RECOMBINANT HOST CELLS AND METHODS FOR THE ANAEROBIC PRODUCTION OF L-ASPARTATE AND BETA-ALANINE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application

Serial No. 62/254,635, filed on November 12, 2015, and entitled "RECOMBINANT HOST CELLS AND METHODS FOR THE ANAEROBIC PRODUCTION OF L-ASPARTATE AND BETA-ALANINE," the complete disclosure of which is expressly incorporated by reference herein.

GOVERNMENT INTEREST

[0002] This invention was made with government support under award number DE-

EE0007565 awarded by the United States Department of Energy. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 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 derived from 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.

[0004] Aspartic acid ("L-aspartate", CAS No. 56-84-8) is currently produced from fumaric acid, a non-renewable, petroleum-derived chemical feedstock. Likewise, beta-alanine (CAS No. 107-96-9) is produced from acrylamide, another non-renewable, petroleum feedstock.

[0005] The current, preferred route for industrial synthesis of L-aspartate and L- aspartate-derived compounds is based on fumaric acid. For example, an enzymatic process in which L-aspartate ammonia lyase catalyzes the formation of L-aspartate from fumaric acid and ammonia (see "Amino Acids," In: Ullmann's Encyclopedia of Industrial Chemistry, Wiley - VCH, Weinheim, New York (2002)).

[0006] The existing, petrochemical-based production routes to L-aspartate and beta- alanine are environmentally damaging, dependent on non-renewable feedstocks, and costly. Thus, there remains a need for methods and materials for biocatalytic conversion of renewable feedstocks into L-aspartate and/or beta-alanine and purification of biosynthetic L-aspartate and/or beta-alanine. SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention provides a recombinant host cell capable of producing L-aspartate or beta-alanine under substantially anaerobic conditions, the host cell comprising one or more heterologous nucleic acids encoding a L-aspartate pathway enzyme and optionally (in the case of beta-alanine producing host cells) a L-aspartate 1 -decarboxylase. In one embodiment, the recombinant host cell has been engineered to produce L-aspartate or beta- alanine under substantially anaerobic conditions.

[0008] Any suitable host cell may be used in practice of the methods of the present invention, and exemplary host cells useful in the compositions and methods provided herein include archaeal, prokaryotic, or eukaryotic cells. In an important embodiment, the recombinant host cell is a yeast cell. In certain embodiments, the recombinant yeast cells provided herein are engineered by the introduction of one or more genetic modifications (including, for example, heterologous nucleic acids encoding enzymes and/or disruption or deletion of native nucleic acids encoding enzymes) into a Crabtree-negative yeast cell. In certain of these embodiments, the host cell belongs to the Pichia/Issatchenkia/Saturnispora/Dekkera clade. 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, and in some of these embodiments the host cell is Pichia kudriavzevii.

[0009] Provided herein in certain embodiments are recombinant host cells having at least one active L-aspartate pathway from phosphoenolpyruvate or pyruvate to L-aspartate. In some embodiments wherein the host cell produces beta-alanine, the recombinant host cell further expresses an L-aspartate 1 -decarboxylase. In certain embodiments, the recombinant host cells provided herein have a L-aspartate pathway that proceeds via phosphoenolpyruvate or pyruvate, and oxaloacetate intermediates. In many embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under substantially anaerobic conditions. In other embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under aerobic conditions. In one embodiment, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of pyruvate carboxylase and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under substantially anaerobic conditions. In one embodiment, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of pyruvate carboxylase and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under aerobic conditions. In certain embodiments, the cell further comprises a heterologous nucleic acid encoding an L-aspartate 1- decarboxylase wherein said heterologous nucleic acid is expressed in sufficient amounts to produce beta-alanine under substantially anaerobic conditions.

[0010] In some embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding a L-aspartate dehydrogenase. In certain embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1) and is capable of producing L-aspartate and/or beta-alanine. In other embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Cupriavidus taiwanensis L- aspartate dehydrogenase (SEQ ID NO: 2) and is capable of producing L-aspartate and/or beta- alanine.

[0011] In various embodiments, the recombinant host cell further comprises a heterologous nucleic acid encoding an L-aspartate 1 -decarboxylase and is capable of producing beta-alanine where cultured under suitable conditions. A L-aspartate 1 -decarboxylase as used herein refers to any protein with L-aspartate decarboxylase activity, meaning the ability to catalyze the decarboxylation of L-aspartate to beta-alanine. In various embodiments, the recombinant host cell provided herein comprises one or more heterologous nucleic acid encoding a L-aspartate 1 -decarboxylase selected from the group consisting of Bacillus subtilis L-aspartate 1 -decarboxylase (SEQ ID NO: 5), Corynebacterium L-aspartate 1 -decarboxylase (SEQ ID NO: 4), and/or Tribolium castaneum L-aspartate 1 -decarboxylase (SEQ ID NO: 3) and is capable of producing beta-alanine. [0012] In various embodiments, L-aspartate dehydrogenase enzymes suitable for use in accordance with the methods of the invention have L-aspartate dehydrogenase activity and comprise an amino acid sequence with at least 55%, at least 60%>, at least 70%, at least 80%>, at least 90%), or at least 95% sequence identity to SEQ ID NO: 14. In various embodiments, L- aspartate 1 -decarboxylase enzymes suitable for use in accordance with the methods of the invention have L-aspartate 1 -decarboxylase activity and comprise an amino acid sequence with at least 40%, at least 45%, at least 50%, or at least 55% sequence identity to SEQ ID NO: 15 and/or 16.

[0013] In a second aspect, the invention provides host cells genetically modified to delete or otherwise reduce the activity of endogenous proteins. Deletion or disruption of ethanol fermentation pathway(s) and nucleic acids encoding ethanol fermentation pathway enzymes is important for engineering a recombinant host cell capable of efficient production of L-aspartate and/or beta-alanine under substantially anaerobic conditions. In various embodiments, recombinant host cells comprising deletion or disruption of one or more nucleic acids encoding ethanol fermentation pathway enzymes decreases ethanol production by at least 10%>, at least 25%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or at least 99% as compared to parental cells that do not comprise this genetic modification.

[0014] In various embodiments, the recombinant host cells comprise a deletion or disruption of one or more nucleic acids encoding an enzyme selected from the group consisting of pyruvate decarboxylase, alcohol dehydrogenase, and/or malate dehydrogenase.

[0015] In a third aspect, methods are provided herein for producing L-aspartate or beta- alanine by recombinant host cells of the invention. In certain embodiments, these methods comprise the step culturing a recombinant host cell described herein in a medium containing at least one carbon source and one nitrogen source under substantially anaerobic conditions such that L-aspartate is produced. In various embodiments, conditions are selected to produce an oxygen uptake rate of around 0-25 mmol/l/hr. In some embodiments, conditions are selected to produce an oxygen uptake rate of around 2.5-15 mmol/l/hr. In other embodiments, these methods comprise the step of culturing a recombinant host cell described herein in a medium containing at least one carbon source and one nitrogen source under aerobic conditions such that L-aspartate is produced. BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 provides a schematic of the L-aspartate pathway enzymes and L-aspartate 1- decarboxylase enzymes provided by the invention. Conversion of oxaloacetate to L-aspartate is catalyzed by L-aspartate dehydrogenase (EC 1.4.1.21) and conversion of L-aspartate to beta- alanine is catalyzed by L-aspartate 1 -decarboxylase (EC 4.1.1.11). Oxaloacetate-forming enzymes provided by the invention include pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and phosphoenolpyruvate carboxykinase (EC 4.1.1.49). Conversion of pyruvate to oxaloacetate is catalyzed by pyruvate carboxylase; conversion of phosphoenolpyruvate to oxaloacetate is catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides recombinant host cells, materials, and methods for the biological production of L-aspartate and/or beta-alanine under substantially anaerobic conditions.

[0018] 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, biosynthetic pathways, 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 steps or steps, in accordance with the invention. All such modifications are within the scope of the claims appended hereto.

[0019] All patents, patent applications, and publications cited herein are incorporated by reference in their entireties.

Section 1: Definitions

[0020] 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.

[0021] 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.

[0022] The term "accession number", and similar terms such as "protein accession number", "UniProt ID", "gene ID", "gene accession number" refer to designations given to specific proteins or genes. These identifiers describe a gene or enzyme sequence in publicly accessible databases, such as NCBI.

[0023] A dash (-) in a consensus sequence indicates that there is no amino acid at the specified position. A plus (+) in a consensus sequence indicates any amino acid may be present at the specified position. Thus, a plus in a consensus sequence herein indicates a position at which the amino acid is generally non-conserved; a homologous enzyme sequence, when aligned with the consensus sequence, can have any amino acid at the indicated "+" position.

[0024] As used herein, the term "express", when used in connection with a nucleic acid encoding an enzyme or an enzyme itself in a cell, means that the enzyme, which may be an endogenous or exogenous (heterologous) enzyme, is produced in the cell. The term "overexpress", in these contexts, means that the enzyme is produced at a higher level, i.e., enzyme levels are increased, as compared to the wild-type, in the case of an endogenous enzyme. Those skilled in the art appreciate that overexpression of an enzyme 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 enzyme, and the like.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The term "homologous", as well as variations thereof, such as "homology", refers to the similarity of a nucleic acid or amino acid sequence, typically in the context of a coding sequence for a gene or the amino acid sequence of a protein. Homology searches can be employed using a known amino acid or coding sequence (the "reference sequence") for a useful protein to identify homologous coding sequences or proteins that have similar sequences and thus are likely to perform the same useful function as the protein defined by the reference sequence. As will be appreciated by those of skill in the art, a protein having greater than 90% identity to a reference protein as determined by, for example and without limitation, a BLAST (blast.ncbi.nlm.nih.gov) search is highly likely to carry out the identical biochemical reaction as the reference protein. In some cases, two enzymes having greater than 20% identity will carry out identical biochemical reactions, and the higher the identity, i.e., 40% or 80% identity, the more likely the two proteins have the same or similar function. As will be appreciated by those skilled in the art, homologous enzymes can be identified by BLAST searching.

[0029] 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.

[0030] 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.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] As used herein, "L-aspartate" is intended to mean an amino acid having the chemical formula C ₄ H ₅ NO ₄ and a molecular mass of 131.10 g/mol (CAS# 56-84-8). L-aspartate as described herein can be a salt, acid, base, or derivative depending on the structure, pH, and ions present. The terms "L-aspartate", "L-aspartic acid", "L-aspartate", and "aspartic acid" are used interchangeably herein.

[0037] As used herein, beta-alanine is intended to mean a beta amino acid having the chemical formula C ₃ H ₆ NO ₂ and a molecular mass of 88.09 g/mol (CAS # 107-95-9). Beta- alanine as described herein can be a salt, acid, base, or derivative depending on the structure, pH, and ions present. Beta-alanine is also referred to as "β-alanine", "3-aminopropionic acid", and "3-aminopropanoate", and these terms are used interchangeably herein.

[0038] As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term is also intended to include sealed chambers of liquid or solid growth medium maintained with an atmosphere of less than about 1% oxygen.

Section 2: Recombinant host cells for production of L-aspartate and beta-alanine 2.1 Host cells

[0039] In one aspect, the invention provides a recombinant host cell capable of producing

L-aspartate or beta-alanine under substantially anaerobic conditions, the host cell comprising one or more heterologous nucleic acids encoding a L-aspartate pathway enzyme and optionally (in the case of beta-alanine producing host cells) a L-aspartate 1 -decarboxylase. In one embodiment, the recombinant host cell has been engineered to produce L-aspartate or beta-alanine under substantially anaerobic conditions. In another embodiment, the recombinant host cell natively produces L-aspartate or beta-alanine under substantially anaerobic conditions. In another embodiment, the recombinant host cell has been engineered to produce L-aspartate or beta- alanine under aerobic conditions.

[0040] Any suitable host cell may be used in practice of the methods of the present invention, and exemplary host cells useful in the compositions and methods provided herein include archaeal, prokaryotic, or eukaryotic cells.

2.1.1 Yeast cells [0041] 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/insertion of nucleic acids into the yeast chromosome by homologous recombination are well established. Yeast also offers a number of advantages as an industrial fermentation host. Yeast cells can generally 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 the desired product under substantially anaerobic conditions provides a number of advantages with regard to the present invention. First, this characteristic results in efficient product biosynthesis when the host cell is supplied with a carbohydrate carbon source. Second, from a process standpoint, the ability to run a fermentation under substantially anaerobic conditions decreases production cost.

[0042] 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.

[0043] 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 metbanosorbosa, Candida sonorensis, Candida tropicalis, Candida utilis, Cryptococcus curvatus, Hansenula polymorpha, Issatchenkia orientalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, omagataella pastoris, Lipomyces starkeyi, Pichia angusta, Pichia deserticola, Pichia galeiformis, Pichia kodamae, Pichia kudriavzevii, Pichia membranaefaciens, Pichia methanolica, Pichia pastoris, Pichia salictaria, Pichia stipitis, Pichia thermotolerans, Pichia trehalophila, 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.

[0044] In certain embodiments, the recombinant yeast cells provided herein are engineered by the introduction of one or more genetic modifications (including, for example, heterologous nucleic acids encoding enzymes and/or disruption or deletion of native nucleic acids encoding enzymes) into a Crabtree-negative yeast cell. In certain of these embodiments, the host cell belongs to the Pichia/Issatchenkia/Saturnispora/Dekkera clade. 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, and in some of these embodiments the host cell is Pichia kudriavzevii.

[0045] 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. In certain of these embodiments, the host cell belongs to the Saccharomyces clad. 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, and in one of these embodiments the host cell is Saccharomyces cerevisiae.

[0046] Members of the Pichia/Issatchenkia/Saturnispora/Dekkera or the Saccharomyces clade are identified by analysis of their 26S ribosomal DNA using the methods described by Kurtzman CP., 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 clade exhibit greater sequence identity in the D1/D2 domain of the 26S ribosomal subunit DNA to other host cells within the clade as compared to host cells outside the clade. Therefore, host cells that are members of a clade (e.g., the Pichia/Issatchenkia/Saturnispora/Dekkera or Saccharomyces clades) can be identified using the methods of Kurtzman and Robnett.

2.1.2 Other host cells

[0047] Recombinant host cells other than yeast cells are also suitable for use in accordance with the methods of the invention so long as the engineered host cell is capable of growth and/or product formation under substantially anaerobic conditions, 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, Schizochytnum, Thraustochytrium, Trichoderma, Xanthophyllomyces. Examples of eukaryotic strains include, but are not limited to: Aspergillus niger, Aspergillus oryzae, Crypthecodiniurn. cohnii, Cunninghamella japonica, Entomophthora coronata, Mortierella alpina, Mucor circinelloides, Neurospora crassa, Pythium uitimum, Schizochytnum limacinum, Thraustochytrium aureum, Trichoderma reesei and Xanthophyllomyces dendrorhous.

[0048] 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 jarmaschii, Methanobacterium thernioautotrophicum, Thermoplasma acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii, Pyrococcus abyssi, and Aeropyrum peraix.

[0049] Illustrative examples of recombinant prokaryotic host ceils provided by the invention include, but are not limited to, ceils belonging to the genera Agrobacterium, Aiicyciobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methyl obacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas. Examples of prokaryotic strains include, but are not limited to Bacillus subtilis, Brevibacterium ammoniagenes, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonic, Pseudomonas pudita, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Staphylococcus aureus.

[0050] Escherichia coli and Corynebacterium glutamicum are particularly good prokaryotic host cells for use in accordance with the methods of the invention. E. coli is capable of growth and/or product (L-aspartate or beta-alanine) formation under substantially anaerobic conditions, 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 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. Corynebacterium glutamicum is well utilized for industrial production of various amino acids. While generally regarded as a strict aerobe, wild type Corynebacterium glutamicum is capable of growth under substantially anaerobic conditions if nitrate is supplied to the fermentation broth as an electron acceptor. In one embodiment, the recombinant host cell is a Corynebacterium host cell.

[0051] In some embodiments, the host cell is a microbe that is capable of growth and/or production of L-aspartate or beta-alanine under substantially anaerobic conditions. Suitable host ceils may natively grow under substantially anaerobic conditions or may be engineered to be capable of growth under substantially anaerobi c conditions,

[0052] Certain of these host ceils, 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. 2.2 L-aspartate pathway enzymes and L-aspartate 1-decarboxylases

[0053] Provided herein in certain embodiments are recombinant host cells having at least one active L-aspartate pathway from phosphoenolpyruvate or pyruvate to L-aspartate. In some embodiments wherein the host cell produces beta-alanine, the recombinant host cell further expresses an L-aspartate 1 -decarboxylase. A recombinant host cell having an active L-aspartate pathway as used herein produces active enzymes necessary to catalyze each metabolic reaction in a L-aspartate fermentation pathway, and therefore is capable of producing L-aspartate and/or beta-alanine in measurable yields and/or titers when cultured under suitable conditions. A recombinant host cell having an active L-aspartate pathway comprises one or more heterologous nucleic acids encoding L-aspartate pathway enzymes.

[0054] In certain embodiments, the recombinant host cells provided herein have a L- aspartate pathway that proceeds via phosphoenolpyruvate or pyruvate, and oxaloacetate intermediates. In many embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under substantially anaerobic conditions. In other embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more L-aspartate pathway enzymes selected from the group consisting of phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and L-aspartate dehydrogenase wherein the heterologous nucleic acid is expressed in sufficient amounts to produce L-aspartate under aerobic conditions. In certain embodiments, the cell further comprises a heterologous nucleic acid encoding an L- aspartate 1 -decarboxylase wherein said heterologous nucleic acid is expressed in sufficient amounts to produce beta-alanine under substantially anaerobic conditions. Thus, one will recognize that recombinant host cells engineered for production of L-aspartate in accordance with the methods of the invention express an L-aspartate pathway, and recombinant host cells engineered for production of beta-alanine express, in addition to an L-aspartate pathway, a L- aspartate 1 -decarboxylase.

[0055] In some embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more enzymes of an L-aspartate pathway. In some embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one L-aspartate pathway enzyme. In some embodiments, said one L-aspartate pathway enzyme is L-aspartate dehydrogenase. In other embodiments, said one L-aspartate pathway enzyme is pyruvate carboxylase. In other embodiments, said one L-aspartate pathway enzyme is phosphoenolpyruvate carboxylase. In still further embodiments, said one L-aspartate pathway enzyme is phosphoenolpyruvate carboxykinase. In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding two L-aspartate pathway enzymes. In some embodiments, said two L-aspartate pathway enzymes are L-aspartate dehydrogenase and pyruvate carboxylase. In other embodiments, said two L-aspartate pathway enzymes are L-aspartate dehydrogenase and phosphoenolpyruvate carboxylase. In other embodiments, said two L-aspartate pathway enzymes are L-aspartate dehydrogenase and phosphoenolpyruvate carboxykinase. In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding three L-aspartate pathway enzymes. In some embodiments, said three L-aspartate pathway enzymes are L-aspartate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase. In other embodiments, said three L-aspartate pathway enzymes are L-aspartate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase. In other embodiments, said three L-aspartate pathway enzymes are L-aspartate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase. In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding all four L-aspartate pathway enzymes (i.e., L-aspartate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase). In certain embodiments, the recombinant host cell further comprises a heterologous nucleic acid encoding L-aspartate 1 -decarboxylase.

[0056] The recombinant host cells of the present invention include microbes that employ combinations of metabolic reactions for biosynthetically producing the compounds of the invention. The biosynthesized compounds can be produced intracellularly and/or secreted into the culture medium. The biosynthesized compounds produced by the recombinant host cells are L-aspartate and/or beta-alanine. The relationship of these compounds with respect to the metabolic reactions described herein are depicted in Figure 1. In one embodiment, the recombinant host cell is engineered to produce L-aspartate under substantially anaerobic conditions. In another embodiment, the recombinant host cell is engineered to produce L- aspartate under aerobic conditions. In another embodiment, the recombinant host cell is engineered to produce beta-alanine under substantially anaerobic conditions. [0057] The production of L-aspartate or beta-alanine via the biosynthetic pathways and recombinant host cells of the invention is particularly useful because L-aspartate and beta- alanine can be produced under substantially anaerobic conditions. Microorganisms generally lack the capacity to produce L-aspartate or beta-alanine (derived from L-aspartate using a L- aspartate 1 -decarboxylase) under substantially anaerobic conditions. As described herein, the recombinant host cells of the invention are engineered to produce L-aspartate and/or beta-alanine when grown under substantially anaerobic conditions and supplied with a carbohydrate as the primary carbon source and an assimilable nitrogen source.

[0058] The L-aspartate pathway and L-aspartate 1 -decarboxylase enzymes and nucleic acids encoding said enzymes may be endogenous or heterologous. In certain embodiments, the recombinant host cells provided herein comprise one or more heterologous nucleic acids encoding L-aspartate pathway and/or L-aspartate 1 -decarboxylase enzymes. In certain embodiments, the recombinant host cell comprises a single heterologous nucleic acid encoding a L-aspartate pathway or L-aspartate 1 -decarboxylase gene. In other embodiments, the cell comprises multiple heterologous nucleic acids encoding L-aspartate pathway and/or L-aspartate 1 -decarboxylase enzymes. In these embodiments, the recombinant host cell may comprise multiple copies of a single heterologous nucleic acid and/or multiple copies of two or more heterologous nucleic acids. Recombinant host cells comprising multiple heterologous nucleic acids may comprise any number of heterologous nucleic acids.

[0059] In certain embodiments, the recombinant host cells provided herein comprise one or more endogenous nucleic acids encoding L-aspartate pathway and/or L-aspartate 1- decarboxylase enzymes. In certain of these embodiments, the cells may be engineered to express more of these endogenous enzymes. In certain of these embodiments, the endogenous enzyme being expressed at a higher level (produced at a higher amount as compared to a parental or control cell) may be operatively linked to one or more exogenous promoters or other regulatory elements.

[0060] In certain embodiments, the recombinant host cells provided herein comprise one or more endogenous nucleic acids encoding L-aspartate pathway and/or L-aspartate 1- decarboxylase enzymes and one or more heterologous nucleic acids encoding L-aspartate pathway and/or L-aspartate 1 -decarboxylase enzymes. In these embodiments, the recombinant host cells may have an active L-aspartate pathway and/or L-aspartate 1 -decarboxylase that comprises one or more endogenous nucleic acids encoding L-aspartate pathway and/or L- aspartate 1 -decarboxylase enzymes and one or more heterologous nucleic acids encoding L- aspartate pathway and/or L-aspartate 1 -decarboxylase enzymes. In certain embodiments, the recombinant host cell may comprise both endogenous and heterologous nucleic acids encoding an L-aspartate pathway or L-aspartate 1 -decarboxylase enzyme.

2.2.1 Oxaloacetate-forming enzymes

[0061] Three enzymes can be used to form oxaloacetate from the glycolytic intermediates phosphoenolpyruvate and/or pyruvate, and Figure 1 provides a schematic showing the biosynthetic relationship of the three oxaloacetate-forming enzymes to the production of L- aspartate and beta-alanine. One oxaloacetate-forming enzyme provided by the invention is pyruvate carboxylase (EC 6.4.1.1), catalyzing conversion of pyruvate and hydrogen carbonate to oxaloacetate along with concomitant hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). Another oxaloacetate-forming enzyme is phosphoenolpyruvate carboxylase (EC 4.1.1.31), catalyzing conversion of phosphoenolpyruvate and hydrogen carbonate to oxaloacetate along with concomitant release of phosphate. The third oxaloacetate-forming enzymes is phosphoenolpyruvate carboxykinase (EC 4.1.1.49), catalyzing formation of oxaloacetate from phosphoenolpyruvate and carbon dioxide along with concomitant formation of ATP from ADP. In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding an oxaloacetate-forming enzyme selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase that results in increased production of L-aspartate or beta-alanine under substantially anaerobic conditions as compared to a parent cell not comprising said one or more heterologous nucleic acids. In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding an oxaloacetate-forming enzyme selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase that results in increased production of L-aspartate or beta- alanine under aerobic conditions as compared to a parent cell not comprising said one or more heterologous nucleic acids.

[0062] Recombinant host cells of the invention engineered for production of L-aspartate or beta-alanine under substantially anaerobic conditions through increased expression of oxaloacetate-forming enzymes generally comprise one or more heterologous nucleic acids encoding at least one oxaloacetate-forming enzyme. In some embodiments, a recombinant host cell engineered for production of L-aspartate or beta-alanine under substantially anaerobic conditions comprises one or more heterologous nucleic acid encoding one oxaloacetate-forming enzyme. In other embodiments, a recombinant host cell engineered for production of L-aspartate or beta-alanine under substantially anaerobic conditions comprises heterologous nucleic acids encoding two oxaloacetate-forming enzymes. In yet a further embodiment, recombinant host cells of the invention engineered for production of L-aspartate or beta-alanine under substantially anaerobic conditions comprise heterologous nucleic acids encoding all three oxaloacetate- forming enzymes.

2.2.1.1 Pyruvate carboxylase

[0063] One oxaloacetate-forming enzyme is pyruvate carboxylase, and in one embodiment, a recombinant host cell of the invention comprises one or more heterologous nucleic acids encoding a pyruvate carboxylase wherein said host cell is capable of producing L- aspartate or beta-alanine under substantially anaerobic conditions. In another embodiment, a recombinant host cell of the invention comprises one or more heterologous nucleic acids encoding a pyruvate carboxylase wherein said host cell is capable of producing L-aspartate or beta-alanine under aerobic conditions.

[0064] In some embodiments, a nucleic acid encoding pyruvate carboxylase is derived from a fungal source. Non-limiting examples of pyruvate carboxylase enzymes derived from fungal sources suitable for use in accordance with the methods of the invention include those selected from the group consisting of Aspergillus niger (UniProt ID: Q9HES8), Aspergillus terreus (UniProt ID: 093918), Aspergillus oryzae (UniProt ID:Q2UGL1; SEQ ID NO: 7), Aspergillus fumigatus (UniProt ID: Q4WP18), Paecilomyces variotii (UniProt ID: V5FWI7), and Saccharomyces cerevisiae (UniProt ID: PI 1154) pyruvate carboxylase. In a specific embodiment, a recombinant host cell of the invention comprises one or more heterologous nucleic acids encoding Aspergillus oryzae pyruvate carboxylase (SEQ ID NO: 7) wherein said host cell is capable of producing L-aspartate or beta-alanine under substantially anaerobic conditions. In another specific embodiment, a recombinant host cell of the invention comprises one or more heterologous nucleic acids encoding Aspergillus oryzae pyruvate carboxylase (SEQ ID NO: 7) wherein said host cell is capable of producing L-aspartate or beta-alanine under aerobic conditions.

2.2.1.2 Phosphoenolpyruvate carboxylase

[0065] Oxaloacetate can also be produced from phosphoenolpyruvate, which serves as the substrate for both phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase enzymes. In some embodiments, a nucleic acid encoding phosphoenolpymvate carboxylase is derived from a fungal source. A specific, non-limiting example of a phosphoenolpyruvate carboxylase enzyme derived from a fungal source suitable for use in accordance with the methods of the invention is Aspergillus niger phosphoenolpyruvate carboxylase (UniProt ID: A2QM99).

[0066] In other embodiments, a nucleic acid encoding phosphoenolpyruvate carboxylase is derived from a bacterial source. Non-limiting examples of phosphoenolpyruvate carboxylase enzymes derived from bacterial sources suitable for use in accordance with the methods of the invention include Escherichia coli (UniProt ID: H9UZE7; SEQ ID NO: 8), Mycobacterium tuberculosis (UniProt ID: P9WIH3), and Cory neb acterium glutamicum (UniProt ID: P12880) phosphoenolpyruvate carboxylase enzymes. In a specific embodiment, said phosphoenolpyruvate carboxylase is Escherichia coli phosphoenolpyruvate carboxylase (SEQ ID NO: 8).

[0067] In various embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding a phosphoenolpyruvate carboxylase that results in increased production of L-aspartate or beta-alanine under substantially anaerobic conditions as compared to a parent cell not comprising said one or more heterologous nucleic acids. In a specific embodiment, said phosphoenolpyruvate carboxylase is Escherichia coli phosphoenolpyruvate carboxylase (SEQ ID NO: 8).

2.2.1.3 Phosphoenolpyruvate carboxylase

[0068] Non-limiting examples of phosphoenolpyruvate carboxykinase enzymes suitable for use in accordance with the methods of the invention include Escherichia coli (UniProt ID: P22259), Anaerobiospirillum succiniciproducens (UniProt ID: 009460), Actinobacillus succinogenes (UniProt ID: A6VKV4), Mannheimia succiniciproducens (SEQ ID NO: 6), and Haemophilus influenzae (UniProt ID: A5UDR5) PEP carboxykinase enzymes. In yet another embodiment, the recombinant host cell comprises one or more heterologous nucleic acids encoding a phosphoenolpyruvate carboxykinase that results in increased production of L- aspartate or beta-alanine under substantially anaerobic conditions as compared to a parent cell not comprising said one or more heterologous nucleic acids. In a specific embodiment, said phosphoenolpyruvate carboxykinase is Mannheimia succiniciproducens phosphoenolpyruvate carboxykinase (SEQ ID NO: 6).

2.2.2 L-aspartate dehydrogenase enzymes [0069] Provided herein is a recombinant host cell capable of producing L-aspartate or beta-alanine, the cell comprising one or more heterologous nucleic acids encoding a L-aspartate dehydrogenase. A L-aspartate dehydrogenase as used herein refers to any protein with L- aspartate dehydrogenase activity, meaning the ability to catalyze the conversion of oxaloacetate to L-aspartate.

[0070] Proteins capable of catalyzing this reaction suitable for use in the compositions and methods provided herein include both NAD-dependent L-aspartate dehydrogenase and NADP-dependent L-aspartate dehydrogenase enzymes. NAD-dependent L-aspartate dehydrogenase enzymes catalyze the conversion of oxaloacetate and ammonia to L-aspartate using NADH as the electron donor. Likewise, NADP-dependent L-aspartate dehydrogenase enzymes catalyze the conversion of oxaloacetate and ammonia to L-aspartate using NADPH as the electron donor. Many L-aspartate dehydrogenase enzymes are capable of using both NADH and NADPH as electron acceptors; as such, an NAD-dependent L-aspartate dehydrogenase may also be an NADP-dependent L-aspartate dehydrogenase (and vice versa). In these cases, usage of either NADH or NADPH as the electron donor is dependent on both the relative concentration of, and affinity constant of the L-aspartate dehydrogenase exhibits for, NADH or NADPH, respectively.

In some embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding an L-aspartate dehydrogenase, which is capable of producing L-aspartate and/or beta-alanine. L-aspartate dehydrogenases suitable for use in accordance with the methods of the invention include those selected from the non-limiting group consisting of Acinetobacter sp. SH024 (UniProt ID: D6JRV1; SEQ ID NO: 22), Arthrobacter aurescens (UniProt ID: A1R621), Burkholderia pseudomallei (UniProt ID: Q3JFK2; SEQ ID NO: 20), Burkholderia thailandensis (UniProt ID: Q2T559; SEQ ID NO: 19), Comamonas testosteroni (UniProt ID: D0IX49), Cupriavidus taiwanensis (UniProt ID: B3R8S4; SEQ ID NO: 2), Dinoroseobacter shibae (UniProt ID: A8LLH8; SEQ ID NO: 24), Klebsiella pneumoniae (UniProt ID: A6TDT8; SEQ ID NO: 23), Ochrobactrum anthropi (UniProt ID: A6X792; SEQ ID NO: 21), Polaromonas sp. (UniProt ID: Q126F5; SEQ ID NO: 18), Pseudomonas aeruginosa (UniProt ID: Q9HYA4; SEQ ID NO: 1), Ralstonia solanacearum (UniProt ID: Q8XRV9; SEQ ID NO: 17), Comamonas testosterone (UniProt ID: D0IX49; SEQ ID NO: 26), Cupriavidus pinatubonensis (UniProt ID: Q46VA0; SEQ ID NO: 27), and Ruegeria pomeroyi (UniProt ID: Q5LPG8; SEQ ID NO: 25) L- aspartate dehydrogenase. In certain embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1), which is capable of producing L-aspartate and/or beta-alanine. In other embodiments, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Cupriavidus taiwanensis L-aspartate dehydrogenase (SEQ ID NO: 2), which is capable of producing L-aspartate and/or beta-alanine. In some embodiments, a recombinant host cell of the present invention comprises a heterologous nucleic acid encoding an L-aspartate dehydrogenase selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, and 27, wherein the recombinant host cell is capable of producing L- aspartate and/or beta-alanine. In some embodiments, a recombinant host cell of the present invention comprises a plurality of heterologous nucleic acids, each encoding an L-aspartate dehydrogenase selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, and 27, wherein the recombinant host cell is capable of producing L-aspartate and/or beta-alanine.

Homologs to L-aspartate dehydrogenase enzymes

[0071] L-aspartate dehydrogenases also useful in the compositions and methods provided herein include those enzymes that are said to be "homologous" to any of the L-aspartate dehydrogenase enzymes described herein. Such homologs have the following characteristics: (1) is capable of catalyzing the conversion of oxaloacetate to L-aspartate; (2) it shares substantial sequence identity with any L-aspartate dehydrogenase described herein; (3) comprises a substantial number of amino acids corresponding to highly conserved amino acids in any L- aspartate dehydrogenase described herein; and (4) comprises one or more specific amino acids corresponding to strictly conserved amino acids in any L-aspartate dehydrogenase described herein.

[0072] A homolog is said to share substantial sequence identity to an L-aspartate dehydrogenase if the amino acid sequence of the homolog is at least 60%, at least 70%, at least 80%), at least 90%>, at least 95%>, or at least 97%> the same as that of a L-aspartate dehydrogenase amino acid sequence set forth herein.

[0073] A number of amino acids in L-aspartate dehydrogenase enzymes provided by the invention are highly conserved, and proteins homologous to an L-aspartate dehydrogenase enzyme of the invention will generally comprise amino acids corresponding to a substantial number of highly conserved amino acids. A homolog is said to comprise a substantial number of amino acids corresponding to highly conserved amino acids in a reference sequence if at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% of the highly conserved amino acids in the reference sequence are found in the homologous protein.

[0074] Highly conserved amino acids in Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1) are G8, G10, Al l, 112, G13, E69, C70, A71, A75, L84, V92, S94, G96, A97, G123, A124, 1125, G126, D129, L131, A134, V142, K148, P149, F174, G176, A178, A181, L184, P186, N188, N190, V191, A192, A193, T194, L197, A198, G201, V207, A211, D212, P213, N218, G226, A227, F228, G229, P239, N243, P244, K245, T246, S247, L249, T250, S253, R256, L258, and N260. In some embodiments, L-aspartate enzymes homologous to Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1) comprise amino acids corresponding to at least a 50% of these highly conserved amino acids. In some embodiments, L-aspartate enzymes homologous to Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1) comprise amino acids corresponding to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of these highly conserved amino acids.

[0075] Highly conserved amino acids in Cupriavidus taiwanensis L-aspartate dehydrogenase (SEQ ID NO: 2) are G8, G10, Al l, 112, G13, C69, A70, A74, L83, V91, S93, G95, A96, S121, G122, A123, 1124, G125, D128, L130, A133, V141, K147, P148, F173, E174, G175, A177, A180, L183, P185, N187, N189, V190, A191, A192, T193, L196, A197, G200, V206, A210, D211, P212, N217, G225, A226, F227, G228, P238, N242, P243, K244, T245, S246, L248, T249, S252, S252, R255, A256, L257, L257, and N259. In some embodiments, L- aspartate enzymes homologous to Cupriavidus taiwanensis L-aspartate dehydrogenase (SEQ ID NO: 2) comprise amino acids corresponding to at least 50% of these highly conserved amino acids. In some embodiments, L-aspartate enzymes homologous to Cupriavidus taiwanensis L- aspartate dehydrogenase (SEQ ID NO: 2) comprise amino acids corresponding to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of these highly conserved amino acids.

Strictly conserved amino acids in L-aspartate dehydrogenase enzymes

[0076] Some amino acids in L-aspartate dehydrogenase enzymes provided by the invention are strictly conserved, and proteins homologous to an L-aspartate dehydrogenase enzyme of the invention must comprise amino acid(s) corresponding to these strictly conserved amino. [0077] Amino acid H220 in SEQ ID NO: 1 functions as a general acid/base (although the invention is not to be limited by any theory of mechanism of action) and is necessary for enzyme activity; thus, an amino acid corresponding to H220 in SEQ ID NO: 1 is present in all enzymes homologous to SEQ ID NO: 1. Amino acid H220 in SEQ ID NO: 1 corresponds to amino acid HI 19 in SEQ ID NO: 2, and L-aspartate dehydrogenase enzymes homologous to SEQ ID NO: 2 must comprise an amino acid corresponding to HI 19 in SEQ ID NO: 2.

Additional L-aspartate dehydrogenase enzymes

[0078] In addition to L-aspartate dehydrogenase enzymes homologous to those described above, another class of L-aspartate dehydrogenase enzymes that can be expressed in engineered P. kudriavzevii to produce L-aspartate from oxaloacetate are L-aspartate transaminase (EC 2.6.1.1) enzymes, which catalyzes reduction of oxaloacetate to L-aspartate along with concomitant oxidation of glutamate to alpha-ketoglutarate. Using this enzyme, it is important to recycle the alpha- ketoglutarate back to glutamate to provide the glutamate substrate necessary for additional rounds of L-aspartate transaminase catalysis. This can be accomplished by expressing a glutamate dehydrogenase (EC 1.4.1.2) that reduces alpha-ketoglutarate back to glutamate using NADH as the electron donor. This alternative metabolic pathway to L-aspartate from oxaloacetate is most useful in cases where L-aspartate dehydrogenase activity is insufficient to produce L-aspartate at the desired rate. In some embodiments of the present invention, the recombinant host cell comprises a heterologous nucleic acid encoding a L-aspartate dehydrogenase that is an L-aspartate transaminase.

[0079] Examples of suitable L-aspartate transaminase enzymes include those selected from the non-limiting group consisting of Saccharomyces cerevisiae AAT2 (UnitProt ID: P23542), Schizosaccharomyces pombe L-aspartate transaminase (UniProt ID: 094320), Escherichia coli AspC (UniProt ID: P00509), Pseudomonas aeruginosa AspC (UniProt ID: P72173), and Rhizobium meliloti AatB (UniProt ID: Q06191), among others.

2.2.3 L-aspartate 1-decarboxylase enzymes

[0080] In various embodiments, the recombinant host cell further comprises a heterologous nucleic acid encoding a L-aspartate 1-decarboxylase. A L-aspartate 1- decarboxylase as used herein refers to any protein with L-aspartate decarboxylase activity, meaning the ability to catalyze the decarboxylation of L-aspartate to beta-alanine.

[0081] Proteins capable of catalyzing this reaction suitable for use in the compositions and methods provided herein include both bacterial L-aspartate 1 -decarboxylases and eukaryotic L-aspartate decarboxylases. Bacterial L-aspartate 1 -decarboxylases are pyruvoyl-dependent decarboxylases where the covalently bound pyruvoyl cofactor is produced by autocatalytic rearrangement of a specific serine residues (e.g., S25 in SEQ IDs NO: 4 and 5). Eukaryotic L- aspartate decarboxylases, in contrast, do not possess a pyruvoyl cofactor and instead possess a pyridoxal 5 '-phosphate cofactor. In some embodiments, the recombinant host cell comprises a heterologous nucleic acid encoding a bacterial L-aspartate 1 -decarboxylase and is capable of producing beta-alanine. In other embodiments, the recombinant host cell comprises a heterologous nucleic acid encoding a eukaryotic L-aspartate 1 -decarboxylase and is capable of producing beta-alanine.

[0082] Bacterial L-aspartate 1 -decarboxylase enzymes suitable for use in accordance with the methods of the invention include those selected from the non-limiting group consisting of Arthrobacter aurescens (UniProt ID: A1RDH3), Bacillus cereus (UniProt ID: A7GN78), Bacillus subtilis (UniProt ID: P52999; SEQ ID NO: 5), Burkholderia xenovorans (UniProt ID: Q143J3), Clostridium acetobutylicum (UniProt ID: P58285), Clostridium beijerinckii (UniProt ID: A6LWN4), Corynebacterium efficiens (UniProt ID: Q8FU86), Corynebacterium glutamicum (UniProt ID: Q9X4N0; SEQ ID NO: 4), Corynebacterium jeikeium (UniProt ID: Q4JXL3), Cupriavidus necator (UniProt ID: Q9ZHI5), Enterococcus faecalis (UniProt ID: Q833S7), Escherichia coli (UniProt ID: Q0TLK2), Helicobacter pylori (UniProt ID: P56065), Lactobacillus plantarum (UniProt ID: Q88Z02), Mycobacterium smegmatis (UniProt ID: A0QNF3), Pseudomonas aeruginosa (UniProt ID: Q9HV68), Pseudomonas fluorescens (UniProt ID: Q848I5), Staphylococcus aureus (UniProt ID: A6U4X7), and Streptomyces coelicolor (UniProt ID: P58286) L-aspartate 1 -decarboxylase. In one embodiment, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Bacillus subtilis L-aspartate 1 -decarboxylase (SEQ ID NO: 5) and is capable of producing beta-alanine. In another embodiment, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Corynebacterium L-aspartate 1 -decarboxylase (SEQ ID NO: 4) and is capable of producing beta-alanine.

[0083] In addition to the bacterial L-aspartate 1 -decarboxylase enzymes, the invention also provides eukaryotic L-aspartate 1 -decarboxylases suitable for use in the compositions and methods of the invention. Eukaryotic L-aspartate 1 -decarboxylase enzymes suitable for use in accordance with the methods of the invention include those selected from the non-limiting group consisting of Tribolium castaneum (UniProt ID: A9YVA8; SEQ ID NO: 3), Aedes aegypti (UniProt ID: Q171 S0), Drosophila mojavensis (UniProt ID: B4KIX9), and Dendroctonus ponderosae (UniProt ID: U4UTD4) L-aspartate 1 -decarboxylase. In one embodiment, the recombinant host cell provided herein comprises a heterologous nucleic acid encoding Tribolium castaneum L-aspartate 1 -decarboxylase (SEQ ID NO: 3) and is capable of producing beta- alanine.

[0084] L-aspartate 1 -decarboxylase enzymes also useful in the compositions and methods provided herein include those enzymes which are said to be "homologous" to any of the L-aspartate 1 -decarboxylase enzymes described herein. Such homologs have the following characteristics: (1) is capable of catalyzing the decarboxylation of L-aspartate to beta-alanine; (2) it shares substantial sequence identity with any L-aspartate 1 -decarboxylase described herein; (3) comprises a substantial number of amino acids corresponding to highly conserved amino acids in any L-aspartate 1 -decarboxylase described herein; and (4) comprises one or more specific amino acids corresponding to strictly conserved amino acids in any L-aspartate 1 -decarboxylase described herein.

Percent sequence identity

[0085] A homolog is said to share substantial sequence identity to an L-aspartate 1- decarboxylase if the amino acid sequence of the homolog is at least 60%, at least 70%, at least 80%), at least 90%, at least 95%, or at least 97% the same as that of a L-aspartate 1- decarboxylase amino acid sequence described herein.

Highly conserved amino acids in L-aspartate 1-decarboxylase enzymes

[0086] A number of amino acids in both bacterial and eukaryotic L-aspartate 1- decarboxylase enzymes provided herein are highly conserved, and proteins homologous to either a bacterial or a eukaryotic L-aspartate dehydrogenase enzyme of the invention will generally comprise amino acids corresponding to a substantial number of highly conserved amino acids. As described above, a homolog is said to comprise a substantial number of amino acids corresponding to highly conserved amino acids in a reference sequence if at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% of the highly conserved amino acids in the reference sequence are found in the homologous protein.

[0087] Highly conserved amino acids in Corynebacterium glutamicum L-aspartate 1- decarboxylase (SEQ ID NO: 4) are K9, Hl l, R12, A13, V15, T16, A18, L20, Y22, G24, S25, D29, E42, N51, G52, R54, T57, Y58, 160, G62, G65, G67, N72, G73, A74, A75, A76, G82, D83, V85, 186, Y90, E97, P103, and N112. In some embodiments, L-aspartate 1-decarboxylase enzymes homologous to Corynebacterium glutamicum L-aspartate 1-decarboxylase (SEQ ID NO: 4) comprise amino acids corresponding to at least a 50% of these highly conserved amino acids. In some embodiments, L-aspartate 1 -decarboxylase enzymes homologous to Corynebacterium glutamicum L-aspartate 1 -decarboxylase (SEQ ID NO: 4) comprise amino acids corresponding to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%), or more than 95% of these highly conserved amino acids.

[0088] Highly conserved amino acids in Bacillus subtilis L-aspartate 1 -decarboxylase

(SEQ ID NO: 5) are K9, Hl l, R12, A13, V15, T16, A18, L20, Y22, G24, S25, D29, E42, N51, G52, R54, T57, Y58, 160, G62, G65, G67, N72, G73, A74, A75, A76, G82, D83, V85, 186, Y90, E97, P103, and N112. In some embodiments, L-aspartate 1 -decarboxylase enzymes homologous to Bacillus subtilis L-aspartate 1 -decarboxylase (SEQ ID NO: 5) comprise amino acids corresponding to at least a 50% of these highly conserved amino acids. In some embodiments, L- aspartate 1 -decarboxylase enzymes homologous to Bacillus subtilis L-aspartate 1 -decarboxylase (SEQ ID NO: 5) comprise amino acids corresponding to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of these highly conserved amino acids.

[0089] Highly conserved amino acids in Tribolium castaneum L-aspartate 1- decarboxylase (SEQ ID NO: 3) are V88, P94, D102, LI 15, S126, V127, T129, H131, P132, F134, N136, Q137, L138, S140, D143, Y145, Q150, T153, D154, L156, N157, P158, S159, Y161, T162, E164, V165, P167, L171, M172, E173, E174, V176, L177, E179, M180, R181, 1183, G185, G191, G193, F195, P197, G198, G199, S200, A202, N203, G204, Y205, 1207, A210, R211, P216, K219, G222, L229, F232, T233, S234, E235, A237, H238, Y239, S240, K243, A245, F247, G249, G251, G264, P285, V288, T291, G293, T294, T295, V296, G298, A299, F300, D301, C310, K312, W316, H318, D320, A321, A322, W323, G324, G325, G326, A327, L328, S330, R334, L336, L337, G339, D344, S345, V346, T347, W348, N349, P350, H351, K352, L353, L354, A356, Q358, Q359, C360, S361, T362, L364, H367, L371, H375, A379, Y381, L382, F383, Q384, D386, K387, F388, Y389, D390, D394, G396, D397, H399, Q401, C402, G403, R404, A406, D407, V408, K410, F411, W412, M414, W415, A417, K418, G419, G422, H426, F431, R444, G446, P454, N458, F461, Y463, P465, R469, L481, A485, P486, K489, E490, M492, G496, M498, T501, Y502, Q503, N510, F511, F512, R513, V515, Q517, S519, L521, D525, M526, E532, E534, L536. In some embodiments, L-aspartate 1- decarboxylase enzymes homologous to Tribolium castaneum L-aspartate 1 -decarboxylase (SEQ ID NO: 3) comprise amino acids corresponding to at least a 50% of these highly conserved amino acids. In some embodiments, L-aspartate 1 -decarboxylase enzymes homologous to Tribolium castaneum L-aspartate 1 -decarboxylase (SEQ ID NO: 3) comprise amino acids corresponding to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%), or more than 95% of these highly conserved amino acids.

L-aspartate 1-decarboxylase strictly conserved amino acids

[0090] Some amino acids in L-aspartate 1-decarboxylase enzymes provided by the invention are strictly conserved, and proteins homologous to an L-aspartate 1-decarboxylase enzyme of the invention must comprise amino acid(s) corresponding to these strictly conserved amino acids.

[0091] Strictly conserved amino acids in both the Bacillus subtilis L-aspartate 1- decarboxylase (SEQ ID NO: 5) and Corynebacterium glutamicum L-aspartate 1-decarobxylase (SEQ ID NO: 4) amino acid sequences are K9, G24, S25, R54, and Y58. The epsilon-amine group on K9 is believed to form an ion pair with alpha-carboxyl group on L-aspartate, R54 is believed to form an ion pair with the gamma-carboxyl group on L-aspartate, and Y58 is believed to donate a proton to an extended enolate reaction intermediate; thus, these three amino acids are important for L-aspartate binding and subsequent decarboxylation. Additionally, proteolytic cleavage between residues G24 and S25 produces an N-terminal pyruvoyl moiety also necessary for decarboxylase activity. Therefore, enzymes homologous to SEQ ID NO: 4 and/or SEQ ID 5 will comprise amino acids corresponding to K9, G24, S25, R54, and Y58 in SEQ ID NOs: 4 and/or 5.

[0092] Strictly conserved amino acids in the Tribolium castaneum L-aspartate 1- decarboxylase (SEQ ID NO: 3) amino acid sequence are Q137, H238, K352, and R513. Q137 and R513 form a salt bridge with the gamma-carboxyl group on L-aspartate, H238 is a base- stacking residue with the pyridine ring of the pyridoxal 5 '-phosphate cofactor, and K352 forms a Schiff base linkage with the pyridoxal 5 '-phosphate cofactor. Thus, these four amino acids are important for L-aspartate or cofactor binding and subsequent L-aspartate decarboxylation, and enzymes homologous to SEQ ID NO: 3 will comprise amino acids corresponding to Q137, H238, K352, and R513 in SEQ ID NO : 3.

2.2.4 Consensus Sequences

[0093] The present invention also provides consensus sequences useful in identifying and/or constructing L-aspartate dehydrogenases and L-aspartate 1 -decarboxylases suitable for use in accordance with the methods of the invention. In various embodiments, these consensus sequences comprise active site amino acid residues believed to be necessary (although the invention is not to be limited by any theory of mechanism of action) for substrate recognition and reaction catalysis, as described below. Thus, a L-aspartate dehydrogenase encompassed by a L- aspartate dehydrogenase consensus sequence provided herein has an enzymatic activity that is identical, or essentially identical, or at least substantially similar with respect to ability to reduce oxaloacetate to L-aspartate to that of one of the enzymes exemplified herein. Likewise, a L- aspartate 1 -decarboxylase encompassed by a L-aspartate 1 -decarboxylase consensus sequence provided herein has an enzymatic activity that is identical, or essentially identical, or at least substantially similar with respect to ability to decarboxylate L-aspartate to beta-alanine to that of one of the enzymes exemplified herein.

[0094] Enzymes also useful in the compositions and methods provided herein include those that are homologous to consensus sequences provided by the invention. As noted above, any enzyme substantially homologous to an enzyme described herein can be used in a host cell of the invention.

[0095] The percent sequence identity of an enzyme relative to a consensus sequence is determined by aligning the enzyme sequence against the consensus sequence. Those skilled in the art will recognize that various sequence alignment algorithms are suitable for aligning an enzyme with a consensus sequence. See, for example, Needleman, SB, 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 enzyme sequence relative to the consensus sequence, the percentage of positions where the enzyme possesses an amino acid (or dash) described by the same position in the consensus sequence determines the percent sequence identity.

2.2.4.1 L-aspartate dehydrogenase consensus sequences

[0096] An L-aspartate dehydrogenase consensus sequence (SEQ ID NO: 14) provides the 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 an L-aspartate dehydrogenase. Those of skill in the art will recognize that fixed amino acids and conserved amino acids in these consensus sequences are identical to (in the case of fixed amino acids) or consistent with (in the case of conserved amino acids) with the wild-type sequence(s) on which the consensus sequence is based. Following alignment of a query protein with a consensus sequence provided herein, the occurrence of a dash in the aligned query protein sequence indicates an amino acid deletion in the query protein sequence relative to the consensus sequence at the indicated position. Likewise, the occurrence of a dash in the aligned consensus sequence indicates an amino acid addition in the query protein sequence relative to the consensus sequence at the indicated position. Amino acid additions and deletions are common to proteins encompassed by consensus sequences of the invention, and their occurrence is reflected as a lower percent sequence identity (i.e., amino acid addition or deletions are treated identically to amino acid mismatches when calculating percent sequence identity).

[0097] In various embodiments, L-aspartate dehydrogenase enzymes suitable for use in accordance with the methods of the invention have L-aspartate dehydrogenase activity and comprise an amino acid sequence with at least 55%, at least 60%>, at least 70%, at least 80%>, at least 90%), or at least 95% sequence identity to SEQ ID NO: 14. For example, the Pseudomonas aeruginosa L-aspartate dehydrogenase (SEQ ID NO: 1) and Cupriavidus taiwanensis L-aspartate dehydrogenase (SEQ ID NO: 2) sequences are 79% and 83%> identical to consensus sequence SEQ ID NO: 14, and are therefore encompassed by consensus sequence SEQ ID NO: 14.

[0098] In enzymes homologous to SEQ ID NO: 14, amino acids that are highly conserved are G8, G10, Al l, 112, G13, E69, A71, G72, H73, A75, H79, P82, L84, G87, S94, G96, A97, L98, A110, Al l l, G114, L120, G123, A124, 1125, G126, D129, A130, A133, A134, G137, G138, L139, V142, Y144, G146, R147, K148, P149, W153, T156, P157, E159, D163, L164, 1173, F174, G176, A178, A181, A182, P186, K187, N188, A189, N190, V191, A192, A193, T194, A198, G199, G201, L202, T205, V207, L209, A211, D212, P213, N218, H220, A224, G226, A227, F228, G229, L233, P239, L240, N243, P244, K245, T246, S247, A248, L249, T250, S253, R256, A257, N260, and 1267. In various embodiments, L-aspartate dehydrogenase enzymes homologous to SEQ ID NO: 14 comprise at least 50%, at least 60%>, at least 70%), at least 80%>, at least 85%>, at least 90%, at least 95%, or sometimes all of these highly conserved amino acids at positions corresponding to the highly conserved amino acids identified in SEQ ID NO: 14. In some embodiments, each of these highly conserved amino acids are found in a desired L-aspartate dehydrogenase, as provided in SEQ ID NOs: 1 and 2.

[0099] Amino acid H220 in SEQ ID NO: 14 functions as a general acid/base (although the invention is not to be limited by any theory of mechanism of action) and is necessary for enzyme activity; thus, an amino acid corresponding to H220 in consensus sequence SEQ ID NO: 14 is found in enzymes homologous to SEQ ID NO: 14. For example, the strictly conserved amino acid corresponding to H220 in consensus sequence SEQ ID NO: 14 is found in L- aspartate dehydrogenases set forth in SEQ ID NOs: 1 and 2.

2.2.4.2 L-aspartate 1-decarboxylase Consensus Sequences

[00100] L-aspartate 1 -decarboxylases also useful in the compositions and methods provided herein include those that are homologous to L-aspartate 1-decarboxylase consensus sequences described herein. Any L-aspartate 1-decarboxylase substantially homologous to an L- aspartate 1-decarboxylase consensus sequence described herein can be used in a host cell of the invention.

[00101] The invention provides two L-aspartate 1-decarboxylase consensus sequences: (i) L- aspartate 1-decarboxylase based on bacterial L-aspartate 1-decarboxylase enzymes (SEQ ID NO: 15), and (ii) L-aspartate 1-decarboxylase based on eukaryotic L-aspartate 1-decarboxylase enzymes (SEQ ID NO: 16). The consensus sequences provide 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 an L-aspartate dehydrogenase of that class. Those of skill in the art will recognize that fixed amino acids and conserved amino acids in these consensus sequences are identical to (in the case of fixed amino acids) or consistent with (in the case of conserved amino acids) with the wild- type sequence(s) on which the consensus sequence is based. Following alignment of a query protein with a consensus sequence provided herein, the occurrence of a dash in the aligned query protein sequence indicates an amino acid deletion in the query protein sequence relative to the consensus sequence at the indicated position. Likewise, the occurrence of a dash in the aligned consensus sequence indicates an amino acid addition in the query protein sequence relative to the consensus sequence at the indicated position. Amino acid additions and deletions are common to proteins encompassed by consensus sequences of the invention, and their occurrence is reflected as a lower percent sequence identity (i.e., amino acid addition or deletions are treated identically to amino acid mismatches when calculating percent sequence identity).

Bacterial L-aspartate 1-decarboxylase Consensus Sequences

[0102] The invention provides a L-aspartate 1-decarboxylase consensus sequence based on bacterial L-aspartate 1-decarboxylase enzymes (SEQ ID NO: 15), and in various embodiments, L-aspartate 1-decarboxylase enzymes suitable for use in accordance with the methods of the invention have L-aspartate 1-decarboxylase activity and comprise an amino acid sequence with at least 40%, at least 45%, at least 50%, or at least 55% sequence identity to SEQ ID NO: 15. The Bacillus subtilis L-aspartate 1-decarboxylase (SEQ ID NO: 5) and Corynebacterium glutamicum L-aspartate 1 -decarboxylase (SEQ ID NO: 4) amino acid sequences are 55% and 79% identical to consensus sequence SEQ ID NO: 15, and are therefore encompassed by consensus sequence SEQ ID NO: 15.

[0103] In enzymes homologous to SEQ ID NO: 15, amino acids that are highly conserved are K9, Hl l, R12, A13, V15, T16, A18, L20, Y22, G24, S25, D29, E42, N51, G52, R54, T57, Y58, 160, G62, G65, G67, N72, G73, A74, A75, A76, G82, D83, V85, 186, Y90, E97, P103, and N112. In various embodiments, L-aspartate 1 -decarboxylase enzymes homologous to SEQ ID NO: 15 comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%), at least 95%, or sometimes all of these highly conserved amino acids at positions corresponding to the highly conserved amino acids identified in SEQ ID NO: 15. For example, all of the highly conserved amino acids are found in the L-aspartate 1 -decarboxylase sequences set forth in SEQ ID NOs: 4 and 5.

[0104] Five strictly conserved amino acids (K9, G24, S25, R54, and Y58) are present in consensus sequence SEQ ID NO: 15, and these residues are important for L-aspartate 1- decarboxylase activity. The function, although the invention is not to be limited by any theory of mechanism of action, of each strictly conserved amino acid is as follows. The epsilon-amine group on K9 forms an ion pair with alpha-carboxyl group on L-aspartate, R54 is forms an ion pair with the gamma-carboxyl group on L-aspartate, and Y58 donates a proton to an extended enolate reaction intermediate. Additional strictly conserved residues in SEQ ID NO: 15 are G24 and S25, and proteolytic cleavage between G24 and S25 results in production of an N-terminal pyruvoyl moiety required for decarboxylase activity. Enzymes homologous to consensus sequence SEQ ID NO: 15 comprise amino acids corresponding to all five of the strictly conserved amino acids identified in consensus sequence SEQ ID NO: 15.

Eukaryotic L-aspartate 1-decarboxylase Consensus Sequences

[0105] The invention provides a second L-aspartate 1-decarboxylase consensus sequence based on eukaryotic L-aspartate 1-decarboxylase enzymes (SEQ ID NO: 16). In various embodiments, L-aspartate 1-decarboxylase enzymes suitable for use in accordance with the methods of the invention have L-aspartate 1-decarboxylase activity and comprise an amino acid sequence with at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% sequence identity to SEQ ID NO: 16. The Tribolium castaneum L-aspartate 1-decarboxylase (SEQ ID NO: 3) amino acid sequence is 70% identical to consensus sequence SEQ ID NO: 16, and is therefore encompassed by consensus sequence SEQ ID NO: 16. [0106] In enzymes homologous to SEQ ID NO: 16, highly conserved amino acids are

V130, P136, D144, L157, S168, V169, T171, H173, P174, F176, N178, Q179, L180, S182, D185, Y187, Q192, T195, D196, L198, N199, P200, S201, Y203, T204, E206, V207, P209, L213, M214, E215, E216, V218, L219, E221, M222, R223, 1225, G227, G234, G236, F238, P240, G241, G242, S243, A245, N246, G247, Y248, 1250, A253, R254, P259, K262, G265, L272, F275, T276, S277, E278, A280, H281, Y282, S283, K286, A288, F290, G292, G294, G307, P328, V331, T334, G336, T337, T338, V339, G341, A342, F343, D344, C353, K355, W359, H361, D363, A364, A365, W366, G367, G368, G369, A370, L371, S373, R377, L379, L380, G382, D387, S388, V389, T390, W391, N392, P393, H394, K395, L396, L397, A399, Q401, Q402, C403, S404, T405, L407, H410, L414, H418, A422, Y424, L425, F426, Q427, D429, K430, F431, Y432, D433, D437, G439, D440, H442, Q444, C445, G446, R447, A449, D450, V451, K453, F454, W455, M457, W458, A460, K461, G462, G465, H469, F474, R487, G489, P497, N501, F504, Y506, P508, R512, L525, A529, P530, K533, E534, M536, G540, M542, T545, Y546, Q547, N554, F555, F556, R557, V559, Q561, S563, L565, D569, M570, E576, E578, and L580. In various embodiments, L-aspartate 1 -decarboxylase enzymes homologous to SEQ ID NO: 16 comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or sometimes all of these highly conserved amino acids at positions corresponding to the highly conserved amino acids identified in SEQ ID NO: 16. All of these highly conserved amino acids are found in the Tribolium castaneum L-aspartate 1- decarboxylases set forth in SEQ ID NO: 3.

[0107] Strictly conserved amino acids in the eukaryotic L-aspartate 1 -decarboxylase consensus sequence (SEQ ID NO: 16) are Q179, H281, K395, and R557. The function, although the invention is not to be limited by any theory of mechanism of action, of each strictly conserved amino acid is as follows. Q179 and R557 form a salt bridge with the gamma-carboxyl group on L-aspartate, H281 is a base-stacking residue with the pyridine ring of the pyridoxal 5'- phosphate cofactor, and K395 forms a Schiff base linkage with the pyridoxal 5 '-phosphate cofactor. Thus, these four amino acids are important for L-aspartate or cofactor binding and subsequent L-aspartate decarboxylation. Enzymes homologous to consensus sequence SEQ ID NO: 16 comprise amino acids corresponding to all four strictly conserved amino acids identified in consensus sequence SEQ ID NO: 16. All four of these strictly conserved amino acids are found in the Tribolium castaneum L-aspartate 1 -decarboxylase set forth in SEQ ID NO: 3.

Section 3: Deletions or disruption of endogenous nucleic acids [0108] In another aspect, the invention provides host cells genetically modified to delete or otherwise reduce the activity of endogenous proteins. Specific nucleic acid sequences are partially, substantially, or completely deleted or disrupted, silenced, inactivated, or down- regulated in order to partially, substantially, or completely reduce or eliminate the activity for which they encode, as in, for example, expression or activity of an enzyme. As used herein, "deletion or disruption" with regard to a nucleic acid means that either all or part of a protein coding region, a promoter, a terminator, and/or other regulatory element is modified (such as by deletion, insertion, or mutation of nucleic acids) such that the nucleic acid no longer produces an protein, produces a reduced quantity of an protein, or produces a protein with reduced activity (e.g., reduced enzymatic activity).

[0109] As used herein, "deletion or disruption" with regard to an enzyme means deletion or disruption of at least one, and often more than one, and sometimes all copies of nucleic acid(s) encoding enzymes with the specified activity. Many host cells suitable for use in the compositions and methods of the invention comprise two or more endogenous nucleic acids encoding two or more enzymes with the same activity. For example, diploid, triploid, and tetraploid microbes comprise two, three, and four sets of chromosomes, respectively, and two nucleic acids encoding for two enzymes with the same enzyme activity are found on each chromosome pair. Likewise, gene duplication events can lead to the occurrence of two or more nucleic acids on the genome of a host cell encoding for two or more enzymes with the same activity. In some embodiments, the recombinant host cells comprise a deletion or disruption of one nucleic acid encoding an enzyme. In other embodiments, the recombinant host cells comprise a deletion or disruption of more than one nucleic acids encoding an enzyme, and sometimes all nucleic acids encoding an enzyme.

[0110] In certain embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more metabolic pathways. As used herein, "deletion or disruption" with regard to a metabolic pathway means that the pathway produces a reduced quantity of one or more end-products of the metabolic pathway. In certain embodiments, deletion or disruption of a metabolic pathway is accomplished by deletion or disruption of one or more nucleic acids encoding metabolic pathway enzymes. In some of these embodiments, the recombinant host cell comprising said deleted or disrupted metabolic pathway no longer produces the end-product of the metabolic pathway, or produces at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% less end-product of the metabolic pathway as compared to a parental cell.

[0111] In certain embodiments, the nucleic acids deleted or disrupted as described herein may be endogenous to the native strain of the microorganism, and may be understood to be "native nucleic acids" or "endogenous nucleic acids". A nucleic acid is thus an endogenous nucleic acid if it has not been genetically modified or manipulated through human intervention in a manner that intentionally alters the genotype and/or phenotype of the microorganism. For example, a nucleic acid of a wild type organism may be considered to be an endogenous nucleic acid. In other embodiments, the nucleic acids targeted for deletion or disruption may be heterologous to the microorganism.

[0112] In certain embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more nucleic acids encoding enzymes. In some of these embodiments, the host cells comprising the one or more deleted or disrupted nucleic acids no longer produce an enzyme, or produce less than 10%>, less than 25%, less than 50%, less than 75%), less than 90%, less than 95%, or less than 97% of the amount of enzyme produced by parental cells. In other embodiments, the recombinant host cells comprising the deleted or disrupted nucleic acid(s) produces the same amount of enzyme as parental cells, but the enzyme exhibits reduced activity as compared to the enzyme encoded by the unmodified nucleic acid. In some of these embodiments, the deleted or disrupted nucleic acid no longer encodes for an active enzyme, or encodes for an enzyme with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% reduced activity as compared to the enzyme encoded by the endogenous nucleic acid. Those skilled in the art will recognize that deletion or disruption of a nucleic acid can simultaneously result in both a decrease in the quantity of an enzyme produced by a recombinant host cell as well as a decrease in the activity of an enzyme encoded by the deleted or disrupted nucleic acid.

3.1. Deletion or disruption of endogenous anaerobic pathways and enzymes encoding endogenous anaerobic pathway enzymes

[0113] The present invention describes the engineering of a recombinant host cell to convert various endogenous anaerobic fermentation pathways into anaerobic L-aspartate, and optionally beta-alanine, pathways. Microbes will not grow under anaerobic growth conditions unless the fermentation pathway is redox balanced (i.e., there is no net accumulation of NADH, NADPH, or other redox cofactor). [0114] Reduction and oxidation (redox) reactions play a key role in anaerobic metabolism, allowing the transfer of electrons from one compound to another, and thereby creating free energy for use in cellular metabolism. Redox co-factors facilitate the transfer of electrons from one chemical to another within the host cell. Several compounds and proteins can function as redox co-factors. During anaerobic catabolism of carbohydrates the most relevant co- factors are nicotinamide adenine dinucleotides (NADH and NADPH), and the iron sulfur protein ferredoxin (Fd). Typically, NADH is the most relevant co-factor in yeast cells during anaerobic catabolism of carbohydrates.

[0115] In order for cellular growth, the redox co-factors must discharge the same number of electrons they accept; thus, the net electron accumulation in the host cell is zero. Electrons are placed onto redox co-factors during carbohydrate catabolism, and must be removed from redox co-factors during end-product formation. In order for an end-product to be produced at high yield under anaerobic conditions the type and number of redox co-factors used during carbohydrate catabolism must match the type and number of redox co-factors used during end-product formation.

[0116] Carbohydrate catabolism ends in the formation of pyruvate, and electrons are removed during the conversion of glyceraldehyde 3-phosphate to 1, 3 -biphosphogly cerate (providing two electrons). This reaction is catalyzed by glyceraldehyde phosphate dehydrogenase (GAPDH; EC 1.2.1.12), and in yeast the endogenous enzyme uses NAD+ is used as the electron acceptor. When using glucose as the carbohydrate, two mols glyceraldehyde 3- phosphate can be theoretically produced per mol glucose, and thus two mols NADH can theoretically be produced per mol glucose in host cells expressing an NAD-dependent GAPDH. GAPDH enzymes may use alternate co-factors, including NADPH; NADP-dependent GAPDH enzymes are categorized under enzyme commission number EC 1.2.1.13, and include those found in Chlamydomonas reinhardtii, Clostridium acetobutylicum, Spinacia oleracea, and Sulfolobus solfataricus, among others. Host cells comprising NAD-dependent GAPDH enzymes can be engineered using standard microbial engineering techniques to express NADP-dependent GAPDH enzymes and thus produce NADPH, or a combination of NADH and NADPH, during carbohydrate catabolism to pyruvate.

[0117] Redox co-factors accepting electrons during catabolism of carbohydrates to pyruvate must discharge those electrons during production of the fermentation end- product to enable anaerobic growth and/or production of the end-product at high yield. Microbes capable of growth under substantially anaerobic conditions comprise one or more endogenous anaerobic fermentation pathways whose activity results in the reconsumption of redox cofactors produced during carbohydrate catabolism. The activity of endogenous anaerobic fermentation pathway(s) reduces the availability of redox cofactors for use by the heterologous L-aspartate pathway enzymes of the invention, thereby decreasing L-aspartate and/or beta-alanine yields from carbohydrates. Therefore, deletion or disruption of endogenous anaerobic fermentation pathways and nucleic acids encoding endogenous anaerobic fermentation pathway enzymes is useful for increasing the yield of L-aspartate and/or beta-alanine produced by recombinant host cells of the invention grown under substantially anaerobic conditions.

[0118] An anaerobic fermentation pathway is any metabolic pathway that: (i) comprises enzymes that reconsume redox cofactors produced during carbohydrate catabolism, and (ii) whose activity results in a detectable level of end-product in host cells grown under substantially anaerobic conditions. Examples of anaerobic fermentation pathways include, but are not limited to, ethanol, glycerol, malate, lactate, 1-butanol, isobutanol, 1,3 -propanediol, and 1,2-propanediol anaerobic fermentation pathways. For example, ethanol is the main fermentation end-product of most wild-type microbes, and especially yeast, grown anaerobically on carbohydrate, and the redox co-factors produced during catabolism of carbohydrates to pyruvate are reconsumed during conversion of pyruvate to ethanol. In the recombinant host cells of the present invention, the endogenous fermentation pathway, typically, but not limited to, an ethanol fermentation pathway, has been deleted or disrupted. Redox cofactors produced during pyruvate formation from glucose are reconsumed during production of L-aspartate through the activity of an L- aspartate dehydrogenase, and the net result is a redox balanced, and thus anaerobic, fermentation pathway capable of producing L-aspartate and/or beta-alanine at high yield.

3.2. Deletion or disruption of ethanol fermentation pathways and nucleic acids encoding ethanol fermentation pathway enzymes

[0119] Deletion or disruption of ethanol fermentation pathway(s) and nucleic acids encoding ethanol fermentation pathway enzymes is important for engineering a recombinant host cell capable of efficient production of L-aspartate and/or beta-alanine under substantially anaerobic conditions.

[0120] In yeast host cells, an ethanol fermentation pathway comprises two enzymes: pyruvate decarboxylase and alcohol dehydrogenase. Pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of pyruvate to acetaldehyde; alcohol dehydrogenase (EC 1.1.1.1) catalyzes the reduction of acetaldehyde to ethanol along with concomitant oxidation of NADH to NAD+ and/or NADPH to NADP+. In yeast cells of the invention, an ethanol fermentation pathway can be deleted or disrupted by deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylase and/or alcohol dehydrogenase. In certain embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more endogenous nucleic acids encoding an ethanol fermentation pathway enzyme. In some embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylase. In some embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more nucleic acids encoding alcohol dehydrogenase. In some embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylase and alcohol dehydrogenase.

[0121] Deletion or disruption of nucleic acids encoding ethanol fermentation pathway enzymes decrease the ability of the recombinant host cell to produce ethanol and/or increases the ability of the recombinant host cell to produce L-aspartate or beta-alanine. In various embodiments, recombinant host cells comprising deletion or disruption of one or more nucleic acids encoding ethanol fermentation pathway enzymes decreases ethanol production by at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or at least 99%) as compared to parental cells that do not comprise this genetic modification. In some embodiments, recombinant host cells comprising deletion or disruption of one or more nucleic acids encoding ethanol fermentation pathway enzymes increase L-aspartate or beta-alanine production by at least 10%>, at least 25%, at least 50%, at least 75%, at least 100%>, or more than 100%) as compared to parental cells that do not comprise this genetic modification.

3.2.1 Deletion or disruption of nucleic acids encoding pyruvate decarboxylase

[0122] In various embodiments, the recombinant host cells comprise a deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylase. In some embodiments, one nucleic acid encoding pyruvate decarboxylase is deleted or disrupted. In other embodiments, two nucleic acids encoding pyruvate decarboxylase are deleted or disrupted. In other embodiments, more than two nucleic acids encoding pyruvate decarboxylase are deleted or disrupted. In still further embodiments, all nucleic acids encoding pyruvate decarboxylase are deleted or disrupted. [0123] In various embodiments, the recombinant host cell comprises a deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylases with an amino acid sequence set forth in SEQ ID NO: 9, or one or more nucleic acids encoding enzymes with an amino sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, at least 97%), or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. In specific embodiments wherein the recombinant host cell of the invention is Pichia kudriavzevii, the recombinant host cell comprises a deletion or disruption of two nucleic acids encoding pyruvate decarboxylases with an amino acid sequence set forth in SEQ ID NO: 9, or two nucleic acids encoding enzymes with amino sequences with at least 50%, at least 60%, at least 70%, at least 80%), at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0124] Some yeast cells have more than one nucleic acid encoding pyruvate decarboxylase, and in these host cells one or more nucleic acids encoding pyruvate decarboxylases may be deleted or disrupted for the purposes of deleting or disrupting the ethanol fermentation pathway. For example, wild type Saccharomyces cerevisiae has three endogenous pyruvate decarboxylases: PDC1 (SEQ ID NO: 10), PDC5, and PDC6. PDC1 is the major isoform (has the highest expression level and/or activity) in S. cerevisiae while PDC5 and PDC6 are minor isoforms. In certain embodiments wherein the recombinant host cell of the invention is Saccharomyces cerevisiae, the recombinant host cell comprises a deletion or disruption of one or more nucleic acids encoding pyruvate decarboxylases with an amino acid sequence set forth in SEQ ID NO: 10, or one or more nucleic acids encoding enzymes with amino acid sequences with at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. For example, S. cerevisiae pyruvate decarboxylases PDC5 and PDC6 have 88% and 84% amino acid sequence identity, respectively, to the amino acid sequence set forth in SEQ ID NO: 10.

3.2.2 Deletion or disruption of nucleic acids encoding alcohol dehydrogenase

[0125] In addition to deletion or disruption of nucleic acid encoding pyruvate decarboxylase, a yeast ethanol fermentation pathway can be deleted or disrupted by deletion or disruption of nucleic acids encoding alcohol dehydrogenase. In various embodiments, the recombinant host cells provided herein comprise a deletion or disruption of one or more nucleic acids encoding alcohol dehydrogenase. In some embodiments, one nucleic acid encoding alcohol dehydrogenase is deleted or disrupted. In other embodiments, two nucleic acids encoding alcohol dehydrogenase are deleted or disrupted. In other embodiments, more than two nucleic acids encoding alcohol dehydrogenase are deleted or disrupted. In still further embodiments, all nucleic acids encoding alcohol dehydrogenase are deleted or disrupted.

[0126] In certain embodiments, the recombinant host cell comprises a deletion or disruption of a nucleic acid encoding an alcohol dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 11, or with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or greater than 97% sequence identity to SEQ ID NO: 11. In specific embodiments wherein the recombinant host cell of the invention is Pichia kudriavzevii, the recombinant host cell comprises a deletion or disruption of two nucleic acids encoding alcohol dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 11, or two nucleic acids encoding enzymes with amino sequences with at least 50%, at least 60%, at least 70%), at least 80%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11.

3.3 Deletion or disruption of malate fermentation pathways and nucleic acids encoding malate dehydrogenase

[0127] A malate fermentation pathway comprises one enzyme, malate dehydrogenase

(EC 1.1.1.37), which catalyzes the formation of malate (the end-product of a malate fermentation pathway) from oxaloacetate along with concomitant oxidation of NADH to NAD+. Those skilled in the art will recognize that malate dehydrogenase and L-aspartate dehydrogenase use the same substrate (oxaloacetate) and will often use the same redox cofactor (NADH or NADPH) to produce their respective products. Thus, the expression of endogenous malate dehydrogenase, and particularly malate dehydrogenase located in the cytosol of yeast cells, can decrease anaerobic production of L-aspartate and/or beta-alanine. Thus, deletion or disruption of a malate fermentation pathway is useful for increasing L-aspartate or beta-alanine production in recombinant host cells of the invention grown under substantially anaerobic conditions. A malate fermentation pathway can be deleted or disrupted by deletion or disruption of nucleic acids encoding malate dehydrogenase.

[0128] In various embodiments, the recombinant host cells comprise a deletion or disruption of one or more nucleic acids encoding malate dehydrogenase. In some embodiments, one nucleic acid encoding malate dehydrogenase is deleted or disrupted. In other embodiments, two nucleic acids encoding malate dehydrogenase are deleted or disrupted. In other embodiments, more than two nucleic acids encoding malate dehydrogenase are deleted or disrupted. In still further embodiments, all nucleic acids encoding malate dehydrogenase are deleted or disrupted.

[0129] In various embodiments, the recombinant host cell comprises a deletion or disruption of one or more nucleic acids encoding malate dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 13, or one or more nucleic acids encoding enzymes with an amino sequence with at least 50%, at least 60%>, at least 70%, at least 80%>, at least 95%, at least 97%), or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13. In specific embodiments wherein the recombinant host cell of the invention is Pichia kudriavzevii, the recombinant host cell comprises a deletion or disruption of two nucleic acids encoding malate dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 13, or two nucleic acids encoding enzymes with amino sequences with at least 50%, at least 60%, at least 70%, at least 80%), at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

3.4 Deletion or disruption of additional byproduct metabolic pathways and nucleic acids encoding byproduct metabolic pathway enzymes

[0130] Besides ethanol and malate, additional byproducts are formed by host cells of the invention, including glycerol, acetic acid, and various four-carbon dicarboxylic acids (e.g., fumarate and succinate). Deletion or disruption of these byproduct metabolic pathways and nucleic acids encoding byproduct metabolic pathway enzymes are also useful for increasing L- aspartate or beta-alanine production by host cells of the invention.

[0131] In certain embodiments, recombinant host cells provided herein comprise a deletion or disruption of a glycerol fermentation pathway. A glycerol fermentation pathway comprises one enzyme, NAD-dependent glycerol-3 -phosphate dehydrogenase (EC 1.1.1.8), which catalyzes the formation of glycerol (the end-product of a glycerol metabolic pathway) from glycerol-3 -phosphate along with concomitant oxidation of NADH to NAD+. Glycerol fermentation pathway activity decreases the pool of NADH available for use L-aspartate dehydrogenase in the production of L-aspartate from oxaloacetate in recombinant host cells of the invention grown under substantially anaerobic conditions. Thus, deletion or disruption of a glycerol fermentation pathway is useful for increasing L-aspartate or beta-alanine production in recombinant host cells of the invention. A glycerol metabolic pathway can be deleted or disrupted by deletion or disruption of nucleic acids encoding NAD-dependent glycerol-3- phosphate dehydrogenase. [0132] In various embodiments, the recombinant host cells comprise a deletion or disruption of one or more nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase. In some embodiments, one nucleic acid encoding NAD-dependent glycerol-3 - phosphate dehydrogenase is deleted or disrupted. In other embodiments, two nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase are deleted or disrupted. In other embodiments, more than two nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase are deleted or disrupted. In still further embodiments, all nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase are deleted or disrupted.

[0133] In various embodiments, the recombinant host cell comprises a deletion or disruption of one or more nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 12, or one or more nucleic acids encoding enzymes with an amino sequence with at least 50%, at least 60%, at least 70%, at least 80%), at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In specific embodiments wherein the recombinant host cell of the invention is Pichia kudriavzevii, the recombinant host cell comprises a deletion or disruption of two nucleic acids encoding NAD-dependent glycerol-3 -phosphate dehydrogenase with an amino acid sequence set forth in SEQ ID NO: 12, or two nucleic acids encoding enzymes with amino sequences with at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12.

Section 4. Genetic modifications to increase L-aspartic acid production

[0134] In another aspect, the invention provides host cells genetically modified to express heterologous nucleic acids encoding enzymes enabling energy efficient L-aspartic acid production. "Energy efficient", as defined herein, refers to production of L-aspartic acid with a lower ATP requirement as compared to a parental, or control strain. Decreasing the expenditure of ATP is important aspect of L-aspartate production under substantially anaerobic conditions. If host cell ATP requirements become sufficiently high, additional oxygen must be provided to the culture to support L-aspartate production. Two enzymes useful for increasing the energy efficiency of L-aspartate production in genetically modified host cells of the invention are ureases (EC 3.5.1.5) and L-aspartate permeases.

Urease enzymes

[0135] Ammonia is a co-substrate necessary for L-aspartate production using an L- aspartate dehydrogenase and a nitrogen source must be provided to the fermentation for L- aspartate production. Two commonly used nitrogen sources for fermentative production of small-molecule products are ammonia and urea. Urea is the preferred source of nitrogen as compared to ammonia for at least three reasons. First, urea is non-toxic and can be added at high concentrations; by comparison, ammonia, another commonly used nitrogen source in industry, is basic and high concentrations are toxic to many host cells. Second, urea is neutrally charged, can diffuse across the host cell plasma membrane (i.e., no energy is expended for transport), and the fermentation pH is unaffected by its addition to the fermentation medium. By comparison, ammonia charged and must be transported into the cell enzymatically. Third, the breakdown of urea also releases C0 ₂ , a co-substrate for enzymes in all L-aspartate biosynthetic pathways. No CO ₂ is released during catabolism of ammonia.

[0136] Urease enzymes catalyze the hydrolysis of one molecule urea to one molecule carbamate and one molecule ammonia; subsequent to enzymatic production, the one molecule carbamate then degrades into a ammonia and carbonic acid. Thus, in sum, urease activity results in production of two molecules urea and one molecule carbon dioxide per catalytic cycle. Importantly, urease performs this reaction without expenditure of ATP. In contrast, alternative metabolic pathways capable of catalyzing conversion of urea to ammonia and carbon dioxide do require expenditure of ATP. For example, many host cells, including many yeast host cells, use a urea catabolic pathway comprising the enzymes urea carboxylase and allophenate hydrolase; using this pathway, one molecule ATP is expended per molecule urea catabolized.

[0137] In many embodiments of the present invention, host cells engineered for production of L-aspartate express heterologous nucleic acids encoding a urease. In many of these embodiments, the expressed urease is a Schizosaccharomyces pombe urease. The Schizosaccharomyces pombe urease is comprised of four protein subunits, namely Ure2, UreD, UreF, and UreG proteins. The Schizosaccharomyces pombe urease also uses nickel metal as a cofactor and in some embodiments the engineered host cell expresses one or more heterologous genes encoding a nickel transporter. One suitable nickel transporter protein is the Schizosaccharomyces pombe Nicl nickel-transporter. Aspartate permeases

[0138] Low-cost L-aspartate production benefits from export of L-aspartate from the cytosol, across the host cell membrane, and into the surrounding culture medium. Likewise, it is desirable to export L-aspartate without ATP expenditure, thereby enabling more energy efficient L-aspartate production. [0139] One L-aspartate transport protein suitable for L-aspartate export in engineered host cells of the invention is Arabidopsis thaliana SIAR1 and its homologs. Another suitable L- aspartate transport protein is Arabidopsis thaliana bidirectional amino acid transporter 1 (BAT1) Section 5. Methods of producing L-aspartate or Beta-alanine

[0140] In another aspect, methods are provided herein for producing L-aspartate or beta- alanine by recombinant host cells of the invention. In certain embodiments, these methods comprise the steps of: (a) culturing a recombinant host cell described herein in a medium containing at least one carbon source and one nitrogen source under substantially anaerobic conditions such that L-aspartate is produced; and (b) recovering said L-aspartate from the medium. In other embodiments, these methods comprise the steps of: (a) culturing a recombinant host cell described herein in a medium containing at least one carbon source and one nitrogen source under aerobic conditions such that L-aspartate is produced; and (b) recovering said L- aspartate from the medium. In other embodiments, these methods comprise the steps of: (a) culturing a recombinant host cell described herein in a medium containing at least one carbon source and one nitrogen source under substantially anaerobic conditions such that beta-alanine is produced; and (b) recovering said beta-alanine from the medium. The L-aspartate or beta-alanine can be secreted into the culture medium.

[0141] It is understood that, in the methods of the invention, any of the one or more heterologous nucleic acids can be introduced into a host cell to produce a recombinant host cell of the invention. For example, the heterologous nucleic acids can be introduced so as to confer a L-aspartate fermentation pathway onto the host cell. The recombinant host cell may further comprise heterologous nucleic acids encoding L-aspartate 1 -decarboxylase so as to confer the ability for the recombinant host cell to produce beta-alanine. Alternatively, heterologous nucleic acids can be introduced to produce an intermediate host cell having the biosynthetic capability to catalyze some of the required metabolic reactions to confer L-aspartate or beta-alanine biosynthetic capability.

[0142] Any of the recombinant host cells described herein can be cultured to produce and/or secrete L-aspartate or beta-alanine. For example, recombinant host cells producing L- aspartate can be cultured for the biosynthetic production of L-aspartate. The L-aspartate can be isolated or treated as described below to produce beta-alanine or polyL-aspartate. Similarly, recombinant host cells producing beta-alanine can be cultured for the biosynthetic production of beta-alanine. The beta-alanine can be isolated and subjected to further treatments for the chemical synthesis of beta-alanine family of compounds, including, but not limited to, pantothenic acid, beta-alanine alkyl esters (e.g., beta-alanine methyl ester, beta-alanine ethyl ester, beta-alanine propyl ester, and the like), and poly(beta-alanine).

[0143] The methods of producing L-aspartate or beta-alanine provided herein may be performed in a suitable fermentation broth in a suitable fermentation vessel, including but not limited to a culture plate, a flask, or a fermentor. Further, the methods of the invention can be performed at any scale of fermentation known in the art to support industrial production of microbially produced small-molecules. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble column fermentor, a fixed bed bioreactor, or any combination thereof.

[0144] In some embodiments, the fermentation broth is any fermentation broth in which a recombinant host cell capable of producing L-aspartate or beta-alanine can subsist (maintain growth and/or viability). In some embodiments, the fermentation broth 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 provided to the fermentation broth incrementally or continuously, and is essential cell nutrient is maintained at essentially the minimum level required for efficient assimilation by growing cells.

[0145] In some embodiments, culturing of the cells provided herein to produce L- aspartate or beta-alanine may be divided up into phases. For example, the cell culture process may be divided up into a growth phase, a production phase, and/or a recovery phase. The following paragraphs provide examples of specific conditions that may be used for these phases. One skilled in the art will recognize that these conditions may be varied based on the host cell used, the desired L-aspartate or beta-alanine yield, titer, and/or productivity, or other factors.

[0146] Carbon source. The carbon source provided to the fermentation can be any carbon source that can be fermented by the host cell. Suitable carbon sources include, but are not limited to, monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, methane, or one or more combinations thereof. Exemplary monosaccharides suitable for use in accordance to the methods of the invention include, but are not limited to, dextrose, fructose, galactose, xylose, arabinose, and combinations thereof. Exemplary disaccharides suitable for use in accordance to the methods of the invention include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Exemplary polysaccharides suitable for use in accordance to the methods of the invention include, but are not limited to, starch, glycogen, cellulose, and combinations thereof. In some embodiments, the carbon source is dextrose. In other embodiments, the carbon source is sucrose.

[0147] Nitrogen. Every molecule of L-aspartate or beta-alanine comprises nitrogen atom, and in order to produce L-aspartate or beta-alanine at a high yield, a suitable source of assimilable nitrogen must be provided to the fermentation during host cell cultivation. As used herein, assimilable nitrogen refers to nitrogen that is capable of being metabolized by the host cell of the invention and used in produce L-aspartate. The nitrogen source may be any assimilable nitrogen source that can be utilized by the host cell, including, but not limited to, anhydrous ammonia, ammonium sulfate, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, sodium nitrate, urea, peptone, protein hydrolysates, and yeast extract. In one embodiment, the nitrogen source is anhydrous ammonia. In another embodiment, the nitrogen source is ammonium sulfate. In yet a further embodiment, the nitrogen source is urea. Those skilled in the art will recognize that the mols assimilable nitrogen is dependent on the nitrogen source, and, for example, one mol of anhydrous ammonia ( H3) comprises 1 mol assimilable nitrogen while one mol of diammonium phosphate ( H ₄ ) ₂ P0 ₄ comprises 2 mols assimilable nitrogen.

[0148] A minimum amount of assimilable nitrogen must be provided to the fermentation during host cell cultivation to achieve high L-aspartate or beta-alanine yields. In certain embodiments of the methods provided herein wherein the carbon source is dextrose, the molar ratio of assimilable nitrogen to dextrose provided to the fermentation during host cell cultivation is at least 0.25 : 1, at least 0.5 : 1, at least 0.75 : 1, 1 : 1, at least 1.25 : 1, at least 1.5 : 1, at least 1.75 : 1, at least 2: 1, or greater than 2: 1. In certain embodiments of the methods provided herein wherein the carbon source is sucrose, the molar ratio of assimilable nitrogen to sucrose is at least 0.1 : 1, at least 0.2: 1, at least 0.3 : 1, at least 0.4: 1, at least 0.5 : 1, at least 0.6: 1, at least 0.7: 1, at least 0.8: 1, at least 0.9: 1, at least 1 : 1, or greater than 1 : 1.

[0149] pH. The pH of the culture medium can be controlled by the addition of acid or base to the culture medium. Preferably, the pH is maintained from about 3.0 to about 8.0.

[0150] Aspartic acid exhibits a relatively low solubility in water and will crystallize from solution. Only about 6 g/1 aspartic acid is soluble at 30°C. Crystallization occurs when the concentration of the fully protonated, aspartic acid, form of L-aspartate increases to above the solubility limit. It is advantageous to crystallize aspartic acid during the fermentation for several reasons. First, crystallization provides an aspartic acid sink, enabling a high concentration gradient to be maintained across the cell membrane and helping to increase the kinetics of product export outside the host cell. Second, the L-aspartic acid that has crystallized from solution in the fermentation can be more readily separated from the majority of the cells and fermentation broth, accomplishing a purification step.

[0151] To facilitate efficient purification, in many cases, it is desirable for the majority of the L-aspartate to be in the insoluble, crystallized form (i.e. crystallized aspartic acid) prior to purification. Preferably, greater than about 50 g/1 aspartic acid is in an insoluble, crystallized form prior to purification of the aspartic acid from the fermentation broth. More preferably, greater than about 75 g/1 of aspartic acid produced is an insoluble, crystallized form prior to purification of the aspartic acid from the fermentation broth.

[0152] To crystallize aspartic acid from the fermentation broth, the pH of the fermentation should be decreased to below pH 3.86, the pKa of aspartic acid R-chain, prior to L-aspartate purification. The broth pH can be decreased during the fermentation (i.e., while the host cells are producing aspartic acid), and/or the broth pH can be decreased at the conclusion of the fermentation. The broth pH can be decreased due to endogenous production of aspartic acid, and/or the broth pH can be decreased due to supplementation of an acid to the fermentation. Non-limiting examples of suitable acids include aspartic acid, acetic acid, hydrochloric acid, and sulfuric acid.

[0153] Temperature. The temperature of the fermentation broth can be any temperature suitable for growth of the recombinant host cells and/or production of L-aspartate or beta- alanine. Preferably, during host cell production of L-aspartate or beta-alanine the fermentation broth is maintained at a temperature in the range of from about 20° C to about 45° C, preferably in the range of from about 25° C to about 37° C, and more preferably in the range from about 28° C to about 32° C.

[0154] Oxygen. During cultivation, aeration and agitation conditions are selected to produce a desired oxygen uptake rate. In various embodiments, conditions are selected to produce an oxygen uptake rate of around 0-25 mmol/l/hr. In some embodiments conditions are selected to produce an oxygen uptake rate of around 2.5-15 mmol/l/hr. Oxygen uptake rate as used herein refers to the volumetric rate at which oxygen is consumed during the fermentation. Inlet and outlet oxygen concentrations can be measured with exhaust gas analysis, for example by mass spectrometers. Oxygen uptake rate can be calculated by one of ordinary skill in the art using the Direct Method described in Bioreaction Engineering Principles 3 ^rd Edition, 2011, Spring Science + Business Media, p. 449.

[0155] While the L-aspartate pathways described herein are preferably used to produce

L-aspartate or beta-alanine under substantially anaerobic conditions, they are capable of producing L-aspartate or beta-alanine under a range of oxygen concentrations. In some embodiments, the L-aspartate pathways produce L-aspartate or beta-alanine under aerobic conditions. In preferred embodiments, the L-aspartate pathways produce L-aspartate or beta- alanine under substantially anaerobic conditions.

[0156] A high yield of either L-aspartate or beta-alanine from the provided carbon and nitrogen source(s) is desirable in order to decrease the production cost. As used herein, yield is calculated as the percentage of the mass of carbon source catabolized by host cells of the invention and used to produce either L-aspartate or beta-alanine. In some cases, only a fraction of the carbon source provided to a fermentation is catabolized by host the cells, and the remainder is found unconsumed in the fermentation broth or is consumed by contaminating microbes in the fermentation. Thus, it is important to both ensure that fermentation is both substantially pure of contaminating microbes and that the concentration of unconsumed carbon source at the completion of the fermentation is measured. For example, if 100 grams of glucose are provided to host cells, and at the end of the fermentation 25 grams of beta-alanine are produced and there remains 10 grams of glucose, the beta-alanine yield is 27.7% (i.e., 10 grams beta-alanine from 90 grams glucose). In certain embodiments of the methods provided herein, the final yield of L-aspartate on the carbon source is at least 10%>, at least 20%, at least 30%>, at least 40%), at least 50%, or greater than 50%. In certain embodiments, the host cells provided herein are capable of producing at least 80%>, at least 85%>, or at least 90% by weight of carbon source to L-aspartate. In certain embodiments of the methods provided herein, the final yield of beta-alanine on the carbon source is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%), or greater than 50%. In certain embodiments, the host cells provided herein are capable of producing at least 80%, at least 85%, or at least 90% by weight of carbon source to beta-alanine.

[0157] In addition to yield, the titer, or concentration, of L-aspartate or beta-alanine produced in the fermentation is another important metric for decreasing production, and, assuming all other metrics are equal, a higher titer is preferred as compared to a lower titer. Generally speaking, titer is provided as grams product (e.g., L-aspartate or beta-alanine) produced per liter of fermentation broth (i.e., g/1). In some embodiments, the L-aspartate titer is at least 1 g/1, at least 5 g/1, at least 10 g/1, at least 15 g/1, at least 20 g/1, at least 25 g/1, at least 30 g/1, at least 40 g/1, at least 50 g/1, at least 60 g/1, at least 70 g/1, at least 80 g/1, at least 90 g/1, at least 100 g/1, or greater than 100 g/1 at some point during the fermentation, and preferably at the conclusion of the fermentation. In other embodiments, the beta-alanine titer is at least 1 g/1, at least 5 g/1, at least 10 g/1, at least 15 g/1, at least 20 g/1, at least 25 g/1, at least 30 g/1, at least 40 g/1, at least 50 g/1, at least 60 g/1, at least 70 g/1, at least 80 g/1, at least 90 g/1, at least 100 g/1, or greater than 100 g/1 at some point during the fermentation, and preferably at the conclusion of the fermentation.

[0158] Further, productivity, or the rate of product (i.e., L-aspartate or beta-alanine) formation, is important for decreasing production cost, and, assuming all other metrics are equal, a higher productivity is preferred over a lower productivity. Generally speaking, productivity is provided as grams product produced per liter of fermentation broth per hour (i.e., g/l/hr). In some embodiments, the L-aspartate productivity is at least 0.1 g/1, at least 0.25 g/1, at least 0.5 g/1, at least 0.75 g/1, at least 1.0 g/1, at least 1.25 g/1, at least 1.25g/l, at least 1.5 g/1, or greater than 1.5 g/1 over some time period during the fermentation. In other embodiments, the beta-alanine productivity is at least 0.1 g/1, at least 0.25 g/1, at least 0.5 g/1, at least 0.75 g/1, at least 1.0 g/1, at least 1.25 g/1, at least 1.25g/l, at least 1.5 g/1, or greater than 1.5 g/1 over some time period during the fermentation.

[0159] Decreasing byproduct formation is also important for decreasing production cost, and, generally speaking, the lower the byproduct concentration the lower the production cost. Byproducts that can occur during production of L-aspartate or beta-alanine producing host cells in accordance with the methods of the invention include ethanol, acetate, and pyruvate. In certain embodiments of the methods provided herein, the recombinant host cells produce ethanol at a low yield from the provided carbon source. In certain embodiments, ethanol may be produced at a yield of 10% or less, and preferably at a yield of 5% or less at the conclusion of the fermentation. In certain embodiments of the methods provided herein, the recombinant host cells produce acetate at a low yield from the provided carbon source. In certain embodiments, acetate may be produced at a yield of 10% or less, and preferably at a yield of 5% or less at the conclusion of the fermentation. In certain embodiments of the methods provided herein, the recombinant host cells produce pyruvate at a low yield from the provided carbon source. In certain embodiments, pyruvate may be produced at a yield of 10% or less, and preferably at a yield of 5% or less at the conclusion of the fermentation. [0160] Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of L-aspartate and/or beta-alanine. Fermentation procedures can be scaled up for manufacturing of L-aspartate or beta-alanine. Exemplary fermentation procedures include, for example, fed-batch fermentation and batch product separation; fed-batch fermentation and continuous product separation; batch fermentation and batch product separation; and continuous fermentation and continuous product separation. All of these processes are well known in the art.

[0161] In addition to the biosynthesis of L-aspartate and beta-alanine as described herein, the recombinant host cells and methods of the invention can also be utilized in various combinations with each other and with other microbes and methods known in the art to achieve product biosynthesis by other routes. For example, one alternative to product beta-alanine other than the use of L-aspartate producing host cell of the invention and chemical conversion or other than the use of a beta-alanine producing host cell of the invention is through addition of a second microbe capable of converting L-aspartate to beta-alanine.

[0162] One such procedure includes, for example, the cultivation of a L-aspartate producing host cell of the invention to produce L-aspartate as described herein. The L-aspartate can then be used as a substrate for a second microbe that converts L-aspartate to beta-alanine. The L-aspartate can be added directly to another culture of the second microbe, or the L- aspartate producing microbes in the original culture can be removed by, for example, cell separation and the second microbe capable of producing beta-alanine from L-aspartate added to the culture in a sufficient amount to enable production of beta-alanine from the L-aspartate in the fermentation broth.

EXAMPLES

Example 1: Construction of engineered Pichia kudriavzevii strains expressing L-aspartate dehydrogenases, and their use in the production of L-aspartate in yeast

[0163] Nucleic acids encoding different L-aspartate dehydrogenases were codon-optimized for yeast, synthesized, and integrated into the Pichia kudriavzevii genome; in vivo expression of the L-aspartate dehydrogenases resulted in production of L-aspartate. Codon optimized DNA encoding for each L- aspartate dehydrogenase was first synthesized by a commercial DNA synthesis company (e.g. , Gen9, Inc.). The synthetic DNA was then amplified by PCR using primers to add DNA sequences aiding molecular cloning of the DNA into expression constructs. The primers used were as follows (listed as UniProt ID for the protein encoded by the template DNA, forward primer name and sequence, reverse primer name and sequence): Q9HYA4 encoding template DNA, YO1504 forward primer (5'- CACAAACAAACACAATTACAAAAAATGTTGAATATCGTTATGATTGGTTG-3 ' ) and YO 1505 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATAGAGATAGCGTGAGCATG); B3R8S4 encoding template DNA, YO 1506 forward primer (5 '- CACAAACAAACACAATTACAAAAAATGTTGCACGTTTCTATGGTTGG-3 ') and YO 1507 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAGATAGAAACGGCGTGGG-3 ' ) ; Q8XRV9 encoding template DNA, YO 1508 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGTTACATGTTTCTATGGTCGG-3 ') and YO 1509 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAGATAGAGACAGC ATGAGCTC-3 ' ) ; Q 126F5 encoding template DNA, YO 1510 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGTTGAAGATCGCTATGATTGG-3 ') and YO 151 1 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATAACCAAAGCTCTACCTCTG-3'); Q2T559 encoding template DNA, Y01512 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGAGAAACGCTCATGCCC-3 ') and Y01513 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATGACACAATGGGAAGCAC-3 '); Q3JFK2 encoding template DNA, Y01514 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGCGTAACGCCCATGCTC-3 ' ) and Y01515 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATAACACAATGGGAGGCTC-3 '); A6X792 encoding template DNA, Y01516 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGTCTGTCTCTGAAACTATCGTC-3 ') and Y01517 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATAACGGTGGTAGCAACTC-3 '); D6JRV1 encoding template DNA, Y01518 forward primer (5 '- CACAAACAAACACAATTACAAAAAATGAAGAAGTTGATGATGATCGG-3 ') and Y01519 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATTTGGATGGCCTCAAC AG-3 ' ) ; A6TDT8 encoding template DNA, YO 1520 forward primer (5 '- CACAAACAAACACAATTACAAAAAATGATGAAGAAGGTCATGTTAATTG-3 ') and Y01521 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAGGCCAATTCTCTACAAGC-3 '); A8LLH8 encoding template DNA, Y01522 forward primer (5 '- CACAAACAAACACAATTACAAAAAATGAGATTGGCTTTGATCGG-3 ' ) and Y01523 reverse primer (5'- GAGTATGGATTTTACTGGCTGGATTAAACAACCCAGGCAGCG-3 '); Q5LPG8 encoding template DNA, Y01524 forward primer (5 '-

CACAAACAAACACAATTACAAAAAATGTGGAAGTTGTGGGGTTC-3 ') and Y01525 reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAGAAGGATGGTCTAATGGCAG-3 '); DOIX49 encoding template DNA, Y01526 encoding forward primer (5 '- CACAAACAAACACAATTACAAAAAATGAAAAACATCGCCTTAATTGG-3 ' ) and Y01527 encoding reverse primer (5 '- GAGTATGGATTTTACTGGCTGGATTAAATAGCCAATGGAGCGAC- 3'). For DNA encoding L-aspartate dehydrogenase Q46VA0, 5'- and 3'- DNA sequences with homology to the adjacent parts needed for molecular cloning was included during synthesis and no PCR amplification step was used when cloning the Q46VA0 encoding DNA.

[0164] The resulting DNA fragments were purified and cloned downstream of the P. kudriavzevii TDHl promoter and upstream of the S. cerevisiae GRE3 terminator, which are flanked in 5' by 473 bp of sequence upstream of the P. kudriavzevii Adh6c gene and in 3' by a non-functional portion of the Ura3 selection marker, in a plasmid vector containing the ampicillin resistance cassette and the pUC origin of replication using conventional molecular cloning methods. The resulting plasmids were transformed into E. coli competent host cells and selected on LB agar plates containing Amp ¹⁰⁰ . Following overnight incubation at 37°C, individual colonies were inoculated in 5 ml of LB-Amp ¹⁰⁰ grown overnight at 37°C on a shaker before the plasmids were isolated and the identity and integrity of the constructs confirmed by sequencing, resulting in plasmids s393-405. The complementary construct for genomic integration containing the remaining part of the Ura3 marker and a region corresponding to 385 bp downstream of the P. kudriavzevii Adh6c gene was constructed similarly to produce plasmid s376.

[0165] P. kudriavzevii strain LPK15434 was used as the background strain for genomic integration of the L-aspartate dehydrogenase expression constructs. LPK15434 is a uracil auxotroph generated from wild type Pichia kudriavzevii through deletion of the URA3 gene. The plasmids encoding the various L-aspartate dehydrogenase expression cassettes (s393-405) were first digested with restriction enzyme Mssl to release the linear integration cassette and co- transformed into the host strains with Mssl-digested s376 using standard procedures and selected on defined agar medium lacking uracil. After 3 days incubation at 30°C, uracil prototroph transformants were re-streaked on selective medium lacking uracil, and correct integration of the L-aspartate dehydrogenase expression cassettes was confirmed by PCR.

[0166] PCR verified transformants (2-6 for each strain) were inoculated in a 96-well plate containing 0.5 ml of medium (YNB, 2% glucose, 100 mM citrate buffer pH 5.0) along with control strain LPK15419 and grown at 30°C for 3 days, shaking at 300 rpm with 50 mm throw in an incubator maintained at 80% r.h. Control strain LPK15419 is identical to LPK15434 with the exception that the URA3 gene has not been deleted. After 3 days, the cultures were pelleted and the medium supernatant was filtered on a 0.2 micron PVDF membrane and stored at 4°C until analysis. [0167] For HPLC analysis, samples and L-aspartate standards were derivatized with one volume of phtaldialdehyde reagent according to standard procedures and immediately analyzed on a Shimadzu HPLC system configured as follows: Agilent C18 Plus (2.1x150mm, 5μπι) column at 40°C, UV detector at 340 nm; 0.4 mL/min isocratic mobile phase (40mM NaH ₂ P0 ₄ , pH = 7.8) flow; 5 μL injection volume; 18 min total run time.

[0168] The control strain LPK15419 did not produce a detectable amount of L-aspartate. In the LPK15434 background engineered for expression of L-aspartate dehydrogenase proteins, a detectable level of L-aspartate was measured. Expression of the following L-aspartate dehydrogenase proteins resulted in the indicate amount of L-aspartate (mean +/- standard deviation): Q9HYA4, 13±2 mg/L; B3R8S4, 9±0 mg/L; Q8XRV9, 13±3 mg/L; Q126F5, 13±1 mg/L; Q2T559, 11±1 mg/L; Q3JFK2, 15±2 mg/L; A6X792, 13±3 mg/L; D6JRV1, 13±4 mg/L; A6TDT8, 12±1 mg/L; A8LLH8, 11±2 mg/L; Q5LPG8, 14±1 mg/L; D0IX49, 12±2 mg/L; and Q46VA0, 10±2 mg/L. Thus, all engineered Pichia kudriavzevii strains expressing heterologous L-aspartate dehydrogenase proteins resulted in production of L-aspartate while no L-aspartate was observed in the parental, control strain. This example demonstrates, in accordance with the present invention, the expression of nucleic acids encoding L-aspartate dehydrogenase proteins in engineered Pichia kudriavzevii for production of L-aspartate.

SEQUENCE LISTINGS

SEQ ID NO: 1. Pseudomonas aeruginosa L-aspartate dehydrogenase.

1- MLNIVMIGCG AIGAGVLELL ENDPQLRVDA VIVPRDSETQ

41- VRHRLASLRR PPRVLSALPA GERPDLLVEC AGHRAIEQHV

81- LPALAQGIPC LVVSVGALSE PGLVERLEAA AQAGGSRIEL

121- LPGAIGAIDA LSAARVGGLE SVRYTGRKPA SAWLGTPGET

161- VCDLQRLEKA RVIFDGSARE AARLYPKNAN VAATLSLAGL

201- GLDRTQVRLI ADPESCENVH QVEASGAFGG FELTLRGKPL

241- AANPKTSALT VYSVVRALGN HAHAISI -267

SEQ ID NO: 2. Cupriavidus taiwanensis L-aspartate dehydrogenase.

1- MLHVSMVGCG AIGRGVLELL KSDPDVVFDV VIVPEHTMDE

41- ARGAVSALAP RARVATHLDD QRPDLLVECA GHHALEEHIV

81- PALERGIPCM VVSVGALSEP GMAERLEAAA RRGGTQVQLL

121- SGAIGAIDAL AAARVGGLDE VIYTGRKPAR AWTGTPAEQL

161- FDLEALTEAT VIFEGTARDA ARLYPKNANV AATVSLAGLG

201- LDRTAVKLLA DPHAVENVHH VEARGAFGGF ELTMRGKPLA

241- ANPKTSALTV FSVVRALGNR AHAVSI -266

SEQ ID NO: 3. Tribolium castaneum L-aspartate 1 -decarboxylase.

1- MPATGEDQDL VQDLIEEPAT FSDAVLSSDE ELFHQKCPKP

41 - APIYSPISKP VSFESLPNRR LHEEFLRSSV DVLLQEAVFE

81 - GTNRKNRVLQ WREPEELRRL MDFGVRGAPS THEELLEVLK

12 1- KVVTYSVKTG HPYFVNQLFS AVDPYGLVAQ WATDALNPSV

16 1- YTYEVSPVFV LMEEVVLREM RAIVGFEGGK GDGIFCPGGS

20 1- IANGYAISCA RYRFMPDIKK KGLHSLPRLV LFTSEDAHYS

24 1- IKKLASFEGI GTDNVYLIRT DARGRMDVSH LVEEIERSLR

28 1- EGAAPFMVSA TAGTTVIGAF DPIEKIADVC QKYKLWLHVD

32 1- AAWGGGALVS AKHRHLLKGI ERADSVTWNP HKLLTAPQQC

36 1- STLLLRHEGV LAEAHSTNAA YLFQKDKFYD TKYDTGDKHI

40 1- QCGRRADVLK FWFMWKAKGT SGLEKHVDKV FENARFFTDC

44 1- IKNREGFEMV IAEPEYTNIC FWYVPKSLRG RKDEADYKDK

48 1- LHKVAPRIKE RMMKEGSMMV TYQAQKGHPN FFRIVFQNSG

52 1- LDKADMVHFV EEIERLGSDL -540

SEQ ID NO: 4. Corynebactenum glutamicum L-aspartate 1 -decarboxylase.

1- MLRTILGSKI HRATVTQADL DYVGSVTIDA DLVHAAGLIE

41- GEKVAIVDIT NGARLETYVI VGDAGTGNIC INGAAAHLIN

81- PGDLVIIMSY LQATDAEAKA YEPKIVHVDA DNRIVALGND

121- LAEALPGSGL LTSRSI -136

SEQ ID NO: 5. Bacillus subtilis L-aspartate 1 -decarboxylase.

1- MYRTMMSGKL HRATVTEANL NYVGSITIDE DLIDAVGMLP

41- NEKVQIVNNN NGARLETYI I PGKRGSGVIC LNGAAARLVQ

81- EGDKVIIISY KMMSDQEAAS HEPKVAVLND QNKIEQMLGN

121- EPARTIL -127

SEQ ID NO: 6. Mannheimia succiniciproducens phosphoenolpyruvate carboxykinase.

1- MTDLNQLTQE LGALGIHDVQ EVVYNPSYEL LFAEETKPGL

41- EGYEKGTVTN QGAVAVNTGI FTGRSPKDKY IVLDDKTKDT 81- VWWTSEKVKN DNKPMSQDTW NSLKGLVADQ LSGKRLFVVD

121- AFCGANKDTR LAVRVVTEVA WQAHFVTNMF IRPSAEELKG

161- FKPDFVVMNG AKCTNPNWKE QGLNSENFVA FNITEGVQLI

201- GGTWYGGEMK KGMFSMMNYF LPLRGIASMH CSANVGKDGD

241- TAIFFGLSGT GKTTLSTDPK RQLIGDDEHG WDDEGVFNFE

281- GGCYAKTINL SAENEPDIYG AIKRDALLEN VVVLDNGDVD

321- YADGSKTENT RVSYPIYHIQ NIVKPVSKAG PATKVIFLSA

361- DAFGVLPPVS KLTPEQTKYY FLSGFTAKLA GTERGITEPT

401- PTFSACFGAA FLSLHPTQYA EVLVKRMQES GAEAYLVNTG

441- WNGTGKRISI KDTRGI IDAI LDGSIDKAEM GSLPIFDFSI

481- PKALPGVNPA ILDPRDTYAD KAQWEEKAQD LAGRFVKNFE

521- KYTGTAEGQA LVAAGPKA -538

SEQ ID NO: 7. Aspergillus oryzae pyruvate carboxylase amino acid sequence.

1- MAAPFRQPEE AVDDTEFIDD HHEHLRDTVH HRLRANSSIM

41- HFQKILVANR GEIPIRIFRT AHELSLQTVA IYSHEDRLSM

81 - HRQKADEAYM IGHRGQYTPV GAYLAGDEII KIALEHGVQL

121- IHPGYGFLSE NADFARKVEN AGIVFVGPTP DTIDSLGDKV

161- SARRLAIKCE VPVVPGTEGP VERYEEVKAF TDTYGFPIII

201- KAAFGGGGRG MRVVRDQAEL RDSFERATSE ARSAFGNGTV

241- FVERFLDKPK HIEVQLLGDS HGNVVHLFER DCSVQRRHQK

281 - VVEVAPAKDL PADVRDRILA DAVKLAKSVN YRNAGTAEFL 321- VDQQNRHYFI EINPRIQVEH TITEEITGID IVAAQIQIAA

361- GASLEQLGLT QDRISARGFA IQCRITTEDP AKGFSPDTGK

401- IEVYRSAGGN GVRLDGGNGF AGAI ITPHYD SMLVKCTCRG 441- STYEIARRKV VRALVEFRIR GVKTNIPFLT SLLSHPTFVD 481- GNCWTTFIDD TPELFSLVGS QNRAQKLLAY LGDVAVNGSS 521- IKGQIGEPKL KGDVIKPKLF DAEGKPLDVS APCTKGWKQI 561- LDREGPAAFA KAVRANKGCL IMDTTWRDAH QSLLATRVRT 601- IDLLNIAHET SYAYSNAYSL ECWGGATFDV AMRFLYEDPW 641- DRLRKMRKAV PNIPFQMLLR GANGVAYSSL PDNAIYHFCK 681- QAKKCGVDIF RVFDALNDVD QLEVGIKAVH AAEGVVEATM 721- CYSGDMLNPH KKYNLEYYMA LVDKIVAMKP HILGIKDMAG 761- VLKPQAARLL VGSIRQRYPD LPIHVHTHDS AGTGVASMIA 801 - CAQAGADAVD AATDSMSGMT SQPSIGAILA SLEGTEQDPG 841- LNLAHVRAID SYWAQLRLLY SPFEAGLTGP DPEVYEHEIP 881- GGQLTNLIFQ ASQLGLGQQW AETKKAYEAA NDLLGDIVKV 921- TPTSKVVGDL AQFMVSNKLT PEDVVERAGE LDFPGSVLEF 961- LEGLMGQPFG GFPEPLRSRA LRDRRKLEKR PGLYLEPLDL 1001- AKIKSQIREK FGAATEYDVA SYAMYPKVFE DYKKFVQKFG 1041- DLSVLPTRYF LAKPEIGEEF HVELEKGKVL ILKLLAIGPL 1081- SEQTGQREVF YEVNGEVRQV AVDDNKASVD NTSRPKADVG 1121- DSSQVGAPMS GVVVEIRVHD GLEVKKGDPL AVLSAMKMEM 1161- VISAPHSGKV SSLLVKEGDS VDGQDLVCKI VKA -1193

SEQ ID NO: 8. Escherichia coli phosphoenolpyruvate carboxylase amino acid sequence.

1 - MNEQYSALRS NVSMLGKVLG ETIKDALGEH ILERVETIRK 41- LSKSSRAGND ANRQELLTTL QNLSNDELLP VARAFSQFLN 81- LANTAEQYHS ISPKGEAASN PEVIARTLRK LKNQPELSED 121- TIKKAVESLS LELVLTAHPT EITRRTLIHK MVEVNACLKQ 161- LDNKDIADYE HNQLMRRLRQ LIAQSWHTDE IRKLRPSPVD

201 - EAKWGFAVVE NSLWQGVPNY LRELNEQLEE NLGYKLPVEF 241- VPVRFTSWMG GDRDGNPNVT ADITRHVLLL SRWKATDLFL 281- KDIQVLVSEL SMVEATPELL ALVGEEGAAE PYRYLMKNLR

321- SRLMATQAWL EARLKGEELP KPEGLLTQNE ELWEPLYACY

361- QSLQACGMGI IANGDLLDTL RRVKCFGVPL VRIDIRQEST

401- RHTEALGELT RYLGIGDYES WSEADKQAFL IRELNSKRPL

441- LPRNWQPSAE TREVLDTCQV IAEAPQGSIA AYVISMAKTP

481- SDVLAVHLLL KEAGIGFAMP VAPLFETLDD LNNANDVMTQ

521- LLNIDWYRGL IQGKQMVMIG YSDSAKDAGV MAASWAQYQA

561- QDALIKTCEK AGIELTLFHG RGGSIGRGGA PAHAALLSQP

601- PGSLKGGLRV TEQGEMIRFK YGLPEITVSS LSLYTGAILE

641- ANLLPPPEPK ESWRRIMDEL SVISCDVYRG YVRENKDFVP

681- YFRSATPEQE LGKLPLGSRP AKRRPTGGVE SLRAIPWIFA

721- WTQNRLMLPA WLGAGTALQK VVEDGKQSEL EAMCRDWPFF

761- STRLGMLEMV FAKADLWLAE YYDQRLVDKA LWPLGKELRN

801- LQEEDIKVVL AIANDSHLMA DLPWIAESIQ LRNIYTDPLN

841- VLQAELLHRS RQAEKEGQEP DPRVEQALMV TIAGIAAGMR

881- NTG -883

SEQ ID NO: 9. Pichia kudriavzevii pyruvate decarboxylase.

1- MTDKISLGTY LFEKLKEAGS YSIFGVPGDF NLALLDHVKE

41- VEGIRWVGNA NELNAGYEAD GYARINGFAS LITTFGVGEL

81- SAVNAIAGSY AEHVPLIHIV GMPSLSAMKN NLLLHHTLGD

121- TRFDNFTEMS KKISAKVEIV YDLESAPKLI NNLIETAYHT

161- KRPVYLGLPS NFADELVPAA LVKENKLHLE EPLNNPVAEE

201- EFIHNVVEMV KKAEKPI ILV DACAARHNIS KEVRELAKLT

241- KFPVFTTPMG KSTVDEDDEE FFGLYLGSLS APDVKDIVGP

281- TDCILSLGGL PSDFNTGSFS YGYTTKNVVE FHSNYCKFKS

321- ATYENLMMKG AVQRLISELK NIKYSNVSTL SPPKSKFAYE

361- SAKVAPEGII TQDYLWKRLS YFLKPRDI IV TETGTSSFGV

401- LATHLPRDSK SISQVLWGSI GFSLPAAVGA AFAAEDAHKQ

441- TGEQERRTVL FIGDGSLQLT VQSISDAARW NIKPYIFILN

481- NRGYTIEKLI HGRHEDYNQI QPWDHQLLLK LFADKTQYEN

521- HVVKSAKDLD ALMKDEAFNK EDKIRVIELF LDEFDAPEIL

561- VAQAKLSDEI NSKAA -575

SEQ ID NO: 10. Saccharomyces cerevisiae PDCl .

1- MSEITLGKYL FERLKQVNVN TVFGLPGDFN LSLLDKIYEV

41- EGMRWAGNAN ELNAAYAADG YARIKGMSCI ITTFGVGELS

81- ALNGIAGSYA EHVGVLHVVG VPSISAQAKQ LLLHHTLGNG

121- DFTVFHRMSA NISETTAMIT DIATAPAEID RCIRTTYVTQ

161- RPVYLGLPAN LVDLNVPAKL LQTPIDMSLK PNDAESEKEV

201- IDTILALVKD AKNPVILADA CCSRHDVKAE TKKLIDLTQF

241- PAFVTPMGKG SIDEQHPRYG GVYVGTLSKP EVKEAVESAD

281- LILSVGALLS DFNTGSFSYS YKTKNIVEFH SDHMKIRNAT

321- FPGVQMKFVL QKLLTTIADA AKGYKPVAVP ARTPANAAVP

361- ASTPLKQEWM WNQLGNFLQE GDVVIAETGT SAFGINQTTF

401- PNNTYGISQV LWGSIGFTTG ATLGAAFAAE EIDPKKRVIL

441- FIGDGSLQLT VQEISTMIRW GLKPYLFVLN NDGYTIEKLI

481- HGPKAQYNEI QGWDHLSLLP TFGAKDYETH RVATTGEWDK

521- LTQDKSFNDN SKIRMIEIML PVFDAPQNLV EQAKLTAATN

561- AKQ -563 SEQ ID NO: 11. Pichia kudriavzevii alcohol dehydrogenase (ADHl).

1- MFASTFRSQA VRAARFTRFQ STFAIPEKQM GVIFETHGGP

41- LQYKEIPVPK PKPTEILINV KYSGVCHTDL HAWKGDWPLP

81- AKLPLVGGHE GAGIVVAKGS AVTNFEIGDY AGIKWLNGSC

121- MSCEFCEQGD ESNCEHADLS GYTHDGSFQQ YATADAIQAA

161- KIPKGTDLSE VAPILCAGVT VYKALKTADL RAGQWVAISG

201- AAGGLGSLAV QYAKAMGLRV LGIDGGEGKK ELFEQCGGDV

241- FIDFTRYPRD APEKMVADIK AATNGLGPHG VINVSVSPAA

281- ISQSCDYVRA TGKVVLVGMP SGAVCKSDVF THVVKSLQIK

321- GSYVGNRADT REALEFFNEG KVRSPIKVVP LSTLPEIYEL

361- MEQGKILGRY VVDTSK -376

SEQ ID NO: 12. Pichia kudriavzevii glycerol 3-phosphate dehydrogenase.

1- MVSPAERLST IASTIKPNRK DSTSLQPEDY PEHPFKVTVV

41- GSGNWGCTIA KVIAENTVER PRQFQRDVNM WVYEELIEGE

81- KLTEI INTKH ENVKYLPGIK LPVNVVAVPD IVEACAGSDL

121- IVFNIPHQFL PRILSQLKGK VNPKARAISC LKGLDVNPNG

161- CKLLSTVITE ELGIYCGALS GANLAPEVAQ CKWSETTVAY

201- TIPDDFRGKG KDIDHQILKS LFHRPYFHVR VISDVAGISI

241- AGALKNVVAM AAGFVEGLGW GDNAKAAVMR IGLVETIQFA

281- KTFFDGCHAA TFTHESAGVA DLITTCAGGR NVRVGRYMAQ

321- HSVSATEAEE KLLNGQSCQG IHTTREVYEF LSNMGRTDEF

361- PLFTTTYRI I YENFPIEKLP ECLEPVED -388

SEQ ID NO: 13. Pichia kudriavzevii cytosolic malate dehydrogenase

1- MSNVKVALLG AAGGIGQPLA LLLKLNPNIT HLALYDVVHV

41- PGVAADLHHI DTDVVITHHL KDEDGTALAN ALKDATFVIV

81- PAGVPRKPGM TRGDLFTINA GICAELANAI SLNAPNAFTL

121- VITNPVNSTV PIFKEIFAKN EAFNPRRLFG VTALDHVRSN

161- TFLSELIDGK NPQHFDVTVV GGHSGNSIVP LFSLVKAAEN

201- LDDEI IDALI HRVQYGGDEV VEAKSGAGSA TLSMAYAANK

241- FFNILLNGYL GLKKTMISSY VFLDDSINGV PQLKENLSKL

281- LKGSEVELPT YLAVPMTYGK EGIEQVFYDW VFEMSPKEKE

321- NFITAIEYID QNIEKGLNFM VR -342

SEQ ID NO: 14. L-aspartate dehydrogenase consensus sequence

1- MLHIAMIGCG AIGAGVLELL KSDPDLRVDA VIVPEESMDA

41- VREAVAALAP VARVLTALPA DARPDLLVEC AGHRAIEEHV

81- VPALERGIPC AVASVGALSE PGLAERLEAA ARRGGTQVQL

121- LSGAIGAIDA LAAARVGGLD SVVYTGRKPP LAWKGTPAEQ

161- VCDLDALTEA TVIFEGSARE AARLYPKNAN VAATLSLAGL

201- GLDRTQVRLI ADPAVTENVH HVEARGAFGG FELTMRGKPL

241- AANPKTSALT VYSVVRALGN RAHALSI -267

SEQ ID NO: 15. Bacterial L-aspartate 1 -decarboxylase consensus sequence

1- MLRTMLKSKI HRATVTQADL HYVGSVTIDA DLLDAADILE

41- GEKVAIVDIT NGARLETYVI AGERGSGVIG INGAAAHLVH

81- PGDLVIIIAY AQMSDAEARA YEPRVVFVDA DNRIVE-LGN

121- DPAEALPGG -129 SEQ ID NO: 16. Eukaryotic L-aspartate 1 -decarboxylase consensus sequence

1- MPANGNFPVA LEVISIFKPY NSAVEDLASM AKTDTSASSS

41- GSDSAGSSED EDVQLFASKG NLLNSKLLKK SNNNNKNNNI

81- NENNNKNAAA GLKRFASLPN RAEHEEFLRD CVDEILKLAV

121- FEGTNRSSKV VEWHDPEELK KLFDFELRAE PDSHEKLLEL

161- LRATIRYSVK TGHPYFVNQL FSSVDPYGLV GQWLTDALNP

201- SVYTYEVAPV FTLMEEVVLR EMRRIVGFPN DGEGDGIFCP

241- GGSIANGYAI SCARYKYAPE VKKKGLHSLP RLVIFTSEDA

281- HYSVKKLASF MGIGSDNVYK IATDEVGKMR VSDLEQEILR

321- ALDEGAQPFM VSATAGTTVI GAFDPLEGIA DLCKKYNLWM

361- HVDAAWGGGA LMSKKYRHLL KGIERADSVT WNPHKLLAAP

401- QQCSTFLTRH EGILSECHST NATYLFQKDK FYDTSYDTGD

441- KHIQCGRRAD VLKFWFMWKA KGTSGFEAHV DKVFENAEYF

481- TDSIKARPGF ELVIEEPECT NICFWYVPPS LRGMERDNAE

521- FYEKLHKVAP KIKERMIKEG SMMITYQPLR DLPNFFRLVL

561- QNSGLDKSDM LYFINEIERL GSDLV -585

SEQ ID NO: 17. Ralstonia solanacearum L- aspartate dehydrogenase.

1- MLHVSMVGCG AIGQGVLELL KSDPDLCFDT VIVPEHGMDR

41- ARAAIAPFAP RTRVMTRLPA QADRPDLLVE CAGHDALREH

81- VVPALEQGID CLVVSVGALS EPGLAERLEA AARRGHAQMQ

121- LLSGAIGAID ALAAARVGGL DAVVYTGRKP PRAWKGTPAE

161- RQFDLDALDR TTVIFEGKAS DAALLFPKNA NVAATLALAG

201- LGMERTHVRL LADPTIDENI HHVEARGAFG GFELIMRGKP

241- LAANPKTSAL TVFSVVRALG NRAHAVSI -268

SEQ ID NO: 18. Polaromonas sp. L-aspartate dehydrogenase.

1- MLKIAMIGCG AIGASVLELL HGDSDVVVDR VITVPEARDR

41- TEIAVARWAP RARVLEVLAA DDAPDLVVEC AGHGAIAAHV

81- VPALERGIPC VVTSVGALSA PGMAQLLEQA ARRGKTQVQL

121- LSGAIGGIDA LAAARVGGLD SVVYTGRKPP MAWKGTPAEA

161- VCDLDSLTVA HCIFDGSAEQ AAQLYPKNAN VAATLSLAGL

201- GLKRTQVQLF ADPGVSENVH HVAAHGAFGS FELTMRGRPL

241- AANPKTSALT VYSVVRALLN RGRALVI -267

SEQ ID NO: 19. Burkholderia thailandensis L-aspartate dehydrogenase.

1- MRNAHAPVDV AMIGFGAIGA AVYRAVEHDA ALRVAHVIVP

41- EHQCDAVRGA LGERVDVVSS VDALAYRPQF ALECAGHGAL

81- VDHVVPLLRA GTDCAVASIG ALSDLALLDA LSEAADEGGA

121- TLTLLSGAIG GVDALAAAKQ GGLDEVQYIG RKPPLGWLGT

161- PAEALCDLRA MTAEQTIFEG SARDAARLYP KNANVAATVA

201- LAGVGLDATK VRLIADPAVT RNVHRVVARG AFGEMSIEMS

241- GKPLPDNPKT SALTAFSAIR ALRNRASHCV I -271

SEQ ID NO: 20. Burkholderia pseudomallei L-aspartate dehydrogenase.

1- MRNAHAPVDV AMIGFGAIGA AVYRAVEHDA ALRVAHVIVP

41- EHQCDAVRGA LGERVDVVSS VDALACRPQF ALECAGHGAL

81- VDHVVPLLKA GTDCAVASIG ALSDLALLDA LSNAADAGGA 121- TLTLLSGAIG GIDALAAARQ GGLDEVRYIG RKPPLGWLGT

161- PAEAICDLRA MAAEQTIFEG SARDAAQLYP RNANVAATIA

201- LAGVGLDATR VCLIADPAVT RNVHRIVARG AFGEMSIEMS

241- GKPLPDNPKT SALTAFSAIR ALRNRASHCV I -271

SEQ ID NO: 21. Ochrobactrum anthropi L-aspartate dehydrogenase.

1- MSVSETIVLV GWGAIGKRVA DLLAERKSSV RIGAVAVRDR

41- SASRDRLPAG AVLIENPAEL AASGASLVVE AAGRPSVLPW

81- GEAALSTGMD FAVSSTSAFV DDALFQRLKD AAAASGAKLI

121- IPPGALGGID ALSAASRLSI ESVEHRIIKP AKAWAGTQAA

161- QLVPLDEISE ATVFFTDTAR KAADAFPQNA NVAVITSLAG

201- IGLDRTRVTL VADPAARLNT HEI IAEGDFG RMHLRFENGP

241- LATNPKSSEM TALNLVRAIE NRVATTVI -268

SEQ ID NO: 22. Acinetobacter sp. SH024 L-aspartate dehydrogenase.

1- MKKLMMIGFG AMAAEVYAHL PQDLQLKWIV VPSRSIEKVQ

41- SQVSSEIQVI SDIEQCDGTP DYVIEVAGQA AVKEHAQKVL

81- AKGWTIGLIS VGTLADSEFL IQLKQTAEKN DAHLHLLAGA

121- IAGIDGISAA KEGGLQKVTY KGCKSPKSWK GSYAEQLVDL

161- DHVVEATVFF TGTAREAATK FPANANVAAT IALAGLGMDE

201- TMVELTVDPT INKNKHTIVA EGGFGQMTIE LVGVPLPSNP

241- KTSTLAALSV IRACRNSVEA IQI -263

SEQ ID NO: 23. Klebsiella pneumoniae L-aspartate dehydrogenase.

1- MMKKVMLIGY GAMAQAVIER LPPQVRVEWI VARESHHAAI

41- CLQFGQAVTP LTDPLQCGGT PDLVLECASQ QAVAQYGEAV

81 - LARGWHLAVI STGALADSEL EQRLRQAGGK LTLLAGAVAG

121- IDGLAAAKEG GLERVTYQSR KSPASWRGSY AEQLIDLSAV

161- NEAQIFFEGS AREAARLFPA NANVAATIAL GGIGLDATRV

201- QLMVDPATQR NTHTLHAEGL FGEFHLELSG LPLASNPKTS

241- TLAALSAVRA CRELA -255

SEQ ID NO: 24. Dinoroseobacter shibae L-aspartate dehydrogenase.

1- MRLALIGLGA INRAVAAGMA GQAEMVALTR SGAEAPGVMA

41- VSDLSALRVF APDLVVEAAG HGAARAYLPG LLAAGIDVLM

81- ASVGVLADPE TEAAFRAAPA HGAQLTIPAG AIGGLDLLAA

121- LPKDSLRAVR YTGVKPPAAW AGSPAADGRD LSALDGPVTL

161 - FEGTARQAAL RFPNNANVAA TLALAGAGFD RTEARLVADP

201 - DAAGNGHAYD VISDTAEMTF SVRARPSDTP GTSATTAMSL

241- LRAIRNRDAA WW - 267

SEQ ID NO: 25. Ruegeria pomeroyi L-aspartate dehydrogenase.

1 - MWKLWGSWPE GDRVRIALIG HGPIAAHVAA HLPVGVQLTG

41- ALCRPGRDDA ARAALGVSVA QALEGLPQRP DLLVDCAGHS

81 - GLRAHGLTAL GAGVEVLTVS VGALADAVFC AELEDAARAG

121- GTRLCLASGA IGALDALAAA AMGTGLQVTY TGRKPPQGWR

161- GSRAEKVLDL KALTGPVTHF TGTARAAAQA YPKNANVAAA

201 - VALAGAGLDA TRAELIADPG AAANIHEIAA EGAFGRFRFQ 241- IEGLPLPGNP RSSALTALSL LAALRQRGAA IRPSF -275

SEQ ID NO: 26. Comamonas testosteroni L-aspartate dehydrogenase.

1- MKNIALIGCG AIGSSVLELL SGDTQLQVGW VLVPEITPAV

41 - RETAARLAPQ AQLLQALPGD AVPDLLVECA GHAAIEEHVL

81- PALARGIPAV IASIGALSAP GMAERVQAAA ETGKTQAQLL

121- SGAIGGIDAL AAARVGGLET VLYTGRKPPK AWSGTPAEQV

161- CDLDGLTEAF CIFEGSAREA AQLYPKNANV AATLSLAGLG

201- LDKTMVRLFA DPGVQENVHQ VEARGAFGAM ELTMRGKPLA

241- ANPKTSALTV YSVVRAVLNN VAPLAI -266

SEQ ID NO: 27. Cupriavidus pinatubonensis L-aspartate dehydrogenase.

1 - MSMLHVSMVG CGAIGRGVLE LLKADPDVAF DVVIVPEGQM

41- DEARSALSAL APNVRVATGL DGQRPDLLVE CAGHQALEEH

81- IVPALERGIP CMVVSVGALS EPGLVERLEA AARRGNTQVQ

121- LLSGAIGAID ALAAARVGGL DEVIYTGRKP ARAWTGTPAA

161- ELFDLEALTE PTVIFEGTAR DAARLYPKNA NVAATVSLAG

201- LGLDRTSVRL LADPNAVENV HHIEARGAFG GFELTMRGKP

241- LAANPKTSAL TVFSVVRALG NRAHAVSI -268