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WO2024233705A2 - Cellule hôte recombinante pour produire de l'aspartate - Google Patents

Cellule hôte recombinante pour produire de l'aspartate Download PDF

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Publication number
WO2024233705A2
WO2024233705A2 PCT/US2024/028433 US2024028433W WO2024233705A2 WO 2024233705 A2 WO2024233705 A2 WO 2024233705A2 US 2024028433 W US2024028433 W US 2024028433W WO 2024233705 A2 WO2024233705 A2 WO 2024233705A2
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seq
aspartic acid
aspartate
salt
host cell
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WO2024233705A3 (fr
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Nick OHLER
Rebecca Lennen
Azadeh ALIKHANI
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Lygos Inc
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Lygos Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • host cells such as recombinant microorganism host cells, for producing aspartic acid, preferably in amounts larger than produced by corresponding wild type cells, culturing them to produce aspartic acid or a salt thereof, and identifying cells that are thus suitable, and uses of aspartic acid produced thereby.
  • BACKGROUND There is a need to produce aspartic acid from sustainable resources, such as by culturing host cells.
  • a recombinant host cell for producing aspartic acid In some embodiments, the recombinant host cells produce aspartic acid, preferably in amounts larger than produced by corresponding wild type cells.
  • the recombinant host cell comprises a heterologous nucleic acid encoding an aspartate racemase or a broad spectrum amino acid racemase. In one embodiment, the recombinant host cell comprises a heterologous nucleic acids encoding an aspartate racemase. In one embodiment, the recombinant host cell comprises a heterologous nucleic acids encoding a broad spectrum amino acid racemase. In another aspect, provided herein is a method of producing aspartic acid or a salt thereof comprising the step of culturing the recombinant host cell provided herein to produce aspartic acid or the salt thereof.
  • a process comprising converting L-aspartic acid or a salt thereof, either non-purified in a broth or purified in a solution, to D-aspartic acid or a salt thereof, comprising contacting the broth or the solution of L-aspartic acid with one or more cells of a microorganism, preferably a heterologous microorganism, expressing an aspartate racemase, thereby converting L-aspartic acid to D-aspartic acid.
  • the microorganism is a recombinant host cell, preferably one provided herein.
  • a process comprising converting L-aspartic acid or a salt thereof, to D-aspartic acid or a salt thereof, comprising contacting a broth or a solution of L- aspartic acid with an aspartate racemase, thereby converting L-aspartic acid to D-aspartic acid.
  • the L -aspartic acid or the salt thereof is either non-purified in a broth.
  • the L -aspartic acid or the salt thereof is purified in a solution.
  • a process comprising heterologously expressing an aspartate racemase in a microorganism that overproduces L-aspartic acid or a salt thereof to diagnose upstream pathway enzyme inhibition by L-aspartic acid or a salt thereof, wherein the total production of D-aspartic acid or a salt thereof and L-aspartic acid or a salt thereof with expression of the aspartate racemase is higher than the concentration of only L-aspartic acid or a salt thereof produced without expression of the aspartate racemase, indicating a feedback inhibition of L-aspartic acid in an upstream pathway.
  • overproduces refers to produces more L-aspartic acid, such as two-fold more, such as five-fold more, such as ten-fold more, such as fifty -fold more, such as 100-fold more, such as 1000-fold more, on a per cell, per time, and/or per volume basis than the nonmodified host.
  • a process comprising heterologously expressing an aspartate 1 -decarboxylase in a microorganism that overproduces L-aspartic acid or a salt thereof to diagnose upstream pathway enzyme inhibition by L-aspartic acid or a salt thereof, wherein the total production of beta alanine or a salt thereof and L-aspartic acid or a salt thereof with expression of the aspartate 1 -decarboxylase higher than the concentration of only L-aspartic acid or a salt thereof produced without expression of the aspartate racemase indicates a feedback inhibition of L-aspartic acid in an upstream pathway.
  • microorganism comprising a feedback inhibition of L-aspartic acid or a salt thereof in an upstream pathway as identified by a process provided herein.
  • microorganism comprising a no or substantially no feedback inhibition of L-aspartic acid or a salt thereof in an upstream pathway as identified by the process provided herein.
  • a process of producing aspartic acid or a salt thereof comprising culturing the microorganism identified herein, thereby producing aspartic acid or a salt thereof.
  • a process of producing aspartic acid or a salt thereof comprising culturing the microorganism identified herein, thereby producing aspartic acid or a salt thereof.
  • process comprising: contacting a recombinant, heterologous microorganism comprising one or more of a polypeptide having: one or more heterologous nucleic acids encoding an oxaloacetate-forming enzyme selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase; and with a feed stock comprising glucose, and optionally a bicarbonate (HCO3 ) salt, to produce at least about 7 g/liter, or at least about 10 g/liter, or at least about 15 g/liter aspartic acid or a salt thereof, after about 1 - about 12 days of contacting.
  • a feed stock comprising glucose, and optionally a bicarbonate (HCO3 ) salt
  • the heterologus microorganism, a host cell further comprises one or more disruptions of one or more genes encoding a succinate dehydrogenase subunit, a malate dehydrogenase, and/or one or more disruptions of one or more genes encoding a lactate dehydrogenase.
  • the process (or the culturing) produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of alanine or a salt thereof after about 1 - about 12 days of contacting. In another embodiment, the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of alpha-ketoglutaric acid or a salt thereof after about 1 - about 12 days of contacting.
  • the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of pyruvic acid or a salt thereof after about 1 - about 12 days of contacting.
  • the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of acetic acid or a salt thereof after about 1 - about 12 days of contacting.
  • a non-naturally occurring microorganism that produces or overproduces L-aspartic acid or a salt thereof anaerobically or microaerobically comprising: (1) an deletion or reduced expression of lactate dehydrogenase; (2) deletion, reduced expression, or conditionally reduced expression of one or more of succinate dehydrogenase, malate dehydrogenase, or fumarase during the production phase; and (3) increased expression of a native or heterologous phosphoenolpyruvate carboxykinase, aspartate-resistant native or heterologous pyruvate carboxylase, and/or aspartate-resistant native or heterologous phosphoenolpyruvate carboxylase.
  • the microorganism further comprises deletion or reduced expression of one or more of acetate kinase, phosphotransacetylase, coenzyme A transferase, and/or pyruvate quinone oxidase.
  • the microorganism further comprises expression of a heterologous NADP-dependent glyceraldehyde 3-phosphate dehydrogenase, and/or modifications that increase carbon flux in the pentose phosphate pathway including but not limited to any combination of one or more of the following: (1) increasing expression or activity of a fructose 1,6-bisphosphatase, (2) increasing expression or activity of a glucose 6-phosphate dehydrogenase, (3) increasing expression or activity of a transaldolase, (4) increasing expression or activity of a transketolase, (5) increasing expression or activity of a 6- phosphonogluconolactonase.
  • a heterologous NADP-dependent glyceraldehyde 3-phosphate dehydrogenase and/or modifications that increase carbon flux in the pentose phosphate pathway including but not limited to any combination of one or more of the following: (1) increasing expression or activity of a fructose 1,6-bisphosphatase, (2) increasing expression or activity of a glucose
  • microorganism further comprises native overexpression or heterologous expression of an aspartate transaminase and/or aspartate dehydrogenase.
  • the microorganism further comprises deletion of one or more alanine transaminases.
  • the microorganism further comprises deletion of aspartate ammonia lyase.
  • the microorganism further comprises native overexpression or heterologous expression of a glucokinase, galactose permease, or glucose permease.
  • the microorganism further comprises overexpression or heterologous expression of a glutamate mechanosensitive channel or other aspartate exporter, or an exporter engineered for enhanced export of L-aspartic acid.
  • the microorganism further comprises deletion or reduced expression of genes in the L-lysine, L-threonine, L-cysteine, S-adenosyl-L-methionine, L- methionine, L-isoleucine, and beta-nicotinamide adenine dinucleotide biosynthesis pathways.
  • the microorganism further comprises native overexpression or heterologous expression of a nucleoside diphosphate kinase and/or an adenylate kinase. In another embodiment, the microorganism further comprises heterologous expression of a membrane-bound or soluble pyridine nucleotide transhydrogenase. In another embodiment, the microorganism further comprises conditional overexpression of a phosphoenolpyruvate carboxykinase when oxygen levels are reduced from those of ambient air, and/or at 37 degrees centigrade. In another embodiment, the microorganism further comprises native overexpression or heterologous expression of a carbonic anhydrase.
  • the microorganism further comprises expression of an aspartate transaminase possessing reduced alanine transaminase activity.
  • the microorganism further comprises heterologous expression of an aspartate racemase.
  • a non-naturally occurring or recombinant microorganism that produces or overproduces L-aspartic acid or a salt thereof aerobically comprising: (1) reduced carbon flux through the oxidative tricarboxylic acid cycle via reduced expression or activity of pyruvate dehydrogenase, isocitrate dehydrogenase, citrate synthase, citrate hydro-lyase, or D-threo-isocitrate hydro-lyase; and optionally (2) increased expression of a native or heterologous native or heterologous pyruvate carboxylase, and/or native or heterologous phosphoenolpyruvate carboxylase; and further optionally (3) deletion or reduced expression of phosphoeno
  • the native or heterologous pyruvate carboxylase and/or phosphoenolpyruvate carboxylase comprises reduced inhibition by aspartate.
  • the microorganism further comprises an expression of a heterologous NADP-dependent glyceraldehyde 3-phosphate dehydrogenase, and/or modifications that increase carbon flux in the pentose phosphate pathway including but not limited to any combination of one or more of the following: (1) increasing expression or activity of a fructose 1,6-bisphosphatase, (2) increasing expression or activity of a glucose 6-phosphate dehydrogenase, (3) increasing expression or activity of a transaldolase, (4) increasing expression or activity of a transketolase, (5) increasing expression or activity of a 6- phosphonogluconolactonase.
  • the microorganism further comprises a deletion or reduced expression of one or more of acetate kinase, phosphotransacetylase, coenzyme A transferase, and/or pyruvate quinone oxidase.
  • microorganism further comprises native overexpression or heterologous expression of an aspartate transaminase and/or aspartate dehydrogenase.
  • the microorganism further comprises a deletion of one or more alanine transaminases.
  • the microorganism further comprises deletion of aspartate ammonia lyase.
  • the microorganism further comprises native overexpression or heterologous expression of a glucokinase, galactose permease, or glucose permease.
  • the microorganism further comprises overexpression or heterologous expression of a glutamate mechanosensitive channel or other aspartate exporter, or an exporter engineered for enhanced export of L-aspartic acid.
  • the microorganism further comprises deletion or reduced expression of genes in the L-lysine, L-threonine, L-cysteine, S-adenosyl-L-methionine, L- methionine, L-isoleucine, and beta-nicotinamide adenine dinucleotide biosynthesis pathways.
  • the microorganism further comprises heterologous expression of a membrane-bound or soluble pyridine nucleotide transhydrogenase.
  • the microorganism further comprises native overexpression or heterologous expression of a carbonic anhydrase.
  • the microorganism further comprises an expression of an aspartate transaminase possessing reduced alanine transaminase activity.
  • the microorganism further comprises a heterologous expression of an aspartate racemase.
  • the microorganism further comprises a deletion or reduced expression of a lysine exporter LysE and/or threonine exporter ThrE, to reduce the accumulation of lysine as a byproduct.
  • the microorganism further comprises deletion or reduced expression of a glutamate dehydrogenase, glutamate synthase, or glutamine synthase to reduce accumulation of glutamic acid as a byproduct.
  • the microorganism further comprises a deletion or reduced expression of transcriptional regulator SugR.
  • the microorganism further comprises a deletion or reduced expression of oxaloacetate decarboxylase.
  • the microorganism further comprises a deletion or reduced expression of malate:quinone oxidoreductase.
  • the microorganism further comprises a deletion or reduced expression of transcriptional regulator PckR.
  • the microorganism further comprises an increased expression or activity of transcriptional regulators RamA and/or RamB.
  • the microorganism is produces aspartic acid or a salt thereof anaerobically. In another embodiment, the microorganism is produces aspartic acid or a salt thereof aerobically. In another embodiment, the microorganism is produces aspartic acid or a salt thereof anaerobically or aerobically.
  • a process for producing aspartic acid or a salt thereof using a nonnatural or recombinant microorganism provided herein comprising culturing the microorganism with a surfactant, such as a polysorbate surfactant such as Tween-20 or Tween- 80, thereby producing aspartic acid or the salt thereof.
  • a surfactant such as a polysorbate surfactant such as Tween-20 or Tween- 80
  • the present disclosure provides recombinant host cells, materials, methods, and embodiments for the biological production and purification of aspartic acid While the present disclosure describes details specific to L-aspartic acid, those of ordinary skill in the art will recognize that various changes may be made, and equivalents may be substituted without departing from the invention
  • the present disclosure is not limited to particular nucleic acids, expression vectors, enzymes, biosynthetic pathways, host microorganisms, processes, or enantiomers, as these may vary.
  • the terminology used herein is for the purposes of describing particular embodiments only and is not to be construed as limiting.
  • aspartic add encompasses two different enantiomers D-aspartic acid (synonymous with R-aspartic acid) and 1, -aspartic acid (synonymous with S-aspartic acid) — many materials, methods, and embodiments disclosed that relate to L-aspartic acid also pertain to D-aspartic acid. In addition, many modifications may be made to adapt to a particular situation, materials, composition of matter, process, process steps or process flows, in accordance with the invention. All such modifications are within the scope of the claims appended hereto.
  • the term ‘'comprising’’ is intended to mean that the compounds, compositions and processes include die recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and processes, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method. “Consisting of' shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
  • a “salt” is derived from a variety of organic and inorganic counter ions well known in the art and include, when the compound contains an acidic functionality, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Salts include acid addition salts formed with inorganic acids or organic acids.
  • Inorganic acids suitable for forming acid addition salts include, by way of example and not limitation, hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.
  • hydrohalide acids e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.
  • sulfuric acid nitric acid
  • phosphoric acid phosphoric acid
  • Organic acids suitable for forming acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4- hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-di sulfonic acid, 2-hydroxyethanesuifonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalene
  • Salts also include salts formed when an acidic proton present in the parent compound is either replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion) or by an ammonium ion (e.g , an ammonium ion derived from an organic base, such as, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, dimethylamine, diethylamine, tri ethylamine, and ammonia)
  • a metal ion e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion
  • an ammonium ion e.g , an ammonium ion derived from an organic base, such as, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, dimethylamine, diethylamine, tri ethylamine, and ammonia
  • accession number and similar terms such as “protein accession number”, “UniProt ID”, “gene ID” and “gene accession number” refer to designations given to specific proteins or genes. These identifiers described a gene or protein sequence in publicly accessible databases such as the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • heterologous refers to a material that is non-native to a cell
  • a nucleic acid is heterologous to a cell, and so is a “heterologous nucleic acid” with respect to that cel I, if at least, one of the following is true: 1 ) the nucleic acid is not naturally found in that cell (that i s, it is an “’exogenous” nucleic acid); 2) 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, 3) 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
  • a protein is heterologous to a host cell if it is produced by translation of RNA or the corresponding RN A is produced by transcription of a heterologous nucleic acid. Further, 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.
  • homologous 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.
  • the reference sequence a known amino acid or coding sequence
  • a protein having homology to a reference protein is determined, for example and without limitation, by a BLAST (htps://blast.ncbi.nlm.nih.gov) search.
  • a protein with high percent homology is highly likely to carry out the identical biochemical reaction as the reference protein. In some cases, two enzymes having greater than 40% homology will carry out identical biochemical reactions, and the higher the homology, i.e., 40%, 50%, 60%, 70%, 80%, 90% or greater than 95% homology, the more likely the two proteins have the same or similar function.
  • a protein with at least 60% homology, and in some cases, at least 40% homology, to its reference protein is defined as substantially homologous. Any protein substantially homologous to a reference sequence can be used in a host cell according to the present disclosure. Generally, homologous proteins share substantial sequence identity. Sets of homologous proteins generally possess one or more specific amino acids that are conserved across all members of the consensus sequence protein class.
  • the percent sequence identity of a protein relative to a consensus sequence is determined by aligning the protein sequence against the consensus sequence. Practitioners in the art will recognized that various sequence alignment algorithms are suitable for aligning a protein with a consensus sequence. See, for example, Needleman, S B, et al., “A general method applicable to the search for similarities in the amino acid sequence of two proteins.” Journal of Molecular Biology 48 (3): 443-53 (1970). Following alignment of the protein sequence relative to the consensus sequence, the percentage of positions where the protein possesses an amino acid described by the same position in the consensus sequence determines the percent sequence identity.
  • any of the amino acids described by the degenerate amino acid may be present in the protein at the aligned position for the protein to be identical to the consensus sequence at the aligned position.
  • the following one-letter symbol is used—“B” refers to aspartic acid or asparagine; “Z” refers to glutamine or glutamic acid; “J” refers to leucine or isoleucine; and “X” or “+” refers to any amino acid.
  • 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.
  • 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.
  • enzymes useful in the compositions and methods provided herein can also be identified by the occurrence of highly conserved amino acid residues in the query protein sequence relative to a consensus sequence. For each consensus sequence provided herein, a number of highly conserved amino acid residues are described.
  • Enzymes useful in the compositions and methods provided herein include those that comprise a substantial number, and sometimes ah, of the highly conserved amino acids at positions aligning with the indicated residues in the consensus sequence. Those skilled in the art will recognize that, as with percent identity, the presence or absence of these highly conserved amino acids in a query protein sequence can be determined following alignment of the query protein sequence relative to a given consensus sequence and comparing the amino acid found in the query protein sequence that aligns with each highly conserved amino acid specified in the consensus sequence.
  • Proteins that share a specific function are not always defined or limited by percent sequence identity. In some cases, a protein with low percent sequence identity with a reference protein is able to carry out. the identical biochemical reaction as the reference protein. Such proteins may share three-dimensional structure which enables shared specific functionality, but not necessarily sequence similarity. Such proteins may share an insufficient amount of sequence similarity to indicate that they are homologous via evolution from a common ancestor and would not be identified by a BLAST search or other sequence-based searches. Thus, in some embodiments of the present disclosure, homologous proteins comprise proteins that, lack substantial sequence similarity but share substantial functional similarity and/or substantial structural si m ilari ty .
  • 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.
  • 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.
  • expression vector refers 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 (i e., 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.
  • an “expression vector” contains nucleic acids (ordinarily DNA) to be expressed by the host cell.
  • the expression vector can be contained in materials to aid in achieving entry' of the nucleic acids 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 disclosure include those into which a nucleic acid sequence can be, or has been, inserted, along with any preferred or required operational elements.
  • an expression vector can be transferred into a host cell and, typically, replicated therein (although, on can also employ, in some embodiments, non-replicable vectors that provide for “transient” expression).
  • an expression vector that integrates into chromosomal, mitochondrial, or plastid DNA is employed.
  • 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.
  • plasmids as well as other expression vectors, are described herein or are well known to those of ordinary skill in the art.
  • ferment refers 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.
  • recombinant host ceil'’ and “recombinant 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 cell or microorganism as described herein may be a prokaryotic cell (e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic cell.
  • a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.
  • 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 Additionally, any reference to a “purified” material is intended to refer to an isolated or pure material.
  • nucleic acid and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), segments of polydeoxyribonucleotides, and segments of polyribonucleotides.
  • 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 non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA.
  • nucleic acid may also be referred to herein with respect to its sequence, the order in which different nucleotides occur in the nucleic acid, as the sequence of nucleotides in a nucleic acid typically defines its biological activity, e.g., as in the sequence of a coding region, the nucleic acid in a gene composed of a promoter and coding region, which encodes the product of a gene, which may be an RNA, e.g., a rRNA, tRNA, or mRNA, or a protein (where a gene encodes a protein, both the mRNA and the protein are “gene products” of that. gene).
  • the term “genetic disruption” refers to several ways of altering genomic, chromosomal or plasmid-based gene expression.
  • Non-limiting examples of genetic disruptions include CRISPR, RNAi, nucleic acid deletions, nucleic acid insertions, nucleic acid substitutions, nucleic acid mutations, knockouts, premature stop codons and transcriptional promoter modifications.
  • “genetic disruption” is used interchangeably with “genetic modification’’, “genetic mutation” and “genetic alteration.’’
  • Genetic disruptions give rise to altered gene expression and or altered protein activity. Altered gene expression encompasses decreased, eliminated and increased gene expression levels. In some examples, gene expression results in protein expression, in which case the term “gene expression” is synonymous with “protein expression.”
  • 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 viuES 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'”)
  • 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.
  • transduce refers to the introduction of one or more nucleic acids into a cell
  • 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.”
  • 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., die 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.
  • 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.
  • amino acid is intended to mean the molecule having the chemical formula C4H7NO4 and a molecular mass of 133.11 g/mol (CAS No. 56-84-8). Aspartic acid as described herein can be a salt, acid, base, or derivative depending on the structure, pH and ions present. The terms “aspartic acid” and “aspartate” are used interchangeably.
  • aspartic acid is deprotonated to the aspartate anion C4H6NO4 •
  • aspartate anion is also used interchangeably with “aspartate”, and practitioners skilled in the art understand that these terms are synonyms
  • the aspartate anion is capable of forming an ionic bond with a cation to produce an aspartate salt
  • aspartate is intended to mean a variety of aspartate salt forms, and is used interchangeably with “aspartate salts”.
  • aspartates comprise sodium aspartate (CAS No. 3792-50-5) and ammonium aspartate (CAS No 130296-88-7).
  • Aspartate salts can crystallize in various states of hydration.
  • sodium aspartate monohydrate is intended to mean C4HsNNaOs with a molecular mass of 173 1 g/mol, wherein a single molecule of sodium aspartate crystallizes with one molecule of water.
  • magnesium aspartate dihydrate is intended to mean CsHi&MgNhOio with a molecular mass of 324.525 g/mol, wherein a single molecule of magnesium aspartate crystallizes with two molecules of water Aspartate salts can also form anhydrous crystals; for example, “anhydrous magnesium aspartate” is intended to mean CsFhcMgNzOs with a molecular mass of 288.495 g/mol.
  • aspartate is also used interchangeably with “aspartic acid” and practitioners in the art understand that these terms are synonyms.
  • the aspartic acid and aspartate salts of the present disclosure are synthesized from biologically produced organic components by a fermenting microorganism.
  • aspartic acid, aspartate salts, or their precursor(s) are synthesized from the fermentation of sugars by recombinant host cells of the present disclosure.
  • bio- or the adjective “bio-based” may be used to distinguish these biologically- produced aspartic, acid and aspartate salts from those that are derived from petroleum feedstocks.
  • “aspartic acid” is defined as “bio-based aspartic acid”
  • “aspartate salt” is defined as “bio-based aspartate salt”.
  • p-alanine is intended to mean the molecule having the chemical formula C3H7NO2 and a molecular mass of 89.09 g/mol (CAS No. 107-95-9). Practitioners of ordinary skill in the art understand that the terms “p-Ala,” “3-aminopropanoate,” and “3- aminopropionic acid” are synonymous with p-alanine and the three terms can be used interchangeably. In conditions with pH values higher than the pKa of p-alanine (e.g., about pH>3.63 when using a base, such as sodium hydroxide), p-alanine is deprotonated to the p- alanine anion CiHsNCb .
  • p-alanine anion is capable of forming an ionic bond with a cation to produce an p-alanine salt.
  • p-alanine salt is intended to mean a variety of p-alanine salt forms.
  • 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.
  • byproduct means an undesired chemical related to the biological production of a target molecule.
  • byproduct is intended to mean any amino acid, amino acid precursor, chemical, chemical precursor, organic acid, organic acid precursor, biofuel, biofuel precursor, or small molecule, that may accumulate during biosynthesis of aspartic acid. In some cases, “byproduct” accumulation may decrease the yields, titers or productivities of the target product (e.g , aspartic acid) in a fermentation.
  • NAD redox cofactor nicotinamide adenine dinucleotide
  • NAD(P) refers to both phosphorylated (NADP) and un-phosphorylated (NAD) forms, and encompasses oxidized versions (NAD’ and NADP”) rind reduced versions (NADH and NADPH) of both forms.
  • NAD(Py” refers to the oxidized versions of phosphorylated and un-phosphorylated NAD, i.e., NAD + and NADPT
  • NA.D(P)H refers to the reduced versions of phosphorylated and un- phosphorylated NAD, i.e., NADH and NADPH.
  • NAD(P)H is used to describe the redox cofactor in an enzyme catalyzed reaction, it indicates that NADH and/or NADPH is used.
  • NAD(P)' f is the notation used, it indicates that NAD* and/or NADP 4 is used.
  • proteins may only bind either a phosphorylated or un-phosphorylated cofactor, there are redox cofactor promiscuous proteins, natural or engineered, that are indiscriminate; in these cases, the protein may use either NADH and/or NADPH.
  • enzymes that preferentially utilize either NAD(P) or NAD may carry' out the same catalytic reaction when bound to either form.
  • temperatures, titers, yields, oxygen uptake rate (OUR), and pH are recited in the description and in the claims. It should be understood that these values are not exact. However, the values can be approximated to the rightmost/last/k-ast significant figure, except where otherwise indicated
  • a temperature range of from about 30° C. to about 42° C. covers the range 25° C. to 44° C
  • numerical ranges recited can also include the recited minimum value and the recited maximum value when the values are approximated to the rightmost/last/Ieast significant figure.
  • a temperature range of from about 25° C. to about 50° C covers the range of 25° C. to 50° C
  • a recombinant host cell comprising: a heterologous nucleic acids encoding an aspartate racemase or a broad spectrum amino acid racemase.
  • the recombinant host cell comprises a heterologous nucleic acids encoding an aspartate racemase. In one embodiment, the recombinant host cell comprises a heterologous nucleic acids encoding a broad spectrum amino acid racemase.
  • the recombinant host cell further comprises one or more heterologous nucleic acids encoding an aspartate-forming enzyme, which is an aspartate dehydrogenase. In one embodiment, the recombinant host cell further comprises one or more heterologous nucleic acids encoding an aspartate-forming enzyme, which is an aspartate transaminase.
  • the recombinant host cell further 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.
  • an oxaloacetate-forming enzyme selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase.
  • the oxaloacetate-forming enzyme is pyruvate carboxylase.
  • the oxaloacetate-forming enzyme is phosphoenolpyruvate carboxylase.
  • the oxaloacetate-forming enzyme is phosphoenolpyruvate carboxykinase.
  • the recombinant host cell further overexpresses a native or heterologous, aspartate inhibition-resistant mutant of the native oxaloacetate-forming enzyme.
  • the recombinant host cell expresses a heterologous pyruvate carboxylase or phosphoenolpyruvate carboxylase that is naturally more resistant to inhibition by aspartate than the native pyruvate carboxylase or phosphoenolpyruvate carboxylase in Corynebacterhim glutamicum .
  • the native pyruvate carboxylase or phosphoenolpyruvate carboxylase is mutated to be resistant to inhibition by aspartate relative to the native enzyme in Corynebacterhim glutamicum .
  • the recombinant host cell further comprises one or more heterologous nucleic acids encoding an aspartate 1 -decarboxylase.
  • the recombinant host cell is capable of producing aspartate under anaerobic conditions.
  • the recombinant host cell is a bacterial cell. In another embodiment, the recombinant host cell is Escherichia coli. In another embodiment, the recombinant host cell is Corynebacterium glutamicum. In another embodiment, the recombinant host cell is Pantoea ananatis.
  • the aspartate dehydrogenase is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
  • the aspartate dehydrogenase has at least 40% amino acid identity with SEQ ID NO: 33.
  • the aspartate transaminase is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the aspartate transaminase has at least 40% amino acid identity with SEQ ID NO: 36.
  • the pyruvate carboxylase is SEQ ID NO: 15.
  • phosphoenolpyruvate carboxykinase is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.
  • the phosphoenolpyruvate carboxylase is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
  • the phosphoenolpyruvate carboxylase has at least 40% amino acid identity with SEQ ID NO: 35.
  • the aspartate 1 -decarboxylase is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 37, and SEQ ID NO: 38.
  • the phosphoenolpyruvate carboxylase has at least 40% amino acid identity with SEQ ID NO: 39 or SEQ ID NO: 40.
  • the recombinant host cell further comprises one or more disruptions of one or more genes encoding a succinate dehydrogenase subunit.
  • the succinate dehydrogenase subunit is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11.
  • the succinate dehydrogenase subunit has at least 40% amino acid identity with SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 11.
  • the recombinant host cell further comprises one or more disruptions of one or more genes encoding a lactate dehydrogenase.
  • the lactate dehydrogenase comprises SEQ ID NO: 1 .
  • the lactate dehydrogenase has at least 40% amino acid identity with SEQ ID NO: 1.
  • the recombinant host cell further comprises one or more genes encoding a NADP + -utilizing GAPDH.
  • the NADP + -utilizing GAPDH is selected from the group comprising UniProt ID: Q97D25 and UniProtID: Q6M0E6.
  • the recombinant host cell further comprises one or more disruptions of one or more genes encoding NAD + -utilizing GAPDH.
  • the NAD + -utilizing GAPDH is selected from the group comprising UniProtID: A0A0U4IQV8 and UniProt ID: P0A9B2.
  • a method of producing aspartic acid, beta-alanine, or a salt of each thereof comprising the step of culturing the recombinant host cell provided herein to produce aspartic acid, beta-alanine, or the salt of each thereof.
  • aspartic acid or a salt thereof is produced.
  • beta alanine is produced.
  • the culturing is performed under anaerobic conditions.
  • the method produces at least 25% yield of 1-aspartic acid or b- alanine per g-glucose.
  • the method further comprises: recovering the aspartic acid and/or the b-alanine from recombinant host cell- containing culture; and purifying the aspartic acid or beta-alanine.
  • a method for isolating aspartic acid or a salt thereof comprising: culturing the recombinant host cell provided herein in a fermentation broth to produce aspartic acid or a salt thereof; separating the recombinant host cell from the fermentation broth to produce a clarified fermentation broth; optionally, concentrating the clarified fermentation broth to provide a concentrated fermentation broth; optionally contacting the concentrated fermentation broth with an ion exchange resin or activated carbon adsorbent; acidifying the clarified fermentation broth or the concentrated fermentation broth to precipitate the aspartic acid or the salt thereof; and isolating the precipitated aspartic acid or the salt thereof.
  • the fermentation broth is maintained at a pH of about 6 to about pH 8.
  • the acidifying is performed with a mineral acid or a resin based acid.
  • the aspartic acid is isolated by filtration.
  • a supernatant is obtained after the acidification, which contains additional aspartic acid to be isolated by subsequent crystallization.
  • the host cell is a bacterial cell. In another embodiment, the host cell is Corynebacterium glutamicum. In another embodiment, the recombinant host cell is capable of producing aspartate under anaerobic conditions.
  • the culturing produce at least about 7 g/liter, or at least about 10 g/liter, or at least about 15 g/liter aspartic acid or a salt thereof, after about 1 - about 12 days of contacting.
  • the recombinant host cell further comprises one or more disruptions of one or more genes encoding a succinate dehydrogenase subunit.
  • the recombinant host cell further comprises one or more disruptions of one or more genes encoding a lactate dehydrogenase.
  • the fermentation broth comprises at least about 20 g/1 of aspartic acid or the salt thereof.
  • the concentrated acidified broth comprises at least about 90 g/1 of aspartic acid or the salt thereof.
  • up to about 90% or up to about 95% of the aspartic acid or the salt thereof present in the fermentation broth is isolated.
  • the isolated aspartic acid or the salt thereof has a purity of about 90% or more.
  • a process comprising converting L-aspartic acid or a salt thereof, either non-purified in a broth or purified in a solution, to D-aspartic acid or a salt thereof, comprising contacting the broth or the solution of L-aspartic acid with one or more cells of a microorganism, preferably a heterologous microorganism, expressing an aspartate racemase, thereby converting L-aspartic acid to D-aspartic acid.
  • the microorganism is a recombinant host cell, preferably one provided herein.
  • a process comprising converting L-aspartic acid or a salt thereof, to D-aspartic acid or a salt thereof, comprising contacting a broth or a solution of L- aspartic acid with an aspartate racemase, thereby converting L-aspartic acid to D-aspartic acid.
  • the L -aspartic acid or the salt thereof is either non-purified in a broth.
  • the L -aspartic acid or the salt thereof is purified in a solution.
  • a solution comprising the aspartate racemase is contacted with immobilized aspartate racemase, preferably comprised in a column.
  • the process comprises flowing the broth or the solution of L-aspartic acid or the salt thereof through a immobilized aspartate racemase, preferably comprised in a column.
  • a process comprising heterologously expressing an aspartate racemase in a microorganism that overproduces L-aspartic acid or a salt thereof to diagnose upstream pathway enzyme inhibition by L-aspartic acid or a salt thereof, wherein the total production of D-aspartic acid or a salt thereof and L-aspartic acid or a salt thereof with expression of the aspartate racemase higher than the concentration of only L-aspartic acid or a salt thereof produced without expression of the aspartate racemase indicates a feedback inhibition of L-aspartic acid in an upstream pathway.
  • microorganism comprising a feedback inhibition of L-aspartic acid or a salt thereof in an upstream pathway as identified by a process provided herein.
  • microorganism comprising a no or substantially no feedback inhibition of L-aspartic acid or a salt thereof in an upstream pathway as identified by the process provided herein.
  • a process of producing aspartic acid or a salt thereof comprising culturing the microorganism identified herein, thereby producing aspartic acid or a salt thereof.
  • process comprising: contacting a recombinant, heterologous microorganism comprising one or more of a polypeptide having: one or more heterologous nucleic acids encoding an oxaloacetate-forming enzyme selected from the group consisting of pyruvate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase; and one or more disruptions of one or more genes encoding a succinate dehydrogenase subunit, a malate dehydrogenase, and/or one or more disruptions of one or more genes encoding a lactate dehydrogenase, with a feed stock comprising glucose, and optionally a bicarbonate (HCO3 ) salt, to produce at least about 7 g/liter, or at least about 10 g/liter, or at least about 15 g/liter aspartic acid or a salt thereof, after about 1 - about 12 days of contacting.
  • the contacting is performed in the absence of, or the contacting does not require, the bicarbonate salt. In another embodiment, the contacting is performed in the presence of the bicarbonate salt. In another embodiment, the contacting is performed under an anaerobic condition. In another embodiment, the contacting is performed under an aerobic condition. In another embodiment, the contacting is performed under an anaerobic condition. In another embodiment, the contacting is performed under an aerobic condition.
  • the contacting is initially performed in the absence of, or initially the contacting does not require, the bicarbonate salt, and then the contacting is performed in the presence of the bicarbonate salt.
  • the initial contacting is performed under an anaerobic condition.
  • the contacting performed in the presence of the bicarbonate salt is performed under an aerobic condition.
  • the process (or the culturing) produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of alanine or a salt thereof after about 1 - about 12 days of contacting.
  • the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of alpha-ketoglutaric acid or a salt thereof after about 1 - about 12 days of contacting.
  • the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of pyruvic acid or a salt thereof after about 1 - about 12 days of contacting. In another embodiment, the process produces less than about 4 g/liter, preferably less than about 3 g/liter, or more preferably, less than about 2 g/liter of acetic acid or a salt thereof after about 1 - about 12 days of contacting.
  • An embodiment which is non-naturally occurring microorganism that produces L- aspartic acid anaerobically or microaerobically composed of: (1) an deletion or reduced expression of lactate dehydrogenase; (2) deletion, reduced expression, or conditionally reduced expression of one or more of succinate dehydrogenase, malate dehydrogenase, or fumarase during the production phase; and (3) increased expression of a native or heterologous phosphoenolpyruvate carboxykinase, aspartate-resistant native or heterologous pyruvate carboxylase, and/or aspartate-resistant native or heterologous phosphoenolpyruvate carboxylase.
  • microorganism in embodiment 1 with additional deletion or reduced expression of one or more of acetate kinase, phosphotransacetylase, coenzyme A transferase, and/or pyruvate quinone oxidase.
  • microorganism in embodiments 1-3 with additional native overexpression or heterologous expression of an aspartate transaminase and/or aspartate dehydrogenase.
  • microorganism in embodiments 1-4 with additional deletion of one or more alanine transaminases.
  • microorganism in embodiments 1-6 with additional native overexpression or heterologous expression of a glucokinase, galactose permease, or glucose permease.
  • the microorganism in embodiments 1 -7 with additional overexpression or heterologous expression of a glutamate mechanosensitive channel or other aspartate exporter, or an exporter engineered for enhanced export of L-aspartic acid.
  • microorganism in embodiments 1-8 with additional deletion or reduced expression of genes in the L-lysine, L-threonine, L-cysteine, S-adenosyl-L-methionine, L- methionine, L-isoleucine, and beta-nicotinamide adenine dinucleotide biosynthesis pathways.
  • the microorganism of embodiments 1-9 with additional native overexpression or heterologous expression of a nucleoside diphosphate kinase and/or an adenylate kinase.
  • the microorganism of embodiments 1-10 with additional heterologous expression of a membrane-bound or soluble pyridine nucleotide transhydrogenase.
  • microorganism of embodiments 1-11 with conditional overexpression of a phosphoenolpyruvate carboxykinase when oxygen levels are reduced from those of ambient air, and/or at 37 degrees Celsius.
  • the microorganism of embodiments 1-12 with native overexpression or heterologous expression of a carbonic anhydrase.
  • the microorganism of embodiments 1-13 with expression of an aspartate transaminase possessing reduced alanine transaminase activity.
  • the microorganism of embodiments 1-14 with additional heterologous expression of an aspartate racemase.
  • An embodiment which is a process for producing aspartic acid using the microorganism of claims 1-15 and 34-38 by cultivating with a surfactant, such as a polysorbate surfactant such as Tween-20 or Tween-80, that enhances aspartic acid production.
  • a surfactant such as a polysorbate surfactant such as Tween-20 or Tween-80
  • An embodiment which is a non-naturally occurring microorganism that produces L- aspartic acid aerobically composed of: (1) reduced carbon flux through the oxidative tricarboxylic acid cycle via reduced expression or activity of pyruvate dehydrogenase, isocitrate dehydrogenase, citrate synthase, citrate hydro-lyase, or D-/Areo-isocitrate hydro-lyase; and optionally (2) increased expression of a native or heterologous native or heterologous pyruvate carboxylase, and/or native or heterologous phosphoenolpyruvate carboxylase; and further optionally (3) deletion or reduced expression of phosphoenolpyruvate carboxykinase.
  • the microorganism in embodiments 17-19 with additional deletion or reduced expression of one or more of acetate kinase, phosphotransacetylase, coenzyme A transferase, and/or pyruvate quinone oxidase.
  • the microorganism in embodiments 17-20 with additional native overexpression or heterologous expression of an aspartate transaminase and/or aspartate dehydrogenase.
  • the microorganism in embodiments 17-22 with additional deletion of aspartate ammonia lyase.
  • microorganism in embodiments 17-23 with additional native overexpression or removal of native regulation (e.g., deletion of the gene encoding lolR), or heterologous expression of a glucokinase, galactose permease, or glucose permease.
  • native regulation e.g., deletion of the gene encoding lolR
  • heterologous expression of a glucokinase, galactose permease, or glucose permease e.g., deletion of the gene encoding lolR
  • heterologous expression of a glucokinase, galactose permease, or glucose permease e.g., deletion of the gene encoding lolR
  • heterologous expression of a glucokinase, galactose permease, or glucose permease e.g., deletion of the gene encoding lolR
  • microorganism in embodiments 17-25 with additional deletion or reduced expression of genes in the L-lysine, L-threonine, L-cysteine, S-adenosyl-L-methionine, L-methionine, L-isoleucine, and beta-nicotinamide adenine dinucleotide biosynthesis pathways.
  • the microorganism of embodiments 17-26 with additional heterologous expression of a membrane-bound or soluble pyridine nucleotide transhydrogenase.
  • microorganism of embodiments 17-30 with deletion or reduced expression of a lysine exporter LysE and/or threonine exporter ThrE, to reduce the accumulation of lysine as a byproduct.
  • An embodiment which is a process for producing aspartic acid using the microorganism of claims 17-32 and 34-36 by cultivating with a surfactant, such as a polysorbate surfactant such as Tween-20 or Tween-80, that enhances aspartic acid production.
  • a surfactant such as a polysorbate surfactant such as Tween-20 or Tween-80
  • the succinate dehydrogenase subunit is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11.
  • the oxaloacetate-forming enzyme is a phosphoenolpyruvate carb oxy kinase.
  • the oxaloacetate-forming enzyme is a phosphoenolpyruvate carboxykinase selected from the group consisting SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.
  • the heterologous microorganism further comprises one or more heterologous nucleic acids encoding an aspartate-forming enzyme selected from the group consisting of aspartate dehydrogenase and aspartate transaminase.
  • the aspartate dehydrogenase has a sequence having at least 95% amino acid identity with SEQ ID NO: 23, AspDH#15, AspDH#17, AspDH#18, or AspDH#20.
  • the recombinant, heterologous microorganism is a recombinant, heterologous Corynebaclerium glutamicum.
  • the aspartate dehydrogenase (AspDH) (EC #1.4.1.21) utilized herein catalyzes the conversion of one molecule of oxaloacetate, one molecule of NAD(P)H, one molecule of NEE and one proton to one molecule of aspartate, one molecule of H2O and one molecule of NAD(P) + .
  • Any enzyme is suitable for use in accordance with the invention so long as the enzyme is capable of catalyzing said AspDH reaction.
  • NADP which consists of reduced and oxidized forms, i.e., NADPH and NADP +
  • NADP which consists of reduced and oxidized forms
  • Native enzyme cofactor specificity can be altered, however, by standard microbial engineering techniques, and recombinant host cells can be designed to express modified enzymes that utilize NADH, or NADH and NADPH non-selectively, instead of NADPH exclusively.
  • AspDH is able to utilize either NADH or NADPH as a cofactor.
  • NADH is produced during the recombinant host cell's glycolytic processes in converting glucose to pyruvate.
  • the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in glycolysis reduces NAD + to NADH; therefore, in embodiments wherein GAPDH produces NADH, the AspDH is NADH- utilizing to ensure AspDH turnover is not impeded as AspDH is able to utilize readily available NADH.
  • GAPDH glyceraldehyde 3-phosphate dehydrogenase
  • natively expressed GAPDH reduces NADP + to generate NADPH.
  • the AspDH is NADPH-utilizing. Details on NADPH-producing/NADP + -utilizing GAPDH are disclosed below in section 2.5.1.2.
  • the AspDHs of the present disclosure comprise: (1) NADH-utilizing AspDH; (2) NADPH-utilizing AspDH; and (3) AspDH that can indiscriminately utilize NADH and NADPH.
  • the recombinant host cells comprise an AspDH that utilizes NADH as a cofactor and is capable of producing aspartic acid and/or P-alanine.
  • the recombinant host cells comprise an AspDH that utilizes NADPH as a cofactor and is capable of producing aspartic acid and/or P-alanine.
  • the recombinant host cells comprise an AspDH that utilizes NADH and/or NADPH as a cofactor and is capable of producing aspartic acid and/or P-alanine.
  • AspDH is capable of utilizing NADH and NADPH
  • recombinant host cells may further comprise a transhydrogenase (EC #1.6.1.1, 1.6.1.2, or 1.6.1.5).
  • the AspDH is derived from a prokaryotic source.
  • the AspDH is derived from a host cell belonging to a genus selected from the group comprising Bradyrhizobium, Escherichia, Thermotoga, Klebsiella, Cupriavidus, Rhodopseudomonas, Pseudomonas, Variovorax, Delftia, Ralstonia, Burkholderia, Ochrobactrum, Acinetobacter, Dinoroseobacter, Ruegeria, Herbaspirillum, and Comamonas.
  • Non-limiting examples of prokaryotic AspDH enzymes include the Pseudomonas aeruginosa UniProt ID: Q9HYA4 (abbv. PaAspDH), Cupriavidus taiwanensis UniProt ID: B3R8S4 (abbv. AspDH #2), the Polar omonas sp. UniProt ID: Q126FS (abbv. AspDH #4), Klebsiella pneumoniae UniProt ID: A6TDT8 (abbv. AspDH #9), Comamonas testosteroni UniProt ID: D0IX49 (abbv. AspDH #12), Delftia acidovarans UniProt ID: S2WWY2 (abbv.
  • AspDH #14) Pariovorax sp. UniProt ID: A0A1C6Q9L7 (abbv. AspDH #16), Thermotoga maritima UniProt ID: Q9X1X6 (abbv. TmAspDH), Ralstonia solanacearum UniProt ID: Q8XRV9 (abbv. AspDH #3), Burkholderia thailandensis UniProt ID: Q2T559 (abbv. AspDH #5), Burkholderia pseudomallei UniProt ID: Q3 JFK2 (abbv. AspDH #6), Ochrobactrum anthropic UniProt ID: A6X792 (abbv.
  • AspDH #7 Acinetobacter sp. UniProt ID: D6JRV1 (abbv. AspDH #8), Dinoroseobacter shibae UniProt ID: A8LLH8 (abbv. AspDH #10), Rugeria pomeroyi UniProt ID: Q5LPG8 (abbv. AspDH #11), Ralstonia eutropha UniProt ID: Q46VA0 (abbv. AspDH #13), Pseudomonase sp. ENNP23 UniProt ID: A0A1E4W5J7 (abbv. AspDH #15), Herbaspirillum frisingense UniProt ID: R0EI78 (abbv.
  • AspDH #17) Burkholderiaceae bacterium 16 UniProt ID: A0A0F0FQG4 (abbv. AspDH #18), Ralstonia sp. GA3-3 UniProt ID: R7XIB1 (abbv. AspDH #19), Cupriavidus sp. SK-3 UniProt ID: A0A069IKY7 (abbv. AspDH #20) and Cupriavidus necator UniProt ID: Q46VA0 (abbv. CnAspDH).
  • the AspDH is derived from an archaeal source. In many of these embodiments, the AspDH is derived from a host cell belonging to the genus Archaeoglobus.
  • archaeal AspDH is the A. fulgidus UniProt ID: 028440.
  • the AspDH is the Cupriavidus taiwanensis AspDH (abbv. AspDH #2; UniProt ID: B3R8S4; SEQ ID NO: 19). In some embodiments, the AspDH is the Polaromonas sp. AspDH (abbv. AspDH #4; UniProt ID: Q126FS; SEQ ID NO: 20). In some embodiments, the AspDH is the Klebsiella pneumoniae AspDH (abbv. AspDH #9; UniProt ID: A6TDT8; SEQ ID NO: 21). In some embodiments, the AspDH is the Comamonas testosteroni AspDH (abbv.
  • the AspDH is the Delftia acidovarans AspDH (abbv. AspDH #14; UniProt ID: S2WWY2; SEQ ID NO: 22).
  • the AspDH is the Variovorax sp. AspDH (abbv. AspDH #16; UniProt ID: A0A1C6Q9L7; SEQ ID NO: 23).
  • the AspDH is the Pseudomonase aeruginosa AspDH (abbv. PaAspDH; UniProt ID: Q9HYA4; SEQ ID NO: 34).
  • the AspDH is the Ralstonia solanacearum UniProt ID: Q8XRV9 (abbv. AspDH #3). In some embodiments, the AspDH is the Burkholderia thailandensis UniProt ID: Q2T559 (abbv. AspDH #5). In some embodiments, the AspDH is the Burkholderia pseudomallei UniProt ID: Q3JFK2 (abbv. AspDH #6). In some embodiments, the AspDH is the Ochrobactrum anthropic UniProt ID: A6X792 (abbv. AspDH #7). In some embodiments, the AspDH is the Acinetobacter sp.
  • the AspDH is the Dinoroseobacter shibae UniProt ID: A8LLH8 (abbv. AspDH #10).
  • the AspDH is the Rugeria pomeroyi UniProt ID: Q5LPG8 (abbv. AspDH #11).
  • the AspDH is the Ralstonia eutropha UniProt ID: Q46VA0 (abbv. AspDH #13).
  • the AspDH is the Pseudomonase sp. ENNP23 UniProt ID: A0A1E4W5J7 (abbv. AspDH #15).
  • the AspDH is the Herbaspirilhim fiisingense UniProt ID: R0EI78 (abbv. AspDH #17). In some embodiments, the AspDH is the Burkholderiaceae bacterium 16 UniProt ID: A0A0F0FQG4 (abbv. AspDH #18). In some embodiments, the AspDH is the Ralstonia sp. GA3-3 UniProt ID: R7XIB1 (abbv. AspDH #19). In some embodiments, the AspDH is the Cupriavidus sp. SK-3 UniProt ID: A0A069IKY7 (abbv. AspDH #20)
  • recombinant host cells comprise one or more heterologous nucleic acids encoding an AspDH wherein said recombinant host cells are capable of producing aspartic acid and/or P-alanine.
  • proteins suitable for use in accordance with methods of the present disclosure have AspDH activity and comprise an amino acid sequence with at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 34.
  • proteins suitable for use in accordance with methods of the present disclosure have AspDH activity and comprise an amino acid sequence with at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with AspDH #3, AspDH #5, AspDH #6, AspDH #7, AspDH #8, AspDH #10, AspDH #11, AspDH #13, AspDH #15, AspDH #17, AspDH #18, AspDH #19, or AspDH #20.
  • the recombinant host cell is a C. glutamicum strain.
  • recombinant host cells comprise one or more heterologous nucleic acids encoding an AspDH wherein the AspDH was mutagenized towards an altered enzyme characteristic such as altered substrate affinity, cofactor affinity, altered reaction rate, and/or altered inhibitor affinity.
  • the AspDH variant is a product of one or more protein engineering cycles.
  • the AspDH variant comprises one or more point mutations.
  • proteins suitable for use in accordance with methods of the present disclosure have AspDH activity and comprise an amino acid sequence with at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 34.
  • proteins suitable for use in accordance with the methods of the present disclosure have AspDH activity and comprise an amino acid sequence with at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with AspDH #3, AspDH #5, AspDH #6, AspDH #7, AspDH #8, AspDH #10, AspDH #11, AspDH #13, AspDH #15, AspDH #17, AspDH #18, AspDH #19, or AspDH #20.
  • the AspDH variant has increased affinity for NADH.
  • the recombinant host cell is a C. ghitamicum strain.
  • the AspDH consensus sequence #2 (SEQ ID NO: 33) 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 specific position in an AspDH.
  • Many amino acids in consensus sequence #2 (SEQ ID NO: 33) are highly conserved and AspDHs suitable for use in accordance with the methods of the present disclosure will comprise a substantial number, and sometimes all, of these highly conserved amino acids at positions aligning with the location of the indicated amino acids in consensus sequence #2 (SEQ ID NO: 33).
  • proteins suitable for use in accordance with the methods of the present disclosure have AspDH activity and comprise an amino acid sequence with at least 40%, at least 50%, at least 60%, at least 65%, or at least 70% sequence identity with consensus sequence #2 (SEQ ID NO: 33).
  • the PaAspDH sequence (SEQ ID NO: 34) is at least 40% identical to consensus sequence #2 (SEQ ID NO: 33), and is therefore encompassed by consensus sequence #2 (SEQ ID NO: 33).
  • amino acids that are highly conserved are G8, G10, All, 112, G13, E69, A71, G72, H73, A75, H79, P82, L84, G87, S94, G96, A97, L98, A110, Al 11, G114, L120, G123, A124, 1125, G126, D129, A 130, 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, L
  • AspDH enzymes homologous to SEQ ID NO: 33 comprise at least 40%, 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: 33.
  • each of these highly conserved amino acids are found in a desired AspDHs as provided, for example, in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 34.
  • Amino acid H220 in SEQ ID NO: 33 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: 33 is found in enzymes homologous to SEQ ID NO: 33.
  • the strictly conserved amino acid corresponding to H220 in consensus sequence SEQ ID NO: 33 is found in AspDHs set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 34.
  • the recombinant microorganism comprises a deletion or suppression of lactate dehydrogenase (Idh). In another embodiment, the recombinant microorganism comprises a deletion or suppression of the genes encoding the 3 subunits of succinate dehydrogenase (sdhA, sdhB, sdhC). In another embodiment, the recombinant microorganism comprises a deletion or suppression of acetate kinase (ackA). In another embodiment, the recombinant microorganism comprises a deletion or suppression of phosphoacetyltransferase (pta).
  • Idh lactate dehydrogenase
  • the recombinant microorganism comprises a deletion or suppression of the genes encoding the 3 subunits of succinate dehydrogenase (sdhA, sdhB, sdhC). In another embodiment, the recombinant microorganism comprises a deletion or suppression of acetate kina
  • the recombinant microorganism comprises a deletion or suppression of malate:quinone oxidoreductase (mqo., Uniprot ID 069282). In another embodiment, the recombinant microorganism comprises a deletion or suppression of alaninevaline aminotransferase (avtA, Uniprot ID Q8NMH4).
  • the recombinant microorganism comprises a deletion or suppression of oxaloacetate decarboxylase activity possessed by 2-keto-4-pentenoate hydratase/2-oxohepta-3-ene-l, 7-dioic acid hydratase (pdx; locus ID Cgll290; Uniprot ID Q8NQY2).
  • the recombinant microorganism comprises phosphoenolpyruvate carboxykinase (PckA).
  • PckA phosphoenolpyruvate carboxykinase
  • the PckA is a native PckA.
  • the PckA is heterologous.
  • the PckA is aspartate feedback-resistant or aspartate resistant.
  • Illustrative and non-limiting examples of PckA utilized herein include those disclosed in the examples section.
  • the PckA is overexpressed.
  • the recombinant microorganism comprises phosphoenolpyruvate carboxykinase (PCK1).
  • the PCK1 is a native PCK1.
  • the PCK1 is heterologus.
  • the PCK1 is aspartate feedback-resistant or aspartate resistant.
  • the PCK1 is overexpressed.
  • Illustrative and nonlimiting examples of PCK1 utilized herein include those disclosed in the examples section.
  • the recombinant microorganism comprises a pyruvate carboxylase (Pyc).
  • the Pyc is overexpressed.
  • the Pyc is native Pyc.
  • the Pyc is heterologous.
  • the pyruvate carboxylase useful herein has an amino acid sequence selected from SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95.
  • the Pyc is heterologous.
  • the pyruvate carboxylase useful herein has an amino acid sequence selected from SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104.
  • Pyes utilized herein include those disclosed in the examples section.
  • a recombinant microorganism comprising overexpression of Corynebacterium glutamicum PckA.
  • the recombinant microorganism comprises overexpression of Herbaspirillum frisingense AspDH.
  • the recombinant microorganism comprises overexpression of an NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum, CaGapC.
  • a recombinant microorganism comprising overexpression of Corynebacterium glutamicum PckA, Herbaspirillum frisingense AspDH, and an NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum, CaGapC.
  • a recombinant microorganism comprising pyruvate carboxylase from Lactococcus lactis.
  • the recombinant microorganism comprises NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum.
  • the recombinant microorganism comprises aspartate dehydrogenase from Herbaspirillum frisingense.
  • a recombinant microorganism comprising pyruvate carboxylase from Lactococcus lactis, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum, aspartate dehydrogenase from Herbaspirillum frisingense .
  • a recombinant microorganism comprising pyruvate carboxylase from Lactococcus lactis.
  • the recombinant microorganism comprises NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum .
  • the recombinant microorganism comprises aspartate dehydrogenase from Herbaspirillum frisingense .
  • a recombinant microorganism comprising pyruvate carboxylase from Lactococcus lactis, NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum, aspartate dehydrogenase from Herbaspirillum frisingense.
  • one or more of pyruvate carboxylase from Lactococcus lactis, NADP-dependent glyceraldehyde- 3 -phosphate dehydrogenase from Clostridium acetobutylicum, and aspartate dehydrogenase from Herbaspirillum frisingense are overexpressed.
  • the recombinant microorganism comprises reduced or suppressed GltA citrate synthase activity (Uniprot ID P42457). In some embodiments, the recombinant microorganism comprises a deletion of a gene encoding methylcitrate synthase 1 (prpCl, Uniprot ID Q8NSH7). In some embodiments, the recombinant microorganism comprises reduced or suppressed citrate synthase activity. Without being bound by theory, recombinants microorganisms comprising reduced or suppressed citrate synthase activity, are, in certain embodiments, useful for producing or overproducing aspartic acid or a salt thereof under aerobic fermentation conditions.
  • Such recombinant microorganisms that are useful for aerobic fermentation do not comprise, in certain embodiments, Idh, sdh, and such other dehydrogenase deletions or suppressions.
  • Recombinant microorganisms that produce or overproduce aspartic acid or a salt thereof under anaerobic fermentation conditions comprise, in certain embodiments, Idh, sdh, and such other dehydrogenase deletions or suppressions.
  • the recombinant microorganism comprises a deletion of a gene encoding methylcitrate synthase 1 (prpCl)
  • a recombinant microorganism comprising pyruvate carboxylase from Lactococcus lactis.
  • a recombinant microorganism comprising aspartate dehydrogenase from Herbaspirillum frisingense.
  • a recombinant microorganism comprising aspartate racemase (EC 5.1.1.3).
  • aspartate racemase useful herein has amino acid sequences selected from SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.
  • a recombinant microorganism comprising aspartate racemase from Enterococcus faecalis.
  • a recombinant microorganism comprising: pyruvate carboxylase from Lactococcus lactis, aspartate dehydrogenase from Herbaspirillum frisingense, and aspartate racemase from Enterococcus faecalis .
  • recombinant microorganisms comprising the following expression or overexpression and/or deletion of genes/proteins are provided, which have background strain genotype selected from and production strain genotype (parent + plasmid-based overexpression) selected from
  • a method of producing aspartic acid or a salt thereof comprising fermenting a recombinant microorganism selected from LCG4-258, LCG4-
  • the aspartic acid or the salt thereof is produced at about 5 - about 40 g/L aspartate. In one embodiment, the aspartic acid or the salt thereof is produced at about 5 - about 30 g/L aspartate.
  • the aspartic acid or the salt thereof is produced at about 10 - about 25 g/L aspartate.
  • the titer is obtained in about 24- about 150 hours. In one embodiment, the titer is obtained in about 48- about 150 hours. In one embodiment, the titer is obtained in about 72- about 150 hours. In one embodiment, the titer is obtained in about 96- about 150 hours. In one embodiment, the titer is obtained in about 136 hours.
  • the maximum productivity is about 0.17 g/L/hr. In one embodiment, the maximum productivity is about 0.2 g/L/hr. In one embodiment, the maximum productivity is about 0.25 g/L/hr.
  • the maximum productivity is about 0.3 g/L/hr. In one embodiment, the maximum productivity is about 0.5 g/L/hr. In one embodiment, the maximum productivity is about 0.6 g/L/hr. In one embodiment, the maximum productivity is about 0.75 g/L/hr.
  • the recombinant microorganism or host cell is Coryne bacterium glutamicum.
  • amino acid or nucleic acid sequences utilized herein, including those in the examples, are useful in accordance with the present disclosure.
  • amino acid sequences of proteins useful in accordance with the present disclosure include, without limitation, the sequences: HfAspDH (protein), VsAspDH (protein), CgPckA (protein), CaGapC (protein), CgPpc-N917G (protein), CgPyc-P458S (protein), EcPckA, EhPckAl, EhPckA2, GtPckA, LmPckA-Y180F, MfPckA, MsPckA, PfPckA, RbPckA, RnPCKl, ScPCKl, and SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:
  • sequences having at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, and at least 90% identity with amino acid or nucleic acid sequences utilized herein, including those in the examples, are useful in accordance with the present disclosure.
  • sequences having at least 99%, identity with amino acid or nucleic acid sequences utilized herein, including those in the examples, are useful in accordance with the present disclosure.
  • sequences having at least 98% identity with amino acid and nucleic acid sequences utilized herein, including those in the examples are useful in accordance with the present disclosure.
  • sequences having at least 97% identity with amino acid and nucleic acid sequences utilized herein, including those in the examples, are useful in accordance with the present disclosure.
  • sequences having at least 96% identity with amino acid and nucleic acid sequences utilized herein, including those in the examples are useful in accordance with the present disclosure.
  • sequences having at least 95% identity with amino acid and nucleic acid sequences utilized herein, including those in the examples are useful in accordance with the present disclosure.
  • sequences having at least 90% identity with amino acid and nucleic acid sequences utilized herein, including those in the examples are useful in accordance with the present disclosure.
  • the polysuccinimide is hydrolyzed to provide polyaspartic acid or a salt thereof.
  • the polysuccinimide is crosslinked to form a crosslinked polymer such as a polysuccinimide.
  • the crosslinker is an alpha, omega diamine.
  • the crosslinker is a Jeffamine diamine.
  • the crosslinker is H2N(CH2)2O(CH2)2O(CH2)2NH2.
  • the crosslinked polysuccinimide is hydrolyzed to provide a crosslinked polyaspartate.
  • Example 1 Construction of aspartic acid production strains with phosphenolpyruvate carboxykinase overexpression.
  • Corynebacterium glutamicum MB001 is a reduced genome strain of C. glutamicum ATCC 13032 with in-frame deletions of 3 large prophages (Baumgart 2013).
  • the gene encoding lactate dehydrogenase (ldh) was first deleted from MB001 to generate strain LCG4-001 by transforming with pLCSac-LDH, which is an E. coli and C.
  • glutamicum shuttle vector containing a ColE1 origin, temperature-sensitive pBL1 origin, NeoR/KanR resistance gene, the gene encoding levansucrase (sacB) from Bacillus subtilis under control of a lacM promoter (Tan 2012), and 750 bp upstream and downstream homologous regions to C. glutamicum ldh that also contain the last 267 bp and 278 bp of the ldh gene, respectively.
  • the plasmid was transformed via electroporation and sucrose counterselection was performed according to the method described by Okibe et al., 2011.
  • LCG4-248 An additional knockout strain, LCG4-248, was also generated by deleting the genes encoding acetate kinase (ackA) and phosphoacetyltransferase (pta) present in the pta-ackA operon in LCG4-009. This was performed by first transforming LCG4-009 with pLASP-001, an E. coli/C. glutamicum shuttle vector containing a ColE1 origin, temperature-sensitive pBL1 origin, NeoR/KanR resistance gene, the gene encoding the lac operon repressor (lacI) from E.
  • lacI lac operon repressor
  • Electrocompetent cells were prepared after inducing the cell culture with 1 mM isopropyl-beta-D-1-galactopyranoside (IPTG) during exponential growth.
  • IPTG isopropyl-beta-D-1-galactopyranoside
  • LCG4-009 containing pLASP-001 was transformed with pLASP-002, which contains an E. coli pMB1 origin, C. glutamicum pCC1 origin, a spectinomycin resistance gene (aadA), a single guide RNA targeting a Cas9 cut site within pta represented by SEQ ID NO: 249 (ATCTTCCATCAAATTCCAGC) fused to a tracr RNA represented by SEQ ID NO: 250 (gttttagagctagaaatagcaagttaaaataaggctagtccg) expressed from a constitutive synthetic J23119 constitutive promoter represented in SEQ ID NO: 251 (ttgacagctagctcagtcctaggtataat), and upstream and downstream regions flanking the pta-ackA operon of 766 bp and 740 bp length, respectively.
  • adA a single guide RNA targeting a Cas9 cut site within pt
  • Expression vector pLASP-004 contains a ColE1 origin, pBL1 origin, NeoR/KanR resistance marker, a codon-optimized gene encoding aspartate dehydrogenase from Herbaspirillum frisingense (Uniprot ID R0EI78; HfAspDH) under a C.
  • glutamicum etfU promoter pCgEftU
  • CgPckA C. glutamicum
  • pCgGapC C. glutamicum gap promoter
  • Expression vector pLASP-058 is identical to pLASP-004 with the exception of HfAspDH being replaced by a gene encoding a codon-optimized aspartate dehydrogenase from Variovorax sp. HW608 (Uniprot A0A1C6Q9L7; VsAspDH).
  • Expression vector pLASP-059 is identical to pLASP-058 with the exception of CaGapC being replaced by a codon-optimized gene encoding an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans serotype c (Uniprot Q59931; SmuGapN).
  • Aspartate production strain LCG4-133 was generated by transforming pL ASP-058 into LCG4- 009 by electroporation.
  • LCG4-244 was generated by transforming pLASP-058 into LCG4-248 by electroporation.
  • LCG4-258 was generated by transforming pLASP-004 into LCG4-248 by electroporation.
  • All plasmids described above were constructed in one or multiple steps via ligation-independent cloning of PCR generated DNA fragments from existing or synthesized templates with T4 polymerase.
  • Example 2 Construction of aspartic acid production strains with aspartate feedback-resistant phosphenolpyruvate carboxylase or pyruvate carboxylase overexpression.
  • Expression vector pLASP-011 is identical to pLASP-004 with the exception of CgPckA being replaced by the native gene encoding phosphoenolpyruvate carboxylase (ppc) from C. glutamicum with an N917G mutation that was previously shown to exhibit reduced feedback inhibition from aspartate and malate (Chen 2014).
  • This vector was constructed by Gibson assembly using HiFi Assembly Master Mix (New England Biolabs, Ipswich, MA) with three PCR amplified DNA fragments.
  • the three fragments were amplified from pLASP-004 using primers YO4160 and YO13403, pLASP-004 using primers YO5556 and YO13356, and MB001 genomic DNA using primers YO13353 and Y013400.
  • the latter two primers incorporated the N917G mutation near the 3’ terminus of ppc and introduced non-coding mutations that altered the codons for L915, R916, S918, and G919. All oligonucleotides are listed in Table 1.
  • Expression vector pLASP-012 is identical to pLASP-011 with the exception of having CgPpc- D299N in place of CgPpc-N917G.
  • the D299N mutation was also previously shown to exhibit reduced feedback inhibition from aspartate and malate (Chen 2014). It was constructed by Gibson assembly of four PCR fragments that were amplified from pLASP-004 using primers YO4160 and YO13403, from pLASP-004 using primers YO5556 and YO13356, from MB001 genomic DNA using primers YO13353 and YO13363, and MB001 genomic DNA using primers YO13565 and YO13566.
  • the D299N mutation was introduced in YO13363 and YO13565 together with non-coding mutations that altered the codons for S296, L297, S298, and R300.
  • Expression vector pLASP-013 is identical to pLASP-004 with the exception of CgPckA being replaced by the native gene encoding pyruvate carboxylase (pyc) from C. glutamicum with a P458S mutation that has previously been shown to exhibit reduced feedback inhibition from aspartate (Ohnishi 2002), plus the replacement of the native GTG start codon with ATG.
  • CgPckA pyruvate carboxylase
  • Expression vector pLASP-016 is identical to pLASP-013 but contain pyc from C. glutamicum with the native GTG start codon.
  • Expression vectors pLASP-020 and pL ASP-021 are identical to pLASP-013 and pLASP-016, respectively, with the exception of containing a T343A mutation that has been shown to also exhibit reduced feedback inhibition from aspartate (Kortmann 2019).
  • Expression vectors described above were electroporated into LCG4-009 (MB001 with Idh and sdhCAB deletions) and LCG4-248 (MB001 with Idh, sdhCAB, and pta-ackA deletions) to generate production strains described in Table 2.
  • Example 3 Hungate tube screening procedure for aspartate production.
  • baffled shake flasks containing 30 mL of brain heart infusion (BHI) medium 37 g/L BHI powder dissolved in deionized water and autoclaved to sterilize
  • 25 mg/L kanamycin were inoculated with single colonies of strains struck from cryogenic glycerol stocks on BHI agar plates containing 25 mg/L kanamycin, and grown with 250 rpm shaking at 30°C.
  • BHI brain heart infusion
  • OD600 was measured and cultures were spun down at 3000 x g for 10 minutes, supernatants applied to a 96-well 0.22 um fdter plate on top of a 96-well HPLC plate and spun down another 3000 x g for 10 minutes. Filtered supernatants were then diluted 10-fold in deionized water into HPLC plates for organic acid and glucose analysis, and an HPLC plate for amino acid analysis.
  • the HPLC plates were thermally sealed with foil using an Agilent Plate-Lok and stored at 4°C until analysis by HPLC with RID or UV detection (with pre-column derivatization by o-phthalylaldehyde for amino acid UV detection).
  • CGXII l contains per liter: 100 mL of 1 M K-MOPS pH 7.0, 100 mL of 200 g/L ammonium sulfate, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1.64 mL of 10 mg/mL iron sulfate heptahydrate, 1 mL of a 30 mg/mL solution of protocatechuic acid in 0.05 M NaOH, 125 mL of 20% (w/v) glucose, 1 mL of Wolfe’s vitamin mix, 408.23 mL of Cg amino acid mix, 0.5 mL of 50 mg/mL kanamycin sulfate, and balance deionized water
  • CGXII_7 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 200 mL of 2 M urea, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1 mL of 10 mg/mL iron sulfate heptahydrate, 200 mL of 20% (w/v) glucose, 1.869 mL of Tween- 80, 0.5 mL of 50 mg/mL kanamycin sulfate, and balance deionized water.
  • 1000X CGXII trace metal solution contains per I L: 10 g magnesium sulfate monohydrate, 1 g zinc sulfate heptahydrate, 0.32 g copper sulfate pentahydrate, and 0.02 g nickel sulfate hexahydrate.
  • Wolfe’s vitamin solution contains per 1 L: 10 mg pyridoxine hydrochloride, 5 mg thiamine hydrochloride, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg calcium D-(+)-pantothenate, 5 mg p- aminobenzoic acid, 5 mg R-(+)-alpha-lipoic acid, 2 mg biotin, 2 mg folic acid, and 1 uL of a 100 g/L stock of vitamin B 12.
  • Cg amino acid mix contains per 1 L: 9.06 mL of 50 g/L L-alanine, 17.9 mL of 50 g/L L-arginine, 23.0 mL of 10 g/L L-asparagine, 94.6 mL of 5 g/L L-aspartic acid, 36.7 mL of 50 g/L L-cysteine, 16.9 mL of 100 g/L sodium L-glutamate, 17.9 mL of 10 g/L L- glutamine, 3.9 mL of 100 g/L L-glycine, 17.9 mL of 10 g/L L-histidine, 160.7 mL of 5 g/L L- isoleucine, 100.4 mL of 20 g/L L-leucine, 10.5 mL of 200 g/L L-lysine, 17.4 mL of 20 g/L L- methionine, 62.7 mL of 20 g/L L-phen
  • Example 4 Aspartate production of selected strains in Hungate tubes in CGXII l and CGXII 7.
  • Figure 1 Aspartate titers in mM in selected strains and media.
  • Figure 2 Aspartate titers in mM normalized to OD600 in selected strains and media.
  • Example 5 Aspartate production of strains overexpressing CgPckA and overexpressing aspartate feedback resistance mutants of CgPpc and CgPyc.
  • Aspartate levels in mM normalized to measured OD 600 are shown in Figure 4. While titers are lower than for CgPckA overexpression, moderate aspartate accumulations are observed in particular in LCG4-379 and LCG4-386, which overexpress CgPpc-N917G in the LCG4-009 and LCG4-248 backgrounds, respectively. Aspartate is detected in all other strains but at lower levels, with the next highest producing strain both for aspartate titer and aspartate titer/OD 600 being from LCG4-381, in which CgPyc-P458S with the ATG start codon replacement is overexpressed in the LCG4-009 background.
  • Figure 3 Aspartate titers in mM in selected strains resuspended in CGXII_1 medium.
  • Figure 4 Aspartate titers in mM normalized to ODeoo in selected strains resuspended in CGXII l medium.
  • Example 6 Aspartate production strains with and without heterologous aspartate dehydrogenase or NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase expression.
  • Expression vector pLASP-022 is identical to pLASP-004 but with HfAspDH and pCgEftU driving its expression being deleted. It was constructed by Gibson assembly of two PCR fragments that were amplified from pLASP-004 using primers YO3707 and YO13387, and from pLASP-004 using primers YO13386 and YO4497.
  • Expression vector pLASP-023 is identical to pLASP-004 but with CgPckA deleted (its promoter and terminator were left intact).
  • Expression vector pLASP-024 is identical to pLASP-004 but with CaGapC deleted (its promoter and terminator were left intact). It was constructed by Gibson assembly of two PCR fragments that were amplified from pLASP-004 using primers YO 13390 and YO2541, and pLASP-004 using primers YO3413 and YO5015.
  • Expression vectors described above were electroporated into LCG4-248 (MB001 with Idh, sdhCAB, and pta-ackA deletions) to generate production strains LCG4-361, LCG4-362, and LCG4-363 described in Table 2.
  • LCG4-375 is LCG4-248 re-transformed with pLASP-004 and should therefore be genotypically equivalent to LCG4-258.
  • Figure 5 Aspartate titers in mM for LCG4-258, LCG4-361 through LCG4-363, and LCG4-375 resuspended in CGXII l medium after a 48 hour production phase.
  • Figure 6 Aspartate in mM divided by ODeoo for LCG4-258, LCG4-361 through LCG4-363, and LCG4-375 resuspended in CGXII 1 medium after a 48 hour production phase.
  • Table 2 List of production strain IDs denoting the strain background plus transformed plasmids.
  • Plasmids overexpressing aspartate racemases are constructed by replacing CaGapC (encoding an NADP + -dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acetobutylicum) under a CgGapA promoter (native NAD + -dependent glyceraldehyde-3 - phosphate dehydrogenase) and synthetic E. coli terminator in pLASP-004, pLASP-202, or pLASP-065 with different genes encoding aspartate racemases shown in Table 1-A. Aspartate racemases were ordered as synthetic gene fragments from Twist Biosciences and were either codon-optimized for C.
  • glutamicum or native sequences are PCR amplified with flanking sequence using Q5 polymerase (New England Biolabs, Ipswich, MA) and assembled using NEB HiFi assembly mix (New England Biolabs) according to the manufacturer’s directions.
  • the assembled products are transformed into chemically competent NEB 5-alpha cells (New England Biolabs) and plated on LB agar containing 25 ug/mL kanamycin sulfate. Correct clones are screened by colony PCR and validated by NGS whole plasmid sequencing.
  • the examples utilize SEQ ID NOs 1-A to 7-A, which are, as apparent, differently numbered from those used in US 2022/0119821 and also utilized herein.
  • Table 1-A Heterologous aspartate racemases Plasmids, which also overexpress CgPckA (from a strong constitutive promoter in pLASP-004 or a native C. glutamicum fumC promoter) or LIPycA (pyruvate carboxylase from Lactococcus lactis, codon-optimized for C. glutamicum) are transformed into LCG4-248 (Corynebacterium glutamicum MB001 Mdh AsdhCAB kpta-ackA) by electroporation of 100 ng plasmid DNA with 50 uL electrocompetent cell aliquots at 1.8 kV.
  • CgPckA from a strong constitutive promoter in pLASP-004 or a native C. glutamicum fumC promoter
  • LIPycA pyruvate carboxylase from Lactococcus lactis, codon-optimized for C. glutamicum
  • Cells are subsequently heat shocked at 46°C for 6 minutes and outgrown at 30°C for at least 1.5 hours before plating on BHI agar containing 91 g/L D-sorbitol and 25 ug/mL kanamycin sulfate.
  • Strains are tested in small-scale screening as described in prior examples, and cell supernatants are then analyzed by HPLC to quantify L-aspartate and D-aspartate, or the sum of L-aspartate and D-aspartate for assessing total production of L-aspartate.
  • Example 8 Construction of aspartic acid production strains with phosphenolpyruvate carboxykinase overexpression Strain LCG4-248 is Corynebacterium glutamicum MB001 with gene knockouts inactivating lactate dehydrogenase (Idh), succinate dehydrogenase (sdhCAB operon), and acetate kinase and phosphotransacetylase (pta-ackA operon), with construction described in prior examples.
  • Idh lactate dehydrogenase
  • sdhCAB operon succinate dehydrogenase
  • pta-ackA operon acetate kinase and phosphotransacetylase
  • the native PEP carboxykinase from C. glutamicum is a GTP-dependent enzyme, producing GTP when operating in the direction of converting phosphoenolpyruvate to oxaloacetate.
  • the PEP carboxykinases tested are alternative ATP- and pyrophosphate-dependent enzymes, since these substrates may be more amenable to energy balancing under anaerobic conditions when produced alongside oxaloacetate. It was desired to test replacement of the native PEP carboxykinase in production plasmid pLASP-004 with heterologous PEP carboxykinases curated from public databases and the scientific literature.
  • Expression vector pLASP-004 contains a ColEl origin, pBLl origin, NeoR/KanR resistance marker, a codon-optimized gene encoding aspartate dehydrogenase from Herbaspirillum frisingense (Uniprot ID R0EI78; HfAspDH) under a C. glutamicum etfU promoter (pCgEftU), the gene encoding phosphoenolpyruvate carboxykinase from C.
  • CgPckA glutamicum
  • pCgEftU pCgEftU
  • pCgGapC Clostridium acetobutylicum
  • pCgGap C. glutamicum gap promoter
  • Alternative expression vectors were constructed that replaced CgPckA with heterologous phosphoenolpyruvate carboxykinases (PEPCKs).
  • Synthetic gene fragments containing the PEPCKs and 40 bp of flanking homology were ordered from Twist Biosciences (San Francisco, CA) for cloning via Gibson assembly using NEB HiFi assembly mix (New England Biolabs, Ipswich, MA) into the pLASP-004 backbone. Codon optimized gene sequences were according to Puigbo et al., 2007 using the HEG-DB C. glutamicum ATCC 13032 codon frequency table (Puigbo et al., 2008). These plasmids were then transformed into LCG4-248 to generate new production strains.
  • Table 1-B shows the gene names, derived organism, and protein sequences of all generated constructs.
  • Table 2-B lists the gene names, organisms, whether producing ATP or pyrophosphate, plasmid IDs, corresponding strain IDs after transformation into LCG4-248, and references (if applicable).
  • Table 2-B Strain IDs for plasmid-transformed C. glutamicum strains with gene names, originating organism for the PEPCK, substrate preference (ATP or pyrophosphate (PPi)), and associated references.
  • Example 9 Construction of aspartic acid production strains with putative heterologous aspartate feedback-resistant pyruvate carboxylase overexpression
  • Expression vector pLASP-004 was modified such that CgPckA was replaced with heterologous single or multi-subunit heterologous pyruvate carboxylases (PYCs) from different organisms.
  • PYCs heterologous single or multi-subunit heterologous pyruvate carboxylases
  • a synthetic artificial operon was generated between subunits derived from the same organism, with the sequence “TAAGGAGGAAATTACAT” added between the stop codon of PycA (subunit A) and start codon of PycB (subunit B).
  • This spacer sequence contains a strong bacterial RBS “AGGAGG” plus an additional 9 bp AT-rich RBS spacer sequence, and was incorporated into split synthetic gene fragments.
  • These synthetic gene fragments additionally contained the PYCs and 40 bp of flanking homology (split into multiple fragments when the size was >1800 bp) were ordered from Twist Biosciences (San Francisco, CA) for cloning via Gibson assembly using NEB HiFi assembly mix (New England Biolabs, Ipswich, MA) into the pLASP-004 backbone. Codon optimized gene sequences were obtained from protein sequences according to Puigbo et al., 2007, using the HEG-DB C. glutamicum ATCC 13032 codon frequency table (Puigbo et al., 2008). Plasmid sequences were validated by whole plasmid sequencing.
  • Table 3-B shows the gene names, derived organism, protein sequence IDsof all generated constructs.
  • Table 4-B lists the C. glutamicum strain IDs expressing the various PYC genes, plasmid IDs, and the Pyc gene(s) expressed from the plasmid.
  • Pyruvate carboxylases from Archaea were selected due to their reported lack of inhibition in the presence of elevated concentrations of aspartate (Mukhopadhyay et al., 2000). All archaeal PYCs have two subunits, PycA and PycB. Two pyruvate carboxylases from lactic acid bacteria (Lactobacillales) were selected, LIPycA and EfPycA, which are in single subunits.
  • LIPycA was not specifically reported to be aspartate feedback inhibition resistant, however it was shown that in a mutant of LIPycA made insensitive to inhibition by cyclic di-AMP, intracellular aspartate levels were increased in a strain with constitutively high cyclic di-AMP levels (Choi et al., 2017), thus it was desired to investigate if lactic acid bacteria could possess alternative allosteric regulation of pyruvate carboxylases that would render them aspartate-resistant in C. glutamicum. Pyruvate carboxylases from the liver of Rattus norvegicus and Gallus gallus were also previously found to exhibit no inhibition in the presence of aspartate (Scrutton and White, 1974).
  • Table 4-B Strain IDs for plasmid-transformed C. glutamicum strains with plasmid and gene names. Note that all Pyc genes are codon-optimized for C. glutamicum, and mitochondrial targeting peptides were removed from some Pyc genes as described in the text. LCG4-437 pLASP-072
  • BHI brain heart infusion
  • 96 well deepwell plates containing 290 uL of BHI + 45 mM potassium 3 -morpholinopropane- 1 -sulfonate (K-MOPS) pH 7.0 + 25 g/L glucose + 25 mg/L kanamycin sulfate were inoculated with 10 uL of preculture and grown with 300 rpm shaking at 30°C in a gastight box (EnzyScreen, Heemstede, The Netherlands), clamped with autoclaved sandwich covers (EnzyScreen) and with the valves left open to the atmosphere.
  • K-MOPS potassium 3 -morpholinopropane- 1 -sulfonate
  • Valves were then closed and the gastight box was incubated at 37°C with 300 rpm shaking for 48 hours. At the end of production, OD600 was measured and plates were spun down at 3000 rpm for 10 minutes. Supernatants were applied to a 96-well 0.22 um filter plate on top of a 96-well HPLC plate and spun down another 3000 x g for 10 minutes. Filtered supernatants were then diluted 10-fold in deionized water into HPLC plates for organic acid and glucose analysis, and an HPLC plate for amino acid analysis.
  • HPLC plates were thermally sealed with foil using an Agilent Plate-Lok and stored at 4°C until analysis by HPLC with RID or UV detection (with pre-column derivatization by o-phthalylaldehyde for amino acid UV detection).
  • CGXII_7 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 200 mL of 2 M urea, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1 mL of 10 mg/mL iron sulfate heptahydrate, 200 mL of 20% (w/v) glucose, 1.869 mL of Tween-80, 0.5 mL of 50 mg/mL kanamycin sulfate, and balance deionized water.
  • 1000X CGXII trace metal solution contains per I L: 10 g magnesium sulfate monohydrate, 1 g zinc sulfate heptahydrate, 0.32 g copper sulfate pentahydrate, and 0.02 g nickel sulfate hexahydrate.
  • Example 11 Aspartate production of C. glutamicum strain LCG4-248 expressing PEPCK and PYC heterologues
  • Measured aspartate levels in mM for PYC heterologues described in Example 2 are shown in Figure 8, using the screening procedure described in Example 3. These values are normalized to the final aerobic growth volume of 0.3 mL prior to addition of sodium bicarbonate, glucose, and additional water. Individual points are biological replicates that originated from different colonies inoculated into the original preculture plate.
  • Example 12 Growth curves of PYC expressing strains compared to LCG4-258
  • Growth curves were measured by growing precultures in 96 well deepwell plates as described in Example 3 only in 2XTY medium (16 g/L tryptone, 10 g/L yeast extract, 2.5 g/L K2HPO4, 10 g/L glucose) + 25 mg/L kanamycin sulfate, and after approximately 24 hours, inoculating a 96 well microtiter plate (Corning) containing 200 uL of CGXII_9 medium plus 25 mg/L kanamycin sulfate with 2.67 uL of preculture. Plates were then run in a kinetic growth assay in a Tecan Ml 000 plate reader with 5 mm amplitude linear shaking at 30°C, and absorbance measurements at 600 nm taken every 15 minutes.
  • 2XTY medium (16 g/L tryptone, 10 g/L yeast extract, 2.5 g/L K2HPO4, 10 g/L glucose
  • 2XTY medium 16 g/L tryptone, 10 g/L yeast extract,
  • CGXII 9 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 100 mL of 200 g/L ammonium sulfate, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1.64 mL of 10 mg/mL iron sulfate heptahydrate, 1 mL of a 30 mg/mL solution of protocatechuic acid in 0.05 M NaOH, 50 mL of 20% (w/v) glucose, 1 mL of Wolfe’s vitamin mix, 392.23 mL of Cg amino acid mix (minus L- cysteine), 15 mL of 50 g/L L-cysteine, 0.5 mL of
  • Cg amino acid mix contains per 1 L: 9.06 mL of 50 g/L L-alanine, 17.9 mL of 50 g/L L-arginine, 23.0 mL of 10 g/L L-asparagine, 94.6 mL of 5 g/L L-aspartic acid, 16.9 mL of 100 g/L sodium L- glutamate, 17.9 mL of 10 g/L L-glutamine, 3.9 mL of 100 g/L L-glycine, 17.9 mL of 10 g/L L- histidine, 160.7 mL of 5 g/L L-isoleucine, 100.4 mL of 20 g/L L-leucine, 10.5 mL of 200 g/L L- lysine, 17.4 mL of 20 g/L L-methionine, 62.7 mL of 20 g/L L-phenylalanine, 3.2 mL of 100 g/L L
  • FIG. 7 Aspartate titers in mM in selected strains expressing heterologous PEP carboxykinases (LCG4-460 through LCG4-471).
  • FIG. 8 Aspartate titers in mM in selected strains expressing heterologous pyruvate carboxylases (LCG4-428 through LCG4-437).
  • Figure 9 Growth curves measured as described in Example 5 for LCG4-258, LCG4-431, and LCG4-432
  • the following publications may disclose processes that can be adapted by one of skill in the art in view of this disclosure to make and use the invention provided herein, each of which is incorporated herein in its entirety by reference.
  • prpCl encoding methylcitrate synthase 1
  • C. glutamicum possesses genes encoding two methylcitrate synthases (prpCl and prpC2 ⁇ and PrpCl was shown to have a higher activity than PrpC2 as a citrate synthase (Radmacher and Eggeling, 2007).
  • Homologous regions upstream and downstream of prpCl were amplified by PCR with Q5 Hot Start DNA Polymerase (New England Biolabs, Ipswich, MA) using oligonucleotides YO14038 and YO14039, and Y014040 and YO14041, respectively.
  • the vector backbone contains a temperature-sensitive pBLl origin of replication, B. subtilis sacB (encoding levansucrase), kanamycin-resistance conferring NeoR marker, and an E. coll ColEl origin of replication, and was amplified from internal vector pL ASP- 169 as described above using YO 14024 and YO 14025.
  • the purified PCR products (vector backbone first treated with FastDigest Dpnl from Thermo Scientific) were assembled via Gibson assembly using NEB HiFi Assembly Mix (New England Biolabs) and transformed into chemically competent E. coli NEB 5-alpha (New England Biolabs) to generate pLASP-273.
  • the sequence was validated using whole plasmid NGS.
  • pLASP-273 was transformed into electrocompetent LCG4-000 as previously described and cells were plated directly at 37°C on BHI agar plates containing 91 g/L sorbitol and 25 mg/L kanamycin sulfate to select for plasmid loop-in at the prpCl locus.
  • Resulting colonies were next cultivated at 30°C overnight in minimal A medium (40 g/L glucose, 2 g/L urea, 7 g/L ammonium sulfate, 0.5 g/L potassium phosphate monobasic, 0.5 g/L potassium phosphate dibasic, 0.5 g/L magnesium sulfate heptahydrate, 6 mg/L iron sulfate heptahydrate, 4.2 mg/L manganese sulfate monohydrate, 0.2 mg/L biotin, 0.2 mg/L thiamine hydrochloride) and cells were plated on minimal A agar containing 10% (w/v) sucrose to select for plasmid loop-outs.
  • a prpCl deletion mutant identified by colony PCR was named LCG4-516.
  • vector pLASP-374 was first generated, containing upstream and downstream homology to the full native gltA promoter region by assembling fragments amplified from LCG4-000 genomic DNA using YO14084 and YO14085, and YO14082 and YO14083, and from vector backbone pLASP-228 (containing the same elements as described above for pLASP-169) using YO13891 and YO13892.
  • a series of mutant promoter containing fragments of the C. glutamicum dapA gene which had been described and characterized for their impact on citrate synthase activity by van Ooyen et al.
  • plasmids pLASP-065 encoding pyruvate carboxylase from Lactococcus lactis, NADP-dependent glyceraldehyde-3 -phosphate dehydrogenase from Clostridium acelobutyhcum, and aspartate dehydrogenase from Herbaspirillum frisingense
  • pLASP-328 encoding pyruvate carboxylase from Lactococcus lactis, aspartate dehydrogenase from Herbaspirillum frisingense, and aspartate racemase from Enterococcus faecalis transformed into electrocompetent cells of each strain to generate putative aspartic acid producing strains.
  • oligonucleotides are listed in Table 1-C and the g/M promoter replacement/deletion strains generated and plasmids used to construct them are listed in Table 2- C. Final production strains and the plasmids they contain are listed in Table 3-C.
  • Table 1-C List of oligonucleotides for aerobic aspartic acid overproducing strain construction
  • Table 2-C List of strains generated with the parent strains and integrative plasmids used to construct them and a description of the genotype
  • Table 3-C List of aerobic aspartic acid production strains generated, with their parent strains and plasmids To screen strains for aspartic acid production (L-aspartic acid using pLASP-065, and both L- and D-aspartic acid using pLASP-328), 96 well deepwell plates containing 300 uL of 2XTY medium (16 g/L tryptone, 10 g/L yeast extract, 2.5 g/L K2HPO4, 10 g/L glucose) + 25 mg/L kanamycin sulfate were inoculated with single colonies of strains struck from cryogenic glycerol stocks on BHI agar plates containing 25 mg/L kanamycin, covered with breathable fdm, and grown with 300 rpm shaking at 30°C in a shaker incubator.
  • 2XTY medium 16 g/L tryptone, 10 g/L yeast extract, 2.5 g/L K2HPO4, 10 g/L glucose
  • samples were pre-column derivatized with o- phthalylaldehyde (OP A) in borate buffer and injected on an Agilent ZORBAX Eclipse Plus Cl 8 RRHD, 3.0 mm diameter x 50 mm length, 1.8 pm particle size column held in an oven at 40°C with an Agilent ZORBAX Eclipse Plus C18 3.0 mm x 1.8 pm guard column.
  • Mobile phase A was 10 mM sodium tetraborate, 10 mM sodium phosphate monobasic, 5 mM sodium azide, pH 8.2 adjusted with HC1.
  • Mobile phase B was a 45:45: 10 (v:v:v) mixture of methanol, acetonitrile, and water.
  • the solvent gradient was 98% A/2% B from 0 to 0.2 minutes, up to 52% A/48% B at 3.5 minutes, 5% A/95% B at 4 minutes, and 98% A/2% B at 4.2 minutes, with a 1.0 mL/min flow rate.
  • UV absorbance was measured at 338 nm with 10 nm bandwidth, and samples were quantified against a standard curve containing different concentrations of L-aspartic acid.
  • CGXII 69 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 100 mL of 200 g/L ammonium sulfate, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1.64 mL of 10 mg/mL iron sulfate heptahydrate, 1 mL of 30 mg/mL protocatechuic acid in 0.05 M sodium hydroxide, 50 mL of 20% (w/v) glucose, 1 mL of Wolfe’s vitamin mix, 22.56 mL of 10 g/L L-asparagine, 17.52 mL of 10 g/L L-glutamine, and 61.44 m
  • 1000X CGXII trace metal solution contains per I L: 10 g magnesium sulfate monohydrate, 1 g zinc sulfate heptahydrate, 0.32 g copper sulfate pentahydrate, and 0.02 g nickel sulfate hexahydrate.
  • Wolfe’s vitamin solution contains per 1 L: 10 mg pyridoxine hydrochloride, 5 mg thiamine hydrochloride, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg calcium D-(+)-pantothenate, 5 mg p-aminobenzoic acid, 5 mg R-(+)-alpha-lipoic acid, 2 mg biotin, 2 mg folic acid, and 1 uL of a 100 g/L stock of vitamin B12.
  • Figures 10 and 11 show total (L and D) aspartate titers measured by UPLC in mM after 24 and 48 hour production as described above, with the median titer indicated by the horizontal bar in each box and each point representing the measurement from each biological replicate culture included in the screen.
  • Control strains are LCG4-248; LCG4-258, which is LCG4-248 containing pLASP-004 (expressing CgPckA from a C.
  • LCG4-431 which is LCG4-248 containing pLASP-065 (expressing LIPycA, HfAspDH, and CaGapC); and LCG4-450, which is LCG4-248 containing pLASP-202 (expressing CgPckA from a C. glutamicum fumC promoter, HfAspDH, and CaGapC).
  • LCG4- 258 and LCG4-450 are two of the best validated aspartate producers with CgPckA, and LCG4- 431 is a direct control strain, containing an identical plasmid with the exception of expressing CaGapC instead of the aspartate racemases.
  • Figure 10 Total (L- and D-) aspartic acid titers in mM after 24 hours aerobic cultivation in 96 well deepwell plates in CGXII_69 medium supplemented with 40 g/L sodium bicarbonate.
  • Figure 11 Total (L- and D-) aspartic acid titers in mM after 48 hours aerobic cultivation in 96 well deepwell plates in CGXII_69 medium supplemented with 40 g/L sodium bicarbonate.
  • CGXII 95 and CGXII 96 were CGXII 95 and CGXII 96.
  • the main difference in these media versus CGXII_69 plus 40 g/L sodium bicarbonate were that they are based on an alternative medium used in screening, CGXII 7, with the addition of yeast extract, casamino acids, 45 g/L sodium bicarbonate, a higher concentration of glucose, and addition of either Tween-80 in CGXII 95 or Tween-20 in CGXII 96.
  • the strains also produce large quantities of glutamic acid (up to 42.5 mM median titer from LCG4-602 grown in CGXII 96), which is indicative of the favorable export via the glutamate/aspartate exporting small mechanosensitive channel MscCG in this medium ( Figure 13).
  • CGXII_95 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 200 mL of 2 M urea, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1 mL of 10 mg/mL iron sulfate heptahydrate, 1 mL of 30 mg/mL protocatechuic acid dissolved in 0.05 M NaOH, 200 mL of 20% (w/v) glucose, 1.869 mL of Tween-80, 50 mL of 20 g/L yeast extract, 35 mL of 10% (w/v) casamino acids, 45 g of sodium bicarbonate, and balance deionized water.
  • 1000X CGXII trace metal solution contains per 1 L: 10 g magnesium sulfate monohydrate, 1 g zinc sulfate heptahydrate, 0.32 g copper sulfate pentahydrate, and 0.02 g nickel sulfate hexahydrate.
  • CGXII 96 has the same composition of CGXII 95, except for 1.869 mL of Tween-20 in place of Tween-80.
  • Figure 12 Total (L- and D-) aspartic acid titers in mM after 48 hours cultivation of selected strains in CGXII 95 and CGXII 96 media.
  • Figure 13 Glutamic acid titers in mM after 48 hours cultivation of selected strains in CGXII 95 and CGXII 96 media.
  • Example 14 Dynamic CgPckA expression strains
  • Plasmid pLASP-023 contained in LCG4-362 possesses a deletion of CgPckA, whereas pLASP-022 and pLASP-024 have deletions in HfAspDH and CaGapC, respectively. As was previously shown in other examples, however, anaerobic production of aspartic acid was completely abolished in strain LCG4-362.
  • CgPckA CgPckA
  • lower expression levels during aerobic growth and induced expression during the conditions of the production phase.
  • These conditions include decreased oxygen levels and increased temperature relative to the growth phase.
  • Control strain LCG4-258 still exhibited the highest titer, however it should be noted that growth was performed in BHI 3, which is a very rich medium and all cultures grew to similar optical densities in this screen before anaerobic transition.
  • LCG4-450, LCG4-452, LCG4-456, and LCG4-457 all exhibited moderate titers approaching that of LCG4-258.
  • LCG4-456 and LCG4-450 exhibited two desirable combination of growth in CGXII 3 and production in CGXII 7 after growth in BHI 3.
  • Figure 14 Growth profiles of plasmid gene deletion strains LCG4-361 (-HfAspDH), LCG4-362 (-CgPckA), and LCG4-363 (-CaGapC) compared to LCG4-248 background strain and LCG4- 258 (no genes deleted on plasmid).
  • Figure 15 Growth of CgPckA promoter replacement strains compared to LCG4-258 (orange line; harboring CgPckA under control of a C. glutamicum eftU promoter) and LCG4-248 background strain.
  • Figure 16 Aspartic acid titers (mM) in CgPckA promoter replacement strains after 49 hours anaerobic production phase.
  • Table 4-C Strain IDs, plasmid IDs in each strain (all LCG4-248 parent), native gene promoter cloned in place of eftU for CgPckA in each plasmid, and primers used to amplify the promoters from LCG4-000 genomic DNA with 5’ overhangs for Gibson assembly.
  • Example 15 Enhancing anaerobic aspartic acid production by addition of Tween surfactants
  • Aspartic acid producing strains LCG4-431 and LCG4-450 colonies on BHI + 25 mg/L kanamycin sulfate plates were inoculated in 300 uL 2XTY medium plus 25 mg/L kanamycin sulfate in 96 well deepwell plates, and after approximately 24 hours growth at 30°C with 300 rpm shaking, 10 uL was transferred into 96 well deepwell plates containing 290 uL CGXII 65 medium and cultivated at 30°C with 300 rpm shaking.
  • Aspartic acid titers were generally enhanced with the addition of Tween surfactants, with the largest increases from the addition of Tween 20 at a concentration of 2.0465 g/L original CGXII_65 volume. Larger increases in titer were also observed for LCG4-431, which overexpresses pyruvate carboxylase from Lactococcus lactis, versus LCG4-450 which overexpresses the native C. glutamicum phosphoenolpyruvate carboxy kinase from a C. glutamicum fumC promoter. This is likely indicating an enhanced effect of increasing export of aspartic acid on releasing feedback inhibition of either the heterologous and/or native pyruvate carboxylase.
  • CGXII 65 contains per liter: 200 mL of 1 M K-MOPS pH 7.0, 200 mL of 2 M urea, 7.35 mL of 1 M potassium phosphate monobasic, 5.74 mL of 1 M potassium phosphate dibasic, 1 mL of 1 M magnesium sulfate, 0.07 mL of 1 M calcium chloride, 0.2 mL of a 1 mg/mL biotin solution, 1 mL of 1000X CGXII trace metals, 1 mL of 10 mg/mL iron sulfate heptahydrate, 1 mL of 30 mg/mL protocatechuic acid dissolved in 0.05 M NaOH, 200 mL of 20% (w/v) glucose, 50 mL of 20 g/L yeast extract, 35 mL of 10% (w/v) casamino acids, and balance deionized water.
  • 1000X CGXII trace metal solution contains per I L: 10 g magnesium sulfate monohydrate, 1 g zinc sulfate heptahydrate, 0.32 g copper sulfate pentahydrate, and 0.02 g nickel sulfate hexahydrate.
  • Figure 17 Aspartic acid titers in mM after 70 hours cultivation of LCG4-450 in CGXII_65 with added sodium bicarbonate, glucose, and Tween surfactants added to the concentrations (per mL of original CGXII 65 medium) shown in the labels.
  • Figure 18 Aspartic acid titers in mM after 70 hours cultivation of LCG4-431 in CGXII_65 with added sodium bicarbonate, glucose, and Tween surfactants added to the concentrations (per mL of original CGXII 65 medium) shown in the labels.
  • Example 16 Additional heterologous pyruvate carboxylases for anaerobic aspartic acid production
  • Additional heterologous pyruvate carboxylases from diverse bacterial species were codon- optimized for Corynebacterium glutamicum, purchased as synthetic gene fragments (Twist Biosciences, San Francisco, CA) with homology flanking the vector backbone for ease of Gibson assembly, and cloned in place of CgPckA in pLASP-004 using methods as previously described.
  • a pyruvate carboxylase consisted of two subunits, the subunits were cloned in an artificial operon also as previously described.
  • Plasmids were transformed into LCG4-248 as previously described to generate new strains, which were tested in small-scale screening as previously described for other heterologous pyruvate carboxylase expressing strains, but with an increased sampling time of approximately 70 hours after onset of the anaerobic production phase.
  • Strain IDs with transformed new plasmid IDs, the gene name given to each pyruvate carboxylase, and the source organism and Uniprot ID are shown in Table 6.
  • Aspartic acid titers are shown in Figure 19 compared to control strains LCG4-258 and LCG4-450 (which overexpress CgPckA in place of a pyruvate carboxylase), and LCG4-431 (which overexpresses the previous high performing pyruvate carboxylase.
  • LCG4-614 expressing pyruvate carboxylase from Lactococcus hircilactis (LhPycA) outperforms LCG4-431.
  • LCG4-612 expressing pyruvate carboxylase from Enterococcus thailandicus, exhibits similar aspartic acid production as LCG4-431.
  • Table 6-C Strain IDs, plasmid IDs in each strain (all LCG4-248 parent), pyruvate carboxylase gene names, source organism and Uniprot ID (where applicable), and protein sequence ID for
  • Figure 19 Aspartic acid titers in mM after approximately 70 hours anaerobic cultivation of heterologous pyruvate carboxylase expressing strains and control strains (LCG4-248 background strain, CgPckA overexpressing strains LCG4-258 and LCG4-450, and previous best performing pyruvate carboxylase expressing strain LCG4-431).
  • Example 17 Fermentation of Aspartic acid Or A Salt Thereof Employing Microorganisms Provided Herein Example 17:
  • a shake flask containing 50 mLTYx2 media 16 g/L tryptone, 10 g/L yeast, 5 g/L sodium chloride, 2.5 g/L di-potassium hydrogen phosphate, 11 g/L glucose monohydrate and 0.025 g/L kanamycine was inoculated with a seed vial containing LCG4-258. Strain grew at 30 C on a rotary shaker (throw: 25 mm, RPM:200) for 24 hours to an OD600 of 3-4.
  • IL Fermentation tanks were filled with 500 mL batch media and supplemented with 0.1 m L/ L antifoam agent (Struktol SB2121). PH was adjusted to 7.4 using 5M NH4OH and 2M phosphoric acid. Starting condition was as listed below:
  • Reactors were inoculated with culture to a starting OD of 0.1-0.5. Aerobic condition was maintained for 24-36 hours by cascading agitation to maintain DO at 40%. Upon reaching an OD of ⁇ 10, fermentation was switched to anaerobic mode. Anaerobic switch occurred as the following: DO set point was linearly reduced from 40% to 0% over the course of 15 minutes and subsequently the tanks were flushed with N2 gas for about 20 minutes, purging three times tank volume with Nitrogen gas. After this step, agitation was fixed at 400 rpm and temperature was set at 37C. Inlet and outlet gas lines were clamped to prevent further gas exchange. 500 mL of freshly prepared sterile 45 g/L sodium bicarbonate was added to the tank.
  • Glucose was added to bring batch glucose concentration to 20 g/L. Production continued for 7-10 days. Summary of metrics: We observed 7 g/L aspartic acid production at 280 hours. Note: addition of the amino acid mixture was necessary for aspartic acid production. We did not observe any aspartic acid production in a media with a similar nutrient composition minus the 20 amino acid mixture.
  • Example 18 • Strain: LCG4-450 Fermentation Process Summary: Seed Train: Similar to Example 17 Media: Batch medium (compared to example 17, the media contained only 7 amino acids and started with reduced batch glucose concentration).
  • PH was adjusted to 7.4 using 5M NH4OH and 2M phosphoric acid.
  • Starting condition was as listed below: • Temperature: 30C • pH set point: 7.4 • Aeration: 100 mL/min (1vvm) • Agitation: 500 rpm; cascaded to maintain 40% DO setpoint during aerobic phase. Reactors were inoculated with culture to a starting OD of 1. Aerobic condition was maintained for 72 hours by cascading agitation to maintain DO at 40%. During aerobic phase, feed was added to culture in pulses. Pulses were added in response to DO spikes and each pulse delivered 2 grams glucose/L at a maximum feed rate of 20 g/L/hr.
  • Anaerobic switch occurred as the following: DO set point was linearly reduced from 40% to 0% over the course of 15 minutes and subsequently the tanks were flushed with N2 gas, purging three times tank volume with Nitrogen gas. After this step, agitation was fixed at 500 rpm and temperature was set at 37C. Sodium bicarbonate was added to the tank to bring the batch concentration to 20 g/L. Glucose was also added in one slug dose addition to bring batch glucose concentration to 20 g/L. A simultaneous flow of CO2 and N2 each at 25 mL/min (0.25 vvm) was maintained through the rest of fermentation time to maintain oxygen levels close to zero. Production continued for additional 4 days in anaerobic phase.
  • Feed medium (Asp-Feed 01): similar to Example 18 • 500 g/L glucose + 2.5 g/L KH2PO4+ 2.5 g/L (NH4)2SO4 + 0.2 mg/L biotin+ 0.02 g/L MnSO4-H2O+ 0.002 g/L ZnSO4-7H2O + 0.00064 g/L CuSO4-5H2O+ 0.00004 g/L NiCl2- 6H2O+ 0.0164 g/L FeSO4-7H2O+ 0.06 g/L protocatechuic acid+ 0.03 g/L kanamycin+ 0.01 mg/L thiotic acid+ 0.01 mg/L thiamin hydrochloride+ 0.02 mg/L pyridoxine hydrochloride+ 0.01 mg/L nicotinic acid+ 0.01 mg/L calcium D-(+) pantothenate+ 0.01 mg/L p-aminobenzoic acid+ 0.004 mg/L folic acid+ 0.00
  • Example 20 • Strain: LCG4-450 Fermentation Process Summary: Seed Train: Similar to Example 17
  • Media Base line batch medium composition: • The following nutrient composition was shared between all batch media tested in Example 20: 18 g/L glucose + 0.01 g/L CaCl2-2H2O+ 1 g/L KH2PO4+ 1 g/L K2HPO4+ 20 g/L (NH4)2SO4 + 0.25 g/L MgSO4-7H2O+ 0.202 mg/L biotin+ 0.01 g/L MnSO4-H2O+ 0.001 g/L ZnSO4-7H2O + 0.00032 g/L CuSO4-5H2O+ 0.00002 g/L NiCl2-6H2O+ 0.0164 g/L FeSO4-7H2O+ 0.03 g/L protocatechuic acid+ 0.03 g/L kanamycin+ 0.005 mg/L thiotic acid+ 0.005 mg/L thiamin hydrochloride
  • Feed medium (Asp-Feed 01): similar to Example 18 • 500 g/L glucose + 2.5 g/L KH2PO4+ 2.5 g/L (NH4)2SO4 + 0.2 mg/L biotin+ 0.02 g/L MnSO4-H2O+ 0.002 g/L ZnSO4-7H2O + 0.00064 g/L CuSO4-5H2O+ 0.00004 g/L NiCl2- 6H2O+ 0.0164 g/L FeSO4-7H2O+ 0.06 g/L protocatechuic acid+ 0.03 g/L kanamycin+ 0.01 mg/L thiotic acid+ 0.01 mg/L thiamin hydrochloride+ 0.02 mg/L pyridoxine hydrochloride+ 0.01 mg/L nicotinic acid+ 0.01 mg/L calcium D-(+) pantothenate+ 0.01 mg/L p-aminobenzoic acid+ 0.004 mg/L folic acid+ 0.00
  • Fermentation run condition Run conditions were similar to Example 18. In this example we tested reducing amino acids from the remaining 7 amino acids down to three most essential amino acids for aspartic acid production. Aerobic phase was reduced to 48 hours for all runs.
  • the amino acid composition for each medium in this example is listed in the table below:
  • Base line batch medium composition (similar to Example 20):
  • Feed medium (Asp-Feed 01): similar to Example 18
  • Fermentation run condition Run conditions were similar to Example 18. One exception was that feed was delivered in constant feeding at 0.25 mL/hr from the beginning of the run for 24 hours. In this example we tested reducing amino acids from the remaining 3 amino acids down to the most essential amino acids for aspartic acid production. Aerobic phase was reduced to 24 hours for all runs.
  • the amino acid composition for each medium in this example is listed in the table below: i Asp-PSA 123 i • 0.219 g/L glutamine+ 1.536 g/L phenylalanine i Asp-PSA 124 i • 0.219 g/L glutamine+ 1.024 g/L phenylalanine
  • Glutamine was identified as the main amino acid required for improved titer. There seems to be a linear correlation between aspartic acid titer and batch glutamine concentration in the range of 0 to 0.2 g/L glutamine. Excess glutamine may potentially be toxic in this process.
  • Trt0 ⁇ Per.Tfrn0.Productivity g/L/hr vs. Run. Time. hr
  • Seed Train Similar to Example 17, with the exception that seed media also contained 2.5 g/L sodium bicarbonate. Seed grew to OD of 8.
  • Feed medium (Asp-Feed 01): similar to Example 18
  • Fermentation run condition Run conditions were similar to Example 17. One exception was that feed was delivered at a constant feed rate of 1.3 g glucose/L/hr for 24 hours in aerobic phase and the feed continued at 1.9 g/L/hr in anaerobic phase for another 24 hours.
  • Base line batch medium composition (similar to Example 22):
  • Feed medium (Asp-Feed 01): similar to Example 18
  • Antifoam Struktol SB2121 (0.1 mL/L) at the beginning of the run Fermentation run condition: Run conditions were similar to Example 21. We screened different strains. We found several strains reaching 20 g/L aspartate titer in 136 hours with maximum productivities reaching 0.17 g/L/hr as shown in the plots below.

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Abstract

Sont prévues dans la présente invention des cellules hôtes, telles que des cellules hôtes de micro-organisme recombinantes, pour produire de l'acide aspartique, préférentiellement en quantités supérieures à celles produites par des cellules de type sauvage correspondantes, en les cultivant pour produire de l'acide aspartique ou un sel associé, et en identifiant des cellules qui sont ainsi appropriées, et des utilisations de l'acide aspartique ainsi produit.
PCT/US2024/028433 2023-05-08 2024-05-08 Cellule hôte recombinante pour produire de l'aspartate Pending WO2024233705A2 (fr)

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