases and Polynucleotides Encoding Pyruvate
Carboxylases
[0116] In some aspects of the recombinant host cells and methods of
use thereof, the host cells have pyruvate carboxylase activity. In
some aspects, the host cells comprise a heterologous polynucleotide
encoding a pyruvate carboxylase. The pyruvate carboxylase can be
any pyruvate carboxylase that is suitable for practicing the
invention. In one aspect, the pyruvate carboxylase is an enzyme
that is present in the cytosol of the host cell.
[0117] In one aspect of the recombinant host cells and methods
described herein, the pyruvate carboxylase is (a) a pyruvate
carboxylase having at least 60% sequence identity to SEQ ID NO: 14
or the mature polypeptide sequence thereof; (b) a pyruvate
carboxylase encoded by a polynucleotide that hybridizes under low
stringency conditions with (i) SEQ ID NO: 13 or the mature
polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ
ID NO: 13 or the mature polypeptide coding sequence thereof, or
(iii) the full-length complementary strand of (i) or (ii); (c) a
pyruvate carboxylase encoded by a polynucleotide having at least
60% sequence identity to (iv) SEQ ID NO: 13 or the mature
polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ
ID NO: 13 or the mature polypeptide coding sequence thereof; or
(vi) the full-length complementary strand of (iv) or (v); (d) a
pyruvate carboxylase variant comprising a substitution, deletion,
and/or insertion of one or more (e.g., two, several) amino acids of
SEQ ID NO: 14 or the mature polypeptide sequence thereof; and (e) a
fragment of a polypeptide of (a), (b), (c), or (d) that has
pyruvate carboxylase activity.
[0118] In one aspect, the pyruvate carboxylase comprises or
consists of 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 to SEQ ID NO: 14, or the mature
polypeptide sequence thereof. In one aspect, the 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: 14 or the mature polypeptide sequence thereof.
[0119] In one aspect, the pyruvate carboxylase comprises or
consists of the amino acid sequence of SEQ ID NO: 14, the mature
polypeptide sequence of SEQ ID NO: 14, an allelic variant thereof,
or a fragment of the foregoing, having pyruvate carboxylase
activity. In another aspect, the pyruvate carboxylase comprises or
consists of the amino acid sequence of SEQ ID NO: 14. In another
aspect, the pyruvate carboxylase comprises or consists of the
mature polypeptide sequence of SEQ ID NO: 14. In another aspect,
the pyruvate carboxylase comprises or consists of amino acids 1 to
1193 of SEQ ID NO: 14.
[0120] In one aspect, the pyruvate carboxylase is 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: 13 or the mature
polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ
ID NO: 13 or the mature polypeptide coding sequence thereof, or
(iii) the full-length complementary strand of (i) or (ii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, supra).
[0121] In one aspect, the pyruvate carboxylase is encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to (iv) SEQ ID NO: 13 or the mature polypeptide
coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 13 or
the mature polypeptide coding sequence thereof; or (vi) the
full-length complementary strand of (iv) or (v).
[0122] In one aspect, the pyruvate carboxylase is encoded by SEQ ID
NO: 13 or the mature polypeptide coding sequence thereof. In one
aspect, the pyruvate carboxylase is encoded by SEQ ID NO: 13. In
one aspect, the pyruvate carboxylase is encoded by the mature
polypeptide coding sequence of SEQ ID NO: 13. In one aspect, the
pyruvate carboxylase is encoded by a subsequence of SEQ ID NO: 13,
wherein the subsequence encodes a polypeptide having pyruvate
carboxylase activity. In one aspect, the subsequence contains at
least 3060 nucleotides, e.g., at least 3240 nucleotides or at least
3420 nucleotides of SEQ ID NO: 13.
[0123] In one aspect, the pyruvate carboxylase is a variant
comprising a substitution, deletion, and/or insertion of one or
more (e.g., two, several) amino acids of SEQ ID NO: 14, or the
mature polypeptide sequence thereof, as described supra. In one
aspect, the pyruvate carboxylase is a variant comprising a
substitution, deletion, and/or insertion of one or more amino acids
of SEQ ID NO: 14. In one aspect, the pyruvate carboxylase is a
variant comprising a substitution, deletion, and/or insertion of
one or more amino acids of the mature polypeptide sequence of SEQ
ID NO: 14. In some aspects, the total number of amino acid
substitutions, deletions and/or insertions of SEQ ID NO: 14 or the
mature polypeptide sequence thereof is not more than 14, e.g., not
more than 1, 2, 3, 4, 5, 6, 7, 8 or 9.
[0124] In another aspect, the pyruvate carboxylase is a fragment of
SEQ ID NO: 14, or the mature polypeptide sequence thereof, wherein
the fragment has pyruvate carboxylase activity. In one aspect, the
fragment contains at least 1020 amino acid residues, e.g., at least
1080 amino acid residues, or at least 1140 amino acid residues of
SEQ ID NO: 14.
[0125] The pyruvate carboxylase may also be an allelic variant or
artificial variant of a pyruvate carboxylase.
[0126] The pyruvate carboxylase can also include fused polypeptides
or cleavable fusion polypeptides, as described supra.
[0127] 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.
[0128] Techniques used to isolate or clone a polynucleotide
encoding a pyruvate carboxylase are described supra.
[0129] The polynucleotide of SEQ ID NO: 13 or a subsequence
thereof, as well as the amino acid sequence of SEQ ID NO: 14 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. 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.
[0130] In one aspect, the nucleic acid probe is SEQ ID NO: 13. In
another aspect, the nucleic acid probe is the mature polypeptide
coding sequence of SEQ ID NO: 13. In another aspect, the nucleic
acid probe is a polynucleotide sequence that encodes SEQ ID NO: 14,
the mature polypeptide sequence thereof, or a fragment of the
foregoing.
[0131] For long probes of at least 100 nucleotides in length, very
low to very high stringency and washing conditions are defined as
described supra. For short probes of about 15 nucleotides to about
70 nucleotides in length, stringency and washing conditions are
defined as described supra.
[0132] The pyruvate carboxylase may be obtained from microorganisms
of any genus. In one aspect, the pyruvate carboxylase may be a
bacterial, a yeast, or a filamentous fungal pyruvate carboxylase
obtained from the microorganisms described herein. In another
aspect, the pyruvate carboxylase is an Aspergillus oryzae pyruvate
carboxylase, e.g., the Aspergillus oryzae pyruvate carboxylase of
SEQ ID NO: 14.
[0133] Other pyruvate carboxylases that can be used to practice the
present invention include, but are not limited to, a Aspergillus
clavatus 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
pombe 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). The invention embraces any aspect of
sequence identity, hybridization, variants and fragments described
herein as applied to the malate dehydrogenase polypeptide sequences
and polynucleotide sequences described above.
[0134] 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.
Nucleic Acid Constructs
[0135] The present invention also relates to nucleic acid
constructs comprising a polynucleotide encoding a C4-dicarboxylic
acid transporter (or other polynucleotides described herein, such
as a polynucleotide encoding a malate dehydrogenase and/or a
pyruvate carboxylase) linked to one or more (e.g., two, several)
control sequences that direct the expression of the coding sequence
in a suitable host cell under conditions compatible with the
control sequences. Such nucleic acid constructs may be used in any
of the host cells and methods describe herein. In one aspect, the
heterologous polynucleotide encoding a C4-dicarboxylic acid
transporter is operably linked to promoter foreign to the
polynucleotide. In one aspect, a second heterologous polynucleotide
encoding a malate dehydrogenase is operably linked to promoter
foreign to the polynucleotide. In one aspect, a third heterologous
polynucleotide encoding a pyruvate carboxylase is operably linked
to promoter foreign to the polynucleotide.
[0136] A polynucleotide may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
[0137] The control sequence may be a promoter sequence, a
polynucleotide that is recognized by a host cell for expression of
a polynucleotide encoding a C4-dicarboxylic acid transporter or
other polynucleotides described herein, such as a polynucleotide
encoding a 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
polynucleotide 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.
[0138] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a bacterial host cell are the promoters obtained from
the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus
licheniformis alpha-amylase gene (amyL), Bacillus licheniformis
penicillinase gene (penP), Bacillus stearothermophilus maltogenic
amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),
Bacillus subtilis xylA and xylB genes, E. coli lac operon,
Streptomyces coelicolor agarase gene (dagA), and prokaryotic
beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad.
Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et
al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Gilbert et al., 1980, Scientific American, 242: 74-94; and in
Sambrook et al., 1989, supra.
[0139] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus nidulans acetamidase, Aspergillus
niger neutral alpha-amylase, Aspergillus niger acid stable
alpha-amylase, Aspergillus niger or Aspergillus awamori
glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus
oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787),
Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium
venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO
00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic
proteinase, 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 modified promoter from a gene encoding a neutral alpha-amylase
in Aspergilli in which the untranslated leader has been replaced by
an untranslated leader from a gene encoding triose phosphate
isomerase in Aspergilli; non-limiting examples include modified
promoters from the gene encoding neutral alpha-amylase in
Aspergillus niger in which the untranslated leader has been
replaced by an untranslated leader from the gene encoding triose
phosphate isomerase in Aspergillus nidulans or Aspergillus oryzael
); and mutant, truncated, and hybrid promoters thereof.
[0140] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0141] The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the polypeptide.
Any terminator that is functional in the host cell of choice may be
used in the present invention.
[0142] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger glucoamylase, Aspergillus niger
alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
[0143] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0144] The control sequence may also be a suitable leader sequence,
when transcribed is 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 polynucleotide encoding
the polypeptide. Any leader sequence that is functional in the host
cell of choice may be used.
[0145] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0146] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0147] The control sequence may also be a polyadenylation sequence;
a sequence operably linked to the 3'-terminus of the polynucleotide
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.
[0148] 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.
[0149] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0150] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
polypeptide and directs the polypeptide into the cell's secretory
pathway. The 5'-end of the coding sequence of the polynucleotide
may inherently contain a signal peptide coding sequence naturally
linked in translation reading frame with the segment of the coding
sequence that encodes the 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 may be used.
[0151] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0152] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0153] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0154] The control sequence may also be a propeptide coding
sequence that encodes a propeptide positioned at the N-terminus of
a polypeptide. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propolypeptide is
generally inactive and can be converted to an active polypeptide by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding sequence may be obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus
subtilis neutral protease (nprT), Myceliophthora thermophila
laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and
Saccharomyces cerevisiae alpha-factor.
[0155] Where both signal peptide and propeptide sequences are
present at the N-terminus of a polypeptide, the propeptide sequence
is positioned next to the N-terminus of a polypeptide and the
signal peptide sequence is positioned next to the N-terminus of the
propeptide sequence.
[0156] 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
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the Aspergillus niger glucoamylase promoter,
Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus
oryzae glucoamylase promoter may be used. 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 polynucleotide encoding the
polypeptide would be operably linked with the regulatory
sequence.
Expression Vectors
[0157] The present invention also relates to recombinant host cells
and methods utilizing recombinant expression vectors comprising a
polynucleotide encoding a C4-dicarboxylic acid transporter (or
other polynucleotides described herein, such as a polynucleotide
encoding a malate dehydrogenase and/or a pyruvate carboxylase), a
promoter, and transcriptional and translational stop signals. The
various nucleotide and control sequences may be joined together to
produce a recombinant expression vector that may include one or
more (e.g., two, several) convenient restriction sites to allow for
insertion or substitution of the polynucleotide encoding the
polypeptide at such sites. Alternatively, the polynucleotide may be
expressed by inserting the polynucleotide 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.
[0158] 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
polynucleotide. 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 vector may be a linear or closed
circular plasmid.
[0159] In one aspect, each polynucleotide encoding a
C4-dicarboxylic acid transporter, a malate dehydrogenase, and/or a
pyruvate carboxylase described herein is contained on an
independent vector. In one aspect, two of the polynucleotides are
contained on a single vector. In one aspect, all the
polynucleotides encoding the C4-dicarboxylic acid transporter, the
malate dehydrogenase, and the pyruvate carboxylase are contained on
a single vector.
[0160] 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.
[0161] The vector preferably contains one or more (e.g., two,
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.
[0162] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, or tetracycline resistance. Suitable markers for yeast
host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
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.
[0163] The vector preferably contains 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.
[0164] 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 non-homologous recombination. Alternatively, the
vector may contain additional polynucleotides 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 should contain a sufficient number of nucleic acids, such
as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 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 polynucleotides. On the other hand, the
vector may be integrated into the genome of the host cell by
non-homologous recombination.
[0165] 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" means a polynucleotide that
enables a plasmid or vector to replicate in vivo.
[0166] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAMR1 permitting replication in Bacillus.
[0167] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0168] 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 Res. 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.
[0169] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of a polypeptide. 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.
[0170] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0171] As described herein, the present invention relates to, inter
alia, recombinant host cells comprising a polynucleotide described
herein (e.g., a polynucleotide encoding a C4-dicarboxylic acid
transporter, a malate dehydrogenase, and/or a pyruvate carboxylase)
operably linked to one or more (e.g., two, several) control
sequences that direct the production of a polypeptides described
herein for the recombinant production of a C4-dicarboxylic acid.
The invention also embraces methods of using such host cells for
the production of a C4-dicarboxylic acid. The host cell may
comprise any one or combination of a plurality of the
polynucleotides described. For example, in one aspect, the
recombinant host cell comprises a heterologous polynucleotide
encoding a C4-dicarboxylic acid transporter; and optionally
comprises a heterologous polynucleotide encoding a heterologous
polynucleotide encoding a malate dehydrogenase, and/or a
heterologous polynucleotide encoding pyruvate decarboxylase;
wherein the host cell produces (or is capable of producing) a
greater amount of a C4-dicarboxylic acid compared to the host cell
without the heterologous polynucleotide encoding the
C4-dicarboxylic acid transporter when cultivated under the same
conditions.
[0172] In one aspect, the recombinant host cell comprises:
[0173] (1) a heterologous polynucleotide encoding a C4-dicarboxylic
acid transporter, such as a C4-dicarboxylic acid transporter
selected from: (a) a C4-dicarboxylic acid transporter having at
least 60% sequence identity to SEQ ID NO: 2 or 4, or the mature
polypeptide sequence thereof; (b) a C4-dicarboxylic acid
transporter encoded by a polynucleotide that hybridizes under low
stringency conditions with SEQ ID NO: 1 or 3, the mature
polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing; (c) a C4-dicarboxylic acid
transporter encoded by a polynucleotide having at least 60%
sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide
coding sequence thereof, or the full-length complementary strand of
the foregoing; (d) a C4-dicarboxylic acid transporter variant
comprising a substitution, deletion, and/or insertion of one or
more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4, or the
mature polypeptide sequence thereof; and (e) a fragment of a
polypeptide of (a), (b), (c), or (d) that has C4-dicarboxylic acid
transporter activity;
[0174] (2) an optional heterologous second polynucleotide encoding
a malate dehydrogenase, such as a malate dehydrogenase selected
from: (a) a malate dehydrogenase having at least 60% sequence
identity to SEQ ID NO: 12 or the mature polypeptide sequence
thereof; (b) a malate dehydrogenase encoded by a polynucleotide
that hybridizes under low stringency conditions with (i) SEQ ID NO:
11 or the mature polypeptide coding sequence thereof, (ii) the cDNA
sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence
thereof, or (iii) the full-length complementary strand of (i) or
(ii); (c) a malate dehydrogenase encoded by a polynucleotide having
at least 60% sequence identity to (iv) SEQ ID NO: 11 or the mature
polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ
ID NO: 11 or the mature polypeptide coding sequence thereof; or
(vi) the full-length complementary strand of (iv) or (v); (d) a
malate dehydrogenase variant comprising a substitution, deletion,
and/or insertion of one or more (e.g., two, several) amino acids of
SEQ ID NO: 12 or the mature polypeptide sequence thereof; and (e) a
fragment of a polypeptide of (a), (b), (c), or (d) that has malate
dehydrogenase activity; and
[0175] (3) an optional heterologous third polynucleotide encoding a
pyruvate carboxylase, such as a pyruvate carboxylase selected from:
(a) a pyruvate carboxylase having at least 60% sequence identity to
SEQ ID NO: 14 or the mature polypeptide sequence thereof; (b) a
pyruvate carboxylase encoded by a polynucleotide that hybridizes
under low stringency conditions with (i) SEQ ID NO: 13 or the
mature polypeptide coding sequence thereof, (ii) the cDNA sequence
of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof,
or (iii) the full-length complementary strand of (i) or (ii); (c) a
pyruvate carboxylase encoded by a polynucleotide having at least
60% sequence identity to (iv) SEQ ID NO: 13 or the mature
polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ
ID NO: 13 or the mature polypeptide coding sequence thereof; or
(vi) the full-length complementary strand of (iv) or (v); (d) a
pyruvate carboxylase variant comprising a substitution, deletion,
and/or insertion of one or more (e.g., two, several) amino acids of
SEQ ID NO: 14 or the mature polypeptide sequence thereof; and (e) a
fragment of a polypeptide of (a), (b), (c), or (d) that has
pyruvate carboxylase activity;
[0176] wherein the host cell produces (or is capable of producing)
a greater amount of a C4-dicarboxylic acid (e.g., malic acid)
compared to the host cell without the one or more polynucleotide(s)
(e.g., without the heterologous polynucleotide encoding a
C4-dicarboxylic acid transporter), when cultivated under the same
conditions.
[0177] In one aspect, the host cell comprises a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter
described herein (e.g., SEQ ID NO: 1 or 3, or any described aspect
thereof) and a heterologous polynucleotide encoding a malate
dehydrogenase. In the present invention, the malate dehydrogenase
can be any malate dehydrogenase that is suitable for practicing the
present invention, as described supra. In another aspect, the host
cell comprises a heterologous polynucleotide encoding a
C4-dicarboxylic acid transporter described herein (e.g., SEQ ID NO:
1 or 3, or any described aspect thereof) and a heterologous
polynucleotide encoding a pyruvate carboxylase. In the present
invention, the pyruvate carboxylase can be any pyruvate carboxylase
that is suitable for practicing the present invention, as described
supra. In particular, the pyruvate carboxylase is preferably an
enzyme that is present in the cytosol of the host cell. In one
aspect, the host cell comprises a heterologous polynucleotide
encoding a C4-dicarboxylic acid transporter described herein (e.g.,
SEQ ID NO: 1 or 3, or any described aspect thereof), a second
heterologous polynucleotide encoding a malate dehydrogenase, and a
third heterologous polynucleotide encoding a pyruvate
carboxylase.
[0178] A construct or vector comprising a polynucleotide is
introduced into a host cell so that the construct or 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. The aspects described
below apply to the host cells, per se, as well as methods using the
host cells.
[0179] The host cell may be any cell capable of the recombinant
production of a polypeptide of the present invention, e.g., a
prokaryote or a eukaryote, and/or any cell (e.g., any filamentous
fungal cell) capable of the recombinant production of a
C4-dicarboxylic acid (e.g., malic acid).
[0180] The prokaryotic host cell may be any gram-positive or
gram-negative bacterium. Gram-positive bacteria include, but not
limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0181] The bacterial host cell may be any Bacillus cell including,
but not limited to, 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,
and Bacillus thuringiensis cells.
[0182] The bacterial host cell may also be any Streptococcus cell
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0183] The bacterial host cell may also be any Streptomyces cell
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0184] The introduction of DNA into a Bacillus cell may, for
instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using
competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol.
81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol.
56: 209-221), by electroporation (see, e.g., Shigekawa and Dower,
1988, Biotechniques 6: 742-751), or by conjugation (see, e.g.,
Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The
introduction of DNA into an E. coli cell may, for instance, be
effected by protoplast transformation (see, e.g., Hanahan, 1983, J.
Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et
al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of
DNA into a Streptomyces cell may, for instance, be effected by
protoplast transformation and electroporation (see, e.g., Gong et
al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation
(see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585),
or by transduction (see, e.g., Burke et al., 2001, Proc. Natl.
Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a
Pseudomonas cell may, for instance, be effected by electroporation
(see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397)
or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl.
Environ. Microbiol. 71: 51-57). The introduction of DNA into a
Streptococcus cell may, for instance, be effected by natural
competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun.
32: 1295-1297), by protoplast transformation (see, e.g., Catt and
Jollick, 1991, Microbios 68: 189-207, by electroporation (see,
e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:
3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol.
Rev. 45: 409-436). However, any method known in the art for
introducing DNA into a host cell can be used.
[0185] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0186] The host cell may be a fungal cell. "Fungi" as used herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota (as defined by Hawksworth et al., In, Ainsworth and
Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the
Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and
all mitosporic fungi (Hawksworth et al., 1995, supra).
[0187] The fungal host cell may be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes). Since the classification of yeast may
change in the future, for the purposes of this invention, yeast
shall be defined as described in Biology and Activities of Yeast
(Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc.
App. Bacteriol. Symposium Series No. 9, 1980).
[0188] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0189] The fungal host cell may be a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
1995, supra). 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.
[0190] The filamentous fungal host cell may be an Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0191] For example, the filamentous fungal host cell may be an
Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,
Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta,
Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis
gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa,
Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium
inops, Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium
queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum,
Coprinus cinereus, Coriolus hirsutus, 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 insolens,
Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,
Neurospora crassa, Penicillium purpurogenum, Phanerochaete
chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia
terrestris, Trametes villosa, Trametes versicolor, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride cell.
[0192] In one aspect, the host cell is an Aspergillus host cell. In
another aspect, the host cell is Aspergillus oryzae.
[0193] 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 EP 238023 and Yelton et al., 1984, Proc.
Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for
transforming Fusarium species are described by Malardier et al.,
1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed
using the procedures described by Becker and Guarente, In Abelson,
J. N. and Simon, M. I., editors, Guide to Yeast Genetics and
Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187,
Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.
153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:
1920.
[0194] In some aspects, the host cell comprises one or more (e.g.,
two, several) polynucleotide(s) described herein, wherein the host
cell secretes (and/or is capable of secreting) an increased level
of C4-dicarboxylic acid compared to the host cell without the one
or more polynucleotide(s) when cultivated under the same
conditions. In some aspects, the host cell secretes and/or is
capable of secreting an increased level of C4-dicarboxylic acid
(e.g., malic acid) of at least 5%, e.g., at least 10%, at least
15%, at least 20%, at least 25%, at least 50%, at least 100%, at
least 150%, at least 200%, at least 300%, or at 500% compared to
the host cell without the one or more polynucleotide(s) (e.g.,
without the heterologous polynucleotide encoding a C4-dicarboxylic
acid transporter), when cultivated under the same conditions.
[0195] In any of the aspects of the recombinant host cells and
methods described herein, the C4-dicarboxylic acid may be 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.
[0196] In any of these aspects, the host cell produces (and/or is
capable of producing) a C4-dicarboxylic acid at a yield of at least
than 10%, e.g., at least than 20%, at least than 30%, at least than
40%, at least than 50%, at least than 60%, at least than 70%, at
least than 80%, or at least than 90%, of theoretical.
[0197] In any of these aspects, the recombinant host has an
C4-dicarboxylic acid volumetric productivity (e.g., malic acid
volumetric productivity) greater than about 0.1 g/L per hour, e.g.,
greater than about 0.2 g/L per hour, 0.5 g/L per hour, 0.6 g/L per
hour, 0.7 g/L per hour, 0.8 g/L per hour, 0.9 g/L per hour, 1.0 g/L
per hour, 1.1 g/L per hour, 1.2 g/L per hour, 1.3 g/L per hour, 1.5
g/L per hour, 1.75 g/L per hour, 2.0 g/L per hour, 2.25 g/L per
hour, 2.5 g/L per hour, or 3.0 g/L per hour; or between about 0.1
g/L per hour and about 2.0 g/L per hour, e.g., between about 0.3
g/L per hour and about 1.7 g/L per hour, about 0.5 g/L per hour and
about 1.5 g/L per hour, about 0.7 g/L per hour and about 1.3 g/L
per hour, about 0.8 g/L per hour and about 1.2 g/L per hour, or
about 0.9 g/L per hour and about 1.1 g/L per hour.
[0198] The recombinant host cells may be cultivated in a nutrient
medium suitable for production of the C4-dicarboxylic acid
transporter, malate dehydrogenase, or pyruvate carboxylase using
methods well known in the art, as described below.
[0199] The C4-dicarboxylic acid transporter, malate dehydrogenase,
and pyruvate carboxylase, and activities thereof, can be detected
using methods known in the art. These detection methods may include
use of specific antibodies, formation of an enzyme product, or
disappearance of an enzyme substrate. See, for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring
Harbor Laboratory, New York (2001); Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1999); and Hanai et al., Appl. Environ. Microbiol. 73:7814-7818
(2007)).
Methods
[0200] The present invention also relates to methods of using the
recombinant host cells described herein for the production of a
C4-dicarboxylic acid. In one aspect, the invention embraces a
method of producing a C4-dicarboxylic acid (e.g., malic acid),
comprising: (a) cultivating any one of the recombinant host cells
described herein (e.g., any host cell with C4-dicarboxylic acid
transporter activity, and optionally, malate dehydrogenase
activity, and/or pyruvate carboxylase activity) in a medium under
suitable conditions to produce the C4-dicarboxylic acid; and (b)
recovering the C4-dicarboxylic acid. In one aspect, the invention
embraces a method of producing a C4-dicarboxylic acid (e.g., malic
acid), comprising: (a) cultivating in a medium any one of the
recombinant host cells described herein, wherein the host cell
comprises a heterologous polynucleotide encoding a C4-dicarboxylic
acid transporter; and optionally, a heterologous polynucleotide
encoding a malate dehydrogenase, and/or a heterologous
polynucleotide encoding a pyruvate decarboxylase under suitable
conditions to produce the C4-dicarboxylic acid; and (b) recovering
the C4-dicarboxylic acid. In one aspect, the medium is a
fermentable medium.
[0201] In one aspect of the methods, the C4-dicarboxylic acid
(e.g., malic acid) is produced at a titer greater than about 10
g/L, e.g., greater than about 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125
g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, 210 g/L,
225 g/L, 250 g/L, 275 g/L, 300 g/L, 325 g/L, 350 g/L, 400 g/L, or
500g/L; or between about 10 g/L and about 500 g/L, e.g., between
about 50 g/L and about 350 g/L, about 100 g/L and about 300 g/L,
about 150 g/L and about 250 g/L, about 175 g/L and about 225 g/L,
or about 190 g/L and about 210 g/L.
[0202] In one aspect of the methods, the amount of produced
C4-dicarboxylic acid (e.g., malic acid) is at least 5%, e.g., at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 50%, or at least 100% greater compared to cultivating the
host cell without the polynucleotide that encodes the
C4-dicarboxylic acid transporter under the same conditions.
[0203] In some aspects of the methods, the C4-dicarboxylic acid is
selected from malic acid, succinic acid, oxaloacetic acid, malonic
acid, and fumaric acid. In one aspect, the C4-dicarboxylic acid is
malic acid.
[0204] The recombinant C4-dicarboxylic acid can be optionally
recovered from the fermentation medium using any procedure known in
the art (see, for example, WO 1998/022611 and U.S. Pat. No.
7,601,865) including, but not limited to, chromatography (e.g.,
size exclusion chromatography, adsorption chromatography, ion
exchange chromatography), electrophoretic procedures, differential
solubility, osmosis, distillation, extraction (e.g., liquid-liquid
extraction), pervaporation, extractive filtration, membrane
filtration, membrane separation, reverse, or ultrafiltration. In
one example, the C4-dicarboxylic acid is recovered from other
material in the fermentation medium by filtration.
[0205] In some aspects of the methods, the recombinant
C4-dicarboxylic acid before and/or after being optionally purified
is substantially pure. With respect to the methods of producing a
C4-dicarboxylic acid (or a specific C4-dicarboxylic acid thereof,
such as malic acid), "substantially pure" intends a recovered
preparation of the C4-dicarboxylic acid that contains no more than
15% impurity, wherein impurity intends compounds other than
C4-dicarboxylic acids. In one variation, a preparation of
substantially pure C4-dicarboxylic acid is provided wherein the
preparation contains no more than 25% impurity, or no more than 20%
impurity, or no more than 10% impurity, or no more than 5%
impurity, or no more than 3% impurity, or no more than 1% impurity,
or no more than 0.5% impurity.
[0206] Suitable assays to test for the production of
C4-dicarboxylic acids for the methods of production and host cells
described herein can be performed using methods known in the art.
For example, the final C4-dicarboxylic acid product (e.g., malic
acid), and other organic compounds, can be analyzed by methods such
as HPLC (High Performance Liquid Chromatography), GC-MS (Gas
Chromatography Mass Spectroscopy) and LC-MS (Liquid
Chromatography-Mass Spectroscopy) or other suitable analytical
methods using routine procedures well known in the art. The release
of C4-dicarboxylic acid in the fermentation broth can also be
tested with the culture supernatant. Byproducts and residual sugar
in the fermentation medium (e.g., glucose) can be quantified by
HPLC using, for example, a refractive index detector for glucose
and alcohols, and a UV detector for organic acids (Lin et al.,
Biotechnol. Bioeng. 90:775 -779 (2005)), or using other suitable
assay and detection methods well known in the art.
[0207] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
[0208] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Strains
[0209] Aspergillus clavatus NRRL1 and Aspergillus fumigatus
(Sartorya fumigata) Af293 were used as the source C4-dicarboxylic
acid transporter genes. Aspergillus oryzae NRRL 3488 (or ATCC
56747) was used as a source of a pyruvate carboxylase gene, a
malate dehydrogenase gene, and for production of the
C4-dicarboxylic acids.
Media
[0210] YEG medium was composed of 20 g glucose, 5 g yeast extract,
and deionized water to 1 liter.
[0211] COVE plates were composed of 1 M sucrose, 2% COVE salt
solution, 10 mM acetamide, 15 mM CsCl, and 25 g/l Agar Noble.
[0212] 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.
[0213] 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.
[0214] Seed medium was composed of 40 g glucose, 6 g Bacto-peptone,
750 mg KH.sub.2PO.sub.4, 750 mg K.sub.2HPO.sub.4, 100 mg
MgSO.sub.4.7H.sub.2O, 100 mg CaCl.sub.2.H.sub.2O, 5 mg
FeSO.sub.4.7H.sub.2O, 5 mg NaC, and deionized water to 1 liter.
[0215] Seed medium B is 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.
[0216] Acid production medium C is 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. 1000.times. Micronutrient Solution is
composed of 5 g NaCl, 5 g FeSO.sub.4.7H.sub.2O, 1 g citric acid,
and deionized water to 1 liter.
[0217] PDA plates are composed of 39 g/l potato dextrose agar.
Example 1
Cloning of the Aspergillus clavatus NRRL1 C4-Dicarboxylic Acid
Transporter Gene and Construction of Expression Vector
pShTh120AcC4T
[0218] The 1179 by C4-dicarboxylic acid transporter gene acc4t
(ACLA.sub.--058030) was synthetically constructed into pAcC4T (FIG.
1; DNA2.0, Menlo Park, Calif., USA). The acc4t gene was amplified
from pAcC4T using primers 069735 and 069736 shown below.
TABLE-US-00001 Primer 069735: (SEQ ID NO: 5)
5'-GTGTGATAGAACATCGTCCATAATGTTCGAAAATCG-3' Primer 069736: (SEQ ID
NO: 6) 5'-GTCAGTCACCTCTAGTTAATTAACTAGTCTGCAGCATCCTCATC-3'
[0219] The PCR reaction mixture was composed of 50 ng pAcC4T
template, 200 .mu.M dNTP mixture, 50 pM primer 069735, 50 pM primer
069736, 1.times. Poll reaction buffer (New England Biolabs, MA,
USA), and 1 unit Vent Polymerase (New England Biolabs) and
deionized water to 50 .mu.l. The PCR reaction was incubated in an
EPPENDORF MASTERCYCLER.RTM. (Eppendorf Scientific Inc., Westbury,
N.Y., USA) programmed for 1 cycle at 94.degree. C. for 3 minutes;
35 cycles 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 (50 mM Tris base-50 mM acetate-0.1 mM
disodium EDTA) and purified using a QIAQUICK.RTM. Gel Extraction
Kit (QIAGEN Inc., Valencia, Calif., USA).
[0220] Plasmid pShTh60 (FIG. 2; see also PCT Application No.
PCT/US10/47002, entitled "Methods for Improving Malic Acid
Production in Filamentous Fungi" filed Aug. 27, 2010) 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 pShTh60 using an In-Fusion.TM. Cloning Kit
(Clontech, Mountain View, Calif., USA) according to the
manufacturer's instructions, resulting in pShTh120AcC4T (FIG. 3).
Plasmid pShTh120AcC4T was isolated using a QIAfilter Maxi Plasmid
Isolation Kit (QIAGEN Inc., Valencia, Calif., USA). DNA sequence
analysis was used to confirm the integrity of the acc4t coding
sequence using primers 996270 and 065067 shown below using an
ABI3130XL DNA Analyzer (Applied Biosystems, Inc., Foster City,
Calif., USA) and the primer walking technique with dye-terminator
chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60).
TABLE-US-00002 Primer 996270: (SEQ ID NO: 7)
5'-CTATAGCGAAATGGATTGATTGTCT-3' Primer 065067: (SEQ ID NO: 8)
5'-TGACCTTCCACGCTGACCAC-3'
[0221] The nucleotide sequence (SEQ ID NO: 1) and deduced amino
acid sequence (SEQ ID NO: 2) of the Aspergillus clavatus acc4t gene
are shown in FIG. 4. The genomic coding sequence of 1179 bp
(including stop codon) encodes a polypeptide of 392 amino acids
with a predicted mass of 43.4 kDa and an isoelectric pH of 7.85.
The gene contains no introns. Using the Vector NIT.RTM. program
(Invitrogen, CA, USA), a signal peptide of 52 residues was
predicted, resulting in a predicted mature protein containing 340
amino acids.
Example 2
Cloning of the Aspergillus fumigates Af293 C4-Dicarboxylic Acid
Transporter Gene and Construction of Expression Vector
pShTh121AfC4T
[0222] The 1182 bp C4-dicarboxylic acid transporter gene sequence
afc4t (AFUA.sub.--8G04630) was synthetically constructed into
pAfC4T (FIG. 5; DNA2.0). The afc4t gene was amplified from pAfC4T
using primers 069737 and 069738 shown below.
TABLE-US-00003 Primer 069737: (SEQ ID NO: 9)
5-GTGTGATAGAACATCGTCCATAATGTTCAACGATCATGATCA-3' Primer 069738: (SEQ
ID NO: 10) 5'-GTCAGTCACCTCTAGTTAATTAATTAATCTAGCACATCCTCGTC-3'
[0223] The PCR reaction mixture was composed of 50 ng pAtC4T
template, 200 .mu.M dNTP mixture, 50 pM primer 069737, 50 pM primer
069738, 1.times. Poll reaction buffer, 1 unit Vent Polymerase and
deionized water to 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 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 (50 mM Tris base-50
mM acetate-0.1 mM disodium EDTA) and purified using a QIAQUICK.RTM.
Gel Extraction Kit.
[0224] Plasmid pShTh60 (FIG. 2) was digested and purified as
described above. The purified PCR product above was then inserted
into the digested pShTh60 using an InFusion Cloning Kit according
to the manufacturer's instructions resulting in plasmid
pShTh121AfC4T (FIG. 6). Plasmid pShTh121AfC4T was isolated using a
QIAfilter Maxi Plasmid Isolation Kit. DNA sequence analysis was
used to confirm the integrity of the afc4t coding sequence using
primers 996270 and 065067 as described above.
[0225] The nucleotide sequence (SEQ ID NO: 3) and deduced amino
acid sequence (SEQ ID NO: 4) of the Aspergillus fumigates afc4t
gene are shown in FIG. 7. The genomic coding sequence of 1182 by
(including stop codon) encodes a polypeptide of 393 amino acids
with a predicted mass of 43.8 kDa and an isoelectric pH of 7.30.
The gene contains no introns.
Example 3
Transformation of Expression Vector Fragments of pShTh120AcC4T and
pShTh121AfC4T into Aspergillus oryzae NRRL3488 (ShTh1200 and
ShTh1210)
[0226] Protoplast preparation and transformation of Aspergillus
oryzae NRRL3488 were performed by inoculating approximately
2.times.10.sup.7 spores into 100 ml 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. (Calbiochem, San Diego, Calif., USA) and
rinsing with 50 ml of 0.7 M KCl. The washed mycelia were
resuspended in a 125 ml flask with 20 ml of protoplasting solution
composed of 5 mg of GLUCANEX.TM. (Novozymes NS, Bagsvrd, 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. The tubes were spun in
the centrifuge at 1300.times.g for 10 minutes at room temperature.
The supernatant was discarded and the protoplast pellet was
resuspended in 20 ml of STC buffer. The protoplasts were washed by
two rounds of resuspending the pellet in 20 ml of STC and
centrifugation at 1300.times.g for 10 minutes at room temperature.
The final pellet was resuspended in 2 ml of STC. The protoplasts
were counted by removing a 10 .mu.l sample and counting them in a
haemocytometer (VWR, West Chester, Pa., USA). The volume was
adjusted with STC to obtain a protoplast concentration of
2.times.10.sup.7 per ml.
[0227] The plasmid vectors were prepared for transformation by
restriction digestion with Pme I. The approximately 5 kb expression
cassette from each construct was separated from the vector
sequences by 0.8% agarose gel electrophoresis in TBE buffer and
purified using a QIAQUICK.RTM. Gel Extraction Kit.
[0228] Four transformation reactions were prepared for each
expression vector. For each reaction, a 100 .mu.l solution of
protoplast preparation was transferred to a 12 ml polypropylene
tube, to which was added 2-5 .mu.g of restriction 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.
Transformants containing the expression vector fragment of
pShTh120AcC4T were designated ShTh1200. Transformants containing
the expression vector fragment of pShTh121AfC4T were designated
ShTh1210.
Example 4
Production of Malic Acid in Shake Flask Cultures of Aspergillus
oryzae Transformants Containing Expression Vector Fragments of
pShTh120AcC4T and pShTh121AfC4T (ShTh1200 and ShTh1210)
[0229] Spores from transformants ShTh1200 and ShTh1210 described
above 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.
[0230] Quantitation of malic acid for the shake flask culture
transformants 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.
[0231] 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 a HPLC vial
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.
[0232] 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 for (RSD) for
replicate injections was 5%. Malic acid shows
R.sup.2.gtoreq.0.9999.
[0233] Aspergillus oryzae transformants containing pShTh120AcC4T
(strains ShTh1200) showed an improvement in malic acid production
over the Aspergillus oryzae NRRL 3488 control strains and
comparable malic acid production to Aspergillus oryzae ShTh1040
strains (see PCT Application No. PCT/US10/47002, filed Aug. 27,
2010). Aspergillus oryzae transformants containing pShTh121AfC4T
(strains ShTh1210) showed a slight improvement in malic acid
production over the Aspergillus oryzae NRRL 3488 control strains
and lower malic acid production compared to Aspergillus oryzae
ShTh1040 strains.
Example 5
Fermentation of Aspergillus oryzae Transformants Containing
Expression Vector Fragments of pShTh120AcC4T (ShTh1200)
[0234] Aspergillus oryzae transformants above and control
transformant Aspergillus oryzae ShTh1040 (see PCT Application No.
PCT/US10/47002, filed Aug. 27, 2010) 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 flasks were then incubated at 32.degree. C. and 180
rpm for about 24 hours. The seed flasks are combined to supply the
144 ml inoculum required per tank.
[0235] Three-liter fermentors containing 1.8 liters of medium were
individually inoculated by introducing 144 ml (8%) of the seed
culture broth from the combined seed flasks of either an
Aspergillus oryzae pShTh120AcC4T transformant or an Aspergillus
oryzae ShTh1040 transformant. 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. Samples were withdrawn daily and analyzed
for malic acid production, and the fermentations were completed
after approximately 7 days.
[0236] Quantitation of malic acid in the fermentations was
performed as described in Example 4. The relative malic acid titer
of Aspergillus oryzae pShTh120AcC4T (ShTh1200) transformants were
comparable to the Aspergillus oryzae ShTh1040 transformants,
indicating that the Aspergillus oryzae pShTh120AcC4T transformants
outperform the Aspergillus oryzae NRRL 3488 control (which lack the
overexpressed C4-dicarboxylic acid transporter gene) based on
ShTh1040 and NRRL 3488 comparisons previously described.
The present invention may be further described by the following
numbered paragraphs: [0237] [1] A method of producing a
C4-dicarboxylic acid, comprising:
[0238] (a) cultivating a host cell comprising a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0239] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof;
[0240] (ii) a C4-dicarboxylic acid transporter 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
SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof,
or a full-length complementary strand of the foregoing; [0241]
(iii) a C4-dicarboxylic acid transporter encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide
coding sequence thereof; [0242] (iv) a C4-dicarboxylic acid
transporter variant comprising a substitution, deletion, and/or
insertion of one or more (e.g., two, several) amino acids of SEQ ID
NO: 2 or 4, or the mature polypeptide sequence thereof; and [0243]
(v) a fragment of a C4-dicarboxylic acid transporter of (i), (ii),
(iii), or (iv) that has C4-dicarboxylic acid transporter activity;
and
[0244] (b) recovering the C4-dicarboxylic acid. [0245] [2] A method
of producing a dicarboxylic acid, comprising:
[0246] (a) cultivating a host cell comprising a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0247] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
SEQ ID NO: 2 or the mature polypeptide sequence thereof; [0248]
(ii) a C4-dicarboxylic acid transporter 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 SEQ ID NO: 1,
the mature polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing; [0249] (iii) a
C4-dicarboxylic acid transporter encoded by a polynucleotide having
at least 65%, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, 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%, at least 99%, or 100% sequence identity to SEQ
ID NO: 1 or the mature polypeptide coding sequence thereof; [0250]
(iv) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 2 or the mature polypeptide
sequence thereof; and [0251] (v) a fragment of a C4-dicarboxylic
acid transporter of (i), (ii), (iii), or (iv) that has
C4-dicarboxylic acid transporter activity; and
[0252] (b) recovering the C4-dicarboxylic acid. [0253] [3] A method
of producing a C4-dicarboxylic acid, comprising:
[0254] (a) cultivating a host cell comprising a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0255] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
SEQ ID NO: 4 or the mature polypeptide sequence thereof; [0256]
(ii) a C4-dicarboxylic acid transporter 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 SEQ ID NO: 3,
the mature polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing; [0257] (iii) a
C4-dicarboxylic acid transporter encoded by a polynucleotide having
at least 65%, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, 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%, at least 99%, or 100% sequence identity to SEQ
ID NO: 3 or the mature polypeptide coding sequence thereof; [0258]
(iv) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 4 or the mature polypeptide
sequence thereof; and [0259] (v) a fragment of a C4-dicarboxylic
acid transporter of (i), (ii), (iii), or (iv) that has
C4-dicarboxylic acid transporter activity; and
[0260] (b) recovering the C4-dicarboxylic acid. [0261] [4] The
method of any one of paragraphs [1]-[3], wherein the
C4-dicarboxylic acid transporter has at least 65%, e.g., 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%, at least 99% or 100%
sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide
sequence thereof. [0262] [5] The method of any one of paragraphs
[1]-[4], wherein the C4-dicarboxylic acid transporter is encoded by
a polynucleotide that hybridizes under low stringency conditions,
low-medium stringency conditions, medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with SEQ ID NO: 1 or 3, the mature
polypeptide coding sequence thereof, or a full-length complementary
strand of the foregoing. [0263] [6] The method of any one of
paragraphs [1]-[5], wherein the C4-dicarboxylic acid transporter is
encoded by a polynucleotide having at least 65%, e.g., 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%, at least 99% or 100%
sequence identity to SEQ ID NO: 1 or 3, or the mature polypeptide
coding sequence thereof. [0264] [7] The method of any one of
paragraphs [1]-[6], wherein the C4-dicarboxylic acid transporter
comprises or consists of SEQ ID NO: 2 or 4. [0265] [8] The method
of any one of paragraphs [1]-[6], wherein the C4-dicarboxylic acid
transporter comprises or consists of the mature polypeptide
sequence of SEQ ID NO: 2 or 4. [0266] [9] The method of paragraph
[8], wherein the mature polypeptide sequence of SEQ ID NO: 2 is
amino acids 53 to 392 of SEQ ID NO: 2. [0267] [10] The method of
paragraph [8] or [9], wherein the mature polypeptide sequence of
SEQ ID NO: 4 is amino acids 1 to 393 of SEQ ID NO: 4. [0268] [11]
The method of any one of paragraphs [1]-[6], wherein the
C4-dicarboxylic acid transporter is a fragment of SEQ ID NO: 2 or
4, wherein the fragment has C4-dicarboxylic acid transporter
activity. [0269] [12] The method of any one of paragraphs [1]-[6],
wherein the C4-dicarboxylic acid transporter is a variant
comprising a substitution, deletion, and/or insertion of one or
more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4, or the
mature polypeptide sequence thereof. [0270] [13] The method of any
one of paragraphs [1]-[12], wherein the heterologous polynucleotide
encoding the C4-dicarboxylic acid transporter is operably linked to
a promoter foreign to the polynucleotide. [0271] [14] The method of
any one of paragraphs [1]-[13], wherein the host cell further
comprises a heterologous second polynucleotide encoding a malate
dehydrogenase (e.g., the malate dehydrogenase of SEQ ID NO: 11, or
any described aspect thereof). [0272] [15] The method of paragraph
[14], wherein the heterologous second polynucleotide encoding a
malate dehydrogenase is operably linked to a promoter foreign to
the polynucleotide. [0273] [16] The method of any one of paragraphs
[1]-[15], wherein the host cell further comprises a heterologous
third polynucleotide encoding a pyruvate carboxylase (e.g., the
pyruvate carboxylase of SEQ ID NO: 13, or any described aspect
thereof). [0274] [17] The method of paragraph [16], wherein the
heterologous third polynucleotide encoding a pyruvate carboxylase
is operably linked to a promoter foreign to the polynucleotide.
[0275] [18] The method of any one of paragraphs [1]-[17], wherein
the host cell is a filamentous fungal host cell. [0276] [19] The
method of paragraph [18], 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. [0277] [20] The method of paragraph
[19], wherein the host cell is an Aspergillus host cell. [0278]
[21] The method of paragraph [20], wherein the Aspergillus host
cell is an Aspergillus oryzae host cell. [0279] [22] The method of
any one of paragraphs [1]-[21], wherein the level of the
C4-dicarboxylic acid is increased by at least 25%, e.g., at least
50%, at least 100%, at least 150%, at least 200%, at least 300%, or
at 500% compared to the host cell without the polynucleotide
encoding the heterologous polynucleotide when cultivated under the
same conditions. [0280] [23] The method of any one of paragraphs
[1]-[22], wherein the C4-dicarboxylic acid is selected from malic
acid, succinic acid, oxaloacetic acid, malonic acid, and fumaric
acid. [0281] [24] The method of paragraph [23], wherein the
C4-dicarboxylic acid is malic acid. [0282] [25] A method for
increasing C4-dicarboxylic acid production, comprising:
[0283] (a) transforming into a host cell a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0284] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof;
[0285] (ii) a C4-dicarboxylic acid transporter 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
SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof,
or a full-length complementary strand of the foregoing; [0286]
(iii) a C4-dicarboxylic acid transporter encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide
coding sequence thereof; [0287] (iv) a C4-dicarboxylic acid
transporter variant comprising a substitution, deletion, and/or
insertion of one or more (e.g., two, several) amino acids of SEQ ID
NO: 2 or 4, or the mature polypeptide sequence thereof; and [0288]
(v) a fragment of a C4-dicarboxylic acid transporter of (i), (ii),
(iii), or (iv) that has C4-dicarboxylic acid transporter activity;
and
[0289] (b) cultivating the transformed host cell in a medium;
and
[0290] (c) recovering the C4-dicarboxylic acid. [0291] [26] A
method for increasing C4-dicarboxylic acid production,
comprising:
[0292] (a) transforming into a host cell a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0293] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
the mature polypeptide sequence of SEQ ID NO: 2; [0294] (ii) a
C4-dicarboxylic acid transporter 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 the mature
polypeptide coding sequence of SEQ ID NO: 1, or the full-length
complementary strand thereof; [0295] (iii) a C4-dicarboxylic acid
transporter encoded by a polynucleotide having at least 65%, e.g.,
at least 70%, at least 75%, at least 80%, at least 85%, 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%,
at least 99%, or 100% sequence identity to the mature polypeptide
coding sequence of SEQ ID NO: 1; [0296] (iv) a C4-dicarboxylic acid
transporter variant comprising a substitution, deletion, and/or
insertion of one or more (e.g., two, several) amino acids of the
mature polypeptide sequence of SEQ ID NO: 2; and [0297] (v) a
fragment of a C4-dicarboxylic acid transporter of (i), (ii), (iii),
or (iv) that has C4-dicarboxylic acid transporter activity; and
[0298] (b) cultivating the transformed host cell in a medium;
and
[0299] (c) recovering the C4-dicarboxylic acid. [0300] [27] A
method for increasing C4-dicarboxylic acid production,
comprising:
[0301] (a) transforming into a host cell a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter; wherein
the transporter is selected from: [0302] (i) a C4-dicarboxylic acid
transporter having at least 65%, e.g., 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%, at least 99%, or 100% sequence identity to
SEQ ID NO: 4 or the mature polypeptide sequence thereof; [0303]
(ii) a C4-dicarboxylic acid transporter 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 SEQ ID NO: 3,
the mature polypeptide coding sequence thereof, or the full-length
complementary strand of the foregoing; [0304] (iii) a
C4-dicarboxylic acid transporter encoded by a polynucleotide having
at least 65%, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, 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%, at least 99%, or 100% sequence identity to SEQ
ID NO: 3 or the mature polypeptide coding sequence thereof; [0305]
(iv) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 4 or the mature polypeptide
sequence thereof; and [0306] (v) a fragment of a C4-dicarboxylic
acid transporter of (i), (ii), (iii), or (iv) that has
C4-dicarboxylic acid transporter activity; and
[0307] (b) cultivating the transformed host cell in a medium;
and
[0308] (c) recovering the C4-dicarboxylic acid. [0309] [28] The
method of any one of paragraphs [25]-[27], wherein the
C4-dicarboxylic acid transporter has at least 65%, e.g., 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%, at least 99% or 100%
sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide
sequence thereof. [0310] [29] The method of any one of paragraphs
[25]-[28], wherein the C4-dicarboxylic acid transporter is encoded
by a polynucleotide that hybridizes under low stringency
conditions, low-medium stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with SEQ ID NO: 1 or
3, the mature polypeptide coding sequence thereof, or a full-length
complementary strand of the foregoing. [0311] [30] The method of
any one of paragraphs [25]-[29], wherein the C4-dicarboxylic acid
transporter is encoded by a polynucleotide having at least 65%,
e.g., 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%, at least
99% or 100% sequence identity to SEQ ID NO: 1 or 3, or the mature
polypeptide coding sequence thereof. [0312] [31] The method of any
one of paragraphs [25]-[30], wherein the C4-dicarboxylic acid
transporter comprises or consists of SEQ ID NO: 2 or 4. [0313] [32]
The method of any one of paragraphs [25]-[30], wherein the
C4-dicarboxylic acid transporter comprises or consists of the
mature polypeptide sequence of SEQ ID NO: 2 or 4. [0314] [33] The
method of paragraph [32], wherein the mature polypeptide sequence
of SEQ ID NO: 2 is amino acids 53 to 392 of SEQ ID NO: 2. [0315]
[34] The method of paragraph [32] or [33], wherein the mature
polypeptide sequence of SEQ ID NO: 4 is amino acids 1 to 393 of SEQ
ID NO: 4. [0316] [35] The method of any one of paragraphs
[25]-[30], wherein the C4-dicarboxylic acid transporter is a
fragment of SEQ ID NO: 2 or 4, wherein the fragment has
C4-dicarboxylic acid transporter activity. [0317] [36] The method
of any one of paragraphs [25]-[30], wherein the C4-dicarboxylic
acid transporter is a variant comprising a substitution, deletion,
and/or insertion of one or more (e.g., two, several) amino acids of
SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof.
[0318] [37] The method of any one of paragraphs [25]-[36], wherein
the heterologous polynucleotide encoding the C4-dicarboxylic acid
transporter is operably linked to a promoter foreign to the
polynucleotide. [0319] [38] The method of any one of paragraphs
[25]-[37], wherein the host cell further comprises a heterologous
second polynucleotide encoding a malate dehydrogenase (e.g., the
malate dehydrogenase of SEQ ID NO: 11, or any described aspect
thereof). [0320] [39] The method of paragraph [38], wherein the
heterologous second polynucleotide encoding a malate dehydrogenase
is operably linked to a promoter foreign to the polynucleotide.
[0321] [40] The method of any one of paragraphs [25]-[39], wherein
the host cell further comprises a heterologous third polynucleotide
encoding a pyruvate carboxylase (e.g., the pyruvate carboxylase of
SEQ ID NO: 13, or any described aspect thereof). [0322] [41] The
method of paragraph [40], wherein the heterologous third
polynucleotide encoding a pyruvate carboxylase is operably linked
to a promoter foreign to the polynucleotide. [0323] [42] The method
of any one of paragraphs [25]-[41], wherein the host cell is a
filamentous fungal host cell. [0324] [43] The method of paragraph
[42], 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. [0325] [44] The
method of paragraph [43], wherein the host cell is an Aspergillus
host cell. [0326] [45] The method of paragraph [44], wherein the
Aspergillus host cell is an Aspergillus oryzae host cell. [0327]
[46] The method of any one of paragraphs [25]-[45], wherein the
level of the C4-dicarboxylic acid is increased by at least 25%,
e.g., at least 50%, at least 100%, at least 150%, at least 200%, at
least 300%, or at 500% compared to the host cell without the
polynucleotide encoding the heterologous polynucleotide when
cultivated under the same conditions. [0328] [47] The method of any
one of paragraphs [25]-[46], wherein the C4-dicarboxylic acid is
selected from malic acid, succinic acid, oxaloacetic acid, malonic
acid, and fumaric acid. [0329] [48] The method of paragraph [47],
wherein the C4-dicarboxylic acid is malic acid. [0330] [49] A host
cell comprising a heterologous polynucleotide encoding a
C4-dicarboxylic acid transporter, wherein the transporter is
selected from:
[0331] (a) a C4-dicarboxylic acid transporter having at least 65%,
e.g., 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 2 or 4, or the mature
polypeptide sequence thereof;
[0332] (b) a C4-dicarboxylic acid transporter 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
SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof,
or a full-length complementary strand of the foregoing;
[0333] (c) a C4-dicarboxylic acid transporter encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 1 or 3, or the mature polypeptide
coding sequence thereof;
[0334] (d) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 2 or 4, or the mature
polypeptide sequence thereof; and
[0335] (e) a fragment of a C4-dicarboxylic acid transporter of (a),
(b), (c), or (d) that has C4-dicarboxylic acid transporter
activity;
[0336] wherein the host cell secretes increased levels of
C4-dicarboxylic acid compared to the host cell without the
heterologous polynucleotide when cultivated under the same
conditions. [0337] [50] A host cell comprising a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter, wherein
the transporter is selected from:
[0338] (a) a C4-dicarboxylic acid transporter having at least 65%,
e.g., 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 2 or the mature
polypeptide sequence thereof;
[0339] (b) a C4-dicarboxylic acid transporter 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
SEQ ID NO: 1, the mature polypeptide coding sequence thereof, or
the full-length complementary strand of the foregoing;
[0340] (c) a C4-dicarboxylic acid transporter encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 1 or the mature polypeptide coding
sequence thereof;
[0341] (d) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 2 or the mature polypeptide
sequence thereof; and
[0342] (e) a fragment of a C4-dicarboxylic acid transporter of (a),
(b), (c), or (d) that has C4-dicarboxylic acid transporter
activity;
[0343] wherein the host cell secretes increased levels of
C4-dicarboxylic acid compared to the host cell without the
heterologous polynucleotide when cultivated under the same
conditions. [0344] [51] A host cell comprising a heterologous
polynucleotide encoding a C4-dicarboxylic acid transporter, wherein
the transporter is selected from:
[0345] (a) a C4-dicarboxylic acid transporter having at least 65%,
e.g., 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%, at least
99%, or 100% sequence identity to SEQ ID NO: 4 or the mature
polypeptide sequence thereof;
[0346] (b) a C4-dicarboxylic acid transporter 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
SEQ ID NO: 3, the mature polypeptide coding sequence thereof, or
the full-length complementary strand of the foregoing;
[0347] (c) a C4-dicarboxylic acid transporter encoded by a
polynucleotide having at least 65%, e.g., at least 70%, at least
75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity to SEQ ID NO: 3 or the mature polypeptide coding
sequence thereof;
[0348] (d) a C4-dicarboxylic acid transporter variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of SEQ ID NO: 4 or the mature polypeptide
sequence thereof; and
[0349] (e) a fragment of a C4-dicarboxylic acid transporter of (a),
(b), (c), or (d) that has C4-dicarboxylic acid transporter
activity;
[0350] wherein the host cell secretes increased levels of
C4-dicarboxylic acid compared to the host cell without the
heterologous polynucleotide when cultivated under the same
conditions. [0351] [52] The host cell of any one of paragraphs
[49]-[51], wherein the C4-dicarboxylic acid transporter has at
least 65%, e.g., 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%,
at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4, or the
mature polypeptide sequence thereof. [0352] [53] The host cell of
any one of paragraphs [49]-[52], wherein the C4-dicarboxylic acid
transporter is encoded by a polynucleotide that hybridizes under
low stringency conditions, low-medium stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions with SEQ
ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or a
full-length complementary strand of the foregoing. [0353] [54] The
host cell of any one of paragraphs [49]-[53], wherein the
C4-dicarboxylic acid transporter is encoded by a polynucleotide
having at least 65%, e.g., 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%, at least 99% or 100% sequence identity to SEQ ID NO:
1 or 3, or the mature polypeptide coding sequence thereof. [0354]
[55] The host cell of any one of paragraphs [49]-[54], wherein the
C4-dicarboxylic acid transporter comprises or consists of SEQ ID
NO: 2 or 4. [0355] [56] The host cell of any one of paragraphs
[49]-[54], wherein the C4-dicarboxylic acid transporter comprises
or consists of the mature polypeptide sequence of SEQ ID NO: 2.
[0356] [57] The host cell of paragraph [56], wherein the mature
polypeptide sequence of SEQ ID NO: 2 is amino acids 53 to 392 of
SEQ ID NO: 2. [0357] [58] The host cell of paragraph [56] or [57],
wherein the mature polypeptide sequence of SEQ ID NO: 4 is amino
acids 1 to 393 of SEQ ID NO: 4. [0358] [59] The host cell of any
one of paragraphs [49]-[54], wherein the C4-dicarboxylic acid
transporter is a fragment of SEQ ID NO: 2 or 4, wherein the
fragment has C4-dicarboxylic acid transporter activity. [0359] [60]
The host cell of any one of paragraphs [49]-[54], wherein the
C4-dicarboxylic acid transporter is a variant comprising a
substitution, deletion, and/or insertion of one or more (e.g., two,
several) amino acids of the mature polypeptide sequence of SEQ ID
NO: 2 or 4. [0360] [61] The host cell of any one of paragraphs
[49]-[60], wherein the heterologous polynucleotide encoding the
C4-dicarboxylic acid transporter is operably linked to a promoter
foreign to the polynucleotide. [0361] [62] The host cell of any one
of paragraphs [49]-[61], wherein the host cell further comprises a
heterologous second polynucleotide encoding a malate dehydrogenase
(e.g., the malate dehydrogenase of SEQ ID NO: 11, or any described
aspect thereof). [0362] [63] The host cell of paragraph [62],
wherein the heterologous second polynucleotide encoding a malate
dehydrogenase is operably linked to a promoter foreign to the
polynucleotide. [0363] [64] The host cell of any one of paragraphs
[49]-[63], wherein the host cell further comprises a heterologous
third polynucleotide encoding a pyruvate carboxylase (e.g., the
pyruvate carboxylase of SEQ ID NO: 13, or any described aspect
thereof). [0364] [65] The host cell of paragraph [64], wherein the
heterologous third polynucleotide encoding a pyruvate carboxylase
is operably linked to a promoter foreign to the polynucleotide.
[0365] [66] The host cell of any one of paragraphs [49]-[64],
wherein the host cell is a filamentous fungal host cell. [0366]
[67] The filamentous fungal host cell of paragraph [66], wherein
the 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. [0367] [68] The filamentous fungal
host cell of paragraph [67], wherein the host cell is an
Aspergillus host cell. [0368] [69] The filamentous fungal host cell
of paragraph [68], wherein the host cell is an Aspergillus oryzae
host cell. [0369] [70] The host cell of any one of paragraphs
[49]-[69], wherein the host cell is capable of secreting an
increased level of the malic acid of at least 25%, e.g., at least
50%, at least 100%, at least 150%, at least 200%, at least 300%, or
at 500% compared to the host cell without the polynucleotide
encoding the heterologous polynucleotide when cultivated under the
same conditions. [0370] [71] The host cell of any one of paragraphs
[49]-[70], wherein the C4-dicarboxylic acid is selected from malic
acid, succinic acid, oxaloacetic acid, malonic acid, and fumaric
acid. [0371] [72] The host cell of paragraph [71], wherein the
C4-dicarboxylic acid is malic acid.
Sequence CWU 1
1
1611179DNAAspergillus clavatus 1atgttcgaaa atcgtatacc gccgacctcg
tctcagtcag actctggctt cctcgagaac 60cagctggaaa aacaacatcg actcagcctc
cgtgagaggt taaggcactt tacctgggcc 120tggtacacat tgaccatgag
cacaggtggg ttggctctcc tgatagcgag ccagccatac 180accttcaagg
ggttgaagac cattggactg gtggtctaca tcgtgaactt gatcttgttt
240ggtcttgtct gttcccttat ggccactagg ttcatcctcc acggtggctt
cctcgactcc 300cttcgccatg agcgcgaggg tcttttcttt cctaccttct
ggctatccgt agcaaccatc 360atcaccggct tgcatcgcta cttcggctcc
gatgctcgag aatcgtacct gattgcactc 420gaagtactct tctgggtcta
ctgtgcctgt acactggcca cagcagtgat ccagtactcc 480ttcatcttct
ctgcgcacag atacggcctc cagaccatga tgccctcctg gattctccca
540gccttcccca tcatgctcag tggcacgatt gcctccgtca tcggcgaagc
tcaacccgca 600cggtcatcga tccccgtcat catggccgga gtcaccttcc
agggcctggg gttctcgatc 660agcttcatga tgtacgccca ctatatcggc
cggctgatgg aatcagggct cccctgccgc 720gagcacagac ccggcatgtt
catctgcgtt ggtcccccgg ctttcacagc cctcgctcta 780gtcgggatgg
ccaagggcct gcccgccgag ttcaagctca tcaacgacgc acacgccctc
840gaagacgcgc ggatcctcga gctgctcgca atcaccgcgg gcatcttcct
ctgggccctg 900agtctgtggt tcttcttcat cgccgtcatc gccgtcctcc
ggtccccgcc tacttccttc 960catctcaact ggtgggcctt ggtcttcccg
aacacgggct tcactttggc caccatcacg 1020cttggaaagg cattgggcag
tcccgggatc ttgggcgttg gttctgccat gtcccttggc 1080atcgttggca
tgtggctgtt tgtttttgtc agccatatcc gtgccatcat caaccaggat
1140atcatgtatc cgggcaaaga tgaggatgct gcagactag
11792392PRTAspergillus clavatus 2Met Phe Glu Asn Arg Ile Pro Pro
Thr Ser Ser Gln Ser Asp Ser Gly 1 5 10 15 Phe Leu Glu Asn Gln Leu
Glu Lys Gln His Arg Leu Ser Leu Arg Glu 20 25 30 Arg Leu Arg His
Phe Thr Trp Ala Trp Tyr Thr Leu Thr Met Ser Thr 35 40 45 Gly Gly
Leu Ala Leu Leu Ile Ala Ser Gln Pro Tyr Thr Phe Lys Gly 50 55 60
Leu Lys Thr Ile Gly Leu Val Val Tyr Ile Val Asn Leu Ile Leu Phe 65
70 75 80 Gly Leu Val Cys Ser Leu Met Ala Thr Arg Phe Ile Leu His
Gly Gly 85 90 95 Phe Leu Asp Ser Leu Arg His Glu Arg Glu Gly Leu
Phe Phe Pro Thr 100 105 110 Phe Trp Leu Ser Val Ala Thr Ile Ile Thr
Gly Leu His Arg Tyr Phe 115 120 125 Gly Ser Asp Ala Arg Glu Ser Tyr
Leu Ile Ala Leu Glu Val Leu Phe 130 135 140 Trp Val Tyr Cys Ala Cys
Thr Leu Ala Thr Ala Val Ile Gln Tyr Ser 145 150 155 160 Phe Ile Phe
Ser Ala His Arg Tyr Gly Leu Gln Thr Met Met Pro Ser 165 170 175 Trp
Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly Thr Ile Ala Ser 180 185
190 Val Ile Gly Glu Ala Gln Pro Ala Arg Ser Ser Ile Pro Val Ile Met
195 200 205 Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile Ser Phe
Met Met 210 215 220 Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
Leu Pro Cys Arg 225 230 235 240 Glu His Arg Pro Gly Met Phe Ile Cys
Val Gly Pro Pro Ala Phe Thr 245 250 255 Ala Leu Ala Leu Val Gly Met
Ala Lys Gly Leu Pro Ala Glu Phe Lys 260 265 270 Leu Ile Asn Asp Ala
His Ala Leu Glu Asp Ala Arg Ile Leu Glu Leu 275 280 285 Leu Ala Ile
Thr Ala Gly Ile Phe Leu Trp Ala Leu Ser Leu Trp Phe 290 295 300 Phe
Phe Ile Ala Val Ile Ala Val Leu Arg Ser Pro Pro Thr Ser Phe 305 310
315 320 His Leu Asn Trp Trp Ala Leu Val Phe Pro Asn Thr Gly Phe Thr
Leu 325 330 335 Ala Thr Ile Thr Leu Gly Lys Ala Leu Gly Ser Pro Gly
Ile Leu Gly 340 345 350 Val Gly Ser Ala Met Ser Leu Gly Ile Val Gly
Met Trp Leu Phe Val 355 360 365 Phe Val Ser His Ile Arg Ala Ile Ile
Asn Gln Asp Ile Met Tyr Pro 370 375 380 Gly Lys Asp Glu Asp Ala Ala
Asp 385 390 31182DNAAspergillus fumigatus 3atgttcaacg atcatgatca
tgttccacca acatcatcac agtcggattc tggctttttt 60gaacaagaaa tgaagaaatc
tcctcgacta agccttcgtg agcgcctacg gcacttcacc 120tgggcgtggt
ataccttgac gatgagtacc ggtggactgg ctcttctgat tgctagtcag
180ccgtatacct tcaatggcat gaagggcatc gggatggtcg tttatatcct
caatcttctg 240ttattcgctc ttgtctgttc tttgatggtg ctgagattcg
ttttgcatgg cggtttcctt 300gacagcttgc gccaccctcg cgagggtctc
ttcttcccta ccttctggct atccattgca 360acgatcatca ctggcttgca
tcgttacttc ggctccgacg acctagagtc gtacctcatc 420gcactcgaag
tcctcttctg ggtctactgt agttgcaccc tcgccacagc tgtgatccag
480tactcattcc tctttgccgc ccactcctac ggcctgcaga caatgatgcc
atcatggatc 540ctaccagcct tccccatcat gctcagcgga accatcgcct
cggtcatcag cgaatcccag 600cccgcgcgat ccgcgatccc catcatcact
gccggcgtta ccttccaggg cctcggcttc 660tcaatcagct tcataatgta
cgcccactac atcggccgac tcatgcagtc agggcttccc 720tgccgcgaac
acagaccagc catgttcatt tgcgtggggc ctccgtcttt caccgcgttg
780gcgctagtag ggatggccaa gggcctgccc gacgaattca agataatcaa
agacgcacac 840gtcgaggacg cccggatcct cgagctgatg gctattatcg
tcggcgtgtt cctgtgggcc 900ctgagtctct ggttcttctt cattgccttt
gttgctgtcg tccggtgccg gcccactgcg 960ttccacctta gctggtgggc
catggtcttc cccaacactg ggttcacgct ggccactatt 1020accctgggga
gggcattggg gagccctggc gtcttgggcg tcggctcggc catgtcggtc
1080ggtgttgtct gcatgtgggt cttcgttttc gtctaccaca ttcgtgctgt
catcaggcaa 1140gacatcatgt acccgggcaa agacgaggat gtgctagatt aa
11824393PRTAspergillus fumigatus 4Met Phe Asn Asp His Asp His Val
Pro Pro Thr Ser Ser Gln Ser Asp 1 5 10 15 Ser Gly Phe Phe Glu Gln
Glu Met Lys Lys Ser Pro Arg Leu Ser Leu 20 25 30 Arg Glu Arg Leu
Arg His Phe Thr Trp Ala Trp Tyr Thr Leu Thr Met 35 40 45 Ser Thr
Gly Gly Leu Ala Leu Leu Ile Ala Ser Gln Pro Tyr Thr Phe 50 55 60
Asn Gly Met Lys Gly Ile Gly Met Val Val Tyr Ile Leu Asn Leu Leu 65
70 75 80 Leu Phe Ala Leu Val Cys Ser Leu Met Val Leu Arg Phe Val
Leu His 85 90 95 Gly Gly Phe Leu Asp Ser Leu Arg His Pro Arg Glu
Gly Leu Phe Phe 100 105 110 Pro Thr Phe Trp Leu Ser Ile Ala Thr Ile
Ile Thr Gly Leu His Arg 115 120 125 Tyr Phe Gly Ser Asp Asp Leu Glu
Ser Tyr Leu Ile Ala Leu Glu Val 130 135 140 Leu Phe Trp Val Tyr Cys
Ser Cys Thr Leu Ala Thr Ala Val Ile Gln 145 150 155 160 Tyr Ser Phe
Leu Phe Ala Ala His Ser Tyr Gly Leu Gln Thr Met Met 165 170 175 Pro
Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly Thr Ile 180 185
190 Ala Ser Val Ile Ser Glu Ser Gln Pro Ala Arg Ser Ala Ile Pro Ile
195 200 205 Ile Thr Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
Ser Phe 210 215 220 Ile Met Tyr Ala His Tyr Ile Gly Arg Leu Met Gln
Ser Gly Leu Pro 225 230 235 240 Cys Arg Glu His Arg Pro Ala Met Phe
Ile Cys Val Gly Pro Pro Ser 245 250 255 Phe Thr Ala Leu Ala Leu Val
Gly Met Ala Lys Gly Leu Pro Asp Glu 260 265 270 Phe Lys Ile Ile Lys
Asp Ala His Val Glu Asp Ala Arg Ile Leu Glu 275 280 285 Leu Met Ala
Ile Ile Val Gly Val Phe Leu Trp Ala Leu Ser Leu Trp 290 295 300 Phe
Phe Phe Ile Ala Phe Val Ala Val Val Arg Cys Arg 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 Thr Leu Gly Arg Ala Leu Gly Ser Pro
Gly Val Leu 340 345 350 Gly Val Gly Ser Ala Met Ser Val Gly Val Val
Cys Met Trp Val Phe 355 360 365 Val Phe Val Tyr His Ile Arg Ala Val
Ile Arg Gln Asp Ile Met Tyr 370 375 380 Pro Gly Lys Asp Glu Asp Val
Leu Asp 385 390 536DNAAspergillus clavatus 5gtgtgataga acatcgtcca
taatgttcga aaatcg 36644DNAAspergillus clavatus 6gtcagtcacc
tctagttaat taactagtct gcagcatcct catc 44725DNAAspergillus clavatus
7ctatagcgaa atggattgat tgtct 25820DNAAspergillus clavatus
8tgaccttcca cgctgaccac 20942DNAAspergillus fumigates 9gtgtgataga
acatcgtcca taatgttcaa cgatcatgat ca 421044DNAAspergillus fumigates
10gtcagtcacc tctagttaat taattaatct agcacatcct cgtc
44111430DNAAspergillus oryzae 11atggtcaaag 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 143012330PRTAspergillus
oryzae 12Met 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 133643DNAAspergillus oryzae
13atggcggctc cgtttcgtca gcctgaggag gcggtcgatg acaccgagtt catcgatgac
60caccatgaac acctccgtga taccgtgcac catcggttgc gcgccaattc ctccattatg
120cacttccaga agatcctcgt cgccaaccgt ggtgagatcc ccattcgtat
cttcagaacg 180gcccacgagc tgtcattgca 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 tcaagtgtac 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 agggcccggc 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
2820ccggaggatg 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 3643141193PRTAspergillus oryzae 14Met 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
151294DNAAspergillus oryzae 15atgttcgctg 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
129416340PRTAspergillus oryzae 16Met 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
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