U.S. patent number 10,385,368 [Application Number 15/321,662] was granted by the patent office on 2019-08-20 for recombinant host cells for the production of malonate.
This patent grant is currently assigned to Lygos, Inc.. The grantee listed for this patent is Lygos, Inc.. Invention is credited to Jeffrey A. Dietrich.
United States Patent |
10,385,368 |
Dietrich |
August 20, 2019 |
Recombinant host cells for the production of malonate
Abstract
Compositions and methods for producing malonate in recombinant
host cells in order to increase malonate titer, yield, and/or
productivity are provided. Recombinant host cells comprising
nucleic acids encoding MAE1 transport proteins that increase
production of malonate by the host cell and vectors for expressing
MAE1 transport proteins that increase production of malonate by the
host cell are also disclosed.
Inventors: |
Dietrich; Jeffrey A. (San
Francisco, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Lygos, Inc. |
Emeryville |
CA |
US |
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Assignee: |
Lygos, Inc. (Berkeley,
CA)
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Family
ID: |
54938789 |
Appl.
No.: |
15/321,662 |
Filed: |
June 24, 2015 |
PCT
Filed: |
June 24, 2015 |
PCT No.: |
PCT/US2015/037530 |
371(c)(1),(2),(4) Date: |
December 22, 2016 |
PCT
Pub. No.: |
WO2015/200545 |
PCT
Pub. Date: |
December 30, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180237810 A1 |
Aug 23, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62017723 |
Jun 24, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
1/16 (20130101); C07K 14/38 (20130101); C07K
14/37 (20130101); C12P 7/46 (20130101); C12N
15/81 (20130101) |
Current International
Class: |
C12P
7/46 (20060101); C12N 1/16 (20060101); C07K
14/38 (20060101); C07K 14/37 (20060101); C12N
15/81 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0827541 |
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Nov 2009 |
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EP |
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2495304 |
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Sep 2012 |
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EP |
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WO 2011/163270 |
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Dec 2011 |
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WO |
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WO 2013/028512 |
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Feb 2013 |
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WO |
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2013134424 |
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Sep 2013 |
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WO |
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Other References
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Primary Examiner: Raghu; Ganapathirama
Attorney, Agent or Firm: Faegre Baker Daniels LLP
Claims
The invention claimed is:
1. A recombinant Pichia kudriavzevii host cell capable of producing
malonate wherein said host cell comprises a heterologous nucleic
acid encoding an Aspergillus MAE1 transport protein, wherein the
Aspergillus MAE1 transport protein is SEQ ID NO: 2, and wherein
said host cell produces an increased level of malonate relative to
a parental host cell not comprising a heterologous nucleic acid
encoding said MAE1 transport protein.
2. A method for producing malonate, said method comprising
fermenting a host cell of claim 1 under conditions such that
malonate is produced and secreted into fermentation medium.
3. The recombinant host cell of claim 1, which is capable of
producing malonate under aerobic conditions.
4. The recombinant host cell of claim 1, which is capable of
producing malonate under anaerobic conditions.
5. The recombinant host cell of claim 1, wherein the host cell
produces greater than 1.25-fold more malonate than the parental
host cell.
6. The method of claim 2, wherein the fermenting is performed at a
pH of about 5.0.
7. The method of claim 2, wherein the fermenting is performed at a
temperature of about 30.degree. C.
Description
BACKGROUND OF THE INVENTION
The long-term economic and environmental concerns associated with
the petrochemical industry has provided the impetus for increased
research, development, and commercialization of processes for
conversion of carbon feedstocks into chemicals that can replace
those petroleum feedstocks. One approach is the development of
biorefining processes to convert renewable feedstocks into products
that can replace petroleum-derived chemicals. Two common goals in
improving a biorefining process include achieving a lower cost of
production and reducing carbon dioxide emissions.
Propanedioic acid ("malonate", CAS No. 141-82-2) is currently
produced from non-renewable, petroleum feedstocks. Mono- or
di-esterification of one or both carboxylic acid moieties of
malonate with an alcohol (e.g. methanol or ethanol) yields the
monoalkyl and dialkyl malonates, respectively.
2,2-dimethyl-1,3-dioxane-4,6-dione ("Meldrum's acid" CAS No.
2033-24-1) is produced from malonate using either acetone in acetic
anhydride or isopropenyl acetate in acid.
Chemical synthesis is currently the preferred route for synthesis
of malonate and malonate derived compounds. For example, dialkyl
malonates are produced through either a hydrogen cyanide or carbon
monoxide process. In the hydrogen cyanide process, sodium cyanide
is reacted with sodium chloroacetate at elevated temperatures to
produce sodium cyanoacetate, which is subsequently reacted with an
alcohol/mineral acid mixture to produce the dialkyl malonate.
Hildbrand et al. report yields of 75-85% (see "Malonic acid and
Derivatives" In: Ullmann's Encyclopedia of Industrial Chemistry,
Wiley-VCH, Weinheim, New York (2002)). In the carbon monoxide
process, dialkyl malonates (also referred to herein as diester
malonates) are produced through cobalt-catalyzed
alkoxycarbonylation of chloroacetates with carbon monoxide in the
presence of an alcohol at elevated temperatures and pressures.
The existing, petrochemical-based production routes to the malonate
and malonate-derived compounds are low yielding, environmentally
damaging, dependent upon non-renewable feedstocks, and require
expensive treatment of wastewater and exhaust gas. Recently, new
methods for producting malonate using biological processes have
been described (see PCT Pub. No. WO 13/134424, incorporated herein
by reference). There remains a need, however, for improved methods
and materials for biocatalytic conversion of renewable feedstocks
into malonate, purification of biosynthetic malonate, and
subsequent preparation of downstream chemicals and products.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for
producing malonate in recombinant host cells. In accordance with
the present invention, increased malonate titer, yield, and/or
productivity can be achieved by genetic modifications that increase
production of malonate by the host cell, and the invention provides
recombinant host cells comprising nucleic acids encoding MAE1
transport proteins that increase production of malonate by the host
cell and vectors for expressing MAE1 transport proteins that
increase production of malonate by the host cell. The invention
also provides methods for the use of recombinant host cells
comprising MAE1 transport proteins for the production of
malonate.
In a first aspect, the invention provides a recombinant host cell
capable of producing malonate comprising a heterologous nucleic
acid encoding a malic acid transport protein (herein referred to as
MAE1 transport protein). In one embodiment, the recombinant host
cell has been engineered to produce malonate (e.g., as per methods
described in PCT Pub. No. WO 13/134424, supra). In another
embodiment, the recombinant host cell natively produces malonate.
These recombinant host cells produce more malonate than counterpart
cells that do not comprise such a MAE1 transport protein. In
various embodiments, the host cells can produce at least 1.5-fold
more malonate under appropriate fermentation conditions relative to
parental or control cells that do not comprise a heterologous
nucleic acid encoding a MAE1 transport protein. In some
embodiments, the recombinant host cell is a yeast cell. In one
embodiment, the recombinant host cell is a Pichia kudriavzevii
cell.
In various embodiments, the heterologous nucleic acid provided by
the invention encodes a MAE1 transport protein. Suitable MAE1
transport proteins can be obtained from various eukaryotic
organisms. In various embodiments, the MAE1 transport protein is
obtained from an Aspergillus species or a Schizosaccharomyces
species. Various constructs of the invention utilize the
Aspergillus niger A2R8T9 MAE1 transport protein sequence (SEQ ID
NO: 1) or variants of it. Thus, in various embodiments, suitable
MAE1 transport proteins for use in the methods of the invention
have at least 25%, at least 50%, at least 75%, at least 95%, or at
least 99% identity to SEQ ID NO: 1. Other MAE1 transport proteins
are also suitable for use in accordance with the methods of the
invention, and in various embodiments recombinant host cells
capable of producing malonate comprise heterologous nucleic acids
encoding MAE1 transporters with at least 25%, at least 60%, at
least 80%, at least 90%, at least 95%, or more than 95% sequence
identity to Aspergillus kawachi G7XR17 (SEQ ID NO: 2) and/or
Aspergillus terreus Q0D1U9 (SEQ ID NO: 3) MAE1 transport
proteins.
The invention also provides a variety of recombinant host cells
comprising heterologous nucleic acids encoding MAE1 transport
proteins homologous to MAE1 transport protein consensus sequences
contained herein. These consensus consequences are broadly useful
for determining if a putative transport protein is an MAE1
transport protein suitable for use in accordance with the methods
of the invention. In various embodiments, recombinant host cells
capable of producing malonate comprise heterologous nucleic acids
encoding MAE1 transporters with at least 45% sequence identity to
Aspergillus MAE1 consensus sequence (SEQ ID NO: 7). In some
embodiments, recombinant host cells capable of producing malonate
comprise heterologous nucleic acids encoding MAE1 transporters with
at least 80% sequence identity to Aspergillus MAE1 consensus
sequence (SEQ ID NO: 7).
In a second aspect, the invention provides recombinant expression
vectors encoding a MAE1 transport protein that increase production
of malonate by the host cell. In some embodiments, the expression
vector is a yeast expression vector. In various embodiments, the
expression vector is a Pichia kudriavzevii expression vector. In
other embodiments, the expression vector is a Saccharomyces
cerevisiae expression vector.
In a third aspect, the invention provides methods for producing
malonate in a recombinant host cell, which methods generally
comprise culturing the recombinant host cell capable of producing
malonate and comprising a heterologous nucleic acid encoding a MAE1
transport protein under conditions that enable the recombinant host
cell to produce malonate.
These and other aspects and embodiments of the invention are
described in more detail below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for
producing malonate in recombinant host cells. In accordance with
the present invention, increased malonate titer, yield, and/or
productivity can be achieved by genetic modifications that increase
production of malonate by the host cell, and the invention provides
recombinant host cells comprising nucleic acids encoding MAE1
transport proteins that increase production of malonate by the host
cell and vectors for expressing MAE1 transport proteins that
increase production of malonate by the host cell. The invention
also provides methods for the use of recombinant host cells
comprising MAE1 transport proteins for the production of
malonate.
While the present invention is described herein with reference to
aspects and specific embodiments thereof, those skilled in the art
will recognize that various changes may be made and equivalents may
be substituted without departing from the invention. The present
invention is not limited to particular nucleic acids, expression
vectors, enzymes, host microorganisms, or processes, as such may
vary. The terminology used herein is for purposes of describing
particular aspects and embodiments only, and is not to be construed
as limiting. In addition, many modifications may be made to adapt a
particular situation, material, composition of matter, process,
process step or steps, in accordance with the invention. All such
modifications are within the scope of the claims appended
hereto.
All patents, patent applications, and publications cited herein are
incorporated herein by reference in their entireties.
Definitions
In this specification and in the claims that follow, reference will
be made to a number of terms that shall be defined to have the
following meanings.
As used in the specification and the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
an "expression vector" includes a single expression vector as well
as a plurality of expression vectors, either the same (e.g., the
same operon) or different; reference to "cell" includes a single
cell as well as a plurality of cells; and the like.
Amino acids in the sequence listing are identified by a
three-letter abbreviation, as follows: Ala is alanine, Arg is
arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine,
Gln is glutamine, Glu is glutamic acid, Gly is glycine, His is
histidine, Leu is leucine, Ile is isoleucine, Lys is lysine, Met is
methionine, Phe is phenylalanine, Pro is proline, Ser is serine,
Thr is threonine, Trp is tryptophan, Tyr is tyrosine, and Val is
valine. At some positions, Xaa indicates that any amino acid may be
present at the specified position. At other positions, Xaa
indicates that one of a subset of amino acids can be present,
namely Xaa may represent Arg, Lys, His, Asp, Glu, Ile, Lys, Val,
Ser, or Thr at the indicated position.
Specific amino acid in protein coding sequences discussed herein
are identified by their respective single-letter abbreviation, as
follows: A is alanine, R is arginine, N is asparagine, D is
aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G
is glycine, H is histidine, L is leucine, I is isoleucine, L is
lysine, M is methionine, F is phenylalanine, P is proline, S is
serine, T is threonine, W is tryptophan, Y is tyrosine, and V is
valine. In some instances, these single-letter abbreviations are
followed by the amino acid position in the protein coding sequence
where 1 corresponds to the amino acid (typically methionine) at the
N-terminus of the protein. For example, E124 in S. cerevisiae wild
type EHD3 refers to the glutamic acid at position 124 from the EHD3
N-terminal methionine (i.e., M1). Amino acid substitutions (i.e.,
point mutations) are indicated by identifying the mutated (i.e.,
progeny) amino acid after the single-letter code and number in the
parental protein coding sequence; for example, E124A in S.
cerevisiae EHD3 refers to substitution of alanine for glutamic acid
at position 124 in the EHD3 protein coding sequence. The mutation
may also be identified in parentheticals, for example EHD3 (E124A).
Multiple point mutations in the protein coding sequence are
separated by a backslash (/); for example, EHD3 E124A/Y125A
indicates that mutations E124A and Y125A are both present in the
EHD3 protein coding sequence. The number of mutations introduced
into some examples has been annotated by a dash followed by the
number of mutations, preceding the parenthetical identification of
the mutation (e.g. A5W8H3-1 (E95Q)). The UniProt IDs with and
without the dash and number are used interchangeably herein (i.e.
A5W8H3-1 (E95Q)=A5W8H3 (E95Q)).
As used herein, the term "express", when used in connection with a
nucleic acid a protein or a protein itself in a cell, means that
the protein, which may be an endogenous or exogenous (heterologous)
protein, is produced in the cell. The term "overexpress", in these
contexts, means that the protein is produced at a higher level,
i.e., protein levels are increased, as compared to the wild type,
in the case of an endogenous protein. Those skilled in the art
appreciate that overexpression of a protein can be achieved by
increasing the strength or changing the type of the promoter used
to drive expression of a coding sequence, increasing the strength
of the ribosome binding site or Kozak sequence, increasing the
stability of the mRNA transcript, altering the codon usage,
increasing the stability of the protein, and the like.
The terms "expression vector" or "vector" refer to a nucleic acid
and/or a composition comprising a nucleic acid that can be
introduced into a host cell, e.g., by transduction, transformation,
or infection, such that the cell then produces ("expresses")
nucleic acids and/or proteins other than those native to the cell,
or in a manner not native to the cell, that are contained in or
encoded by the nucleic acid so introduced. Thus, an "expression
vector" contains nucleic acids (ordinarily DNA) to be expressed by
the host cell. Optionally, the expression vector can be contained
in materials to aid in achieving entry of the nucleic acid into the
host cell, such as the materials associated with a virus, liposome,
protein coating, or the like. Expression vectors suitable for use
in various aspects and embodiments of the present invention include
those into which a nucleic acid sequence can be, or has been,
inserted, along with any preferred or required operational
elements. Thus, an expression vector can be transferred into a host
cell and, typically, replicated therein (although, one can also
employ, in some embodiments, non-replicable vectors that provide
for "transient" expression). In some embodiments, an expression
vector that integrates into chromosomal, mitochondrial, or plastid
DNA is employed. In other embodiments, an expression vector that
replicates extrachromasomally is employed. Typical expression
vectors include plasmids, and expression vectors typically contain
the operational elements required for transcription of a nucleic
acid in the vector. Such plasmids, as well as other expression
vectors, are described herein or are well known to those of
ordinary skill in the art.
The terms "ferment", "fermentative", and "fermentation" are used
herein to describe culturing microbes under conditions to produce
useful chemicals, including but not limited to conditions under
which microbial growth, be it aerobic or anaerobic, occurs.
The term "heterologous" as used herein refers to a material that is
non-native to a cell. For example, a nucleic acid is heterologous
to a cell, and so is a "heterologous nucleic acid" with respect to
that cell, if at least one of the following is true: (a) the
nucleic acid is not naturally found in that cell (that is, it is an
"exogenous" nucleic acid); (b) the nucleic acid is naturally found
in a given host cell (that is, "endogenous to"), but the nucleic
acid or the RNA or protein resulting from transcription and
translation of this nucleic acid is produced or present in the host
cell in an unnatural (e.g., greater or lesser than naturally
present) amount; (c) the nucleic acid comprises a nucleotide
sequence that encodes a protein endogenous to a host cell but
differs in sequence from the endogenous nucleotide sequence that
encodes that same protein (having the same or substantially the
same amino acid sequence), typically resulting in the protein being
produced in a greater amount in the cell, or in the case of an
enzyme, producing a mutant version possessing altered (e.g. higher
or lower or different) activity; and/or (d) the nucleic acid
comprises two or more nucleotide sequences that are not found in
the same relationship to each other in the cell. As another
example, a protein is heterologous to a host cell if it is produced
by translation of RNA or the corresponding RNA is produced by
transcription of a heterologous nucleic acid; a protein is also
heterologous to a host cell if it is a mutated version of an
endogenous protein, and the mutation was introduced by genetic
engineering.
The terms "host cell" and "host microorganism" are used
interchangeably herein to refer to a living cell that can be (or
has been) transformed via insertion of an expression vector. A host
microorganism or cell as described herein may be a prokaryotic cell
(e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic
cell. As will be appreciated by one of skill in the art, a
prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic
cell has a membrane-bound nucleus.
The terms "isolated" or "pure" refer to material that is
substantially, e.g. greater than 50% or greater than 75%, or
essentially, e.g. greater than 90%, 95%, 98% or 99%, free of
components that normally accompany it in its native state, e.g. the
state in which it is naturally found or the state in which it
exists when it is first produced.
A carboxylic acid as described herein can be a salt, acid, base, or
derivative depending on the structure, pH, and ions present. The
terms "malonate" and "malonic acid" are used interchangeably
herein. Malonic acid is also called propanedioic acid
(C.sub.3H.sub.4O.sub.4; CAS#141-82-2).
The term "malonate-derived compounds" as used herein refers to
mono-alkyl malonate esters, including, for example and without
limitation, mono-methyl malonate (also referred to as monomethyl
malonate, CAS#16695-14-0), mono-ethyl malonate (also referred to as
monoethyl malonate, CAS#1071-46-1), mono-propyl malonate,
mono-butyl malonate, mono-tert-butyl malonate (CAS#40052-13-9), and
the like; di-alkyl malonate esters, for example and without
limitation, dimethyl malonate (CAS#108-59-8), diethyl malonate
(CAS#105-53-3), dipropyl malonate (CAS#1117-19-7), dibutyl malonate
(CAS#1190-39-2), and the like, and Meldrum's acid (CAS#2033-24-1).
The malonate-derived compounds can be produced synthetically from
malonate and are themselves valuable compounds but are also useful
substrates in the chemical synthesis of a number of other valuable
compounds.
As used herein, the term "nucleic acid" and variations thereof
shall be generic to polydeoxyribonucleotides (containing
2-deoxy-D-ribose) and to polyribonucleotides (containing D-ribose).
"Nucleic acid" can also refer to any other type of polynucleotide
that is an N-glycoside of a purine or pyrimidine base, and to other
polymers containing nonnucleotidic backbones, provided that the
polymers contain nucleobases in a configuration that allows for
base pairing and base stacking, as found in DNA and RNA. As used
herein, the symbols for nucleotides and polynucleotides are those
recommended by the IUPAC-IUB Commission of Biochemical Nomenclature
(Biochem. 9:4022, 1970). A "nucleic acid" may also be referred to
herein with respect to its sequence, the order in which different
nucleotides occur in the nucleic acid, as the sequence of
nucleotides in a nucleic acid typically defines its biological
activity, e.g., as in the sequence of a coding region, the nucleic
acid in a gene composed of a promoter and coding region, which
encodes the product of a gene, which may be an RNA, e.g. a rRNA,
tRNA, or mRNA, or a protein (where a gene encodes a protein, both
the mRNA and the protein are "gene products" of that gene).
The term "operably linked" refers to a functional linkage between a
nucleic acid expression control sequence (such as a promoter,
ribosome-binding site, and transcription terminator) and a second
nucleic acid sequence, the coding sequence or coding region,
wherein the expression control sequence directs or otherwise
regulates transcription and/or translation of the coding
sequence.
The terms "optional" or "optionally" as used herein mean that the
subsequently described feature or structure may or may not be
present, or that the subsequently described event or circumstance
may or may not occur, and that the description includes instances
where a particular feature or structure is present and instances
where the feature or structure is absent, or instances where the
event or circumstance occurs and instances where it does not.
As used herein, "recombinant" refers to the alteration of genetic
material by human intervention. Typically, recombinant refers to
the manipulation of DNA or RNA in a cell or virus or expression
vector by molecular biology (recombinant DNA technology) methods,
including cloning and recombination. Recombinant can also refer to
manipulation of DNA or RNA in a cell or virus by random or directed
mutagenesis. A "recombinant" cell or nucleic acid can typically be
described with reference to how it differs from a naturally
occurring counterpart (the "wild-type"). In addition, any reference
to a cell or nucleic acid that has been "engineered" or "modified"
and variations of those terms, is intended to refer to a
recombinant cell or nucleic acid.
The terms "transduce", "transform", "transfect", and variations
thereof as used herein refers to the introduction of one or more
nucleic acids into a cell. For practical purposes, the nucleic acid
must be stably maintained or replicated by the cell for a
sufficient period of time to enable the function(s) or product(s)
it encodes to be expressed for the cell to be referred to as
"transduced", "transformed", or "transfected". As will be
appreciated by those of skill in the art, stable maintenance or
replication of a nucleic acid may take place either by
incorporation of the sequence of nucleic acids into the cellular
chromosomal DNA, e.g., the genome, as occurs by chromosomal
integration, or by replication extrachromosomally, as occurs with a
freely-replicating plasmid. A virus can be stably maintained or
replicated when it is "infective": when it transduces a host
microorganism, replicates, and (without the benefit of any
complementary virus or vector) spreads progeny expression vectors,
e.g., viruses, of the same type as the original transducing
expression vector to other microorganisms, wherein the progeny
expression vectors possess the same ability to reproduce.
Recombinant Host Cells
In one aspect, the invention provides a recombinant host cell
capable of producing malonate, the host cell comprising a
heterologous nucleic acid encoding a malic acid transport protein
(herein referred to as a MAE1 transport protein or a MAE1
transporter). In one embodiment, the recombinant host cell has been
engineered to produce malonate. In another embodiment, the
recombinant host cell natively produces malonate.
The present invention results in part from the discovery that a
host cell expressing a MAE1 transport protein results in increased
production of malonate relative to a parental host cell that does
not express the MAE1 transport protein. Any suitable host cell may
be used in practice of the methods of the present invention. In
some embodiments, the host cell is a recombinant host microorganism
capable of producing malonate that comprises a nucleic acid
encoding a MAE1 transport protein that results in expression of the
transport protein and provides an increase in the yield, titer,
and/or productivity of malonate relative to a "control cell" or
"reference cell" that does not express the transport protein, or
produces less of it. A "control cell" is thus used for comparative
purposes, and can be a recombinant parental cell that does not
contain one or more of the modification(s) that result in MAE1
transport protein expression (or increased expression) in the host
cell of the invention. Malonate is not naturally produced at high
concentrations in naturally occurring microbes (i.e.
non-recombinant microbes).
A variety of recombinant host cells are useful in accordance with
the methods of the invention. In an important embodiment, the
recombinant host cell is a yeast cell. Yeast cells are excellent
host cells for construction of recombinant metabolic pathways
comprising heterologous enzymes catalyzing production of small
molecule products. There are established molecular biology
techniques and nucleic acids encoding genetic elements necessary
for construction of yeast expression vectors, including, but not
limited to, promoters, origins of replication, antibiotic
resistance markers, auxotrophic markers, terminators, and the like.
Second, techniques for integration of nucleic acids into the yeast
chromosome are well established. Yeast also offers a number of
advantages as an industrial fermentation host. Yeast cells can
tolerate high concentrations of organic acids and maintain cell
viability at low pH and can grow under both aerobic and anaerobic
culture conditions, and there are established fermentation broths
and fermentation protocols. The ability of a strain to propagate
and/or produce desired product under low pH provides a number of
advantages with regard to the present invention. First, this
characteristic provides tolerance to the environment created by the
production of malonate. Second, from a process standpoint, the
ability to maintain a low pH environment limits the number of
organisms that are able to contaminate and spoil a batch.
In various embodiments, yeast cells useful in the method of the
invention include yeasts of a genera selected from the non-limiting
group consisting of Aciculoconidium, Ambrosiozyma, Arthroascus,
Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma,
Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces,
Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara,
Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus,
Eremothecium, Erythrobasidium, Fellomyces, Filobasidium,
Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora,
Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphopichia,
Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa,
Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces,
Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia, Nakazawaea,
Nematospora, Ogataea, Oosporidium, Pachysolen, Phachytichospora,
Phaffia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces,
Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia,
Saturnospora, Schizoblastosporion, Schizosaccharomyces,
Schwanniomyces, Sporidiobolus, Sporobolomyces, Sporopachydermia,
Stephanoascus, Sterigmatomyces, Sterigmatosporidium,
Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora,
Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea,
Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis,
Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis,
and Zygozyma, among others.
In various embodiments, the yeast cell is of a species selected
from the non-limiting group consisting of Candida albicans, Candida
ethanolica, Candida guilliermondii, Candida krusei, Candida
lipolytica, Candida methanosorbosa. Candida sonorensis, Candida
tropicalis, Candida utilis, Cryptococcus curvatus, Hansenula
polymorpha, Issatchenkia orientalis, Kluyveromyces lactis,
Kluyveronmyces marxianus, Kluyveromyces thermotolerans,
Komagataella pastoris, Lipomyces starkeyi, Pichia angusta, Pichia
deserticola, Pichia galeiformis, Pichia kodarnae, Pichia
kudriavzevii, Pichia membranaefaciens, Pichia methanolica, Pichia
pastoris, Pichia salictaria, Pichia stipitis, Pichia
thermotolerans, Pichia trehalophfla, Rhodosporidium toruloides,
Rhodotorula glutinis, Rhodotorula graminis, Saccharomyces bayanus,
Saccharomyces boulardi, Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe and Yarrowia lipolytica. One
skilled in the art will recognize that this list encompasses yeast
in the broadest sense, including both oleaginous and non-oleaginous
strains.
In certain embodiments, the recombinant yeast cells provided herein
are engineered by the introduction of one or more genetic
modifications (including, for example, introduction of heterologous
nucleic acids encoding MAE1 transport proteins) into a
Crabtree-negative yeast cell. As used herein, "a Crabtree-negative
yeast cell" refers to a yeast cell that does not undergo immediate
aerobic alcohol fermentation in response to addition of excess
sugar following growth under sugar-limited conditions. In certain
of these embodiments, the host cell belongs to the
Pichia/Issatchenkia/Saturnispora/Dekkera Glade. In certain of these
embodiments, the host cell belongs to the genus selected from the
group consisting of Pichia, Issatchenkia, or Candida. In certain
embodiments, the host cell belongs to the genus Pichia. In one
embodiment, the recombinant host cell is a Pichia kudriavzevii host
cell. Examples 1 and 2, below, illustrate the use of Pichia
kudriavzevii in accordance with the invention.
In certain embodiments, the recombinant host cells provided herein
are engineered by introduction of one or more genetic modifications
into a Crabtree-positive yeast cell. As used herein, "a
Crabtree-positive yeast cell" refers to a yeast cell that undergoes
immediate aerobic alcohol fermentation in response to addition of
excess sugar following growth under sugar-limited conditions. In
certain of these embodiments, the host cell belongs to the
Saccharomyces Glade. In certain of these embodiments, the host cell
belongs to a genus selected from the group consisting of
Saccharomyces, Hanseniaspora, and Kluyveromyces. In certain
embodiments, the host cell belongs to the genus Saccharomyces. In
one embodiment, the host cell is Sachcharomyces kluyveri. In
another embodiment, the recombinant host cell is a Saccharomyces
cerevisiae host cell.
Members of the Pichia/Issatchenkia/Saturnispora/Dekkera or the
Saccharomyces Glade are identified by analysis of their 26S
ribosomal DNA using the methods described by Kurtzman C. P., and
Robnett C. J., ("Identification and Phylogeny of Ascomycetous
Yeasts from Analysis of Nuclear Large Subunit (26S) Ribosomal DNA
Partial Sequences", Atonie van Leeuwenhoek 73(4):331-371; 1998).
Kurtzman and Robnett report analysis of approximately 500
ascomycetous yeasts were analyzed for the extent of divergence in
the variable D1/D2 domain of the large subunit (26S) ribosomal DNA.
Host cells encompassed by a Glade exhibit greater sequence identity
in the D1/D2 domain of the 26S ribosomal subunit DNA to other host
cells within the Glade as compared to host cells outside the Glade.
Therefore, host cells that are members of a Glade (e.g., the
Pichia/Issatchenkia/Saturnispora/Dekkera or Saccharomyces clades)
can be identified using the methods of Kurtzman and Robnett.
Recombinant host cells other than yeast cells are also suitable for
use in accordance with the methods of the invention. Illustrative
examples include various eukaryotic, prokaryotic, and archaeal host
cells. Illustrative examples of eukaryotic host cells provided by
the invention include, but are not limited to cells belonging to
the genera Aspergillus, Crypthecodinium, Cunninghamella,
Entomophthora, Mortierella, Mucor, Neurospora, Pythium,
Schizochytrium, Thraustochytrium Trichoderma, Xanthophyllomyces.
Examples of eukaryotic strains include, but are not limited to:
Aspergillus niger, Aspergillus oryzae, Crypthecodiniurri cohnii,
Cunninghamella japonica, Entomophthora coronata, Mortierella
alpina, Mucor circinelloides, Neurospora crassa, Pythium ultimum,
Schizochytrium limacinum, Thraustochytrium aureurri, Trichoderma
reesei, and Xanthophyllomyces dendrorhous.
Illustrative examples of recombinant archaea host cells provided by
the invention include, but are not limited to, cells belonging to
the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus,
Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma.
Examples of archae strains include, but are not limited to
Archaeoglobus fulgidus, Halobacterium sp., Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Thermoplasma
acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii,
Pyrococcus abyssi, and Aeropyrum pernix.
Illustrative examples of recombinant prokaryotic host cells
provided by the invention include, but are not limited to, cells
belonging to the genera Agrobacterium, Alicyclobacillus, Alnabaena,
Anacystis, Arthrobacter, Azobacter, Bacillcus, Brevibacterium,
Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia,
Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,
Methylobacterium, Microbacterium, Phomndium, Pseudomonas,
Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus,
Salmonella, Scenedesmun, Serratia, Shigella, Staphiococcus,
Strepromyces, Synnecoccus, and Zymomonas. Examples of prokaryotic
strains include, but are not limited to Bacillus subtilis,
Brevibacterium ammoniagenes, Bacillus arnyloliquefacines,
Brevibacterium ammoniagenes, Brevibacterium immariophilum,
Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli,
Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loci,
Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudita,
Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum
mbrum, Salmonella enterica, Salmonella typhi, Salmonella
typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella
sonnei, and Staphylococcus aureus.
Escherichia coli is a particularly good prokaryotic host cell for
use in accordance with the methods of the invention. E. coli is
well utilized in industrial fermentation of small-molecule products
and can be readily engineered. Unlike most wild type yeast strains,
wild type E. coli can catabolize both pentose and hexose sugars as
carbon sources. The present invention provides a wide variety of
recombinant E. coli host cells suitable for use in the methods of
the invention. In one embodiment, the recombinant host cell is an
Escherichia coli host cell.
Certain of these host cells, including Saccharomyces cerevisiae,
Bacillus subtilis, Lactobacillus acidophilus, have been designated
by the Food and Drug Administration as Generally Regarded As Safe
(or GRAS) and so are employed in various embodiments of the methods
of the invention. While desirable from public safety and regulatory
standpoints, GRAS status does not impact the ability of a host
strain to be used in the practice of this invention; hence,
non-GRAS and even pathogenic organisms are included in the list of
illustrative host strains suitable for use in the practice of this
invention.
MAE1 Transport Proteins
In accordance with the present invention, certain MAE1 transport
proteins have the capacity to transport malonate and increase
malonate production in host cells, including naturally occurring
host cells but especially recombinant host cells engineered to
produce malonate (as per PCT Pub. No. WO 13/134424, supra). An MAE1
transport protein may secrete malonate in an ionic form and in a
protonated form. An MAE1 transport protein may transport other
species, for example a hydrogen ion, together with malonate.
Malonate transport as used herein may be export of malonate from
the interior of a cell to the exterior, and/or import of malonate
from the exterior to the interior.
As described below, a variety of methods and assays may be used by
those skilled in the art to determine if a putative transport
protein is a MAE1 transport protein capable of increasing malonate
production by a recombinant host cell. For example, the percent
sequence identity of a putative MAE1 transport protein relative to
a reference MAE1 transport protein sequence is used to determine if
a putative transport protein is an MAE1 transport protein. Percent
sequence identity is determined by aligning the protein sequence
against a reference sequence. The reference sequence can be a
consensus sequence or a specific protein sequence. Those skilled in
the art will recognize that various sequence alignment algorithms
are suitable for aligning a protein with a reference sequence. See,
for example, Needleman, S B, et al "A general method applicable to
the search for similarities in the amino acid sequence of two
proteins." Journal of Molecular Biology 48 (3): 443-53 (1970).
Following alignment of the protein sequence relative to the
reference sequence, the percentage of positions where the protein
possesses an amino acid (or a dash where no amino acid is present)
described by the same position in the reference sequence determines
the percent sequence identity. When a degenerate amino acid
(represented by Xaa or X) is present in a reference sequence, any
of the amino acids described by the degenerate amino acid may be
present in the protein at the aligned position for the protein to
be identical to the reference sequence at the aligned position.
The Aspergillus niger A2R8T9 MAE1 reference sequence (SEQ ID NO: 1)
is useful for determining the percentage sequence identity between
a putative MAE1 transport protein and a MAE1 transport protein
useful in accordance with the present invention. Suitable MAE1
transport proteins will have at least 25% amino acid sequence
identity to SEQ ID NO: 1, and may, for example and without
limitation, have at least 50%, 75%, 95%, or greater identity to SEQ
ID NO: 1. Thus, proteins G7XR17 (SEQ ID NO: 2, 96% identity),
Q0D1U9 (SEQ ID NO: 3, 61% sequence identity), P50537 (SEQ ID NO: 4,
30% sequence identity), and O59815 (SEQ ID NO: 5, 25% sequence
identity) have the requisite identity to SEQ ID NO: 1. In contrast,
Saccharomyces cerevisiae proteins PDRS (UniProt ID: P33302, 4%
identity), PDR10 (UniProt ID: P51533, 4% identity), PDR11 (UniProt
ID: P40550, 4% identity), PDR12 (UniProt ID: Q02785, 5% identity),
PDR15 (UniProt ID: Q04182, 4% identity), and PDR18 (UniProt ID:
P53756, 5% identity) all have less than 25% amino acid identity to
SEQ ID NO: 1, and are not MAE1 transport proteins. Example 1
further provides methods for determining if a putative transporter
is an MAE1 transport protein and increases malonate production in a
recombinant microbe.
Generally, homologous proteins share substantial sequence identity.
Any protein substantially homologous to a protein specifically
described herein can be used in a host cell of the invention. One
protein is homologous to another (the "reference protein") when it
exhibits the same activity of interest and can be used for
substantially similar purposes. If a protein shares substantial
homology to a reference sequence herein but has suboptimal,
including no, MAE1 transport protein activity, then, in accordance
with the invention, it can be mutated to conform to a reference
sequence provided herein to provide a MAE1 transport protein of the
invention.
Source of MAE1 Transport Proteins
The heterologous nucleic acids encoding a MAE1 transporter may be
obtained from microorganisms of any genus. For purposes of the
present invention, the term "obtained from" as used herein in
connection with a given source shall mean that the MAE1 transporter
encoded by a nucleic acid is produced by the source, or by a cell
in which the nucleic acid from the source has been inserted. It
will be understood that for the organisms indicated below, the
invention encompasses taxonomic equivalents (e.g., anamorphs and
teleomorphs) regardless of the species name by which they are
known. Those skilled in the art will recognize the identity of
appropriate equivalents.
In one embodiment, the recombinant host cell capable of producing
malonate comprises a nucleic acid encoding a eukaryotic MAE1
transport protein that results in expression of the transport
protein and provides an increase in the yield, titer, and/or
productivity of malonate relative to a control cell that does not
express the transport protein, or produces less of it.
MAE1 Transport Proteins Obtained from Aspergillus Species or
Homologous Thereto
In some embodiments, the recombinant host cell capable of producing
malonate comprises a nucleic acid encoding a MAE1 transport protein
obtained from an Aspergillus species (or significantly homologous
thereto) that results in expression of the transport protein and
provides an increase in the yield, titer, and/or productivity of
malonate relative to a control cell that does not express the
transport protein, or produces less of it. In various embodiments,
the nucleic acid encoding a MAE1 transport protein is obtained from
an organism selected from the group consisting of, but not limited
to, Aspergillus clavatus, Aspergillus flavus, Aspergillus
fumigatus, Aspergillus kawachii, Aspergillus nidulans, Aspergillus
niger, Aspergillus oryzae, and Aspergillus terreus (or is
homologous to such a nucleic acid). In various embodiments, the
nucleic acid encodes a MAE1 transport protein selected from the
group consisting of Aspergillus niger A2R8T9 (SEQ ID NO: 1),
Aspergillus kawachi G7XR17 (SEQ ID NO: 2), and Aspergillus terreus
Q0D1U9 (SEQ ID NO: 3). Example 1 demonstrates, in accordance with
the methods of the invention, how expression of various MAE1
transport proteins obtained from Aspergillus species (SEQ ID NOs:
1, 2, and 3) by recombinant host cells increases malonate yields
and titers relative to parental, control cells that do not express
said MAE1 transport proteins. These recombinant host cells have
been engineered to express said MAE1 transport proteins through
transformation with heterologous nucleic acids encoding the MAE1
transporters. Likewise, Example 3 demonstrates, in accordance with
the methods of the invention, how malonate productivity is
increased through fermentation of recombinant host cells expressing
MAE1 transport proteins obtained from Aspergillus species and that
are capable of producing malonate.
In one embodiment, the recombinant host cell capable of producing
malonate comprises a nucleic acid encoding an Aspergillus niger
A2R8T9 MAE1 transport protein (SEQ ID NO: 1) and provides an
increase in the yield, titer, and/or productivity of malonate
relative to a control cell that does not express the Aspergillus
niger A2R8T9 MAE1 transport protein, or produces less of it. In
some embodiments of the invention, the recombinant host cell
capable of producing malonate and comprising a nucleic acid
encoding an Aspergillus niger A2R8T9 MAE1 transport protein (SEQ ID
NO: 1) is a Pichia kudriavzevii host cell. In other embodiments of
the invention, the recombinant host cell capable of producing
malonate and comprising a nucleic acid encoding an Aspergillus
niger A2R8T9 MAE1 transport protein (SEQ ID NO: 1) is a
Sacharromyces cerevisiae host cell.
MAE1 transport proteins useful in the compositions and methods
provided herein include proteins that are "homologous" to the MAE1
transport proteins obtained from Aspergillus species and described
herein. Such homologs have the following characteristics: (1)
capable of transporter activity that is identical, or essentially
identical, or at least substantially similar with respect to
ability to transport malonate across the cell membrane to that of
one of the MAE1 transport proteins exemplified herein; (2) shares
substantial sequence identity with an MAE1 transport protein
described herein; and/or (3) comprises a substantial number of
amino acids corresponding to highly conserved amino acids in a MAE1
transport protein described herein.
A "homolog" as used herein refers to a protein that shares
substantial sequence identity to a reference protein, such as an
MAE1 transport protein, if the amino acid sequence of the homolog
is at least 25%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, or at least 97% the same as that
of an MAE1 transport protein set forth herein.
A number of amino acids in the MAE1 transport proteins provided by
the invention are highly conserved across MAE1 transport proteins
generally, and proteins homologous to a MAE1 transport protein of
the invention will generally possess a substantial number of these
highly conserved amino acids. The presence of a highly conserved
amino acid in the query protein is determined by first aligning the
query protein against the reference sequence; once aligned, the
amino acid residue at the highly conserved position in the
reference protein is compared to the amino acid residue in the
corresponding location in the query protein. If the amino acid
residues are the same, then the query protein is said to possess
this conserved amino acid. A homolog is said to comprise a
substantial number of highly conserved amino acids if at least a
majority, often more than 90%, and sometimes all of the highly
conserved amino acids are found in the homologous protein.
MAE1 transport proteins suitable for use in accordance with the
methods of the invention include those that are homologous to the
Aspergillus niger A2R8T9 MAE1 transport protein sequence (SEQ ID
NO: 1). In one embodiment, suitable MAE1 transport proteins for use
in accordance with the methods of the invention have at least 25%
identity to this MAE1 transport protein reference sequence. In
other embodiments, suitable MAE1 transport proteins have at least
60% identity to SEQ ID NO: 1. In various embodiments, the MAE1
transport protein has malonate transporter activity and comprises
an amino acid sequence having at a percentage sequence identity to
SEQ ID NO: 1 of at least 50%, for example, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 92%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
amino acid sequence identity to SEQ ID NO: 1.
In some embodiments, a MAE1 transport protein with equal to or
greater than 25% identity to the reference sequence SEQ ID NO: 1 is
expressed in a recombinant host cell capable of producing malonate
and is used to increase the production of malonate in said host
cell relative the parental host cell. In other embodiments, a MAE1
transport protein with equal to or greater than 60% identity to the
reference sequence SEQ ID NO: 1 is expressed in a recombinant host
cell capable of producing malonate and is used to increase the
production of malonate in said host cell relative the parental host
cell. MAE1 proteins possessing substantial sequence homology to SEQ
ID NO: 1 and, when expressed in a host cell capable of producing
malonate, increase production of malonate include, but are not
limited to, G7XR17 (SEQ ID NO: 2; 96% identity), Q0D1U9 (SEQ ID NO:
3; 61% identity), P50537 (SEQ ID NO: 4; 30% identity), and O59815
(SEQ ID NO: 5; 25% identity). As illustrated in Example 1, nucleic
acids encoding A2R8T9, G7XR17, Q0D1U9, P50537, and O59815 MAE1
transport proteins were heterologously expressed in a recombinant
Pichia kudriavzevii host cell comprising a malonyl-CoA hydrolase
and increased malonate titers in the fermentation broth.
As illustrated in Example 2, recombinant expression vectors of the
invention comprising nucleic acids encoding the A2R8T9 MAE1
transport protein are heterologously expressed in a genetically
modified Pichia kudriavzevii host cell comprising a malonyl-CoA
hydrolase and increase malonate productivity. As illustrated in
Example 3, fermentation of recombinant host cells capable of
producing malonate and expressing MAE1 transport proteins in
accordance with the methods of the invention increased malonate
productivity.
There are 80 highly conserved amino acids in Aspergillus niger
A2R8T9 MAE1 transport protein (SEQ ID NO: 1): R57, H60, F61, T62,
W63, W65, M70, G73, G74, F86, G88, L89, R114, F115, I116, E130,
F133, T136, L139, I141, T143, I145, L148, L167, I170, F187, T196,
P199, L203, P204, F206, P207, M209, G212, I214, A215, Q222, P223,
A224, G234, F237, Q238, G239, L240, G241, F242, A250, R255, G260,
L261, R267, P268, G269, M270, F271, V274, P276, P277, F279, L282,
L284, G299, F320, L324, C330, A332, F344, W348, A350, F353, N355,
G357, S371, R398, A399, P408, G409, D411, E412, D413. MAE1
transport proteins homologous to SEQ ID NO: 1 generally possess a
majority, often more than 90%, and sometimes all of these highly
conserved amino acids. In various embodiments, host cells of
invention express a MAE1 transport protein that has at least 95% of
these highly conserved amino acids. For example, Q0D1U9 (SEQ ID NO:
3) possess all 80 (i.e., 100%) of the highly conserved amino acids
in SEQ ID NO: 1. The location of these amino acids in SEQ ID NO: 3
are as follows (the corresponding location in SEQ ID NO: 1 is
provided in parentheses): R34 (R57), H37 (H60), F38 (F61), T39
(T62), W40 (W63), W42 (W65), M47 (M70), G50 (G73), G51 (G74), F63
(F86), G65 (G88), L66 (L89), R91 (R114), F92 (F115), I93 (I116),
E107 (E130), F110 (F133), T113 (T136), L116 (L139), I118 (I141),
T120 (T143), I122 (I145), L125 (L148), L144 (L167), I147 (I170),
F164 (F187), T173 (T196), P176 (P199), L180 (L203), P181 (P204),
F183 (F206), P184 (P207), M186 (M209), G189 (G212), I191 (I214),
A192 (A215), Q199 (Q222), P200 (P223), A201 (A224), G211 (G234),
F214 (F237), Q215 (Q238), G216 (G239), L217 (L240), G218 (G241),
F219 (F242), A227 (A250), R232 (R255), G237 (G260), L238 (L261),
R244 (R267), P245 (P268), G246 (G269), M247 (M270), F248 (F271),
V251 (V274), P253 (P276), P254 (P277), F256 (F279), L259 (L282),
L261 (L284), G276 (G299), F297 (F320), L301 (L324), C307 (C330),
A309 (A332), F321 (F344), W325 (W348), A327 (A350), F330 (F353),
N332 (N355), G334 (G357), 5348 (S371), R375 (R398), A376 (A399),
P385 (P408), G386 (G409), D388 (D411), E389 (E412), and D390
(D413). Thus, MAE1 transport protein QOD1U9 has over 95% of the
highly conserved amino acids found in SEQ ID NO: 1 and is thus
homologous to SEQ ID NO: 1. Other MAE1 transport proteins
homologous to SEQ ID NO: 1 include those encoded by the protein
sequences set forth in SEQ ID NOs: 1, 2, 4, and 5.
Other MAE1 transport protein sequences in addition to the
Aspergillus niger A2R8T9 MAE1 transport protein (SEQ ID NO: 1) are
also useful in identifying and/or constructing other MAE1 transport
proteins (and nucleic acids that encode them) suitable for use in
accordance with the methods of the invention. In various
embodiment, a suitable MAE1 transport protein for use in accordance
with the methods of the invention has malonate transporter activity
and comprises an amino acid sequence having a percentage identity
to SEQ ID NO: 2 of at least 33%, for example, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 92%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% amino acid sequence identity to SEQ ID NO: 2. In
other embodiments, a suitable MAE1 transport protein for use in
accordance with the methods of the invention has malonate
transporter activity and comprises an amino acid sequence having a
percentage identity to SEQ ID NO: 3 of at least 50%, for example,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 92%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100% amino acid sequence identity to SEQ ID NO:
3.
MAE1 Transport Proteins Obtained from Schizosaccharomyces Species
or Homologous Thereto
In addition to MAE1 transport proteins obtained from Aspergillus
species and their homologous counterparts, the invention also
provides MAE1 transport proteins obtained from Schizosaccharomyces
species (and homologous counterpart proteins) suitable for use in
accordance with the methods of the invention. In some embodiments,
the recombinant host cell capable of producing malonate comprises a
nucleic acid encoding a MAE1 transport protein obtained from a
Schizosaccharomyces species that results in expression of the
transport protein and provides an increase in the yield, titer,
and/or productivity of malonate relative to a control cell that
does not express the transport protein, or produces less of it. In
various embodiments, the nucleic acid encoding a MAE1 transport
protein is obtained from an organism selected from the group
consisting of, but not limited to, Schizosaccharomyces cryophilus,
Schizosaccharomyces japonica, Schizosaccharomyces octosporus, and
Schizosaccharomyces pombe.
In one embodiment, the recombinant host cell capable of producing
malonate comprises a nucleic acid encoding a Schizosaccharomyces
pombe P50537 MAE1 transport protein (SEQ ID NO: 4) and provides an
increase in the yield, titer, and/or productivity of malonate
relative to a control cell that does not express the
Schizosaccharomyces pombe P50537 MAE1 transport protein, or
produces less of it. In another embodiment, the recombinant host
cell capable of producing malonate comprises a nucleic acid
encoding a Schizosaccharomyces pombe O59815 MAE1 transport protein
(SEQ ID NO: 5) and provides an increase in the yield, titer, and/or
productivity of malonate relative to a control cell that does not
express the Schizosaccharomyces pombe O59815 MAE1 transport
protein. Example 1 demonstrates how practice of the invention using
Schizosaccharomyces pombe P50537 and O59815 MAE1 transport proteins
(SEQ ID NOs: 4 and 5) in recombinant Pichia host cells increased
malonate titer and yield relative to control cells not expressing
these MAE1 transporters.
Suitable MAE1 transport proteins for use in accordance with the
methods of the invention include those that are homologous to the
Schizosaccharomyces pombe P50537 MAE1 transport protein sequence
(SEQ ID NO: 4). In one embodiment, suitable MAE1 transport proteins
for use in accordance with the methods of the invention have at
least 33% identity to this MAE1 transport protein reference
sequence. In another embodiment, suitable MAE1 transport proteins
for use in the methods of invention have at least 50% identity to
SEQ ID NO: 4. In various embodiments, the MAE1 transport protein
has malonate transporter activity and comprises an amino acid
sequence having at a percentage sequence identity to SEQ ID NO: 4
of at least 50%, for example, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 92%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100% amino acid
sequence identity to SEQ ID NO: 4. In some embodiments, a MAE1
transport protein with equal to or greater than 33% identity to the
reference sequence SEQ ID NO: 4 is expressed in a recombinant host
cell capable of producing malonate and is used to increase the
production of malonate in said host cell relative the parental host
cell.
The invention also provides expression vectors for expressing
Schizosaccharomyces pombe O59815 MAE1 transport protein (SEQ ID NO:
5). The natural coding sequence can be used in identifying and/or
constructing MAE1 transport protein coding sequences suitable for
use in accordance with the methods of the invention. In various
embodiments, a suitable MAE1 transport protein for use in
accordance with the methods of the invention has malonate
transporter activity and comprises an amino acid sequence having a
percentage identity to SEQ ID NO: 5 of at least 50%, for example,
at at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 92%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 5.
In some embodiments, a MAE1 transport protein with equal to or
greater than 50% identity to the reference sequence SEQ ID NO: 5 is
expressed in a recombinant host cell capable of producing malonate
and provides an increase in malonate production relative to a
parental cell not expressing said MAE1 transport protein.
MAE1 Consensus Sequences
MAE1 transport proteins suitable for use in the compositions and
methods of the invention include those MAE1 transport proteins
homologous to MAE1 consensus sequences described herein. A
consensus sequence provides a sequence of amino acids in which each
position identifies the amino acid (if a specific amino acid is
identified) or a subset of amino acids (if a position is identified
as variable) most likely to be found at a specified position in a
MAE1 transport protein. At positions in a consensus sequence where
one of a subset of amino acids can be present, the following
abbreviations are used below when referring to subsets of amino
acids: B represents that one of the amino acids R, K, or H is
present at the indicated position; J represents that one of the
amino acids D or E is present at the indicated position; O
represents that one of the amino acids I, L, or V is present at the
indicated position. The percent sequence identity of a protein
relative to a consensus sequence is determined by aligning the
query protein sequence against the consensus sequence.
Proteins homologous to MAE1 consensus sequences have the following
characteristics: (1) is capable of transporter activity that is
identical, or essentially identical, or at least substantially
similar with respect to ability to transport malonate across the
cell membrane to that of one of the MAE1 transport proteins
exemplified herein; (2) it shares substantial sequence identity
with a MAE1 consensus sequence described herein; and/or (3) it
possesses a substantial number of highly conserved amino acids
found in a MAE1 consensus sequence described herein.
Two MAE1 consensus sequences provided herein are useful in
identifying and constructing nucleic acids that encode MAE1
proteins suitable for use in the methods of the invention: (i) an
MAE1 consensus sequence based on Aspergillus MAE1 transport
proteins and referred to herein as an "Aspergillus MAE1 consensus
sequence" (SEQ ID NO: 7); and (ii), a MAE1 consensus sequence based
on both Aspergillus and Schizosaccharomyces pombe MAE1 transport
proteins (SEQ ID NO: 8).
In various embodiments, a recombinant host cell capable of
producing malonate expresses an MAE1 transport protein with at
least 45% sequence identity to SEQ ID NO: 7 and provides an
increase in malonate yield, titer, and/or productivity relative to
a control cell that does not express said MAE1 transport protein.
In some embodiments, the recombinant host cell expresses a protein
with at least 80% identity to SEQ ID NO: 7. In still further
embodiments, the recombinant host cell expresses a protein with a
least 85%, at least 90%, at least 95%, or greater than 95% sequence
identity to SEQ ID NO: 7. For example, the Aspergillus niger A2R8T9
(SEQ ID NO: 1), Aspergillus kawachi G7XR17 (SEQ ID NO: 2),
Aspergillus terreus Q0D1U9 (SEQ ID NO: 3), Schizosaccharomyces
pombe P50537 (SEQ ID NO: 4), and Schizosaccharomyces pombe O59815
(SEQ ID NO: 5) sequences are 100%, 100%, 94%, 52%, and 49%
identical to the Aspergillus MAE1 consensus sequence (SEQ ID NO:
7); therefore, all five of these sequence are homologous to
consensus sequence SEQ ID NO: 7. Additional proteins homologous to
consensus sequence SEQ ID NO: 7 include, but are not limited to,
those selected from the group consisting of: UniProt ID Q0D1U9 (94%
identity to Seq ID NO: 7), UniProt ID B8N8E0 (89% identity to Seq
ID NO: 7), UniProt ID I7ZSL4 (89% identity to Seq ID NO: 7),
UniProt ID S8AYC2 (88% identity to Seq ID NO: 7), UniProt ID A1C406
(87% identity to Seq ID NO: 7), UniProt ID Q2UHT6 (87% identity to
Seq ID NO: 7), UniProt ID A1DB74 (86% identity to Seq ID NO: 7),
UniProt ID K9GWN1 (86% identity to Seq ID NO: 7), UniProt ID K9GI69
(86% identity to Seq ID NO: 7), UniProt ID Q5BDA8 (86% identity to
Seq ID NO: 7), UniProt ID W6Q7W6 (86% identity to Seq ID NO: 7),
UniProt ID B6HF90 (85% identity to Seq ID NO: 7), UniProt ID V5I2J6
(85% identity to Seq ID NO: 7), UniProt ID B0YA01 (84% identity to
Seq ID NO: 7), and UniProt ID Q4WCF3 (84% identity to Seq ID NO:
7); any of these proteins are suitable for expression in
recombinant host cells capable of producing malonate in order to
provide an increase in malonate yield, titer, and/or
productivity.
A number of amino acids in consensus sequence SEQ ID NO: 7 are
highly conserved, and a majority of these amino acids, often more
than 90%, and sometimes all of these amino acids are found in MAE1
transport proteins homologous to consensus sequence SEQ ID NO: 7.
There are 279 highly conserved amino acids in SEQ ID NO: 7; namely:
T5, P8, G9, S10, S11, S13, D14, O40, P49, G50, V52, G53, R55, E56,
R57, O58, R59, H60, F61, T62, W63, A64, W65, Y66, T67, L68, T69,
M70, S71, G73, G74, L75, A76, L77, L78, O79, Q82, P83, F86, G88,
L89, B90, J91, 192, V96, Y97, L99, N100, O101, F103, F104, O106,
V107, U109, M111, A112, R114, F115, I116, L117, H118, J123, S124,
L125, H127, J128, R129, E130, G131, O132, F133, F134, P135, T136,
F137, W138, L139, S140, I141, A142, T143, I145, T146, G147, L148,
Y149, B150, F152, G153, J154, D155, F160, L164, L167, F168, W169,
I170, Y171, C172, T175, O177, A179, V180, Q182, Y183, S184, O186,
F187, K191, Y192, L194, T196, M198, P199, W201, I202, L203, P204,
A205, F206, P207, V208, M209, L210, S211, G212, T213, I214, A215,
S216, V217, I218, Q222, P223, A224, I228, P229, O231, O232, A233,
G234, T236, F237, Q238, G239, L240, G241, F242, S243, I244, S245,
M248, Y249, A250, H251, Y252, O253, G254, R255, L256, M257, E258,
G260, L261, P262, E265, H266, R267, P268, G269, M270, F271, 1272,
V274, G275, P276, P277, A278, F279, T280, A281, L282, A283, L284,
V285, G286, M287, K289, L291, P292, D294, F295, Q296, O297, O298,
G299, D300, A303, D306, R308, O309, L313, A314, O315, O319, F320,
L321, W322, A323, L324, 5325, W327, F328, F329, C330, 1331, A332,
O334, A335, V336, V337, R338, S339, P340, P341, F344, H345, L346,
W348, A350, M351, V352, F353, P354, N355, T356, G357, F358, T359,
L360, A361, T362, I363, L365, S371, G373, O374, G376, V377, T379,
A380, M381, S382, O383, O.sub.385, O386, F389, O390, F391, V392,
O394, S395, O397, R398, A399, V400, I401, R402, K403, D404, I405,
M406, P408, G409, D411, E412, D413, V414, and E416. In various
embodiments, a recombinant host cell capable of producing malonate
expresses an MAE1 transport protein having at least 50% of these
highly conserved amino acids, and wherein said host cell produces
an increased amount (yield, titer, and/or productivity) of malonate
as compared to a control cell that does not express said MAE1
transport protein. For example, Schizosaccharomyces pombe P50537
(SEQ ID NO: 4) and Schizosaccharomyces pombe O59815 (SEQ ID NO: 5)
have 72% and 66% of these highly conserved amino acids,
respectively, and thus have a substantial number of these highly
conserved amino acids. In some embodiments, a recombinant host cell
capable of producing malonate expresses an MAE1 transport protein
having at least 90% of these highly conserved amino acids, and
wherein said host cell produces an increased amount (yield, titer,
and/or productivity) of malonate as compared to a control cell that
does not express said MAE1 transport protein. MAE1 transport
proteins A2R8T9 (SEQ ID NO: 1) and G7XR17 (SEQ ID NO: 2) both have
100% of these highly conserved amino acids, and MAE1 transport
protein Q0D1U9 (SEQ ID NO: 3) has 94% of these highly conserved
amino acids; therefore, these proteins also possess a substantial
number of these highly conserved amino acids.
In addition to the Aspergillus MAE1 consensus sequence, a MAE1
consensus sequence based on both Aspergillus and
Schizosaccharomyces pombe MAE1 transport proteins (SEQ ID NO: 8) is
also useful for identifying and constructing nucleic acids that
encode MAE1 transport proteins suitable for use in accordance with
the compositions and methods of the invention. In various
embodiments, a recombinant host cell capable of producing malonate
expresses an MAE1 transport protein with at least 80% sequence
identity to SEQ ID NO: 8 and provides an increase in malonate
yield, titer, and/or productivity relative to a control cell that
does not express said MAE1 transport protein. In other embodiments,
the recombinant host cell expresses a protein with a least 90%, at
least 95%, or greater than 95% sequence identity to SEQ ID NO: 8.
For example, the Aspergillus niger A2R8T9 (SEQ ID NO: 1),
Aspergillus kawachi G7XR17 (SEQ ID NO: 2), Aspergillus terreus
Q0D1U9 (SEQ ID NO: 3), Schizosaccharomyces pombe P50537 (SEQ ID NO:
4), and Schizosaccharomyces pombe O59815 (SEQ ID NO: 5) sequences
are 95%, 95%, 98%, 85%, and 84% identical to MAE1 consensus
sequence SEQ ID NO: 8, respectively; therefore, all five of these
sequence are homologous to consensus sequence SEQ ID NO: 8.
A number of amino acids in consensus sequence SEQ ID NO: 8 are
highly conserved, and a majority of these amino acids, often more
than 90%, and sometimes all of these amino acids are found in MAE1
transport proteins homologous to consensus sequence SEQ ID NO: 8.
There are 118 highly conserved amino acids in SEQ ID NO: 8; namely:
O23, R40, O41, H43, F44, T45, W46, W48, M53, G56, G57, O58, O61,
F69, G71, L72, O75, O79, O84, O89, R97, F98, I99, U106, E112, O114,
F115, T118, L121, I123, T125, I127, L130, O146, L149, I152, O159,
O162, O167, F168, O175, T177, P180, O183, L184, P185, F187, P188,
M190, O191, G193, I195, A196, O199, Q203, P204, A205, O212, O213,
G215, F218, Q219, G220, L221, G222, F223, O225, A231, R236, G241,
L242, R248, P249, G250, M251, F252, V255, P257, P258, F260, U261,
L263, L265, O266, O279, G280, O294, O300, F301, L305, C311, O312,
A313, O315, O318, F325, W329, A331, O333, F334, N336, G338, O346,
S352, O364, O366, O371, O373, O378, R379, A380, J385, O386, P389,
G390, D392, E393, and D394. In various embodiments, a recombinant
host cell capable of producing malonate expresses an MAE1 transport
protein having at least 95% of these highly conserved amino acids,
and wherein said host cell produces an increased amount (yield,
titer, and/or productivity) as compared to a control cell that does
not express said MAE1 transport protein. In some embodiments, a
recombinant host cell capable of producing malonate expresses an
MAE1 transport protein having all of these highly conserved amino
acids, and wherein said host cell produces an increased amount
(yield, titer, and/or productivity) as compared to a control cell
that does not express said MAE1 transport protein. For example,
100% of the highly conserved amino acids in consensus sequence SEQ
ID NO: 8 are found in MAE1 transport proteins encoded by SEQ ID
NOs: 1, 2, 3, 4, and 5; thus, all five of these proteins have a
substantial number of the highly conserved amino acids in SEQ ID
NO: 8.
Additional Sources of MAE1 Transport Proteins
As described above, nucleic acids encoding MAE1 transport proteins
suitable for use in accordance with the methods of the invention
may be obtained from organisms other than Aspergillus and
Schizosaccharomyces species. In various embodiments, the
recombinant host cell capable of producing malonate comprises a
nucleic acid encoding a MAE1 transport protein obtained from an
organism selected from the group consisting of, but not limited to,
Ajellomyces capsulatus, Arthrobotrys oligospora, Arthroderma
benhamiae, Arthroderma gypseum, Arthroderma otae, Baudoinia
compniacensis, Beauveria bassiana, Bipolaris oryzae, Bipolaris
victoriae, Bipolaris zeicola, Blumeria graminis, Botryosphaeria
parva, Botryotinia fuckeliana, Byssochlamys spectabilis, Capronia
coronata, Capronia epimyces, Chaetomium globosum, Chaetomium
thermophilum, Cladophialophora carrionii, Cladophialophora
psammophila, Cladophialophora yegresii, Claviceps purpurea,
Coccidioides immitis, Coccidioides posadasii, Cochliobolus
heterostrophus, Cochliobolus sativus, Colletotrichum
gloeosporioides, Colletotrichum graminicola, Colletotrichum
higginsianum, Colletotrichum orbiculare, Coniosporium apollinis,
Cordyceps militaris, Cyphellophora europaea, Dactylellina
haptotyla, Emericella nidulans, Eutypa lata, Exophiala
dermatitidis, Fusarium oxysporum, Fusarium pseudograminearum,
Gaeumannomyces graminis, Gibberella fujikuroi, Gibberella
moniliformis, Gibberella zeae, Glarea lozoyensis, Grosmannia
clavigera, Hypocrea atroviridis, Hypocrea jecorina, Hypocrea
virens, Leptosphaeria maculans, Macrophomina phaseolina,
Magnaporthe oryzae, Magnaporthe poae, Malassezia sympodialis,
Marssonina brunnea, Metarhizium acridum, Metarhizium anisopliae,
Mycosphaerella fijiensis, Mycosphaerella graminicola,
Mycosphaerella pini, Nectria haematococca, Neosartorya fischeri,
Neosartorya fumigata, Neurospora crassa, Neurospora tetrasperma,
Ophiocordyceps sinensis, Penicillium chrysogenum, Penicillium
digitatum, Penicillium oxalicum, Penicillium roqueforti,
Pestalotiopsis fici, Phaeosphaeria nodorum, Podospora anserina,
Pyrenophora teres, Pyrenophora tritici-repentis, Saccharomyces
cerevisiae, Sclerotinia borealis, Sclerotinia sclerotiorum,
Setosphaeria turcica, Sordaria macrospora, Sphaerulina musiva,
Thielavia heterothallica, Thielavia terrestris, Togninia minima,
Trichophyton equinum, Trichophyton rubrum, Trichophyton tonsurans,
Trichophyton verrucosum, Verticillium alfalfae, and Verticillium
dahlia.
Expression Vectors
In a second aspect, the invention provides recombinant expression
vectors encoding one or more MAE1 transport protein(s) that results
in expression of the transport protein and provides an increase in
the yield, titer, and/or productivity of malonate relative to a
control cell that does not express the transport protein, or
produces less of it. In various embodiments of the invention, the
recombinant host cell has been modified by "genetic engineering" to
produce a recombinant MAE1 transport protein and secrete malonate.
The host cell is typically engineered via recombinant DNA
technology to express heterologous nucleic acids that encode a MAE1
transport protein, which is either a mutated version of a naturally
occurring MAE1 transport protein, or a non-naturally occurring MAE1
transport protein prepared in accordance with one of the reference
sequences provided herein, or is a naturally occurring MAE1
transport protein with MAE1 transport protein activity that is
either overexpressed in the host cell in which it naturally occurs
or is heteroloccously expressed in a host cell in which it does not
naturally occur.
Nucleic acid constructs of the present invention include expression
vectors that comprise nucleic acids encoding one or more MAE1
transport proteins. The nucleic acids encoding the proteins are
operably linked to promoters and optionally other control sequences
such that the subject proteins are expressed in a host cell
containing the expression vector when cultured under suitable
conditions. The promoters and control sequences employed depend on
the host cell selected for the production of malonate. Thus, the
invention provides not only expression vectors but also nucleic
acid constructs useful in the construction of expression vectors.
Methods for designing and making nucleic acid constructs and
expression vectors generally are well known to those skilled in the
art and so are only briefly reviewed herein.
Nucleic acids encoding the MAE1 transport protein can be prepared
by any suitable method known to those of ordinary skill in the art,
including, for example, direct chemical synthesis and cloning.
Further, nucleic acid sequences for use in the invention can be
obtained from commercial vendors that provide de novo synthesis of
the nucleic acids.
A nucleic acid encoding the desired protein can be incorporated
into an expression vector by known methods that include, for
example, the use of restriction enzymes to cleave specific sites in
an expression vector, e.g., plasmid, thereby producing an
expression vector of the invention. Some restriction enzymes
produce single stranded ends that may be annealed to a nucleic acid
sequence having, or synthesized to have, a terminus with a sequence
complementary to the ends of the cleaved expression vector. The
ends are then covalently linked using an appropriate enzyme, e.g.,
DNA ligase. DNA linkers may be used to facilitate linking of
nucleic acids sequences into an expression vector.
A set of individual nucleic acid sequences can also be combined by
utilizing polymerase chain reaction (PCR)-based methods known to
those of skill in the art. For example, each of the desired nucleic
acid sequences can be initially generated in a separate PCR.
Thereafter, specific primers are designed such that the ends of the
PCR products contain complementary sequences. When the PCR products
are mixed, denatured, and reannealed, the strands having the
matching sequences at their 3' ends overlap and can act as primers
for each other. Extension of this overlap by DNA polymerase
produces a molecule in which the original sequences are "spliced"
together. In this way, a series of individual nucleic acid
sequences may be joined and subsequently transduced into a host
cell simultaneously. Thus, expression of each of the plurality of
nucleic acid sequences is effected.
A typical expression vector contains the desired nucleic acid
sequence preceded and optionally followed by one or more control
sequences or regulatory regions, including a promoter and, when the
gene product is a protein, ribosome binding site, e.g., a
nucleotide sequence that is generally 3-9 nucleotides in length and
generally located 3-11 nucleotides upstream of the initiation codon
that precede the coding sequence, which is followed by a
transcription terminator in the case of E. coli or other
prokaryotic hosts. See Shine et al., Nature 254:34 (1975) and
Steitz, in Biological Regulation and Development: Gene Expression
(ed. R. F. Goldberger), vol. 1, p. 349 (1979) Plenum Publishing,
N.Y. In the case of eukaryotic hosts like yeast a typical
expression vector contains the desired nucleic acid coding sequence
preceded by one or more regulatory regions, along with a Kozak
sequence to initiate translation and followed by a terminator. See
Kozak, Nature 308:241-246 (1984).
Regulatory regions or control sequences include, for example, those
regions that contain a promoter and an operator. A promoter is
operably linked to the desired nucleic acid coding sequence,
thereby initiating transcription of the nucleic acid sequence via
an RNA polymerase. An operator is a sequence of nucleic acids
adjacent to the promoter, which contains a protein-binding domain
where a transcription factor can bind. Transcription factors
activate or repress transcription initiation from a promoter. In
this way, control of transcription is accomplished, based upon the
particular regulatory regions used and the presence or absence of
the corresponding transcription factor. Non-limiting examples for
prokaryotic expression include lactose promoters (LacI repressor
protein changes conformation when contacted with lactose, thereby
preventing the Lad repressor protein from binding to the operator)
and tryptophan promoters (when complexed with tryptophan, TrpR
repressor protein has a conformation that binds the operator; in
the absence of tryptophan, the TrpR repressor protein has a
conformation that does not bind to the operator). Non-limiting
examples of promoters to use for eukaryotic expression include
pACH11, pACO11, pADH1, pADH2, pALD4, pCIT1, pCUP1, pENO2, pFBA1,
pGAL1, pGAPD, pHSP15, pHXK21, pHXT7, pJEN11, pMDH21, pMET3, pPDC1,
pPGI1, pPGK1, pPHO5, pPOX11, pPRB1, pPYK1, pREV1, pRNR2, pRPL1,
pSCT1, pSDH1, pTDH2, pTDH3, pTEF1, pTEF2, pTPI1, and pTPI11. As
will be appreciated by those of ordinary skill in the art, a
variety of expression vectors and components thereof may be used in
the present invention.
Although any suitable expression vector may be used to incorporate
the desired sequences, readily available expression vectors
include, without limitation: plasmids, such as pESC, pTEF,
p414CYC1, p414GALS, pSC101, pBR322, pBBR1MCS-3, pUR, pEX, pMR100,
pCR4, pBAD24, pUC19, pRS series; and bacteriophages, such as M13
phage and .lamda. phage. Of course, such expression vectors may
only be suitable for particular host cells or for expression of
particular MAE1 transport proteins. One of ordinary skill in the
art, however, can readily determine through routine experimentation
whether any particular expression vector is suited for any given
host cell or protein. For example, the expression vector can be
introduced into the host cell, which is then monitored for
viability and expression of the sequences contained in the vector.
In addition, reference may be made to the relevant texts and
literature, which describe expression vectors and their suitability
to any particular host cell. In addition to the use of expression
vectors, strains are built where expression cassettes are directly
integrated into the host genome.
The expression vectors are introduced or transferred, e.g. by
transduction, transfection, or transformation, into the host cell.
Such methods for introducing expression vectors into host cells are
well known to those of ordinary skill in the art. For example, one
method for transforming S. cerevisiae with an expression vector
involves a lithium acetate/polyethylene glycol treatment wherein
the expression vector is introduced into the host cell following
treatment with a solution comprising lithium acetate and
polyethylene glycol.
For identifying whether a nucleic acid has been successfully
introduced or into a host cell, a variety of methods are available.
For example, a culture of potentially transformed host cells may be
separated, using a suitable dilution, into individual cells and
thereafter individually grown and tested for expression of a
desired gene product of a gene contained in the introduced nucleic
acid. For example, an often-used practice involves the selection of
cells based upon antibiotic resistance that has been conferred by
antibiotic resistance-conferring genes in the expression vector,
such as the beta lactamase (amp), aminoglycoside phosphotransferase
(neo), and hygromycin phosphotransferase (hyg, hph, hpt) genes.
Typically, a host cell of the invention will have been transformed
with at least one expression vector. Once the host cell has been
transformed with the expression vector, the host cell is cultured
in a suitable medium containing a carbon source, such as a sugar
(e.g., glucose). As the host cell is cultured, expression of the
enzyme for producing malonate and secretion of malonate into the
fermentation broth occurs.
If a host cell of the invention is to include more than one
heterologous gene, then multiple genes can be expressed from one or
more vectors. For example, a single expression vector can comprise
one, two, or more genes encoding one, two, or more MAE1 transport
protein(s) and/or other proteins providing some useful function,
e.g. producing malonate. The heterologous genes can be contained in
a vector replicated episomally or in a vector integrated into the
host cell genome, and where more than one vector is employed, then
all vectors may replicate episomally (extrachromasomally), or all
vectors may integrate, or some may integrate and some may replicate
episomally. Chromosomal integration is typically used for cells
that will undergo sustained propagation, e.g., cells used for
production of malonate for industrial applications. While a "gene"
is generally composed of a single promoter and a single coding
sequence, in certain host cells, two or more coding sequences may
be controlled by one promoter in an operon. In some embodiments, a
two or three operon system is used.
In some embodiments, the coding sequences employed have been
modified, relative to some reference sequence, to reflect the codon
preference of a selected host cell. Codon usage tables for numerous
organisms are readily available and are used to guide sequence
design. The use of prevalent codons of a given host organism
generally improves translation of the target sequence in the host
cell. As one non-limiting example, in some embodiments the subject
nucleic acid sequences will be modified for yeast codon preference
(see, for example, Bennetzen et al., J. Biol. Chem. 257: 3026-3031
(1982)). In some embodiments, the nucleotide sequences are modified
to include codons optimized for S. cerevisiae codon preference.
Nucleic acids can be prepared by a variety of routine recombinant
techniques. Briefly, the subject nucleic acids can be prepared from
genomic DNA fragments, cDNAs, and RNAs, all of which can be
extracted directly from a cell or recombinantly produced by various
amplification processes including but not limited to PCR and
rt-PCR. Subject nucleic acids can also be prepared by a direct
chemical synthesis.
The nucleic acid transcription levels in a host microorganism can
be increased (or decreased) using numerous techniques. For example,
the copy number of the nucleic acid can be increased through use of
higher copy number expression vectors comprising the nucleic acid
sequence, or through integration of multiple copies of the desired
nucleic acid into the host microorganism's genome. Non-limiting
examples of integrating a desired nucleic acid sequence onto the
host chromosome include recA-mediated recombination, lambda phage
recombinase-mediated recombination and transposon insertion.
Nucleic acid transcript levels can be increased by changing the
order of the coding regions on a polycistronic mRNA or breaking up
a polycistronic operon into multiple poly- or mono-cistronic
operons each with its own promoter. RNA levels can be increased (or
decreased) by increasing (or decreasing) the strength of the
promoter to which the protein-coding region is operably linked.
The translation level of a desired polypeptide sequence in a host
microorganism can also be increased in a number of ways.
Non-limiting examples include increasing the mRNA stability,
modifying the ribosome binding site (or Kozak) sequence, modifying
the distance or sequence between the ribosome binding site (or
Kozak sequence) and the start codon of the nucleic acid sequence
coding for the desired polypeptide, modifying the intercistronic
region located 5' to the start codon of the nucleic acid sequence
coding for the desired polypeptide, stabilizing the 3'-end of the
mRNA transcript, modifying the codon usage of the polypeptide,
altering expression of low-use/rare codon tRNAs used in the
biosynthesis of the polypeptide. Determination of preferred codons
and low-use/rare codon tRNAs can be based on a sequence analysis of
genes obtained from the host microorganism.
The polypeptide half-life, or stability, can be increased through
mutation of the nucleic acid sequence coding for the desired
polypeptide, resulting in modification of the desired polypeptide
sequence relative to the control polypeptide sequence. When the
modified polypeptide is an enzyme, the activity of the enzyme in a
host may be altered due to increased solubility in the host cell,
improved function at the desired pH, removal of a domain inhibiting
enzyme activity, improved kinetic parameters (lower Km or higher
Kcat values) for the desired substrate, removal of allosteric
regulation by an intracellular metabolite, and the like.
Altered/modified enzymes can also be isolated through random
mutagenesis of an enzyme, such that the altered/modified enzyme can
be expressed from an episomal vector or from a recombinant gene
integrated into the genome of a host microorganism.
Methods for Producing Malonate
In a third aspect, the invention provides a method for the
production of malonate, the method comprising the steps of: (a)
culturing a population of any of the recombinant host cells
described herein in a fermentation broth with a carbon source under
conditions suitable for making malonate; and (b) recovering said
malonate from the fermentation broth.
In various embodiments, a recombinant host cell capable of
producing malonate and comprising a heterologous nucleic acid
encoding a MAE1 transport protein provides an increased yield,
titer, and/or productivity of malonate compared to a parent cell
not comprising the heterologous nucleic acid encoding the MAE1
transport protein, but is otherwise genetically identical. In some
embodiments, the increased amount is at least 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100% or greater than 100%, as measured, for example, in
yield, titer, productivity, on a per unit volume of cell culture
basis, on a per unit dry cell weight basis, on a per unit volume of
cell culture per unit time basis, or on a per unit dry cell weight
per unit time basis.
In some embodiments, the host cell produces an elevated level of
malonate that is at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 1.25-fold, at least about 1-fold, at
least about 2-fold, at least about 4-fold, or more, higher than the
level of malonate produced by a parent cell, on a per unit volume
of cell culture basis. For example, as described in Example 1,
strain LPK15010 comprising Aspergillus niger A2R8T9 MAE1 transport
protein produced greater than 2-fold more malonate than the
parental control strain, LPK15003 (i.e., 36.+-.5.6 mM in LPK15003
versus 75.+-.10.3 mM in LPK15010).
In some embodiments, the host cell produces an elevated level of
malonate that is at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 1.25-fold, at least about 2-fold, at
least about 4-fold, at least about 10-fold, or more, higher than
the level of malonate produced by the parent cell, on a per unit
volume of cell culture per unit time basis.
Materials and methods for the maintenance and growth of microbial
cultures are well known to those skilled in the art of microbiology
or fermentation science (see, for example, Bailey et al.,
Biochemical Engineering Fundamentals, second edition, McGraw Hill,
New York, 1986). Consideration must be given to appropriate culture
medium, pH, temperature, and requirements for aerobic,
microaerobic, or anaerobic conditions, depending on the specific
requirements of the host cell, the fermentation, and the
process.
The methods of producing malonate provided herein may be performed
in a suitable culture medium, in a suitable container, including
but not limited to a cell culture plate, a flask, or a fermenter.
Further, the methods can be performed at any scale of fermentation
known in the art to support industrial production of microbial
products. Any suitable fermenter may be used including a stirred
tank fermenter, an airlift fermenter, a bubble fermenter, or any
combination thereof.
In some embodiments, the culture medium is any culture medium in
which a genetically modified microorganism capable of producing
malonate can subsist. In some embodiments, the culture medium is an
aqueous medium comprising assimilable carbon, nitrogen and
phosphate sources. Such a medium can also include appropriate
salts, minerals, metals and other nutrients. In some embodiments,
the carbon source and each of the essential cell nutrients are
added incrementally or continuously to the fermentation media, and
each required nutrient is maintained at essentially the minimum
level needed for efficient assimilation by growing cells.
The invention, having been described in detail, is illustrated by
the following examples, which should not be construed as limiting
the invention, given its diverse aspects, embodiments, and
applications.
EXAMPLES
Example 1: Increasing Malonate Titer in Recombinant Pichia
kudriavzevii Through Heterologous Expression of MAE1 Transport
Proteins
In this example, recombinant P. kudriavzevii host cells capable of
producing malonate were transformed with nucleic acids encoding
MAE1 transport proteins and were shown to increase malonate
production.
Recombinant P. kudriavzevii LPK15001 was used as the base strain,
and harbors a single copy of a malonyl-CoA hydrolase at the GPD1
locus. The following strains were constructed by chromosomal
integration at the FAS1 locus with nucleic acids encoding the
indicated protein, LPK15003: F6A-4; LPK15004: F6A-4 and A1C406;
LPK15005: F6A-4 and G7XR17; LPK15006: F6A-4 and O59815; LPK15009:
F6A-4 and P50537; LPK15010: F6A-4 and A2R8T9; and LPK15011: F6A-4
and Q0DIU9. Protein F6A-4 is a malonyl-CoA hydrolase (UniProt ID
F6AA82 containing point mutations E95N/F304R/Q348A); thus, strain
LPK15003 is the control strain establishing the baseline level of
malonate production in the absence of heterologous expression of a
MAE1 transport protein. In all strains, the pTDH1 promoter was used
to control transcription of the gene encoding the F6A-4 malonyl-CoA
hydrolase. The pPGK1 promoter was used to control transcription of
the genes encoding the G7XR17, O59815, P50537, A2R8T9, and Q0DIU9
proteins. TEF1 terminators were inserted behind all heterologous
genes described above to stop transcription. A kanamycin resistance
marker was included in the assembled nucleic acid to enable
selection for positive integrants. The 5' and 3' ends of the
nucleic acid contained between 962 and 976 basepairs of DNA
sequence homologous to the P. kudriavzevii FAS1 gene were included
to target nucleic acid insertion into the FAS1 locus of the host
genome.
Nucleic acids were transformed into P. kudriavzevii LPK15001 using
a lithium acetate/PEG protocol. In brief, a colony LPK15001 was
inoculated into 50 mL of YNB yeast medium in a culture flask, and
incubated at 30.degree. C. and 85% relative humidity with shaking
(200 rpm) for approximately 4 hours. The culture was then placed on
ice for approximately 15 minutes, centrifuged (.times.6000 g, 1
min), the supernatant removed, and the pellet resuspended in 50 ml
of ice-cold, sterile water. The cells were then resuspended in
approximately 3 ml of ice cold, sterile water and centrifuged
(.times.6000 g, 1 min). The resulting pellet was resuspended in 500
.mu.l of 30% glycerol, 0.1M lithium acetate at 4.degree. C. The
resuspended cells were then aliquoted into 50 .mu.l volumes to
which 5 .mu.l ssDNA (salmon sperm ssDNA, 10 mg/ml), 145 .mu.l 50%
PEG (MW-6,500), and approximately 20 .mu.l of the heterologous
nucleic acid(s) encoding the expression cassettes were added. The
mixture was incubated for 30 minutes at 30.degree. C. and then for
45 minutes at 42.degree. C. The transformations were then plated on
YNB plates containing G418 antibiotic (500 .mu.g/ml) to select for
the presence of the kanamycin resistance cassette.
For production assays, individual colonies were next inoculated
into 500 YNB growth medium supplemented with 8% w/v glucose. All
cultures were inoculated into 2.2-ml volume 96-well plates. The
culture plates were then incubated at 30.degree. C. with shaking
(250 rpm) for 5 days, at which point the fermentation broth was
sampled.
Samples were centrifuged (x6000 g, 1 min) and the supernatant
analyzed for malonate concentration. The separation of malonate was
conducted on a Shimadzu Prominence XR HPLC connected to a
refractive index detector and UV detector monitoring 210 nm.
Product separation was performed on a Bio-Rad Aminex HPX-87h
Fermentation Monitoring column. The UPLC was programmed to run
isocratically using 5 mM H.sub.2SO.sub.4 as the eluent with a flow
rate of 800 .mu.L per minute. 10 .mu.l were injected per sample,
and the sample plate temperature was held at 4.degree. C. Malonate
standards began eluting at .about.8.0 minutes. Malonate
concentrations (mM) in the fermentation broth were calculated by
comparison to a standard curve prepared from authentic malonate
prepared in water.
The results of this production assay were as follows: LPK15003:
36.+-.5.6 mM, LPK15004: 36.+-.5.4 mM, LPK15005: 76.+-.10.6 mM,
LPK15006: 50.+-.7.6 mM, LPK15009: 73.+-.7.0 mM, LPK15010:
75.+-.10.3 mM, and LPK15011: 68.+-.14.4 mM. Thus, strains LPK15006
and LPK15009, which expressed the Schizosaccharomyces pombe MAE1
transport proteins, provided approximately 1.4-fold and 2.0-fold
increases in malonate production, respectively, relative to the
LPK15003 control strain. Strains LPK15005, LPK15010, and LPK15011,
which expressed the MAE1 transport proteins obtained from
Aspergillus species, provided approximately 2.1-fold, 2.1-fold, and
1.9-fold increases in malonate production, respectively, relative
to the LPK15003 control strain.
Upon sequencing of the transformed heterologous nucleic acid in
strain LPK15004 a nucleotide deletion resulting in a frameshift
mutation during translation was identified. Thus, strain LPK15004
in this example did not express the A1C406 MAE1 transport
protein.
This example demonstrates, in accordance with the invention, that
heterologous expression of nucleic acids encoding a wide variety of
MAE1 transport proteins (i.e. A2R8T9, G7XR17, QOD1U9, P50537, and
O59815) increased malonate production in recombinant yeast cells
capable of producing malonate. Moreover, this example provides a
readily conducted, efficient method to determine if a putative MAE1
transport protein is an MAE1 transport protein and efficiently
secretes malonate into the fermentation broth.
Example 2: Increasing Malonate Productivity in Recombinant Pichia
kudriavzevii Through Heterologous Expression of MAE1 Transport
Proteins
In this example, yeast strains LPK15004 and LPK15010 (see Example 1
for strain construction details) is grown in fed-batch control in a
0.5 L bioreactor (Infors, Sixfors system). A single colony of
LPK15004 is isolated from a YPD plate and cultured in 6 mL of YNB4
2% media (20 g/L glucose, 6.7 g/L YNB without amino acids (Sigma),
and 150 mM succinic acid buffer pH 4.0). The culture is maintained
at 30.degree. C. for 24 hours, shaking at 200 rpm. The 6 mL of
culture is combined with 4 mL of 50% (v/v) glycerol and aliquoted
in 1 mL volumes into cryo-vials. Cyro-vials are frozen and
maintained at -80c. One vial is used to inoculate 50 mL of fresh
YNB4 2% media in a 250 mL baffled flask and grown for 24 hrs at
30.degree. C., 200 rpm. This culture is used to inoculate 200 mL of
YNB4 2% media with 0.1% antifoam. The fermentation is maintained at
30.degree. C., at a pH of 5.0 maintained by the addition of
ammonium hydroxide, potassium hydroxide, or sodium hydroxide.
Oxygen transfer is controlled through two rushton impellers run at
1000 rpm, and using a sparger an air flow rate of 30 NL/hr using
compressed air is maintained. The culture is grown overnight
(.about.20 h) to allow for glucose consumption prior to starting
the fed-batch phase. The feed (150 g/L glucose, 13.4 g/L YNB
without amino acids (Sigma)) is initiated automatically when the
dissolved oxygen spikes sharply indicating depletion of glucose.
Feed is delivered for 2 s, every 200 s. Samples are taken daily.
Growth is monitored by measuring optical density at 600 nm (OD600).
Concentration of glucose is measured using a glucose monitor (YSI
Life Sciences). Production of malonic acid, acetic acid, succinic
acid, and pyruvic acid is measured via HPLC as described in Example
1. Productivity is calculated as the malonate formation rate per
unit volume over time, and is expressed as g/l/hr.
Strain LPK15010 provides a higher productivity relative to
LPK15004, demonstrating that in accordance with the invention, that
heterologous expression of the Aspergillus niger A2R8T9 MAE1
transport protein increases malonate productivity in recombinant
yeast cells capable of producing malonate. Moreover, this example
provides another readily conducted, efficient method to determine
if a putative MAE1 transport protein is an MAE1 transport protein
and efficiently secretes malonate into the fermentation broth.
Example 3: Increasing Malonate Productivity in Recombinant Pichia
kudriavzevii Through Heterologous Expression of MAE1 Transport
Proteins
In this example, recombinant P. kudriavzevii host cells capable of
producing malonate were engineered to express a MAE1 transport
protein resulting in increased malonate productivity relative to
control cells that did not express an MAE1 transport protein.
The yeast used in this example was Pichia Kudriavzevii (ARS Culture
Collection strain Y-134). Engineered yeast LPK3013 served as a
control strain, and comprised two malonyl-CoA hydrolase expression
cassettes: (i) one malonyl-CoA hydrolase expression cassette
comprising a pTDH1 promoter, F6A-4 malonyl-CoA hydrolase (see
Example 1 for a description of F6A-4), CYC1 terminator, and a
hygromycin selection marker; this expression cassette was
integrated at the GPD1 locus; and (ii) a second malonyl-CoA
hydrolase expression cassette identical to the first with the
exception that this cassette was integrated at the FAS1 locus and
included a SUC2 selection marker in place of the hygromycin
selection marker. Engineered strain LPK3020 was genetically
identical to LPK3013 with the exception that LPK3020 was
additionally engineered to express the Aspergillus kawachi G7XR17
MAE1 transport protein (SEQ ID NO: 2). The MAE1 transporter
expression cassette comprised a pPGK1 promoter controlling
expression of the Aspergillus kawachi G7XR17 MAE1 transport protein
(SEQ ID NO: 2), and also included a TEF1 terminator cloned
downstream of the gene; this expression cassette was integrated,
along with the above-described malonyl-CoA hydrolase expression
cassette, at the FAS1 locus using a kanamycin resistance marker.
Thus, in this example, strain LPK3013 was the parental, control
strain used to establish the baseline level of malonate
productivity in the absence of expression of a MAE1 transport
protein.
Nucleic acids were transformed into P. kudriavzevii strains using a
lithium acetate/PEG protocol as described in Example 1. Single
colonies of LPK30013 and LPK3020 were isolated from YPD solid media
plates and cultured in 6 mL of YNB4 2% media (20 g/L glucose, 6.7
g/L YNB without amino acids (Sigma), and 150 mM succinic acid
buffer pH 4.0). The culture was maintained at 30.degree. C. for 24
hours, shaking at 200 rpm. The 6 mL of culture was combined with 4
mL of 50% (v/v) glycerol and aliquoted in 1 mL volumes into
cryo-vials. Cyro-vials were frozen and maintained at -80.degree. C.
One vial of each strain was used to inoculate 50 mL of fresh YNB4
2% media in a 250 mL baffled flask and grown for 24 hrs at
30.degree. C., 200 rpm. These cultures were used to inoculate 200
mL of YNB4 2% media with 0.1% antifoam. The fermentation was
maintained at 30.degree. C., and the pH was maintained at 5.0 by
addition of ammonium hydroxide. Oxygen transfer was controlled
through two rushton impellers run at 1000 rpm, and using a sparger
an air flow rate of 30 NL/hr using compressed air was maintained.
The culture was grown overnight (.about.20 h) to allow for glucose
consumption prior to starting the fed-batch phase. The feed (300
g/L glucose, 13.4 g/L Yeast Nitrogen Base without amino acids
(Sigma)) was initiated automatically when the dissolved oxygen
spiked sharply, indicating depletion of glucose. Feed was delivered
for 2 s, every 200 s. Samples were taken daily. Growth was
monitored by measuring optical density at 600 nm (OD600). Samples
were centrifuged (.times.6000 g, 1 min) and the glucose and
malonate concentrations in the supernatant analyzed. Glucose
concentration was measured using a glucose monitor (YSI Life
Sciences). The separation of malonate was conducted on a Shimadzu
Prominence XR HPLC as described in Example 1.
Control strain LPK3013 provided a malonate productivity of 0.17
g/L/hr. Strain LPK3020, expressing the Aspergillus kawachi G7XR17
MAE1 transport protein, provided a malonate productivity of 0.424
g/L/hr. Thus, malonate productivity was increased nearly 2.5-fold
in yeast expressing a MAE1 transport protein as compared to a
parental, control strain that did not express said MAE1 transport
protein.
This example demonstrates that in accordance with the invention,
expression of an MAE1 transport protein in a host cell capable of
producing malonate increases malonate productivity as compared to a
host cell that do not express said MAE1 transport protein.
Moreover, this example provides another readily conducted,
efficient method to determine if a putative MAE1 transport protein
is an MAE1 transport protein and efficiently secretes malonate into
the fermentation broth.
Pursuant to 37 C.F.R. 1.821(c), a sequence listing was submitted
via EFS-Web as an ASCII compliant text file named
20181130_LYGOS_0006_02_US_Sequence_listing_ST25, that was created
on Nov. 30, 2018 and having a size of 46.8 kilobytes. The contents
of the aforementioned file are hereby incorporated by reference in
their entirety.
SEQUENCE LISTINGS
1
81416PRTAspergillus niger 1Met Asn Val Glu Thr Ser Leu Pro Gly Ser
Ser Gly Ser Asp Leu Glu1 5 10 15Thr Phe His His Glu Thr Lys Lys His
Ala Asn His Asp Ser Gly Ile 20 25 30Ser Val Asn His Glu Ala Glu Ile
Gly Val Asn His Thr Phe Glu Lys 35 40 45Pro Gly Pro Val Gly Ile Arg
Glu Arg Leu Arg His Phe Thr Trp Ala 50 55 60Trp Tyr Thr Leu Thr Met
Ser Cys Gly Gly Leu Ala Leu Leu Ile Val65 70 75 80Asn Gln Pro His
Asp Phe Lys Gly Leu Lys Asp Ile Ala Arg Val Val 85 90 95Tyr Cys Leu
Asn Leu Ala Phe Phe Val Ile Val Thr Ser Leu Met Ala 100 105 110Ile
Arg Phe Ile Leu His Lys Asn Met Trp Glu Ser Leu Gly His Asp 115 120
125Arg Glu Gly Leu Phe Phe Pro Thr Phe Trp Leu Ser Ile Ala Thr Met
130 135 140Ile Thr Gly Leu Tyr Lys Cys Phe Gly Asp Asp Ala Asn Glu
Lys Phe145 150 155 160Thr Lys Cys Leu Gln Val Leu Phe Trp Ile Tyr
Cys Gly Cys Thr Met 165 170 175Ile Thr Ala Val Gly Gln Tyr Ser Phe
Val Phe Ala Thr His Lys Tyr 180 185 190Glu Leu His Thr Met Met Pro
Ser Trp Ile Leu Pro Ala Phe Pro Val 195 200 205Met Leu Ser Gly Thr
Ile Ala Ser Val Ile Gly Ser Gly Gln Pro Ala 210 215 220Ser Asp Gly
Ile Pro Ile Ile Ile Ala Gly Ile Thr Phe Gln Gly Leu225 230 235
240Gly Phe Ser Ile Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu
245 250 255Met Glu Val Gly Leu Pro Ser Pro Glu His Arg Pro Gly Met
Phe Ile 260 265 270Cys Val Gly Pro Pro Ala Phe Thr Ala Leu Ala Leu
Val Gly Met Ala 275 280 285Lys Ala Leu Pro Asp Asp Phe Gln Ile Val
Gly Asp Pro His Ala Val 290 295 300Ile Asp Gly Arg Val Met Leu Phe
Leu Ala Val Ser Ala Ala Ile Phe305 310 315 320Leu Trp Ala Leu Ser
Phe Trp Phe Phe Cys Ile Ala Val Val Ala Val 325 330 335Val Arg Ser
Pro Pro Lys Gly Phe His Leu Asn Trp Phe Ala Met Val 340 345 350Phe
Pro Asn Thr Gly Phe Thr Leu Ala Thr Ile Thr Leu Ala Asn Met 355 360
365Phe Glu Ser Pro Gly Val Lys Gly Val Ala Thr Ala Met Ser Leu Cys
370 375 380Val Ile Ile Met Phe Ile Phe Val Leu Val Ser Ala Ile Arg
Ala Val385 390 395 400Ile Arg Lys Asp Ile Met Trp Pro Gly Gln Asp
Glu Asp Val Ser Glu 405 410 4152416PRTAspergillus kawachii 2Met Asn
Val Glu Thr Ser Leu Pro Gly Ser Ser Gly Ser Asp Leu Glu1 5 10 15Thr
Phe His His Glu Thr Lys Lys His Ala Asn His Asp Ser Glu Ile 20 25
30Thr Val Asn His Glu Ala Asp Leu Gly Val Asn His Thr Phe Glu Lys
35 40 45Pro Gly Arg Val Gly Leu Arg Glu Arg Leu Arg His Phe Thr Trp
Ala 50 55 60Trp Tyr Thr Leu Thr Met Ser Cys Gly Gly Leu Ala Leu Leu
Ile Val65 70 75 80Asn Gln Pro His Asp Phe Lys Gly Leu Lys Asp Ile
Ala Arg Val Val 85 90 95Tyr Cys Leu Asn Ile Ala Phe Phe Val Ile Val
Thr Thr Leu Met Ala 100 105 110Ile Arg Phe Ile Leu His Lys Asn Met
Leu Glu Ser Leu Gly His Asp 115 120 125Arg Glu Gly Leu Phe Phe Pro
Thr Phe Trp Leu Ser Ile Ala Thr Met 130 135 140Ile Thr Gly Leu Tyr
Lys Cys Phe Gly Asp Asp Ser Asn Glu Lys Phe145 150 155 160Thr Lys
Cys Leu Gln Val Leu Phe Trp Ile Tyr Cys Gly Phe Thr Met 165 170
175Ile Thr Ala Val Ala Gln Tyr Ser Phe Val Phe Ala Thr His Lys Tyr
180 185 190Glu Leu His Thr Met Met Pro Ser Trp Ile Leu Pro Ala Phe
Pro Val 195 200 205Met Leu Ser Gly Thr Ile Ala Ser Val Ile Ala Ser
Gly Gln Pro Ala 210 215 220Ser Asp Gly Ile Pro Met Val Ile Ala Gly
Ile Thr Phe Gln Gly Leu225 230 235 240Gly Phe Ser Ile Ser Phe Met
Met Tyr Ala His Tyr Ile Gly Arg Leu 245 250 255Met Glu Val Gly Leu
Pro Ser Ser Glu His Arg Pro Gly Met Phe Ile 260 265 270Cys Val Gly
Pro Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ala 275 280 285Lys
Ala Leu Pro Asp Asp Phe Gln Ile Val Gly Asp Pro His Ala Val 290 295
300Ile Asp Gly Arg Val Met Leu Phe Leu Ala Val Ser Ala Ala Ile
Phe305 310 315 320Leu Trp Ala Leu Ser Phe Trp Phe Phe Cys Ile Ala
Val Val Ala Val 325 330 335Val Arg Ser Pro Pro Lys Gly Phe His Leu
Asn Trp Phe Ala Met Val 340 345 350Phe Pro Asn Thr Gly Phe Thr Leu
Ala Thr Ile Thr Leu Ala Asn Met 355 360 365Phe Glu Ser Pro Gly Val
Lys Gly Val Ala Thr Ala Met Ser Leu Cys 370 375 380Val Ile Ile Met
Phe Ile Phe Val Leu Val Ser Ala Ile Arg Ala Val385 390 395 400Ile
Arg Lys Asp Ile Met Trp Pro Gly Gln Asp Glu Asp Val Ser Glu 405 410
4153393PRTAspergillus terreus 3Met Phe Glu Asn Thr Ala Pro Pro Gly
Ser Ser Arg Ser Asp Ser Gly1 5 10 15Ile Leu Asp His Glu Phe Glu Lys
Gln Pro Gly Ser Val Gly Met Arg 20 25 30Glu Arg Ile Arg His Phe Thr
Trp Ala Trp Tyr Thr Leu Thr Met Ser 35 40 45Ala Gly Gly Leu Ala Leu
Leu Leu Gly Ser Gln Pro Asn Thr Phe Thr 50 55 60Gly Leu Arg Glu Ile
Gly Leu Ala Val Tyr Leu Leu Asn Leu Leu Phe65 70 75 80Phe Ala Leu
Val Cys Ser Thr Met Ala Gly Arg Phe Ile Leu His Gly 85 90 95Gly Leu
Val Asp Ser Leu Arg His Glu Arg Glu Gly Ile Phe Phe Pro 100 105
110Thr Phe Trp Leu Ser Ile Ala Thr Ile Ile Thr Gly Leu Tyr Arg Tyr
115 120 125Phe Gly Glu Asp Ala Gly Arg Pro Phe Val Leu Ala Leu Glu
Ala Leu 130 135 140Phe Trp Ile Tyr Cys Ala Cys Thr Leu Leu Val Ala
Val Ile Gln Tyr145 150 155 160Ser Trp Leu Phe Ser Gly Pro Lys Tyr
Arg Leu Gln Thr Ala Met Pro 165 170 175Gly Trp Ile Leu Pro Ala Phe
Pro Val Met Leu Ser Gly Thr Ile Ala 180 185 190Ser Val Ile Ala Glu
Gln Gln Pro Ala Arg Ala Ala Ile Pro Ile Ile 195 200 205Val Ala Gly
Thr Thr Phe Gln Gly Leu Gly Phe Ser Ile Ser Met Ile 210 215 220Met
Tyr Ala His Tyr Val Gly Arg Leu Met Glu Ser Gly Leu Pro Cys225 230
235 240Arg Glu His Arg Pro Gly Met Phe Ile Ala Val Gly Pro Pro Ala
Phe 245 250 255Thr Ala Leu Ala Leu Val Gly Met Thr Lys Gly Leu Pro
His Asp Phe 260 265 270Gln Leu Ile Gly Asp Asp Phe Ala Phe Glu Asp
Ala Arg Ile Leu Gln 275 280 285Leu Leu Ala Ile Ala Val Gly Val Phe
Leu Trp Ala Leu Ser Leu Trp 290 295 300Phe Phe Cys Ile Ala Ala Ile
Ala Val Val Arg Ser Pro Pro Thr Ala305 310 315 320Phe His Leu Ser
Trp Trp Ala Met Val Phe Pro Asn Thr Gly Phe Thr 325 330 335Leu Ala
Thr Ile Asn Leu Gly Thr Ala Leu Lys Ser Glu Gly Ile Gln 340 345
350Gly Val Gly Thr Ala Met Ser Ile Gly Ile Val Ser Ile Phe Leu Phe
355 360 365Val Phe Ile Ser His Val Arg Ala Val Ile Arg Lys Asp Ile
Met Tyr 370 375 380Pro Gly Lys Asp Glu Asp Val Val Glu385
3904438PRTSchizosaccharomyces pombe 4Met Gly Glu Leu Lys Glu Ile
Leu Lys Gln Arg Tyr His Glu Leu Leu1 5 10 15Asp Trp Asn Val Lys Ala
Pro His Val Pro Leu Ser Gln Arg Leu Lys 20 25 30His Phe Thr Trp Ser
Trp Phe Ala Cys Thr Met Ala Thr Gly Gly Val 35 40 45Gly Leu Ile Ile
Gly Ser Phe Pro Phe Arg Phe Tyr Gly Leu Asn Thr 50 55 60Ile Gly Lys
Ile Val Tyr Ile Leu Gln Ile Phe Leu Phe Ser Leu Phe65 70 75 80Gly
Ser Cys Met Leu Phe Arg Phe Ile Lys Tyr Pro Ser Thr Ile Lys 85 90
95Asp Ser Trp Asn His His Leu Glu Lys Leu Phe Ile Ala Thr Cys Leu
100 105 110Leu Ser Ile Ser Thr Phe Ile Asp Met Leu Ala Ile Tyr Ala
Tyr Pro 115 120 125Asp Thr Gly Glu Trp Met Val Trp Val Ile Arg Ile
Leu Tyr Tyr Ile 130 135 140Tyr Val Ala Val Ser Phe Ile Tyr Cys Val
Met Ala Phe Phe Thr Ile145 150 155 160Phe Asn Asn His Val Tyr Thr
Ile Glu Thr Ala Ser Pro Ala Trp Ile 165 170 175Leu Pro Ile Phe Pro
Pro Met Ile Cys Gly Val Ile Ala Gly Ala Val 180 185 190Asn Ser Thr
Gln Pro Ala His Gln Leu Lys Asn Met Val Ile Phe Gly 195 200 205Ile
Leu Phe Gln Gly Leu Gly Phe Trp Val Tyr Leu Leu Leu Phe Ala 210 215
220Val Asn Val Leu Arg Phe Phe Thr Val Gly Leu Ala Lys Pro Gln
Asp225 230 235 240Arg Pro Gly Met Phe Met Phe Val Gly Pro Pro Ala
Phe Ser Gly Leu 245 250 255Ala Leu Ile Asn Ile Ala Arg Gly Ala Met
Gly Ser Arg Pro Tyr Ile 260 265 270Phe Val Gly Ala Asn Ser Ser Glu
Tyr Leu Gly Phe Val Ser Thr Phe 275 280 285Met Ala Ile Phe Ile Trp
Gly Leu Ala Ala Trp Cys Tyr Cys Leu Ala 290 295 300Met Val Ser Phe
Leu Ala Gly Phe Phe Thr Arg Ala Pro Leu Lys Phe305 310 315 320Ala
Cys Gly Trp Phe Ala Phe Ile Phe Pro Asn Val Gly Phe Val Asn 325 330
335Cys Thr Ile Glu Ile Gly Lys Met Ile Asp Ser Lys Ala Phe Gln Met
340 345 350Phe Gly His Ile Ile Gly Val Ile Leu Cys Ile Gln Trp Ile
Leu Leu 355 360 365Met Tyr Leu Met Val Arg Ala Phe Leu Val Asn Asp
Leu Cys Tyr Pro 370 375 380Gly Lys Asp Glu Asp Ala His Pro Pro Pro
Lys Pro Asn Thr Gly Val385 390 395 400Leu Asn Pro Thr Phe Pro Pro
Glu Lys Ala Pro Ala Ser Leu Glu Lys 405 410 415Val Asp Thr His Val
Thr Ser Thr Gly Gly Glu Ser Asp Pro Pro Ser 420 425 430Ser Glu His
Glu Ser Val 4355431PRTSchizosaccharomyces pombe 5Met Asp Ile Val
Lys Gln Arg Tyr His Glu Leu Phe Asp Leu Arg Val1 5 10 15Arg Gly Pro
Arg Met Lys Met Ala Asp Arg Leu Glu His Phe Thr Trp 20 25 30Ala Trp
Phe Ala Ser Ala Met Gly Thr Gly Gly Ile Gly Met Val Thr 35 40 45Ser
Leu Tyr His Phe Arg Phe Tyr Gly Leu Asn Thr Leu Gly Lys Ile 50 55
60Ile Phe Ile Phe Gln Leu Ser Ile Leu Thr Leu Tyr Ile Cys Cys Ile65
70 75 80Thr Phe Arg Phe Ile Arg Tyr Pro Gly Thr Leu Ser Lys Thr Trp
Lys 85 90 95Asn Pro Ser Glu Val Leu Phe Met Pro Thr Ala Leu Leu Ala
Ile Ala 100 105 110Thr Ser Ile Ser Asn Leu Tyr Pro Tyr Ala Phe Pro
Tyr Thr Gly Glu 115 120 125Trp Met Val Trp Leu Ile Arg Ile Leu Tyr
Trp Ile Phe Val Ala Val 130 135 140Ala Cys Ile Phe Val Ile Ser Leu
Phe Tyr Ser Leu Phe His Ala His145 150 155 160Pro Phe Arg Ile Asn
Thr Ile Ile Pro Ala Leu Val Leu Pro Ile Phe 165 170 175Pro Cys Met
Ile Cys Gly Val Ile Ala Ser Ala Ile Val Glu Ser Gln 180 185 190Pro
Ala Arg Gln Ala Lys Asn Met Val Val Ala Gly Ile Ala Phe Gln 195 200
205Gly Leu Gly Phe Trp Ile Tyr Ile Ile Val Tyr Ala Val Asn Met Cys
210 215 220Arg Phe Phe Thr Val Gly Leu Gln Pro Ala Ala Asp Arg Pro
Gly Met225 230 235 240Phe Ile Leu Val Ser Pro Pro Ser Phe Thr Gly
Leu Thr Leu Leu Asp 245 250 255Leu Ala Phe Gly Ala Lys Ala Lys Arg
Pro Tyr Ile Phe Val Gly Asp 260 265 270Asn Ser Ser Glu Tyr Leu Glu
Phe Val Ala Thr Phe Met Ala Leu Phe 275 280 285Met Ile Gly Leu Gly
Ile Phe Asn Phe Cys Leu Ala Phe Val Ser Val 290 295 300Val Ala Gly
Phe Cys Thr Arg Gln Arg Ile Lys Phe Lys Val Ser Trp305 310 315
320Phe Ala Met Ile Phe Ala Asn Val Gly Leu Val Met Asp Val Gln Glu
325 330 335Leu Gly Arg Ala Ile Asp Ser Lys Ala Val Cys Ile Val Gly
Gln Val 340 345 350Cys Gly Val Thr Ile Thr Ile Val Trp Ile Ile Leu
Ile Leu Leu Thr 355 360 365Leu Arg Ala Val Tyr Val Gln Glu Leu Leu
Tyr Pro Gly Lys Asp Glu 370 375 380Asp Ile Asp Thr Ile Leu Pro Asn
Val Leu Glu Tyr Tyr Arg His Leu385 390 395 400Glu Glu Glu Glu Lys
Asp Glu Ala Glu Arg Ser Lys Arg Lys Ala Glu 405 410 415Glu Ser Asp
Gly Lys Thr Thr Arg Glu Leu Thr Ser Gly Gly Leu 420 425
4306392PRTAspergillus clavatus 6Met Phe Glu Asn Arg Ile Pro Pro Thr
Ser Ser Gln Ser Asp Ser Gly1 5 10 15Phe Leu Glu Asn Gln Leu Glu Lys
Gln His Arg Leu Ser Leu Arg Glu 20 25 30Arg Leu Arg His Phe Thr Trp
Ala Trp Tyr Thr Leu Thr Met Ser Thr 35 40 45Gly Gly Leu Ala Leu Leu
Ile Ala Ser Gln Pro Tyr Thr Phe Lys Gly 50 55 60Leu Lys Thr Ile Gly
Leu Val Val Tyr Ile Val Asn Leu Ile Leu Phe65 70 75 80Gly Leu Val
Cys Ser Leu Met Ala Thr Arg Phe Ile Leu His Gly Gly 85 90 95Phe Leu
Asp Ser Leu Arg His Glu Arg Glu Gly Leu Phe Phe Pro Thr 100 105
110Phe Trp Leu Ser Val Ala Thr Ile Ile Thr Gly Leu His Arg Tyr Phe
115 120 125Gly Ser Asp Ala Arg Glu Ser Tyr Leu Ile Ala Leu Glu Val
Leu Phe 130 135 140Trp Val Tyr Cys Ala Cys Thr Leu Ala Thr Ala Val
Ile Gln Tyr Ser145 150 155 160Phe Ile Phe Ser Ala His Arg Tyr Gly
Leu Gln Thr Met Met Pro Ser 165 170 175Trp Ile Leu Pro Ala Phe Pro
Ile Met Leu Ser Gly Thr Ile Ala Ser 180 185 190Val Ile Gly Glu Ala
Gln Pro Ala Arg Ser Ser Ile Pro Val Ile Met 195 200 205Ala Gly Val
Thr Phe Gln Gly Leu Gly Phe Ser Ile Ser Phe Met Met 210 215 220Tyr
Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly Leu Pro Cys Arg225 230
235 240Glu His Arg Pro Gly Met Phe Ile Cys Val Gly Pro Pro Ala Phe
Thr 245 250 255Ala Leu Ala Leu Val Gly Met Ala Lys Gly Leu Pro Ala
Glu Phe Lys 260 265 270Leu Ile Asn Asp Ala His Ala Leu Glu Asp Ala
Arg Ile Leu Glu Leu 275 280 285Leu Ala Ile Thr Ala Gly Ile Phe Leu
Trp Ala Leu Ser Leu Trp Phe 290 295 300Phe Phe Ile Ala Val Ile Ala
Val Leu Arg Ser Pro Pro Thr Ser Phe305 310 315 320His Leu Asn Trp
Trp Ala Leu Val Phe Pro Asn Thr Gly Phe Thr Leu 325 330 335Ala Thr
Ile Thr Leu Gly Lys Ala Leu Gly Ser Pro Gly Ile Leu Gly 340 345
350Val Gly Ser Ala Met Ser Leu Gly Ile Val Gly
Met Trp Leu Phe Val 355 360 365Phe Val Ser His Ile Arg Ala Ile Ile
Asn Gln Asp Ile Met Tyr Pro 370 375 380Gly Lys Asp Glu Asp Ala Ala
Asp385 3907416PRTAspergillisVARIANT(1)..(4)Xaa can be any naturally
occurring amino acidVARIANT(6)..(7)Xaa can be any naturally
occurring amino acidVARIANT(12)..(12)Xaa can be any naturally
occurring amino acidVARIANT(15)..(39)Xaa can be any naturally
occurring amino acidVARIANT(40)..(40)Ile, Leu,
ValVARIANT(41)..(48)Xaa can be any naturally occurring amino
acidVARIANT(51)..(51)Xaa can be any naturally occurring amino
acidVARIANT(54)..(54)Xaa can be any naturally occurring amino
acidVARIANT(58)..(58)Ile, Leu, ValVARIANT(72)..(72)Xaa can be any
naturally occurring amino acidVARIANT(79)..(79)Ile, Leu,
ValVARIANT(80)..(80)Xaa can be any naturally occurring amino
acidVARIANT(81)..(81)Xaa can be any naturally occurring amino
acidVARIANT(84)..(85)Xaa can be any naturally occurring amino
acidVARIANT(87)..(87)Xaa can be any naturally occurring amino
acidVARIANT(90)..(90)Arg, Lys, HisVARIANT(91)..(91)Asp,
GluVARIANT(93)..(95)Xaa can be any naturally occurring amino
acidVARIANT(98)..(98)Xaa can be any naturally occurring amino
acidVARIANT(101)..(101)Ile, Leu, ValVARIANT(102)..(102)Xaa can be
any naturally occurring amino acidVARIANT(105)..(105)Xaa can be any
naturally occurring amino acidVARIANT(106)..(106)Ile, Leu,
ValVARIANT(108)..(108)Xaa can be any naturally occurring amino
acidVARIANT(109)..(109)Ser, ThrVARIANT(110)..(110)Xaa can be any
naturally occurring amino acidVARIANT(113)..(113)Xaa can be any
naturally occurring amino acidVARIANT(119)..(120)Xaa can be any
naturally occurring amino acidVARIANT(121)..(122)Xaa can be any
naturally occurring amino acidVARIANT(123)..(123)Asp,
GluVARIANT(126)..(126)Xaa can be any naturally occurring amino
acidVARIANT(128)..(128)Asp, GluVARIANT(132)..(132)Ile, Leu,
ValVARIANT(144)..(144)Xaa can be any naturally occurring amino
acidVARIANT(150)..(150)Arg, Lys, HisVARIANT(151)..(151)Xaa can be
any naturally occurring amino acidVARIANT(154)..(154)Asp,
GluVARIANT(156)..(159)Xaa can be any naturally occurring amino
acidVARIANT(161)..(163)Xaa can be any naturally occurring amino
acidVARIANT(165)..(166)Xaa can be any naturally occurring amino
acidVARIANT(173)..(174)Xaa can be any naturally occurring amino
acidVARIANT(176)..(176)Xaa can be any naturally occurring amino
acidVARIANT(177)..(177)Ile, Leu, ValVARIANT(178)..(178)Xaa can be
any naturally occurring amino acidVARIANT(181)..(181)Xaa can be any
naturally occurring amino acidVARIANT(185)..(185)Xaa can be any
naturally occurring amino acidVARIANT(186)..(186)Ile, Leu,
ValVARIANT(188)..(190)Xaa can be any naturally occurring amino
acidVARIANT(193)..(193)Xaa can be any naturally occurring amino
acidVARIANT(195)..(195)Xaa can be any naturally occurring amino
acidVARIANT(197)..(197)Xaa can be any naturally occurring amino
acidVARIANT(200)..(200)Xaa can be any naturally occurring amino
acidVARIANT(219)..(221)Xaa can be any naturally occurring amino
acidVARIANT(225)..(227)Xaa can be any naturally occurring amino
acidVARIANT(230)..(230)Xaa can be any naturally occurring amino
acidVARIANT(231)..(232)Ile, Leu, ValVARIANT(235)..(235)Xaa can be
any naturally occurring amino acidVARIANT(246)..(247)Xaa can be any
naturally occurring amino acidVARIANT(253)..(253)Ile, Leu,
ValVARIANT(259)..(259)Xaa can be any naturally occurring amino
acidVARIANT(263)..(264)Xaa can be any naturally occurring amino
acidVARIANT(273)..(273)Xaa can be any naturally occurring amino
acidVARIANT(288)..(288)Xaa can be any naturally occurring amino
acidVARIANT(290)..(290)Xaa can be any naturally occurring amino
acidVARIANT(293)..(293)Xaa can be any naturally occurring amino
acidVARIANT(297)..(298)Ile, Leu, ValVARIANT(301)..(302)Xaa can be
any naturally occurring amino acidVARIANT(304)..(305)Xaa can be any
naturally occurring amino acidVARIANT(307)..(307)Xaa can be any
naturally occurring amino acidVARIANT(309)..(309)Ile, Leu,
ValVARIANT(310)..(312)Xaa can be any naturally occurring amino
acidVARIANT(315)..(315)Ile, Leu, ValVARIANT(316)..(318)Xaa can be
any naturally occurring amino acidVARIANT(319)..(319)Ile, Leu,
ValVARIANT(326)..(326)Xaa can be any naturally occurring amino
acidVARIANT(333)..(333)Xaa can be any naturally occurring amino
acidVARIANT(334)..(334)Ile, Leu, ValVARIANT(342)..(343)Xaa can be
any naturally occurring amino acidVARIANT(347)..(347)Xaa can be any
naturally occurring amino acidVARIANT(349)..(349)Xaa can be any
naturally occurring amino acidVARIANT(364)..(364)Xaa can be any
naturally occurring amino acidVARIANT(366)..(370)Xaa can be any
naturally occurring amino acidVARIANT(372)..(372)Xaa can be any
naturally occurring amino acidVARIANT(374)..(374)Ile, Leu,
ValVARIANT(375)..(375)Xaa can be any naturally occurring amino
acidVARIANT(378)..(378)Xaa can be any naturally occurring amino
acidVARIANT(383)..(383)Ile, Leu, ValVARIANT(384)..(384)Xaa can be
any naturally occurring amino acidVARIANT(385)..(386)Ile, Leu,
ValVARIANT(387)..(388)Xaa can be any naturally occurring amino
acidVARIANT(390)..(390)Ile, Leu, ValVARIANT(393)..(393)Xaa can be
any naturally occurring amino acidVARIANT(394)..(394)Ile, Leu,
ValVARIANT(396)..(396)Xaa can be any naturally occurring amino
acidVARIANT(397)..(397)Ile, Leu, ValVARIANT(407)..(407)Xaa can be
any naturally occurring amino acidVARIANT(410)..(410)Xaa can be any
naturally occurring amino acidVARIANT(415)..(415)Xaa can be any
naturally occurring amino acid 7Xaa Xaa Xaa Xaa Thr Xaa Xaa Pro Gly
Ser Ser Xaa Ser Asp Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Pro Gly Xaa Val Gly Xaa
Arg Glu Arg Xaa Arg His Phe Thr Trp Ala 50 55 60Trp Tyr Thr Leu Thr
Met Ser Xaa Gly Gly Leu Ala Leu Leu Xaa Xaa65 70 75 80Xaa Gln Pro
Xaa Xaa Phe Xaa Gly Leu Xaa Xaa Ile Xaa Xaa Xaa Val 85 90 95Tyr Xaa
Leu Asn Xaa Xaa Phe Phe Xaa Xaa Val Xaa Xaa Xaa Met Ala 100 105
110Xaa Arg Phe Ile Leu His Xaa Xaa Xaa Xaa Xaa Ser Leu Xaa His Xaa
115 120 125Arg Glu Gly Xaa Phe Phe Pro Thr Phe Trp Leu Ser Ile Ala
Thr Xaa 130 135 140Ile Thr Gly Leu Tyr Xaa Xaa Phe Gly Xaa Asp Xaa
Xaa Xaa Xaa Phe145 150 155 160Xaa Xaa Xaa Leu Xaa Xaa Leu Phe Trp
Ile Tyr Cys Xaa Xaa Thr Xaa 165 170 175Xaa Xaa Ala Val Xaa Gln Tyr
Ser Xaa Xaa Phe Xaa Xaa Xaa Lys Tyr 180 185 190Xaa Leu Xaa Thr Xaa
Met Pro Xaa Trp Ile Leu Pro Ala Phe Pro Val 195 200 205Met Leu Ser
Gly Thr Ile Ala Ser Val Ile Xaa Xaa Xaa Gln Pro Ala 210 215 220Xaa
Xaa Xaa Ile Pro Xaa Xaa Xaa Ala Gly Xaa Thr Phe Gln Gly Leu225 230
235 240Gly Phe Ser Ile Ser Xaa Xaa Met Tyr Ala His Tyr Xaa Gly Arg
Leu 245 250 255Met Glu Xaa Gly Leu Pro Xaa Xaa Glu His Arg Pro Gly
Met Phe Ile 260 265 270Xaa Val Gly Pro Pro Ala Phe Thr Ala Leu Ala
Leu Val Gly Met Xaa 275 280 285Lys Xaa Leu Pro Xaa Asp Phe Gln Xaa
Xaa Gly Asp Xaa Xaa Ala Xaa 290 295 300Xaa Asp Xaa Arg Xaa Xaa Xaa
Xaa Leu Ala Xaa Xaa Xaa Xaa Xaa Phe305 310 315 320Leu Trp Ala Leu
Ser Xaa Trp Phe Phe Cys Ile Ala Xaa Xaa Ala Val 325 330 335Val Arg
Ser Pro Pro Xaa Xaa Phe His Leu Xaa Trp Xaa Ala Met Val 340 345
350Phe Pro Asn Thr Gly Phe Thr Leu Ala Thr Ile Xaa Leu Xaa Xaa Xaa
355 360 365Xaa Xaa Ser Xaa Gly Xaa Xaa Gly Val Xaa Thr Ala Met Ser
Xaa Xaa 370 375 380Xaa Xaa Xaa Xaa Phe Xaa Phe Val Xaa Xaa Ser Xaa
Xaa Arg Ala Val385 390 395 400Ile Arg Lys Asp Ile Met Xaa Pro Gly
Xaa Asp Glu Asp Val Xaa Glu 405 410 4158397PRTArtificial
SequenceSequence based on Aspergillus and Schizosaccharomyces pombe
MAE1 transport proteins.VARIANT(1)..(22)Xaa can be any naturally
occurring amino acidVARIANT(23)..(23)Ile, Leu,
ValVARIANT(24)..(39)Xaa can be any naturally occurring amino
acidVARIANT(41)..(41)Ile, Leu, ValVARIANT(42)..(42)Xaa can be any
naturally occurring amino acidVARIANT(47)..(47)Xaa can be any
naturally occurring amino acidVARIANT(49)..(52)Xaa can be any
naturally occurring amino acidVARIANT(54)..(55)Xaa can be any
naturally occurring amino acidVARIANT(58)..(58)Ile, Leu,
ValVARIANT(59)..(60)Xaa can be any naturally occurring amino
acidVARIANT(61)..(61)Ile, Leu, ValVARIANT(62)..(68)Xaa can be any
naturally occurring amino acidVARIANT(70)..(70)Xaa can be any
naturally occurring amino acidVARIANT(73)..(74)Xaa can be any
naturally occurring amino acidVARIANT(75)..(75)Ile, Leu,
ValVARIANT(76)..(78)Xaa can be any naturally occurring amino
acidVARIANT(79)..(79)Ile, Leu, ValVARIANT(80)..(83)Xaa can be any
naturally occurring amino acidVARIANT(84)..(84)Ile, Leu,
ValVARIANT(85)..(88)Xaa can be any naturally occurring amino
acidVARIANT(89)..(89)Ile, Leu, ValVARIANT(90)..(96)Xaa can be any
naturally occurring amino acidVARIANT(100)..(105)Xaa can be any
naturally occurring amino acidVARIANT(106)..(106)Ser,
ThrVARIANT(107)..(111)Xaa can be any naturally occurring amino
acidVARIANT(113)..(113)Xaa can be any naturally occurring amino
acidVARIANT(114)..(114)Ile, Leu, ValVARIANT(116)..(117)Xaa can be
any naturally occurring amino acidVARIANT(119)..(120)Xaa can be any
naturally occurring amino acidVARIANT(122)..(122)Xaa can be any
naturally occurring amino acidVARIANT(124)..(124)Xaa can be any
naturally occurring amino acidVARIANT(126)..(126)Xaa can be any
naturally occurring amino acidVARIANT(128)..(129)Xaa can be any
naturally occurring amino acidVARIANT(131)..(145)Xaa can be any
naturally occurring amino acidVARIANT(146)..(146)Ile, Leu,
ValVARIANT(147)..(148)Xaa can be any naturally occurring amino
acidVARIANT(150)..(151)Xaa can be any naturally occurring amino
acidVARIANT(153)..(158)Xaa can be any naturally occurring amino
acidVARIANT(159)..(159)Ile, Leu, ValVARIANT(160)..(161)Xaa can be
any naturally occurring amino acidVARIANT(162)..(162)Ile, Leu,
ValVARIANT(163)..(166)Xaa can be any naturally occurring amino
acidVARIANT(167)..(167)Ile, Leu, ValVARIANT(169)..(174)Xaa can be
any naturally occurring amino acidVARIANT(175)..(175)Ile, Leu,
ValVARIANT(176)..(176)Xaa can be any naturally occurring amino
acidVARIANT(178)..(179)Xaa can be any naturally occurring amino
acidVARIANT(181)..(182)Xaa can be any naturally occurring amino
acidVARIANT(183)..(183)Ile, Leu, ValVARIANT(186)..(186)Xaa can be
any naturally occurring amino acidVARIANT(189)..(189)Xaa can be any
naturally occurring amino acidVARIANT(191)..(191)Ile, Leu,
ValVARIANT(192)..(192)Xaa can be any naturally occurring amino
acidVARIANT(194)..(194)Xaa can be any naturally occurring amino
acidVARIANT(197)..(198)Xaa can be any naturally occurring amino
acidVARIANT(199)..(199)Ile, Leu, ValVARIANT(200)..(202)Xaa can be
any naturally occurring amino acidVARIANT(206)..(211)Xaa can be any
naturally occurring amino acidVARIANT(212)..(213)Ile, Leu,
ValVARIANT(214)..(214)Xaa can be any naturally occurring amino
acidVARIANT(216)..(217)Xaa can be any naturally occurring amino
acidVARIANT(224)..(224)Xaa can be any naturally occurring amino
acidVARIANT(225)..(225)Ile, Leu, ValVARIANT(226)..(230)Xaa can be
any naturally occurring amino acidVARIANT(232)..(235)Xaa can be any
naturally occurring amino acidVARIANT(237)..(240)Xaa can be any
naturally occurring amino acidVARIANT(243)..(247)Xaa can be any
naturally occurring amino acidVARIANT(253)..(254)Xaa can be any
naturally occurring amino acidVARIANT(256)..(256)Xaa can be any
naturally occurring amino acidVARIANT(259)..(259)Xaa can be any
naturally occurring amino acidVARIANT(261)..(261)Ser,
ThrVARIANT(262)..(262)Xaa can be any naturally occurring amino
acidVARIANT(264)..(264)Xaa can be any naturally occurring amino
acidVARIANT(266)..(266)Ile, Leu, ValVARIANT(267)..(278)Xaa can be
any naturally occurring amino acidVARIANT(279)..(279)Ile, Leu,
ValVARIANT(281)..(293)Xaa can be any naturally occurring amino
acidVARIANT(294)..(294)Ile, Leu, ValVARIANT(295)..(299)Xaa can be
any naturally occurring amino acidVARIANT(300)..(300)Ile, Leu,
ValVARIANT(302)..(304)Xaa can be any naturally occurring amino
acidVARIANT(306)..(310)Xaa can be any naturally occurring amino
acidVARIANT(312)..(312)Ile, Leu, ValVARIANT(314)..(314)Xaa can be
any naturally occurring amino acidVARIANT(315)..(315)Ile, Leu,
ValVARIANT(316)..(317)Xaa can be any naturally occurring amino
acidVARIANT(318)..(318)Ile, Leu, ValVARIANT(319)..(324)Xaa can be
any naturally occurring amino acidVARIANT(326)..(328)Xaa can be any
naturally occurring amino acidVARIANT(330)..(330)Xaa can be any
naturally occurring amino acidVARIANT(332)..(332)Xaa can be any
naturally occurring amino acidVARIANT(333)..(333)Ile, Leu,
ValVARIANT(335)..(335)Xaa can be any naturally occurring amino
acidVARIANT(337)..(337)Xaa can be any naturally occurring amino
acidVARIANT(339)..(345)Xaa can be any naturally occurring amino
acidVARIANT(346)..(346)Ile, Leu, ValVARIANT(347)..(351)Xaa can be
any naturally occurring amino acidVARIANT(353)..(363)Xaa can be any
naturally occurring amino acidVARIANT(364)..(364)Ile, Leu,
ValVARIANT(365)..(365)Xaa can be any naturally occurring amino
acidVARIANT(366)..(366)Ile, Leu, ValVARIANT(367)..(370)Xaa can be
any naturally occurring amino acidVARIANT(371)..(371)Ile, Leu,
ValVARIANT(372)..(372)Xaa can be any naturally occurring amino
acidVARIANT(373)..(373)Ile, Leu, ValVARIANT(374)..(377)Xaa can be
any naturally occurring amino acidVARIANT(378)..(378)Ile, Leu,
ValVARIANT(381)..(384)Xaa can be any naturally occurring amino
acidVARIANT(385)..(385)Asp, GluVARIANT(386)..(386)Ile, Leu,
ValVARIANT(387)..(388)Xaa can be any naturally occurring amino
acidVARIANT(391)..(391)Xaa can be any naturally occurring amino
acidVARIANT(395)..(397)Xaa can be any naturally occurring amino
acid 8Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa His Phe Thr
Trp Xaa Trp 35 40 45Xaa Xaa Xaa Xaa Met Xaa Xaa Gly Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Phe Xaa Gly Leu Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Arg Phe Ile Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu 100 105 110Xaa Xaa Phe Xaa Xaa Thr
Xaa Xaa Leu Xaa Ile Xaa Thr Xaa Ile Xaa 115 120 125Xaa Leu Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140Xaa Xaa
Xaa Xaa Leu Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa145 150 155
160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
165 170 175Thr Xaa Xaa Pro Xaa Xaa Xaa Leu Pro Xaa Phe Pro Xaa Met
Xaa Xaa 180 185 190Gly Xaa Ile Ala Xaa Xaa Xaa Xaa Xaa Xaa Gln Pro
Ala Xaa Xaa Xaa 195 200 205Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Phe
Gln Gly Leu Gly Phe Xaa 210 215 220Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa
Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa225 230 235 240Gly Leu Xaa Xaa Xaa
Xaa Xaa Arg Pro Gly Met Phe Xaa Xaa Val Xaa 245 250 255Pro Pro Xaa
Phe Xaa Xaa Leu Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280
285Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa
290 295 300Leu Xaa Xaa Xaa Xaa Xaa Cys Xaa
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa305 310 315 320Xaa Xaa Xaa Xaa Phe
Xaa Xaa Xaa Trp Xaa Ala Xaa Xaa Phe Xaa Asn 325 330 335Xaa Gly Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 340 345 350Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360
365Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Ala Xaa Xaa Xaa Xaa
370 375 380Xaa Xaa Xaa Xaa Pro Gly Xaa Asp Glu Asp Xaa Xaa Xaa385
390 395
* * * * *