EP1633851A2 - Verfahren und zusammesetzungen zur aminosäureproduktion - Google Patents

Verfahren und zusammesetzungen zur aminosäureproduktion

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Publication number
EP1633851A2
EP1633851A2 EP04754180A EP04754180A EP1633851A2 EP 1633851 A2 EP1633851 A2 EP 1633851A2 EP 04754180 A EP04754180 A EP 04754180A EP 04754180 A EP04754180 A EP 04754180A EP 1633851 A2 EP1633851 A2 EP 1633851A2
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Prior art keywords
polypeptide
variant
bacterium
nucleic acid
bacterial
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EP04754180A
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English (en)
French (fr)
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EP1633851A4 (de
Inventor
Richard B. Bailey
Paul Blomquist
Reed Doten
Edward M. Driggers
Kevin T. Madden
Jessica O'leary
George A. O'toole
Joshua Trueheart
Michael J. Walbridge
Peter Yorgey
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Microbia Inc
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Microbia Inc
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Publication of EP1633851A2 publication Critical patent/EP1633851A2/de
Publication of EP1633851A4 publication Critical patent/EP1633851A4/de
Withdrawn legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/10—Transferases (2.)
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    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
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    • C12N9/10—Transferases (2.)
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • This invention relates to microbiology and molecular biology, and more particularly to methods and compositions for amino acid production.
  • genomic information for production strains and related bacterial organisms provides an opportunity to construct new production strains by the introduction of cloned nucleic acids into na ⁇ ve, unmanipulated host strains, thereby allowing amino acid production in the absence of deleterious mutations (Ohnishi, J., et al. Appl Microbiol Biotechnol. 58:217-223, 2002). Similarly, this information provides an opportunity for identifying and overcoming the limitations of existing production strains.
  • the present invention relates to compositions and methods for production of amino acids and related metabolites in bacteria.
  • the invention features bacterial strains that are engineered to increase the production of amino acids and related metabolites of the aspartic acid family.
  • the strains can be engineered to harbor one or more nucleic acid molecules (e.g., recombinant nucleic acid molecules) encoding a polypeptide (e.g., a polypeptide that is heterologous or homologous to the host cell) and/or they may be engineered to increase or decrease expression and/or activity of polypeptides (e.g., by mutation of endogenous nucleic acid sequences).
  • polypeptides which can be expressed by various methods familiar to those skilled in the art, include variant polypeptides, such as variant polypeptides with reduced feedback inhibition. These variant polypeptides may exhibit reduced feedback inhibition by a product or intermediate of an amino acid biosynthetic pathway, such as S-adenosylmethionine, lysine, threonine or methionine, relative to wild type forms of the proteins. Also featured are the variant polypeptides encoded by the nucleic acids, as well as bacterial cells comprising the nucleic acids and the polypeptides. Combinations of nucleic acids, and cells that include the combinations of nucleic acids, are also provided herein. The invention also relates to improved bacterial production strains, including, without limitation, strains of coryneform bacteria and Enterobacteriaceae (e.g., Escherichia coli (E. coli)).
  • E. coli Escherichia coli
  • Bacterial polypeptides that regulate the production of an amino acid from the aspartic acid family of amino acids or related metabolites include, for example, polypeptides involved in the metabolism of methionine, threonine, isoleucine, aspartate, lysine, cysteine and sulfur, such as enzymes that catalyze the conversion of intermediates of amino acid biosynthetic pathways to other intermediates and/or end product, and polypeptides that directly regulate the expression and/or function of such enzymes.
  • Heterologous proteins may be encoded by genes of any bacterial organism other than the host bacterial species.
  • the heterologous genes can be genes from the following, non-limiting list of bacteria: Mycobacterium smegmatis; Amycolatopsis mediterranei; Streptomyces coelicolor; Tiiermobifidafusca; Erwinia chrysanthemi; Shewanella oneidensis; Lactobacillus plantarum; Bifidobacterium longum; Bacillus sphaericus; and Pectobacterium chrysanthemi.
  • heterologous genes for host strains from the Enterobacteriaceae family also include genes from coryneform bacteria.
  • heterologous genes for host strains of coryneform bacteria also include genes from Enterobacteriaceae family members.
  • the host strain is Escherichia coli and the heterologous gene is a gene of a species other than a coryneform bacteria
  • the host strain is a coryneform bacteria and the heterologous gene is a gene of a species other than Escherichia coli.
  • the host strain is Escherichia coli and the heterologous gene is a gene of a species other than Corynebacterium glutarnicum.
  • the host strain is Corynebacterium glutamicum and the heterologous gene is a gene of a species other than Escherichia coli.
  • the polypeptide is encoded by a gene obtained from an organism of the order Actinomycetales.
  • the heterologous nucleic acid molecule is obtained from Mycobacterium smegmatis, Streptomyces coelicolor, Thermobifida fusca, Amycolatopsis mediterranei, or a coryneform bacteria, hi various embodiments, the heterologous protein is encoded by a gene obtained from an organism of the family Enterobacteriaceae.
  • the heterologous nucleic acid molecule is obtained from Erwinia chysanthemi or Escherichia coli.
  • the host bacterium e.g., coryneform bacterium or bacterium of the family Enterobacteriaceae
  • the host bacterium also has increased levels of a polypeptide encoded by a gene from the host bacterium (e.g., from a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium).
  • Increased levels of a polypeptide encoded by a gene from the host bacterium may result from one of the following: introduction of additional copies of a gene from the host bacterium under the naturally occurring promoter; introduction of additional copies of a gene from the host bacterium under the control of a promoter, e.g., a promoter more optimal for amino acid production than the naturally occurring promoter, either from the host or a heterologous organism; or the replacement of the naturally occurring promoter for the gene from the host bacterium with a promoter more optimal for amino acid production, either from the host or a heterologous organism.
  • Vectors used to generate increased levels of a protein may be integrated into the host genome or exist as an episomal plasmid.
  • the host bacterium has reduced activity of a polypeptide (e.g., a polypeptide involved in amino acid synthesis, e.g., an endogenous polypeptide) (e.g., decreased relative to a control). Reducing the activity of particular polypeptides involved in amino acid synthesis can facilitate enhanced production of particular amino acids and related metabolites.
  • a polypeptide e.g., a polypeptide involved in amino acid synthesis, e.g., an endogenous polypeptide
  • Reducing the activity of particular polypeptides involved in amino acid synthesis can facilitate enhanced production of particular amino acids and related metabolites.
  • expression of a dihydrodipicolinate synthase polypeptide is deficient in the bacterium (e.g., an endogenous dapA gene in the bacterium is mutated or deleted), hi various embodiments, expression of one or more of the following polypeptides is deficient: an mchR gene product, homoserine dehydrogenase, homoserine kinase, methionine adenosyltransferase, homoserine O-acetyltransferase, and phosphoenolpyruvate carboxykinase.
  • the nucleic acid molecule comprises a promoter, including, for example, the lac, trc, trcRBS,phoA, tac, or XPjJ ⁇ P R promoter from E. coli (or derivatives thereof) or the phoA, gpd, rplM, or rpsJ promoter from a coryneform bacteria.
  • the invention features a host bacterium (e.g., a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an.
  • Escherichia coli bacterium comprising at least one (two, three, or four) of: (a) a nucleic acid molecule comprising a sequence encoding a heterologous bacterial aspartokinase polypeptide or a functional variant thereof; (b) a nucleic acid molecule comprising a sequence encoding a heterologous bacterial aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof; (c) a nucleic acid molecule comprising a sequence encoding a heterologous bacterial phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof; (d) a nucleic acid molecule comprising a sequence encoding a heterologous bacterial pyruvate carboxylase polypeptide or a functional variant thereof; (e) a nucleic acid molecule comprising a sequence encoding a heterologous bacterial dihydrodipicolinate synthase polypeptide or a
  • the nucleic acid molecule is free of nucleotide sequences that naturally flank the sequence in the organism from which the nucleic acid molecule is derived, e.g., the nucleic acid molecule is a recombinant nucleic acid molecule).
  • the bacterium comprises nucleic acid molecules comprising sequences encoding two or more distinct heterologous bacterial polypeptides, wherein each of the heterologous polypeptides encodes the same type of polypeptide (e.g., the bacterium comprises nucleic acid molecules comprising sequences encoding an aspartokinase from a first species, and sequences encoding an aspartokinase from a second species.)
  • the polypeptide is selected from an Enterobacteriaceae polypeptide, an Actinomycetes polypeptide, or a variant thereof.
  • the polypeptide is a polypeptide of one of the following Actinomycetes species: Mycobacterium smegmatis, Streptomyces coelicolor, Thermobifida fusca, Amycolatopsis mediterranei and coryneform bacteria, including Corynebacterium glutamicum.
  • the polypeptide is a polypeptide of one of the following Enterobacteriaceae species: Erwinia chysanthemi and Escherichia coli.
  • the polypeptide is a variant polypeptide with reduced feedback inhibition (e.g., relative to a wild-type form of the polypeptide).
  • the bacterium further comprises additional heterologous bacterial gene products involved in amino acid production, hi various embodiments, the bacterium further comprises a nucleic acid molecule encoding a heterologous bacterial polypeptide described herein (e.g., a nucleic acid molecule encoding a heterologous bacterial homoserine dehydrogenase polypeptide).
  • the bacterium further comprises a nucleic acid molecule encoding a homologous bacterial polypeptide (i.e., a bacterial polypeptide that is native to the host species or a functional variant thereof), such as a bacterial polypeptide described herein.
  • the homologous bacterial polypeptide can be expressed at high levels and/or conditionally expressed.
  • the nucleic acid encoding the homologous bacterial polypeptide can be operably linked to a promoter that allows expression of the polypeptide over wild-type levels, and/or the nucleic acid may be present in multiple copies in the bacterium.
  • the heterologous bacterial aspartokinase or functional variant thereof is chosen from: (a) a Mycobacterium smegmatis aspartokinase polypeptide or a functional variant thereof, (b) an Amycolatopsis mediterranei aspartokinase polypeptide or a functional variant thereof, (c) a Streptomyces coelicolor aspartokinase polypeptide or a functional variant thereof, (d) a Thermobifida fusca aspartokinase polypeptide or a functional variant thereof, (e) an Erwinia chrysanthemi aspartokinase polypeptide or a functional variant thereof, and (f) a Shewanella oneidensis aspartokinase polypeptide or a functional variant thereof.
  • the heterologous bacterial aspartokinase polypeptide is an Escherichia coli aspartokinase polypeptide or a functional variant thereof, h certain embodiments, the heterologous bacterial aspartokinase polypeptide is a Corynebacterium glutamicum aspartokinase polypeptide or a functional variant thereof. In certain embodiments the heterologous bacterial asparatokinase polypeptide or functional variant thereof has reduced feedback inhibition.
  • the heterologous bacterial aspartate semialdehyde dehydrogenase polypeptide or functional variant thereof is chosen from: (a) a Mycobacterium smegmatis aspartate semialdehyde dehydrogenase polypeptide r a functional variant thereof, (b) an Amycolatopsis mediterranei aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof, (c) a Streptomyces coelicolor aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof, and (d) a Thermobifida fusca aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof.
  • the heterologous bacterial aspartate semialdehyde dehydrogenase polypeptide is an Escherichia coli aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof.
  • the heterologous bacterial aspartate semialdehyde dehydrogenase polypeptide is a Corynebacterium glutamicum aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof
  • the heterologous bacterial phosphoenolpyruvate carboxylase polypeptide or functional variant thereof is chosen from: (a) a Mycobacterium smegmatis phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof, (b) a Streptomyces coelicolor phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof, (c) a Thermobifida fusca phosphoen
  • the heterologous bacterial phosphoenolpyruvate carboxylase polypeptide is an Escherichia coli phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof. In certain embodiments, the heterologous bacterial phosphoenolpyruvate carboxylase polypeptide is a Corynebacterium glutamicum phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof.
  • the heterologous bacterial pyruvate carboxylase polypeptide or functional variant thereof is chosen from: (a) a Mycobacterium smegmatis pyruvate carboxylase polypeptide or a functional variant thereof, (b) a Streptomyces coelicolor pyruvate carboxylase polypeptide or a functional variant thereof, and (c) a Tliermobifida fusca pyruvate carboxylase polypeptide or a functional variant thereof.
  • the heterologous bacterial pyruvate carboxylase polypeptide is a Corynebacterium glutamicum pyruvate carboxylase or a functional variant thereof.
  • the bacterium is chosen from a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium.
  • Coryneform bacteria include, without limitation, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Brevibacterium lactofermentum, Brevibacterium lactis, and Brevib ⁇ cterium fl ⁇ vum.
  • the Mycobacterium smegmatis aspartokinase polypeptide comprises SEQ ID NO:l or a variant sequence thereof
  • the Amycolatopsis mediterranei aspartokinase polypeptide comprises SEQ ID NO:2 or a variant sequence thereof
  • the Streptomyces coelicolor aspartokinase polypeptide comprises SEQ ID NO:3 or a variant sequence thereof
  • the Thermobifida fusca aspartokinase polypeptide comprises SEQ ID NO:4 or a variant sequence thereof
  • the Erwinia chrysanthemi aspartokinase polypeptide comprises SEQ ID NO:
  • the Shewanella oneidensis aspartokinase polypeptide comprises SEQ ID NO:6 or a variant sequence thereof
  • the Escherichia coli aspartokinase polypeptide comprises SEQ ID NO: 203 or a variant sequence thereof
  • the Corynebacterium glutamicum aspartokinase polypeptide comprises SEQ ID NO: 202 or a variant sequence thereof
  • the Corynebacterium glutamicum aspartate semialdehyde dehydrogenase polypeptide comprises SEQ ID NO:204 or a variant sequence thereof
  • the Escherichia coli aspartate semialdehyde dehydrogenase polypeptide comprises SEQ ID NO: 205 or a variant sequence thereof
  • the Mycobacterium smegmatis phosphoenolpyruvate carboxylase polypeptide or functional variant thereof comprises an amino acid sequence at least 80% identical to SEQ ID NO: 8 (M.
  • the Streptomyces coelicolor phosphoenolpyruvate carboxylase polypeptide comprises SEQ ID NO: 9 or a variant sequence thereof
  • the Thermobifida fusca phosphoenolpyruvate carboxylase polypeptide comprises SEQ ID NO:7 or a variant sequence thereof
  • the Erwinia chrysanthemi phosphoenolpyruvate carboxylase polypeptide comprises SEQ ID NO: 10 or a variant sequence thereof
  • the Mycobacterium smegmatis pyruvate carboxylase polypeptide comprises SEQ ID NO: 13 or a variant sequence thereof
  • the Streptomyces coelicolor pyruvate carboxylase polypeptide comprises SEQ ID NO: 12 or
  • the Mycobacterium smegmatis aspartokinase polypeptide comprises at least one amino acid change chosen from: an ⁇ alanine changed to a Group 1 amino acid residue at position 279; a serine changed to a Group 6 amino acid residue at position 301; a threonine changed to a Group 2 amino acid residue at position 311; and a glycine changed to a Group 3 amino acid residue at position 345;
  • the Mycobacterium smegmatis aspartokinase comprises at least one amino acid change chosen from: an alanine changed to a proline at position 279, a serine changed to a tyrosine at position 301, a tlireonine changed to an isoleucine at position 311, and a glycine changed to an aspartate at position 345.
  • the Amycolatopsis mediterranei aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a Group 1 amino acid residue at position 279; a serine changed to a Group 6 amino acid residue at position 301 ;a threonine changed to a Group 2 amino acid residue at position 311; and a glycine changed to a
  • Amycolatopsis mediterranei aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a proline at position 279; a serine changed to a tyrosine at position 301; a threonine changed to an isoleucine at position 311; and a glycine changed to an aspartate at position 345.
  • the Streptomyces coelicolor aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a Group 1 amino acid residue at position 282; a serine changed to a Group 6 amino acid residue at position 304; a serine changed to a Group 2 amino acid residue at position 314; and a glycine changed to a Group 3 amino acid residue at position 348.
  • the Streptomyces coelicolor aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a proline at position 282; a serine changed to a tyrosine at position 304; a serine changed to an isoleucine at position 314; and a glycine changed to an aspartate at position 348.
  • the Erwinia chrysanthemi aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycine changed to a Group 3 amino acid residue at position 328; a leucine changed to a Group 6 amino acid residue at position 330; a serine changed to a Group 2 amino acid residue at position 350; and a valine changed to a Group 2 amino acid residue other than valine at position 352.
  • the Erwinia chrysanthemi aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycine changed to an aspartate at position 328; a leucine changed to a phenylalanine at position 330; a serine changed to an isoleucine at position
  • Shewanella oneidensis aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycine changed to a Group 3 amino acid residue at position 323; a leucine changed to a Group 6 amino acid residue at position 325; a serine changed to a Group 2 amino acid residue at position 345; and a valine changed to a Group 2 amino acid residue other than valine at position 347.
  • Shewanella oneidensis aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycme changed to an aspartate at position 323; a leucine changed to a phenylalanine at position 325; a serine changed to an isoleucine at position
  • the Corynebacterium glutamicum aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a Group 1 amino acid other than alanine at position 279; a serine changed to a Group 6 amino acid residue at position 301; a tlireonine changed to a Group 2 amino acid residue at position 311; and a glycine changed to a Group 3 amino acid residue at position 345.
  • the Corynebacterium glutamicum aspartokinase polypeptide comprises at least one amino acid change chosen from: an alanine changed to a proline at position 279; a serine changed to a tyrosine at position 301; a threonine changed to an isoleucine at position 311; and a glycine changed to an aspartate at position 345.
  • the Escherichia coli aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycine changed to a Group 3 amino acid residue at position 323; a leucine changed to a Group 6 amino acid residue at position 325; a serine changed to a Group 2 amino acid residue at position 345; and a valine changed to a Group 2 amino acid residue other than valine at position 347.
  • the Escherichia coli aspartokinase polypeptide comprises at least one amino acid change chosen from: a glycine changed to an aspartate at position 323; a leucine changed to a phenylalanine at position 325; a serine changed to an isoleucine at position 345; and a valine changed to a methionine at position 347.
  • the Corynebacterium glutamicum pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to Group 4 amino acid residue at position 458.
  • the Corynebacterium glutamicum pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to a serine at position 458.
  • the Mycobacterium smegmatis pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to Group 4 amino acid residue at position 448.
  • the Mycobacterium smegmatis pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to a serine at position 448.
  • the Streptomyces coelicolor pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to Group 4 amino acid residue at position 449.
  • the Streptomyces coelicolor pyruvate carboxylase polypeptide or variant thereof comprises a proline changed to a serine at position 449.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial dihydrodipicolinate synthase or a functional variant thereof.
  • the heterologous bacterial dihydrodipicolinate synthase polypeptide or functional variant thereof is chosen from: a Mycobacterium smegmatis dihydrodipicolinate synthase polypeptide or a functional variant thereof; a Streptomyces coelicolor dihydrodipicolinate synthase polypeptide or a functional variant thereof; a Thermobifida fusca dihydrodipicolinate synthase polypeptide or a functional variant thereof; and an Erwinia chrysanthemi dihydrodipicolinate synthase polypeptide or a functional variant thereof.
  • the heterologous bacterial dihydrodipicolinate synthase polypeptide or functional variant thereof with reduced feedback inhibition is an Escherichia coli dihydrodipicolinate synthase polypeptide or a functional variant thereof. In certain embodiments the heterologous bacterial dihydrodipicolinate synthase polypeptide or functional variant thereof has reduced feedback inhibition.
  • the Mycobacterium smegmatis dihydrodipicolinate synthase polypeptide is at least 80% identical to SEQ ID NO:15 or SEQ ID NO:16 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:15 or SEQ ID NO:16);
  • the Streptomyces coelicolor dihydrodipicolinate synthase polypeptide comprises SEQ ID NO: 17 or a variant sequence thereof;
  • the Thermobifida fusca dihydrodipicolinate synthase polypeptide comprises SEQ ID NO: 14 or a variant sequence thereof;
  • the Erwinia chrysanthemi dihydrodipicolinate synthase polypeptide comprises SEQ ID NO: 18 or a variant sequence thereof.
  • the Erwinia chrysanthemi dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to a Group 2 amino acid residue at position 80; a leucine changed to a Group 6 amino acid residue at position 88; and a histidine changed to a Group 6 amino acid residue at position 118.
  • the Erwinia chrysanthemi dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to an isoleucine at position 80; a leucine changed to a phenylalanine at position 88; and a histidine changed to a tyrosine at position 118.
  • the Streptomyces coelicolor dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to a Group 2 amino acid residue at position 89; a.leucine changed to a Group 6 amino acid residue at position 97; and a histidine changed to a Group 6 amino acid residue at position 127.
  • the Streptomyces coelicolor dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to an isoleucine at position 89; a leucine changed to a phenylalanine at position 97; and a histidine changed to a tyrosine at position 127.
  • the Mycobacterium smegmatis dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an amino acid residue corresponding to tyrosine 90 of SEQ ID NO: 16 changed to a Group 2 amino acid residue; an amino acid residue corresponding to leucine 98 of SEQ TD NO:16 changed to a Group 6 amino acid residue; and an amino acid residue corresponding to histidine 128 of SEQ ID NO: 16 changed to a Group 6 amino acid residue.
  • the Mycobacterium smegmatis dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an amino acid residue corresponding to tyrosine 90 of SEQ ID NO: 16 changed to an isoleucine; an amino acid residue corresponding to leucine 98 of SEQ ID NO:16 changed to a phenylalanine; and an amino acid residue corresponding to histidine 128 of SEQ ID NO: 16 changed to a histidine.
  • the Escherichia coli dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to a Group 2 amino acid residue at position 80; an alanine changed to a Group 2 amino acid residue at position 81; a glutamatate changed to a Group 5 amino acid residue at position 84; a leucine changed to a Group 6 amino acid residue at position 88; and a histidine changed to a Group 6 amino acid at position 118.
  • the Escherichia coli dihydrodipicolinate synthase polypeptide comprises at least one amino acid change chosen from: an asparagine changed to an isoleucine at position 80; an alanine changed to a valine at position 81; a glutamate changed to a lysine at position 84; a leucine changed to a phenylalanine at position 88; and a histidine changed to a tyrosine at position 118.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial homoserine dehydrogenase or a functional variant thereof.
  • the heterologous bacterial homoserine dehydrogenase polypeptide is chosen from: (a) a Mycobacterium smegmatis homoserine dehydrogenase polypeptide or functional variant thereof; (b) a Streptomyces coelicolor homoserine dehydrogenase polypeptide or a functional variant thereof; (c) a Thermobifida fusca homoserine dehydrogenase polypeptide or a functional variant thereof; and (d) an Erwinia chrysanthemi homoserine dehydrogenase polypeptide or a functional variant thereof, h certain embodiments, the heterologous bacterial homoserine dehydrogenase polypeptide is a homoserine dehydrogenase polypeptide from a coryneform bacteria or a functional variant thereof (e.g., a Corynebacterium glutamicum homoserine dehydrogenase polypeptide or functional
  • the heterologous homoserine dehydrogenase polypeptide or functional variant thereof is an Escherichia coli homoserine dehydrogenase polypeptide or a functional variant thereof, hi certain embodiments the heterologous homoserine dehydrogenase polypeptide or functional variant thereof has reduced feedback inhibition.
  • the heterologous bacterial homoserine dehydrogenase polypeptide is a Streptomyces coelicolor homoserine dehydrogenase polypeptide or functional variant thereof with reduced feedback inhibition;
  • the Streptomyces coelicolor homoserine dehydrogenase polypeptide comprises SEQ ID NO: 19 or a variant sequence thereof;
  • the Thermobifida fusca homoserine dehydrogenase polypeptide comprises SEQ ID NO:21 or a variant sequence thereof;
  • the Corynebacterium glutamicum and Brevibacterium lactofermentum homoserine dehydrogenases polypeptide comprise SEQ ID NO:209 or a variant sequence thereof;
  • the Escherichia coli homoserine dehydrogenase polypeptide comprises either SEQ ID NO:210, SEQ TD NO:211, or a variant sequence thereof h various embodiments the Corynebacterium glutamicum or Brevibacterium lactoferment
  • Corynebacterium glutamicum or Brevibacterium lactofermentum homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a leucine changed to a phenylalanine at position 23; valine changed to an alanine at position 59; a valine changed to an isoleucine at position 104; and a glycine changed to a glutamic acid at position 378.
  • the Mycobacterium smegmatis homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a valine change to a Group 6 amino acid residue at position 10; a valine changed to a Group 1 amino acid residue at position 46; and a glycine changed to Group 3 amino acid residue at position 364.
  • the Mycobacterium smegmatis homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a valine changed to a phenylalanine at position 10; valine changed to an alanine at position 46; and a glycine changed to a glutamic acid at position 378.
  • the Streptomyces coelicolor homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a leucine change to a Group 6 amino acid residue at position 10; a valine changed to a Group 1 amino acid residue at position 46; a glycine changed to Group 3 amino acid residue at position 362; an alteration that truncates the homoserine dehydrogenase protein after the arginine amino acid residue at position 412m
  • the Streptomyces coelicolor homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a leucine changed to a phenylalanine at position 10; a valine changed to an alanine at position 46; and a glycine changed to a glutamic acid at position 362.
  • the Thermobifida fusca homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a leucine change to a Group 6 amino acid residue at position 192; a valine changed to a Group 1 amino acid residue at position 228; a glycine changed to Group 3 amino acid residue at position 545.
  • the Thermobifida fusca homoserine dehydrogenase polypeptide is truncated after the arginine amino acid residue at position 595.
  • the Thermobifida fusca homoserine dehydrogenase polypeptide comprises at least one amino acid change chosen from: a leucine changed to a phenylalanine at position 192; valine changed to an alanine at position 228; and a glycine changed to a glutamic acid at position 545.
  • the Escherichia coli homoserine dehydrogenase polypeptide comprises at least one amino acid change in SEQ ID NO:211 chosen from: a glycine changed to a Group 3 amino acid residue at position 330; and a serine changed to a Group 6 amino acid residue at position 352.
  • the Escherichia coli homoserine dehydrogenase polypeptide comprises at least one amino acid change in SEQ ID NO:211, ,chosen from: a glycine changed to an aspartate at position 330; and a serine changed to a phenylalanine at position 352.
  • the invention also features: a'coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid that encodes a heterologous bacterial O-homoserine acetyltransferase polypeptide or a functional variant thereof.
  • the heterologous bacterial O-homoserine acetyltransferase polypeptide is chosen from: a Mycobacterium smegmatis O-homoserine acetyltransferase polypeptide or functional variant thereof; a Streptomyces coelicolor O-homoserine acetyltransferase polypeptide or a functional variant thereof; a Thermobifida fusca O-homoserine acetyltransferase polypeptide or a functional variant thereof; and an Erwinia chrysanthemi O- homoserine acetyltransferase polypeptide or a functional variant thereof.
  • the heterologous bacterial O-homoserine acetyltransferase polypeptide is an O- homoserine acetyltransferase polypeptide from Corynebacterium glutamicum or a functional variant thereof. In certain embodiments the heterologous O-homoserine acetyltransferase polypeptide or functional variant thereof has reduced feedback inhibition.
  • the Mycobacterium smegmatis O-homoserine acetyltransferase polypeptide is at least 80% identical to SEQ ID NO:22 or SEQ ID NO:23 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:22 or SEQ ID NO:23);
  • the heterologous bacterial O-homoserine acetyltransferase polypeptide is a Thermobifida fusca O-homoserine acetyltransferase polypeptide or functional variant thereof;
  • the Thermobifida fusca O-homoserine acetyltransferase polypeptide comprises SEQ ID NO:24 or a variant sequence thereof;
  • the heterologous bacterial O-homoserine acetyltransferase polypeptide is a
  • glutamicum O-homoserine acetyltransferase polypeptide comprises SEQ ID NO:212 or a variant sequence thereof; or the heterologous bacterial O-homoserine acetyltransferase polypeptide is a Escherichia coli O-homoserine acetyltransferase polypeptide or functional variant thereof; the Escherichia coli O-homoserine acetyltransferase polypeptide comprises SEQ ID NO :213 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial O-acetylhomoserine sulfhydrylase or a functional variant thereof.
  • the heterologous bacterial O-acetylhomoserine sulfhydrylase polypeptide is chosen from: (a) a Mycobacterium smegmatis O-acetylhomoserine sulfhydrylase polypeptide or functional variant thereof; (b) a Streptomyces coelicolor O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof; and (c) a Thermobifida fusca O- acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof, h certain embodiments, the heterologous bacterial O-acetylhomoserine sulfhydrylase polypeptide is an O- acetylhomoserine sulfhydrylase polypeptide from Corynebacterium glutamicum or a functional variant thereof, certain embodiments the heterologous O-acetylhomos
  • the Mycobacterium smegmatis O-acetylhomoserine sulfhydrylase polypeptide is at least 80% identical to SEQ ID NO:26 (e.g., a sequence at least
  • the Thermobifida fusca O-acetylhomoserine sulfhydrylase polypeptide comprises SEQ ID NO:25 or a variant sequence thereof; and the Corynebacterium glutamicum heterologous bacterial O- acetylhomoserine sulfhydrylase polypeptide comprises SEQ ID NO:214 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial methionine adenosyltransferase or a functional variant thereof.
  • the heterologous bacterial methionine adenosyltransferase polypeptide is chosen from: a Mycobacterium smegmatis methionine adenosyltransferase polypeptide or functional variant thereof; a Streptomyces coelicolor methionine adenosyltransferase polypeptide or a functional variant thereof; a Thermobifida fusca methionine adenosyltransferase polypeptide or a functional variant thereof; and an Erwinia chrysanthemi methionine adenosyltransferase polypeptide or a functional variant thereof, h certain embodiments, the heterologous bacterial methionine adenosyltransferase polypeptide is a methionine adenosyltransferase polypeptide from Corynebacterium glutamicum or a functional variant thereof.
  • the heterologous bacterial methionine adenosyltransferase polypeptide is a methionine adenosyltransferase polypeptide from Escherichia coli or a functional variant thereof. In certain embodiments the heterologous methionine adenosyltransferase polypeptide or functional variant thereof has reduced feedback inhibition
  • the Mycobacterium smegmatis O- methionine adenosyltransferase polypeptide is at least 80% identical to SEQ ID NO:27 or SEQ ID NO:28 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:27 or SEQ ID NO:28);
  • the Streptomyces coelicolor methionine adenosyltransferase polypeptide comprises SEQ ID NO:30 or a variant sequence thereof;
  • the heterologous bacterial methionine adenosyltransferase polypeptide is a Thermobifida fusca methionine adenosyltransferase or functional variant thereof;
  • the Thermobifida fusca methionine adenosyltransferase polypeptide comprises SEQ ID
  • Corynebacterium glutamicum heterologous bacterial methionine adenosyltransferase comprises SEQ ID NO:215 or a variant sequence thereof; and the Escherichia coli heterologous bacterial methionine adenosyltransferase polypeptide comprises SEQ ID NO:216 or a variant sequence thereof.
  • the bacterium further comprises a nucleic acid molecule encoding a heterologous bacterial dihydrodipicolinate synthase polypeptide or a functional variant thereof.
  • the heterologous bacterial dihydrodipicolinate synthase polypeptide or a functional variant thereof is chosen from: a Mycobacterium smegmatis dihydrodipicolinate synthase polypeptide or a functional variant thereof; a Streptomyces coelicolor dihydrodipicolinate synthase polypeptide or a functional variant thereof; a Thermobifida fusca dihydrodipicolinate synthase polypeptide or a functional variant thereof; an Erwinia chrysanthemi dihydrodipicolinate synthase polypeptide or a functional variant thereof; an Escherichia coli dihydrodipicolinate synthase polypeptide or a functional variant thereof; and a Corynebacterium glutamicum dihydrodipicolinate synthase polypeptide or a functional variant thereof.
  • the heterologous dihydrodipicolinate synthase polypeptide or functional variant thereof is chosen from:
  • the bacterium further comprises at least one of: (a) a nucleic acid molecule encoding a heterologous bacterial homoserine dehydrogenase polypeptide or a functional variant thereof; (b) a nucleic acid molecule encoding a heterologous bacterial O- homoserine acetyltransferase polypeptide or a functional variant thereof; (c) a nucleic acid molecule encoding a heterologous O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof. In certain embodiments one or more of the heterologous polypeptides or functional variants thereof has reduced feedback inhibition.
  • the heterologous bacterial homoserine dehydrogenase polypeptide is chosen from: a Mycobacterium smegmatis homoserine dehydrogenase polypeptide or functional variant thereof; a Streptomyces coelicolor homoserine dehydrogenase polypeptide or a functional variant thereof; a Thermobifida fusca homoserine dehydrogenase polypeptide or a functional variant thereof; an Escherichia coli homoserine dehydrogenase polypeptide or a functional variant thereof; a Corynebacterium glutamicum homoserine dehydrogenase polypeptide or a functional variant thereof; and an Erwinia chrysanthemi homoserine dehydrogenase polypeptide or a functional variant thereof.
  • the heterologous homoserine dehydrogenase polypeptide or functional variant thereof has reduced feedback inhibition.
  • the heterologous bacterial O-homoserine acetyltransferase polypeptide is chosen from: a Mycobacterium smegmatis O-homoserine acetyltransferase polypeptide or functional variant thereof; a Streptomyces coelicolor O-homoserine acetyltransferase polypeptide or a functional variant thereof; a Thermobifida fusca O-homoserine acetyltransferase polypeptide or a functional variant thereof; an Erwinia chrysanthemi O- homoserine acetyltransferase polypeptide or a functional variant thereof; an Escherichia coli O- homoserine acetyltransferase polypeptide or a functional variant thereof ; and
  • the heterologous O-homoserine acetyltransferase polypeptide or functional variant thereof has reduced feedb ack inhibition.
  • the heterologous bacterial O-acetylhomoserine sulfhydrylase polypeptide is chosen from: a Mycobacterium smegmatis O-acetylhomoserine sulfhydrylase or functional variant thereof; a Streptomyces coelicolor O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof; a Thermobifida fusca O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof; and a Corynebacterium glutamicum O- acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof, hi certain embodiments the heterologous O-acetylhomoserine s
  • Mycobacterium smegmatis methionine adenosyltransferase polypeptide or functional variant thereof a Streptomyces coelicolor methionine adenosyltransferase polypeptide or a functional variant thereof; a Thermobifida fusca methionine adenosyltransferase polypeptide or a functional variant thereof; an Erwinia chrysanthemi methionine adenosyltransferase polypeptide or a functional variant thereof; an Escherichia coli methionine adenosyltransferase polypeptide or a functional variant thereof; or a Corynebacterium glutamicum methionine adenosyltransferase polypeptide or a functional variant thereof).
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising at least two of: (a) a nucleic acid molecule encoding a heterologous bacterial homoserine dehydrogenase polypeptide or a functional variant thereof; (b) a nucleic acid molecule encoding a heterologous bacterial O- homoserine acetyltransferase polypeptide or a functional variant thereof; and (c) a nucleic acid molecule encoding a heterologous bacterial O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof, hi certain embodiments one or more of the heterologous bacterial polypetides or functional variants thereof has reduced feedback inhibition
  • the invention features an Escherichia coli or coryneform bacterium comprising at least one or two of: (a) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartokinase polypeptide or a functional variant thereof; (b) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof; (c) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof; and (d) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial dihydrodipicolinate synthase polypeptide or a functional variant thereof, hi various embodiments, the genetically altered nucleic acid molecule is a genomic nucleic acid molecule (e.g., a genomic nu
  • the bacterium comprises (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d). h one embodiment, the bacterium comprises at least three of (a)-(e).
  • the bacterium has reduced activity of one or more of the following polypeptides, relative to a control: (a) a homoserine dehydrogenase polypeptide; (b) a homoserine kinase polypeptide; and (c) a phosphoenolpyruvate carboxykinase polypeptide.
  • the bacterium comprises a mutation in an endogenous horn gene or an endogenous thrB gene (e.g., a mutation that reduces activity of the polypeptide encoded by the gene (e.g., a mutation in a catalytic region) or a mutation that reduces expression of the polypeptide encoded by the gene (e.g., the mutation causes premature termination of the polypeptide), or a mutation which decreases transcript or protein stability or half life.
  • the bacterium comprises a mutation in an endogenous horn gene and an endogeous thrB gene, hi one embodiment,the bacterium comprises a mutation in an endogenous pck gene.
  • the invention features an Escherichia coli or coryneform bacterium comprising at least one or two of: (a) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof; (b) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartokinase polypeptide or a functional variant thereof: (c) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof; (d) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial homoserine dehydrogenase polypeptide or a functional variant thereof; (e) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial homoserine O-acet
  • the bacterium comprises (a) and at least one of (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), and (1).
  • the bacterium comprises (b) and at least one of (c), (d), (e), (f), (g), (h), (i), (j), (k) > and (1).
  • the bacterium comprises (c) and at least one of (d), (e), (f), (g), (h), (i), (j), (k), and (1).
  • the bacterium comprises (d) and at least one of (e), (f), (g), (h), (i), (j), (k), and (1). In various embodiments, the bacterium comprises (e) and at least one of (f), (g), (h), (i), (j), (k), and (1). In various embodiments, the bacterium comprises (f) and at least one of (g), (h), (i), (j), (k), and (1). h various embodiments, the bacterium comprises (g) and at least one of (h), (i), (j), (k), and (1). In various embodiments, the bacterium comprises
  • the bacterium comprises (i) and at least one of (j) (k), and (1). In various embodiments, the bacterium comprises (j) and at least one of (k), and (1). In various embodiments, the bacterium comprises (k) and (1). various embodiments, the bacterium comprises at least three of (a)-(l).
  • the bacterium has reduced activity of one or more of the following polypeptides, relative to a control: (a) a homoserine kinase polypeptide; (b) a phosphoenolpyruvate carboxykinase polypeptide; (c) a homoserine dehydrogenase polypeptide; and (d) a rncbR gene product polypeptide, e.g., the bacterium comprises a mutation in an endogenous horn gene, an endogenous thrB gene, an endogenous pck gene, or an endogenous mcbR gene, or combinations thereof.
  • the invention features an Escherichia coli or coryneform bacterium comprising at least two of: (a) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof; (b) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartokinase polypeptide or a functional variant thereof; (c) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial aspartate semialdehyde dehydrogenase polypeptide or a functional variant thereof (d) a genetically altered nucleic acid molecule comprising a sequence encoding a bacterial homoserine dehydrogenase polypeptide or a functional variant thereof.
  • at least one of the at least two polypeptides encodes a heterologous polypeptide.
  • the bacterium comprises (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d); or the bacterium comprises at least three of (a)-(d).
  • the bacterium has reduced activity of one or more of the following polypeptides, relative to a control: (a) a phosphoenolpyruvate carboxykinase polypeptide; and (b) a mcbR gene product polypeptide, e.g., the bacterium comprises a mutation in an endogenous pck gene or an endogenous mcbR gene, e.g.,the bacterium comprises a mutation in an endogenous pck gene and an endogenous mcbR gene.
  • the invention also features a method of producing an amino acid or a related metabolite, the method comprising: cultivating a bacterium (e.g., a bacterium described herein) according to under conditions that allow the amino acid the metabolite to be produced, and collecting a composition that comprises the amino acid or related metabolite from the culture.
  • the method can further include fractionating at least a portion of the culture to obtain a fraction enriched in the amino acid or the metabolite.
  • the invention features a method for producing L-lysine, the method comprising: cultivating a bacterium described herein under conditions that allow L-lysine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-lysine).
  • the invention features a method for the preparation of animal feed additives comprising an aspartate-derived amino acid(s), the method comprising two or more of the following steps:
  • the substances that can be added include, e.g., conventional organic or inorganic auxiliary substances or carriers, such as gelatin, cellulose derivatives (e.g., cellulose ethers), silicas, silicates, stearates, grits, brans, meals, starches, gums, alginates sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • auxiliary substances or carriers such as gelatin, cellulose derivatives (e.g., cellulose ethers), silicas, silicates, stearates, grits, brans, meals, starches, gums, alginates sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • the composition that is collected lacks bacterial cells, hi various embodiments, the composition that is collected contains less than 10%, 5%, 1%, 0.5% of the bacterial cells that result from cultivating the bacterium, i various embodiments, the composition comprises at least 1% (e.g., at least 1%, 5%, 10%, 20%, 40%, 50%, 75%, 80%, 90%, 95%, or to 100%) of that bacterial cells that result from cultivating the bacterium.
  • the invention features a method for producing L-methionine, the method comprising: cultivating a bacterium described herein under conditions that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features a method for producing S-adenosyl-L-methionine (S-AM), the method comprising: cultivating a bacterium described herein under conditions that allow S- adenosyl-L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in S-AM).
  • the invention features a method for producing L-threonine or L-isoleucine, the method comprising: cultivating a bacterium described herein under conditions that allow L-threonine or L-isoleucine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-threonine or L-isoleucine).
  • the invention also features methods for producing homoserine, O-acetylhomoserine, and derivatives thereof, the method comprising: cultivating a bacterium described herein under conditions that allow homoserine, O- acetylhomoserine, or derivatives thereof to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in homoserine, O- acetylhomoserine, or derivatives thereof).
  • the invention features a coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial cystathionine beta-lyase polypeptide (e.g., a Mycobacterium smegmatis cystathionine beta-lyase polypeptide or functional variant thereof; a Bifidobacterium longurn cystathionine beta-lyase polypeptide or a functional variant thereof; a Lactobacillus plantarurn cystathionine beta-lyase polypeptide or a functional variant thereof; a Corynebacterium glutamicum cystathionine beta-lyase polypeptide or a functional variant thereof; an Escherichia coli cystathionine beta-lyase polypeptide or a functional variant thereof) or a functional variant thereof.
  • a heterologous bacterial cystathionine beta-lyase polypeptide e.g.,
  • the Mycobacterium smegmatis cystathionine beta-lyase polypeptide comprises a sequence at least 80% identical to SEQ ID NO:59 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:59), or a variant sequence thereof;
  • the Bifidobacterium longum cystathionine beta-lyase polypeptide comprises SEQ ID NO:60 or a variant sequence thereof;
  • the Lactobacillus plantarum cystathionine beta-lyase polypeptide comprises SEQ ID NO:61 or a variant sequence thereof;
  • the Corynebacterium glutamicum cystathionine beta-lyase polypeptide comprises SEQ ID NO:217 or a variant sequence thereof; and the Escherichia coli cystathionine beta-lyase polypeptide comprises SEQ LD NO:218 or a variant
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial glutamate dehydrogenase polypeptide (e.g., a Streptomyces coelicolor glutamate dehydrogenase or functional variant thereof; a Thermobifida fusca glutamate dehydrogenase polypeptide or a functional variant thereof; a Lactobacillus plantarum glutamate dehydrogenase polypeptide or a functional variant thereof; a Corynebacterium glutamicum glutamate dehydrogenase polypeptide or a functional variant thereof; a Escherichia coli glutamate dehydrogenase polypeptide or a functional variant thereof) or a functional variant thereof.
  • a heterologous bacterial glutamate dehydrogenase polypeptide
  • the Mycobacterium smegmatis glutamate dehydrogenase polypeptide comprises SEQ ID NO:62 or a variant sequence thereof; the Thermobifida fusca glutamate dehydrogenase polypeptide comprises SEQ ID NO:63 or a variant sequence thereof; the Lactobacillus plantarum glutamate dehydrogenase polypeptide comprises SEQ ID NO: 65 or a variant sequence thereof; the Corynebacterium glutamicum glutamate dehydrogenase polypeptide comprises SEQ ID NO:219 or a variant sequence thereof; and the Escherichia coli glutamate dehydrogenase polypeptide comprises SEQ ID NO:220 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial diaminopimelate dehydrogenase polypeptide or a functional variant thereof (e.g., a Bacillus sphaericus diaminopimelate dehydrogenase polypeptide or a functional variant thereof; a Corynebacterium glutamicum glutamate dehydrogenase polypeptide or a functional variant thereof).
  • a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial diaminopimelate dehydrogenase polypeptide or a functional variant thereof (e.g., a Bacillus sphaericus dia
  • the Bacillus sphaericus diaminopimelate dehydrogenase polypeptide comprises SEQ ID NO: 65 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial detergent sensitivity rescuer polypeptide (e.g., a Mycobacterium smegmatis detergent sensitivity rescuer polypeptide or functional variant thereof; a Streptomyces coelicolor detergent sensitivity rescuer polypeptide or a functional variant thereof; a Thermobifida fusca detergent sensitivity rescuer polypeptide or a functional variant thereof; a Corynebacterium glutamicum detergent sensitivity rescuer polypeptide or a functional variant thereof) or a functional variant thereof .
  • the Mycobacterium smegmatis detergent sensitivity rescuer polypeptide comprises a sequence at least 80% identical to either SEQ ID NO:68, SEQ ID NO:69 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical), or a variant sequence thereof;
  • the heterologous bacterial detergent sensitivity rescuer polypeptide is a Streptomyces coelicolor detergent sensitivity rescuer polypeptide or functional variant thereof;
  • the Streptomyces coelicolor detergent sensitivity rescuer polypeptide comprises SEQ ID NO:67 or a variant sequence thereof;
  • the Tliermobifida fusca detergent sensitivity rescuer polypeptide comprises SEQ ID NO:66 or a variant sequence thereof;
  • the Corynebacterium glutamicum detergent sensitivity rescuer polypeptide comprises SEQ ID NO:221 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as a Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide (e.g., a Mycobacterium smegmatis 5- methyltetrahydrofolate homocysteine methyltransferase polypeptide or functional variant thereof; a Streptomyces coelicolor 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide or a functional variant thereof; a Thermobifida fusca 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide or a functional variant thereof; a Lactobacillus plantarum 5-methyltetrahydrofolate homocysteine methyltransferase poly
  • the Mycobacterium smegmatis 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide comprises a sequence at least 80% identical to SEQ ID NO:72, SEQ ID NO:73 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical), or a variant sequence thereof;
  • the Streptomyces coelicolor 5- methyltetrahydrofolate homocysteine methyltransferase polypeptide comprises SEQ ID NO:71 or a variant sequence thereof;
  • the Thermobifida fusca 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide comprises SEQ LD NO:70 or a variant sequence thereof;
  • the Lactobacillus plantarum 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide comprises SEQ ID NO:
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide (e.g., a Mycobacterium smegmatis 5- methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide or functional variant thereof; a Streptomyces coelicolor 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide or functional variant thereof; a Corynebacterium glutamicum 5- methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide or a functional variant thereof; an Escherichia
  • the Mycobacterium smegmatis 5- methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide is at least 80% identical to SEQ ID NO:75 or SEQ ID NO:76 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:75 or SEQ ID NO:76);
  • the Streptomyces coelicolor 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide comprises SEQ ID NO:77 or a variant sequence thereof;
  • the Corynebacterium glutamicum 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase polypeptide comprises SEQ LD NO:224 or a variant sequence thereof; and the Escherichia coli 5- methyltetrahydroptero
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial serine hydroxymethyltransferas polypeptide (e.g., a
  • the Mycobacterium smegmatis serine hydroxymethyltransferase polypeptide is at least 80% identical to SEQ ID NO:80 or SEQ ID NO:81 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:80 or SEQ LD NO:81);
  • the Streptomyces coelicolor serine hydroxymethyltransferase polypeptide comprises SEQ ID NO:78 or a variant sequence thereof;
  • the Thermobifida fusca serine hydroxymethyltransferase polypeptide comprises SEQ ID NO:79 or a variant sequence thereof;
  • the Lactobacillus plantarum serine hydroxymethyltransferase polypeptide comprises
  • the Corynebacterium glutamicum serine hydroxymethyltransferase polypeptide comprises SEQ ID NO:226 or a variant sequence thereof; and the Escherichia coli serine hydroxymethyltransferase polypeptide comprises SEQ LD NO:227 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial 5,10-methylenetetrahydrofolate reductase polypeptide (e.g., a Streptomyces coelicolor 5,10-methylenetetrahydrofolate reductase polypeptide or a functional variant thereof; a Thermobifida fusca 5,10-methylenetetrahydrofolate reductase polypeptide or a functional variant thereof; a Corynebacterium glutamicum 5,10-methylenetetrahydrofolate reductase polypeptide or a functional variant thereof; an. Escherichia coli 5,10- methylenetetrahydrofolate reductase polypeptide or a functional variant thereof) or a functional variant thereof.
  • the Streptomyces coelicolor 5,10-methylenetetrahydrofolate reductase polypeptide comprises SEQ ID NO: 84 or a variant sequence thereof;
  • the Tliemiobifida fusca 5,10-methylenetetrahydrofolate reductase polypeptide comprises SEQ ID NO: 83 or a variant sequence thereof;
  • the Corynebacterium glutamicum 5,10-methylenetetrahydrofolate reductase polypeptide comprises SEQ ID NO: 228 or a variant sequence thereof;
  • the Escherichia coli 5,10-methylenetetrahydrofolate reductase polypeptide comprises SEQ ID NO: 229or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial serine O-acetyltransferase polypeptide (e.g., a Mycobacterium smegmatis serine O-acetyltransferase polypeptide or functional variant thereof; a Lactobacillus plantarum serine O-acetyltransferase polypeptide or a functional variant thereof; a Corynebacterium glutamicum serine O-acetyltransferase polypeptide or a functional variant thereof; an Escherichia coli serine O-acetyltransferase polypeptide or a functional variant thereof) or a functional variant thereof.
  • a heterologous bacterial serine O-acetyltransferase polypeptide e.g., a Mycobacterium smegmatis serine O-acetyltransfera
  • the Mycobacterium smegmatis serine O-acetyltransferase polypeptide is at least 80% identical to SEQ ID NO:85 or SEQ ID NO:86 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ DD
  • Lactobacillus plantarum serine O-acetyltransferase polypeptide comprises SEQ ID NO:87 or a variant sequence thereof
  • the Corynebacterium glutamicum serine O-acetyltransferase polypeptide comprises SEQ ID NO:230 or a variant sequence thereof
  • the Escherichia coli serine O-acetyltransferase polypeptide comprises SEQ ID NO:231 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial D-3-phosphoglycerate dehydrogenase polypeptide (e.g., a Mycobacterium smegmatis D-3-phosphoglycerate dehydrogenase polypeptide or functional variant thereof; a Streptomyces coelicolor D-3-phosphoglycerate dehydrogenase polypeptide or a functional variant thereof; a Thermobifida fusca D-3-phosphoglycerate dehydrogenase polypeptide or a functional variant thereof; a Lactobacillus plantarum D-3-phosphoglycerate dehydrogenase polypeptide or a functional variant thereof; a Corynebacterium glutamicum D-3- phosphoglycerate dehydrogenase polypeptid
  • the Mycobacterium smegmatis D-3 -phosphoglycerate dehydrogenase polypeptide is at least 80% identical to SEQ ID NO:88 or SEQ ID NO:89 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:88 or SEQ ID NO:89);
  • the Streptomyces coelicolor D-3- ⁇ hosphoglycerate dehydrogenase polypeptide comprises SEQ ID NO:91 or a variant sequence thereof;
  • Tliermobifida fusca D-3-phosphoglycerate dehydrogenase polypeptide comprises SEQ ID NO:90 or a variant sequence thereof; the Lactobacillus plantarum D-3 -phosphoglycerate dehydrogenase polypeptide comprises SEQ ID NO:92 or a variant sequence thereof; the Corynebacterium glutamicum serine O-acetyltransferase polypeptide comprises SEQ ID NO:232 or a variant sequence thereof; and the Escherichia coli serine O-acetyltransferase polypeptide comprises SEQ ID NO:233 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a heterologous bacterial lysine exporter polypeptide (e.g., a Corynebacterium glutamicum lysine exporter polypeptide or functional variant thereof; a Mycobacterium smegmatis lysine exporter polypeptide or functional variant thereof; a Streptomyces coelicolor lysine exporter polypeptide or a functional variant thereof; an Escherichia coli lysine exporter polypeptide or functional variant thereof or a Lactobacillus plantarum lysine exporter protein or a functional variant thereof) or functional variant thereof.
  • a heterologous bacterial lysine exporter polypeptide e.g., a Corynebacterium glutamicum lysine exporter polypeptide or functional variant
  • the Mycobacterium smegmatis lysine exporter polypeptide is at least 80% identical to SEQ ID NO:93 or SEQ ID NO:94 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:93 or SEQ ID NO:94);
  • the Streptomyces coelicolor lysine exporter polypeptide comprises SEQ ID NO:95 or a variant sequence thereof;
  • the Lactobacillus plantarum lysine exporter polypeptide comprises SEQ ID NO:96 or a variant sequence thereof;
  • the Corynebacterium glutamicum lysine exporter polypeptide comprises SEQ ID NO:234 or a variant sequence thereof; and the Escherichia coli lysine exporter polypeptide comprises SEQ ID NO:237 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a bacterial O-succinylhomoserine (thio)-lyase/O-acetylhomoserine (thio)-lyase polypeptide (e.g., a Corynebacterium glutamicum O-succinylhomoserine (thio)-lyase polypeptide or functional variant thereof; a Mycobacterium smegmatis O-succinylhomoserine (thio)-lyase polypeptide or functional variant thereof; a Streptomyces coelicolor O- succinylhomoserine (thio)-lyase polypeptide or a functional variant thereof; a Thermobifida fusca O-succinylhom
  • the Mycobacterium smegmatis O-succinylho oserine (thio)- lyase polypeptide is at least 80% identical to SEQ ID NO:97 or SEQ ID NO:98 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:97 or SEQ ID NO:98);
  • the Streptomyces coelicolor O-succinylhomoserine (thio)-lyase polypeptide comprises SEQ ID NO:99 or a variant sequence thereof;
  • the Thermobifida fusca O- succinylhomoserine (thio)-lyase polypeptide comprises SEQ ID NO: 100 or a variant sequence thereof;
  • the Lactobacillus plantarum O-succinylhomoserine (thio)-lyase polypeptide comprises
  • the Corynebacterium glutamicum O- succinylhomoserine (thio)-lyase polypeptide comprises SEQ ID NO:235 or a variant sequence thereof
  • the Escherichia coli O-succinylhomoserine (thio)-lyase polypeptide comprises SEQ ID NO:236 or a variant sequence thereof.
  • the invention features a coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes a threonine efflux polypeptide (e.g. a Corynebacterium glutamicum threonine efflux polypeptide or a functional variant thereof; a homolog of the Corynebacterium glutamicum threonine efflux polypeptide or a functional variant thereof; a Streptomyces coelicolor putative threonine efflux polypeptide or a functional variant thereof) or functional variant thereof.
  • a threonine efflux polypeptide e.g. a Corynebacterium glutamicum threonine efflux polypeptide or a functional variant thereof; a homolog of the Corynebacterium glutamicum threonine efflux polypeptide or a functional variant thereof; a Streptomyces coelicolor putative threonine efflux polypeptide or a functional variant
  • the Corynebacterium glutamicum threonine efflux polypeptide comprises SEQ ID NO: 196 or a variant sequence thereof; the homolog of the Corynebacterium glutamicum threonine efflux polypeptide comprises a homolog of SEQ ID NO: 196 or a variant sequence thereof; and the Streptomyces coelicolor putative threonine efflux polypeptide comprises SEQ ID NO: 102 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum hypothetical polypeptide (SEQ ID NO: 198), a bacterial homolog of C.
  • a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum hypothetical polypeptide (SEQ ID NO: 198), a bacterial homolog of C.
  • glutamicum hypothetical polypeptide (SEQ ID NO: 198), (e.g., a Mycobacterium smegmatis hypothetical polypeptide or functional variant thereof; a Streptomyces coelicolor hypothetical polypeptide or a functional variant thereof; a Thermobifida fusca hypothetical polypeptide or a functional variant thereof; an Escherichia coli hypothetical polypeptide or a functional variant thereof; ox a Lactobacillus plantarum hypothetical polypeptide or a functional variant thereof) or a functional variant thereof.
  • SEQ ID NO: 198 glutamicum hypothetical polypeptide
  • the bacterial homolog is: a Mycobacterium smegmatis hypothetical polypeptide at least 80% identical to SEQ ID NO:104 or SEQ ID NO:105 (e.g., a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 104 or SEQ LD NO: 105);
  • the Streptomyces coelicolor hypothetical polypeptide comprises SEQ ID NO: 103 or a variant sequence thereof;
  • the Thermobifida fusca hypothetical polypeptide comprises SEQ ID NO 106 or a variant sequence thereof;
  • the Lactobacillus plantarum hypothetical polypeptide comprises SEQ ID NO: 107 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum putative membrane polypeptide (SEQ ID NO:201), a bacterial homolog of C. glutamicum putative membrane polypeptide (SEQ ID NO:201), (e.g., a
  • Streptomyces coelicolor putative membrane polypeptide or a functional variant thereof a Thermobifida fusca putative membrane polypeptide or a functional variant thereof; an Erwinia chrysanthemi putative membrane polypeptide or a functional variant thereof; an Escherichia coli putative membrane polypeptide or a functional variant thereof; a Lactobacillus plantarum putative membrane polypeptide or a functional variant thereof; or a Pectobacterium chrysanthemi putative membrane polypeptide or a functional variant thereof) or a functional variant thereof.
  • the Streptomyces coelicolor putative membrane polypeptide comprises SEQ ED NO: 111, SEQ ID NO: 112, SEQ ED NO: 113, SEQ ID NO: 114, or a variant sequence thereof;
  • the Thermobifida fusca putative membrane polypeptide comprises SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or a variant sequence thereof;
  • the Erwinia chrysanthemi putative membrane polypeptide comprises SEQ ED NO:l 15 or a variant sequence thereof;
  • the Pectobacterium chrysanthemi putative membrane polypeptide comprises SEQ ID NO:l 16 or a variant sequence thereof;
  • the Lactobacillus plantarum putative membrane polypeptide comprises SEQ ID NO:117, SEQ DD NO:118, SEQ ID NO:119, or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum drug permease polypeptide (SEQ DD NO: 199), a bacterial homolog of C.
  • a coryneform bacterium or a bacterium of the family Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum drug permease polypeptide (SEQ DD NO: 199), a bacterial homolog of C.
  • glutamicum drug permease polypeptide (SEQ DD tSTO:199), (e.g., a Streptomyces coelicolor drug permease polypeptide o a functional variant thereof; a Thermobifida fusca drug permease polypeptide or a functional variant thereof; an Escherichia coli drug permease polypeptide or a functional variant thereof; or a Lactobacillus plantarum drug permease polypeptide or a functional variant thereof) or a functional variant thereof.
  • SEQ DD tSTO:199 glutamicum drug permease polypeptide
  • the Streptomyces coelicolor drug permease polypeptide comprises SEQ DD NO: 120, SEQ DD NO: 121, or a variant sequence thereof;
  • the Thermobifida fusca drug permease polypeptide comprises SEQ D NO: 122, SEQ DD NO: 123, or a variant sequence thereof;
  • the Lactobacillus plantarum drug permease polypeptide comprises SEQ DD NO: 124 or a variant sequence thereof.
  • the invention also features a coryneform bacterium or a bacterium of the family
  • Enterobacteriaceae such as an Escherichia coli bacterium comprising a nucleic acid molecule that encodes C. glutamicum hypothetical membrane polypeptide (SEQ DD NO: 197), a bacterial homolog of C. glutamicum hypothetical membrane polypeptide (SEQ DD NO:197), (e.g., a Thermobifida fusca hypothetical membrane polypeptide or a functional variant thereof).
  • the Thermobifida fusca hypothetical membrane polypeptide comprises SEQ DD NO: 125 or a variant sequence thereof.
  • the invention also provides nucleic acids encoding variant bacterial proteins.
  • Nucleic acids that include sequences encoding variant bacterial polypeptides can be expressed in the organism from which the sequence was derived, or they can be expressed in an organism other than the organism from which they were derived (e.g., heterologous organisms).
  • the invention features an isolated nucleic acid (e.g., a nucleic acid expression vector) that encodes a variant of a bacterial polypeptide (e.g., a variant of a wild-type bacterial polypeptide) that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites.
  • the bacterial polypeptide can include, for example, the following amino acid sequence: G ⁇ -X 2 -K 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X ⁇ -X 12 -X 13 - l3a-Xl3 - l3c"Xl3d- l3e- l3rXl3g-Xl3h-Xl3r l3j-Xl3k- l31-Fl4- l5-Zi6-X 1 7-Xl8-Xl9-X20- 21-
  • X2it-D 22 (SEQ DD NO: ), wherein each of X 2 , X 4 -X 13 , X 15 , and X 17 -X 20 is, independently, any amino acid, wherein each of X 13a -X 13 ⁇ is, independently, any amino acid or absent, wherein each of X2i a -X2i t is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine.
  • the variant of the bacterial polypeptide includes an amino acid change relative to the bacterial protein, e.g., at one or more of G ls K 3 , F 1 , Z 16 , or D2 2 of SEQ DD NO: , or at an amino acid within 8, 5, 3, 2, or 1 residue of G ls K 3 , F 14 , Z 16 , or D 22 of
  • variant of the bacterial polypeptide is otherwise identical in amino acid sequence to the bacterial protein, or at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the bacterial polypeptide, e.g., the variant comprises fewer than 50, 40, 25, 15, 10, 7, 5, 3, 2, or 1 changes relative to the bacterial polypeptide.
  • the bacterial polypeptide includes the following amino acid sequence: L 1 -X 2 -X 3 -G 4 -G 5 -X 6 -F 7 -X 8 -X 9 - Xio-Xn (SEQ DD NO:__), wherein each of X 2 , X4-X13, X 15 , and X 1 -X 2 o is, independently, any amino acid, herein X 8 is selected from valine, leucine, isoleucine, and aspartate, and wherein X ⁇ is selected from valine, leucine, isoleucine, phenylalanine, and methionine; and the variant of the bacterial protein includes an amino acid change e.g., at one or more of L l5 G , X 8 , X lls or at an amino acid residue within 8, 5, 3, 2, or 1 residue of Li, G 4 , X 8 , or X n of SEQ DD NO: ___
  • feedback inhibition of the variant of the bacterial polypeptide by S-adenosylmethionine is reduced, e.g., relative to the bacterial polypeptide (e.g., relative to a wild-type bacterial protein) or relative to a reference protein.
  • Amino acid changes in the variant of the bacterial polypeptide can be changes to alanine (e.g., wherein the original residue is other than an alanine) or non-conservative changes. The changes can be conservative changes.
  • the invention also features polypeptides encoded by the nucleic acids described herein, e.g., a polypeptide encoded by a nucleic acid that encodes a variant of a bacterial polypeptide
  • a variant of a wild-type bacterial polypeptide that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites, wherein the bacterial polypeptide includes SEQ DD NO: or SEQ ED NO: , and wherein the variant includes an amino acid change relative to the bacterial polypeptide.
  • a method for making a nucleic acid encoding a variant of a bacterial polypeptide that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites is also provided.
  • the method includes, for example, identifying a motif in the amino acid sequence of a wild-type form of the bacterial polypeptide, and constructing a nucleic acid that encodes a variant wherein one or more amino acid residues (e.g., one, two, three, four, or five residues) within and/or near (e.g., within 10, 8, 7, 5, 3, 2, or 1 residues) the motif is changed.
  • one or more amino acid residues e.g., one, two, three, four, or five residues
  • near e.g., within 10, 8, 7, 5, 3, 2, or 1 residues
  • the motif in the bacterial polypeptide includes the following amino acid sequence: G ⁇ -X2-K 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 1 o-X 11 -Xi2-Xi3-Xi3a-Xi3b-Xi3c-Xi3d-Xi3e-
  • one or more of G ls K 3 , F 1 , Z 16 , or D 2 2 of SEQ ED NO: is changed, h one embodiment, the variant of the bacterial polypeptide is otherwise identical in amino acid sequence to the bacterial polypeptide.
  • the motif in the bacterial polypeptide includes the following amino acid sequence: L 1 -X 2 -X 3 -G 4 -G 5 -X 6 -F 7 -X 8 -X 9 - Xio-Xn (SEQ DD NO:__), wherein each of X 2 , X4-X13, X15, and X17-X 2 Q is, independently, any amino acid, wherein X 8 is selected from valine, leucine, isoleucine, and aspartate, and wherein X ⁇ is selected from valine, leucine, isoleucine, phenylalanine, and methionine.
  • one or more of L ls G 4 , X 8 , X ⁇ of SEQ DD NO: is changed, hi one embodiment, the variant of the bacterial polypeptide is otherwise identical in amino acid sequence to the bacterial protein.
  • the invention also features' a bacterium that includes a nucleic acid described herein, e.g., a nucleic acid that encodes a variant of a bacterial polypeptide (e.g., a variant of a wild-type bacterial polypeptide) that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites, wherein the bacterial polypeptide includes
  • the bacterium can be a genetically modified bacterium, e.g., a bacterium that has been modified to include the nucleic acid (e.g., by transformation of the nucleic acid, e.g., wherein the nucleic acid is episomal, or wherein the nucleic acid integrates into the genome of the bacterium, either at a random location, or at a specifically targeted location), and/or that has been modified within its genome (e.g., modified such that an endogenous gene has been altered by mutagenesis or replaced by recombination, or modified to include a heterologous promoter upstream of an endogenous gene.
  • the invention also features a method for producing an amino acid or a related metabolite.
  • the methods can include, for example: cultivating a bacterium (e.g., a genetically modified bacterium) that includes a nucleic acid encoding a variant of a bacterial polypeptide (e.g., a variant of a wild-type bacterial polypeptide) that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites, wherein the bacterial polypeptide includes SEQ DD NO: or SEQ ED NO: , and wherein the variant includes an amino acid change relative to the bacterial polypeptide.
  • a bacterium e.g., a genetically modified bacterium
  • a nucleic acid encoding a variant of a bacterial polypeptide (e.g., a variant of a wild-type bacterial polypeptide) that regulates the production of one or more amino acids from the aspartic acid family of amino acids or related metabolites
  • the bacterial polypeptide includes SEQ DD NO: or SEQ ED NO
  • the bacterium is cultivated under conditions in which the nucleic acid is expressed and that allow the amino acid (or related metabolite(s)) to be produced, and a composition that includes the amino acid (or related metabolite(s)) is collected.
  • the composition can include, for example, culture supematants, heat or otherwise killed cells, or purified amino acid.
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide.
  • the variant bacterial homoserine O-acetyltransferase polypeptide exhibits reduced feedback inhibition, e.g. , relative to a wild-type form of the bacterial homoserine O-acetyltransferase polypeptide.
  • the nucleic acid encodes a homoserine O-acetyltransferase polypeptide with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial homoserine O-acetyltransferase polypeptide is chosen from: a Corynebacterium glutamicum homoserine O-acetyltransferase polypeptide, a Mycobacterium smegmatis homoserine O- acetyltransferase polypeptide, a Thermobifida fusca homoserine O-acetyltransferase polypeptide, an Amycolatopsis mediterranei homoserine O-acetyltransferase polypeptide, a Streptomyces coelicolor homoserine O-acetyltransferase polypeptide, an Erwinia chrysanthemi homoserine O- acetyltransferase polypeptide, a Shewanella oneidensis homoserine O-acetyltransferase polypeptide, a Mycobacterium tub
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is a variant of a homoserine O-acetyltransferase polypeptide including the following amino acid sequence: G ⁇ -X2-K 3 -X 4 -X5-X 6 -X 7 -X 8 -X 9 -X 1 o-X ⁇ -X 1 2-Xi3-
  • X21a-X21b-X21c-X21d-X21e-X2irX21g-X21h-X21i-X21j-X21k-X211-X 2 lm-X21n-X21o-X21 -X21q-X21r-X21s- X2i t -D 22 (SEQ DD NO: ), wherein each of X 2 , X 4 -X 13 , X 15 , and X 1 -X2o is, independently, any amino acid, wherein each of X 13a -X 131 is, independently, any amino acid or absent, wherein each of X 1a -X 21t is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine; wherein the variant homoserine O-acetyltransferase polypeptide includes an amino acid change at one or more of G l5 K 3 ,
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is a C. glutamicum homoserine O-acetyltransferase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 231, Lysine 233, Phenylalanine 251, Valine 253, and Aspartate 269.
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is a T fusca homoserine O-acetyltransferase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 81, Aspartate 287, Phenylalanine 269.
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is an E. coli homoserine O-acetyltransferase polypeptide including an amino acid change at Glutamate 252 of S ⁇ Q ID NO: .
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is a mycobacterial homoserine O-acetyltransferase polypeptide including an amino acid change in a residue corresponding to one or more of the following residues of M. leprae homoserine O-acetyltransferase polypeptide set forth in S ⁇ Q DD NO: : Glycine 73, Aspartate 278, and Tyrosine 260.
  • the variant bacterial homoserine O-acetyltransferase polypeptide is a variant of a M. smegmatis homoserine O- acetyltransferase polypeptide.
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O- acetyltransferase polypeptide is an M. tuberculosis homoserine O-acetyltransferase polypeptide including an amino acid change in one or more of the following residues of S ⁇ Q D NO: :
  • Glycine 73 Glycine 73, Tyrosine 260, and Aspartate 278.
  • the invention also features polypeptides encoded by, and bacteria including, the nucleic acids encoding variant bacterial homoserine O-acetyltransferases.
  • the bacteria are coryneform bacteria.
  • the bacteria can further include nucleic acids encoding other variant bacterial proteins (e.g., variant bacterial proteins involved in amino acid production, e.g., variant bacterial proteins described herein).
  • the invention features a method for producing L-methionine or related intermediates such as O-acetyl homoserine, cystathionine, homocysteine, methionine, SAM and derivatives thereof, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase under conditions in which the nucleic acid is expressed and that allow L-methionine (or related intermediate) to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide.
  • the variant bacterial homoserine O-acetylhomoserine sulfhydrylase polypeptide exhibits reduced feedback inhibition, e.g., relative to a wild-type form of the bacterial O-acetylhomoserine sulfhydrylase polypeptide.
  • the nucleic acid encodes an O-acetylhomoserine sulfhydrylase polypeptide with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial O-acetylhomoserine sulfhydrylase polypeptide is chosen from: a Corynebacterium glutamicum homoserine O-acetylhomoserine sulfhydrylase polypeptide, a Mycobacterium smegmatis homoserine O-acetylhomoserine sulfhydrylase polypeptide, a Thermobifida fusca O-acetylhomoserine sulfhydrylase polypeptide, an
  • Amycolatopsis mediterranei O-acetylhomoserine sulfhydrylase polypeptide a Streptomyces coelicolor O-acetylhomoserine sulfhydrylase polypeptide, an Erwinia chrysanthemi homoserine O-acetylhomoserine sulfhydrylase polypeptide, a Shewanella oneidensis O-acetylhomoserine sulfhydrylase polypeptide, a Mycobacterium tuberculosis O-acetylhomoserine sulfhydrylase polypeptide, an Escherichia coli O-acetylhomoserine sulfhydrylase polypeptide, a Corynebacterium acetoglutamicum O-acetylhomoserine sulfhydrylase polypeptide, a Corynebacterium melassecola O-acety
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is a variant of an O-acetylhomoserine sulfhydrylase polypeptide including the following amino acid sequence: G ⁇ -X2- ⁇ 3 -X -X 5 -X6-X 7 -X 8 -X9-X ⁇ 0 -X ⁇ -Xi2- ⁇ 3 -
  • each of X 2 , X4-X 13 , Xis, and X 1 -X 20 is, independently, any amino acid, wherein each of X 13a -X 131 is, independently, any amino acid or absent, wherein each of X 2 a -X 2 n is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine; wherein the variant O- acetylhomoserine sulfhydrylase polypeptide includes an amino acid change at one or more of G la K 3 , F 14 , Zi 6 , or D 22 of SEQ DD NO:_.
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is a variant of a O-acetylhomoserine sulfhydrylase polypeptide including the following amino acid sequence: L 1 -X 2 -X 3 -G 4 -G 5 -X 6 -F -X 8 -X 9 - X 10 -X ⁇ (SEQ DD
  • X is any amino acid
  • X 8 is selected from valine, leucine, isoleucine, and aspartate
  • X ⁇ is selected from valine, leucine, isoleucine, phenylalanine, and methionine
  • the variant of the bacterial polypeptide includes an amino acid change at one or more of Li, G 4 , X 8 , X ⁇ of SEQ DD NO: _.
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is a C. glutamicum O-acetylhomoserine sulfhydrylase polypeptide including an amino acid change in one or more of the following residues of SEQ ED NO: :
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is a T. fusca O-acetylhomoserine sulfhydrylase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 240,
  • the invention features an isolated nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is M. smegmatis O-acetylhomoserine sulfhydrylase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: :
  • the invention features a polypeptide encoded by a nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase.
  • the invention features a bacterium comprising the nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., a variant bacterial polypeptide described herein).
  • the invention features a method for producing L-methionine or related intermediates (e.g., homocysteine, methionine, S-AM, or derivatives thereof), the method comprising: cultivating a genetically modified bacterium comprising the nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide under conditions in which the nucleic acid is expressed and that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L- methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial mcbR gene product, hi various embodiments, the variant bacterial mcbR gene product exhibits reduced feedback inhibition relative to a wild-type form of the mcbR gene product, h various embodiments, the nucleic acid encodes a mcbR gene product with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial mcbR gene product is chosen from: a Corynebacterium glutamicum mcbR gene product, a Corynebacterium acetoglutamicum mcbR gene product, a Corynebacterium melassecola mcbR gene product, and a Corynebacterium thermoaminogenes mcbR gene product.
  • the invention features an isolated nucleic acid encoding a variant bacterial mcbR gene product, wherein the variant mcbR gene product is a variant of an mcbR gene product including the following amino acid sequence: G ⁇ -x 2 -K3-X4-x 5 -x 6 -X7-x 8 -X9-X ⁇ o- ll- l2"-Xl3-Xl3a- l3 -X 13c-Xl3d-Xl3e _ Xl 3 f"-Xl3g _ Xl3h _ Xl3i -X 13j -X 13 _ Xl31 ⁇ Fi4-X ⁇ 5 -Zi6-Xl - X 18 _ Xl9 _ X20 ⁇ X21-X21a-X21b " -X21c- 21d- 21e- 21f ⁇ X21g- " X21h _ X21i-X21j ⁇ X21k
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial mcbR gene product, wherein the variant mcbR gene product is a C. glutamicum mcbR gene product including an amino acid change in one or more of the following residues of SEQ ID NO: 1
  • the amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by the nucleic acids encoding a variant bacterial mcbR gene product.
  • the invention also features a bacterium including the nucleic acids encoding a variant bacterial mcbR gene product.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features methods for producing L-methionine, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial mcbR gene product under conditions in which the nucleic acid is expressed and that allow L- methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial aspartokinase polypeptide.
  • the variant bacterial aspartokinase polypeptide exhibits reduced feedback inhibition relative to a wild-type form of the bacterial aspartokinase polypeptide.
  • the nucleic acid encodes an aspartokinase polypeptide with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial aspartokinase polypeptide is chosen from: a Corynebacterium glutamicum aspartokinase polypeptide, a Mycobacterium smegmatis aspartokinase polypeptide, a Thermobifida fusca aspartokinase polypeptide, an Amycolatopsis mediterranei aspartokinase polypeptide, a Streptomyces coelicolor aspartokinase polypeptide, an Erwinia chrysanthemi aspartokinase polypeptide, a Shewanella oneidensis aspartokinase polypeptide, a Mycobacterium tuberculosis aspartokinase polypeptide, an Escherichia coli aspartokinase polypeptide, a
  • the invention features an isolated nucleic acid encoding a variant bacterial aspartokinase polypeptide, wherein the variant aspartokinase polypeptide is a variant of an aspartokinase polypeptide including the following amino acid sequence: G 1 -x 2 -K3-x 4 -x 5 -
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial aspartokinase polypeptide, wherein the aspartokinase polypeptide is a C. glutamicum aspartokinase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 208, Lysine 210, Phenylalanine 223, Valine 225, and
  • the amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by the nucleic acid encoding a variant bacterial aspartokinase polypeptide.
  • the invention also features a bacterium including the nucleic acid encoding a variant bacterial aspartokinase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the bacterium further comprises one or more nucleic acid molecules (e.g., recombinant nucleic acid molecules) encoding a polypeptide involved in amino acid production (e.g., a polypeptide that is heterologous or homologous to the host cell, or a variant thereof), hi various embodiments, the bacterium further comprises mutations in an endogenous sequence that result in increased or decreased activity of a polypeptide involved in amino acid production (e.g., by mutation of an endogenous sequence encoding the polypeptide involved in amino acid production or a sequence that regulates expression of the polypeptide, e.g., a promoter sequence).
  • nucleic acid molecules e.g., recombinant nucleic acid molecules
  • a polypeptide involved in amino acid production e.g., a polypeptide that is heterologous or homologous to the host cell, or a variant thereof
  • the bacterium further comprises mutations in an endogenous sequence that result in increased or decreased activity of
  • the invention also features a method for producing an amino acid, the method including: cultivating a genetically modified bacterium including the nucleic acid encoding a variant bacterial aspartokinase polypeptide under conditions in which the nucleic acid is expressed and that allow the amino acid to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in the amino acid).
  • the invention features an isolated nucleic acid encoding a variant bacterial O-succinylhomoserine/acetylhomoserine (thiol)-lyase polypeptide (O- succinylhomoserine (thiol)-lyase).
  • the variant O-succinylhomoserine (thiol)-lyase exhibits reduced feedback inhibition relative to a wild-type form of the O- succinylhomoserine (thiol)-lyase polypeptide.
  • the nucleic acid encodes an O-succinylhomoserine (thiol)-lyase polypeptide with reduced feedback inhibition by S- adenosylmethionine.
  • the bacterial O-succinylhomoserine (thiol)-lyase polypeptide is chosen from: a Corynebacterium glutamicum O-succinylhomoserine (thiol)-lyase polypeptide, a Mycobacterium smegmatis O-succinylhomoserine (thiol)-lyase polypeptide, a Tixermobifida fusca O-succinylhomoserine (thiol)-lyase polypeptide, an Amycolatopsis mediterranei O-succinylhomoserine (thiol)-lyase polypeptide, a Streptomyces coelicolor O- succinylhomoserine (thiol)-lyase polypeptide, an Erwinia chrysanthemi O-succinylhomoserine (thiol)
  • the invention features an isolated nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide, wherein the variant O- succinylhomoserine (thiol)-lyase polypeptide is a variant of an O-succinylhomoserine (thiol)- lyase polypeptide including the following amino acid sequence: G ⁇ -X 2 -K 3 -X 4 -x 5 -x 6 -X 7 -x 8 -x 9 -
  • each of X 2 , X4-X13, X15, and X1 7 -X2 0 is, independently, any amino acid
  • each of X 13a -X 131 is, independently, any amino acid or absent
  • each of X 21a -X2it is, independently, any amino acid or absent
  • Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine
  • the variant O-succinylhomoserine (thiol)-lyase polypeptide includes an amino acid change at one or more of Gi, K 3 , F 14 , Z 16 , or D 22 of SEQ DD NO: .
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide, wherein the variant O- succinylhomoserine (thiol)-lyase polypeptide is a C. glutamicum O-succinylhomoserine (thiol)- lyase polypeptide including an amino acid change in one or more of the following residues of
  • amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by a nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide.
  • the invention also features a bacterium including a nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features a method for producing L-methionine, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide under conditions in which the nucleic acid is expressed and that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial cystathionine beta-lyase polypeptide.
  • the variant cystathionine beta-lyase polypeptide exhibits reduced feedback inhibition relative to a wild-type form of the cystathionine beta-lyase polypeptide.
  • the nucleic acid encodes a cystathionine beta-lyase polypeptide with reduced feedback inhibition by S- adenosylmethionine.
  • the bacterial cystathionine beta-lyase polypeptide is chosen from: a Corynebacterium glutamicum cystathionine beta-lyase polypeptide, a Mycobacterium smegmatis cystathionine beta-lyase polypeptide, a Thermobifida fusca cystathionine beta-lyase polypeptide, an Amycolatopsis mediterranei cystathionine beta-lyase polypeptide, a Streptomyces coelicolor cystathionine beta-lyase polypeptide, an Erwinia chrysanthemi cystathionine beta-lyase polypeptide, a Shewanella oneidensis cystathionine beta- lyase polypeptide, a Mycobacterium tuberculosis cystathionine beta-lyase polypeptide, an Escherichia coli cystathionine beta
  • the invention features an isolated nucleic acid encoding a variant bacterial cystathionine beta-lyase polypeptide, wherein the variant cystathionine beta-lyase polypeptide is a variant of a cystathionine beta-lyase polypeptide including the following amino acid sequence: G ⁇ -X 2 -K 3 -X4-X 5 -X6-X 7 -X8-X9-X ⁇ o-X ⁇ 1-Xi2-Xi3-Xi3a-Xi3b-Xi3c-Xi3d-Xi3e-Xi3r
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial cystathionine beta-lyase polypeptide, wherein the variant cystathionine beta-lyase polypeptide is a C. glutamicum cystatliionine beta-lyase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 296, Lysine 298,
  • amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by a nucleic acid encoding a variant bacterial cystathionine beta-lyase.
  • the invention also features a bacterium including a nucleic acid encoding a variant bacterial cystathionine beta-lyase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features a method for producing L-methionine, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial cystathionine beta-lyase polypeptide under conditions in which the nucleic acid is expressed and that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide.
  • the variant 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide exhibits reduced feedback inhibition relative to a wild-type fonn of the 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide.
  • the nucleic acid encodes a 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide with reduced feedback inhibition by S-adenosylmethionine polypeptide.
  • the bacterial 5- methyltetrahydrofolate homocysteine methyltransferase polypeptide is chosen from: a
  • the invention features an isolated nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide, wherein the variant 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide is a variant of a 5- methyltetrahydrofolate homocysteine methyltransferase polypeptide including the following amino acid sequence: G ⁇ -X2-K3-X4-x 5 -x 6 -x 7 -x 8 -X9-X ⁇ o-X ⁇ -Xi2-Xi3-Xi3a-X ⁇ 3 b-Xi3c-Xi3d- l3e ⁇ X l3f - l3 g _ Xl 3 _ Xl3i-Xl3j ⁇ X l3 k _ l3 1"-Fl4-Xi5-Zi6 (SEQ DD NO: ), wherein X is any
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide, wherein the variant 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide is a C. glutamicum 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide including an amino acid change in one or more of the following residues of SEQ D NO: :
  • the amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by the nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase.
  • the invention also features a bacterium including a nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features a method for producing L-methionine, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial 5-methyltetrahydrofolate homocysteine methyltransferase polypeptide under conditions in which the nucleic acid is expressed and that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide.
  • the variant S- adenosylmethionine synthetase polypeptide exhibits reduced feedback inhibition relative to a wild-type form of the S-adenosylmethionine synthetase polypeptide.
  • the nucleic acid encodes an S-adenosylmethionine synthetase polypeptide with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial S- adenosylmethionine synthetase polypeptide is bhosen from: a Corynebacterium glutamicum S- adenosylmethionine synthetase polypeptide, a Mycobacterium smegmatis S-adenosylmethionine synthetase polypeptide, a Thermobifida fusca S-adenosylmethionine synthetase polypeptide, an Amycolatopsis mediterranei S-adenosylmethionine synthetase polypeptide, a Streptomyces coelicolor S-adenosylmethionine synthetase polypeptide, an Erwinia chrysanthemi S- adenosylmethionine synthetase polypeptide, a Shewanella oneidensis S
  • the invention features an isolated nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide, wherein the variant S- adenosylmethionine synthetase polypeptide is a variant of an S-adenosylmetliionine synthetase polypeptide including the following amino acid sequence: G ⁇ -x 2 -K3-X 4 -X 5 -x 6 -x 7 -x 8 -X 9 -x 10 -
  • each of X 2 , X -X13, X15, and X 17 - X 20 is, independently, any amino acid, wherein each of X 13a -X 131 is, independently, any amino acid or absent, wherein each of X 21a -X 21t is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine; wherein the variant S-adenosylmethionine synthetase polypeptide includes an amino acid change at one or more of G ls K 3 , F 14 , Z 16 , orD 2 of SEQ ID NO: .
  • the amino acid change is a change to an alanine.
  • the invention features an isolated nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide, wherein the variant S- adenosylmethionine synthetase polypeptide is a C. glutamicum S-adenosylmethionine synthetase polypeptide including an amino acid change in one or more of the following residues of SEQ ED NO: : Glycine 263, Lysine 265, Phenylalanine 282, Glycine 284, and Aspartate 291.
  • the amino acid change is a change to an alanine.
  • the invention also features a polypeptide encoded by a nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide.
  • the invention also features a bacterium including a nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further comprise one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features a method for producing L-methionine, the method including: cultivating a genetically modified bacterium including a nucleic acid encoding a variant bacterial S-adenosylmethionine synthetase polypeptide under conditions in which the nucleic acid is expressed and that allow L-methionine to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in L-methionine).
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine kinase polypeptide.
  • the variant homoserine kinase polypeptide exhibits reduced feedback inhibition relative to a wild-type form of the bacterial homoserine kinase polypeptide.
  • the nucleic acid encodes a homoserine kinase polypeptide with reduced feedback inhibition by S-adenosylmethionine.
  • the bacterial homoserine kinase polypeptide is chosen from: a Corynebacterium glutamicum homoserine kinase polypeptide, a Mycobacterium smegmatis homoserine kinase polypeptide, a Thermobifida fusca homoserine kinase polypeptide, an Amycolatopsis mediterranei homoserine kinase polypeptide, a Streptomyces coelicolor homoserine kinase polypeptide, an Er-winia chrysanthemi homoserine kinase polypeptide, a Shewanella oneidensis homoserine kinase polypeptide, a Mycobacterium tuberculosis homoserine kinase polypeptide, an Escherichia coli homoserine kinase polypeptide, a Corynebacterium aceto
  • the invention features an isolated nucleic acid encoding a variant bacterial homoserine kinase polypeptide, wherein the homoserine kinase polypeptide is a C. glutamicum homoserine kinase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 160, Lysine 161, Phenylalanine 186,
  • the amino acid change is a change to an alanine, wherein the original residue is other than an alanine.
  • the invention also features a polypeptide encoded by the nucleic acid encoding a variant bacterial homoserine kinase.
  • the invention also features a bacterium including the nucleic acid encoding a variant bacterial homoserine kinase polypeptide.
  • the bacterium is a coryneform bacterium.
  • the bacterium can further include one or more nucleic acids encoding other variant bacterial polypeptides (e.g., variant bacterial polypeptides involved in amino acid production, e.g., variant bacterial polypeptides described herein).
  • the invention also features a method for producing an amino acid, the method including: cultivating a genetically modified bacterium including the nucleic acid encoding a variant bacterial homoserine kinase polypeptide under conditions in which the nucleic acid is expressed and that allow the amino acid to be produced, and collecting the culture.
  • the culture can be fractionated (e.g., to remove cells and/or to obtain fractions enriched in the amino acid).
  • the invention features a bacterium including two or more of the following: a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide; a nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase; a nucleic acid encoding a variant bacterial McbR gene product polypeptide; a nucleic acid encoding a variant bacterial aspartokinase polypeptide; a nucleic acid encoding a variant bacterial O-succinylhomoserine (thiol)-lyase polypeptide; a nucleic acid encoding a variant bacterial cystathione beta-lyase polypeptide; a nucleic acid encoding a variant bacterial 5- methyltetrahydrofolate homocysteine methyltransferase polypeptide; and a nucleic acid encoding a variant bacterial variant
  • the bacterium comprises a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase and a nucleic acid encoding a variant bacterial O- acetylhomoserine sulfhydrylase.
  • at least one of the variant bacterial polypeptides have reduced feedback inhibition (e.g., relative to a wild-type fonn of the polypeptide).
  • the invention features a bacterium including two or more of the following: (a) a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O-acetyltransferase polypeptide is a variant of a homoserine O-acetyltransferase polypeptide including the following amino acid sequence: G
  • each of X 2 , X4- X 13 , X 15 , and X 17 -X2 0 is, independently, any amino acid, wherein each of X 13 a-Xi 3 i is, independently, any amino acid or absent, wherein each of X 21a -X 2 i t is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine; wherein the variant homoserine O-acetyltransferase polypeptide includes an amino acid change at one or more of Gi, K 3 , F 14 , Z 16 , or D 22 of SEQ ED NO: ; (b) a nucleic acid
  • each of X 2 , X 4 -X 13 , Xis, and X ⁇ 7 -X 20 is, independently, any amino acid, wherein each of X 13a -X 13 ⁇ is, independently, any amino acid or absent, wherein each of X 21a -X2i t is, independently, any amino acid or absent, and wherein Z 16 is selected from valine, aspartate, glycine, isoleucine, and leucine; wherein the variant O- acetylhomoserine sulfhydrylase polypeptide includes an amino acid change at one or more of G 1;
  • variant O-acetylhomoserine sulfhydrylase polypeptide is a variant of a O-acetylhomoserine sulfhydrylase polypeptide including the following amino acid sequence: L 1 -X 2 -X 3 -G 4 -G 5 -X 6 -F -X 8 -X 9 - X 10 -X ⁇ (SEQ ED)
  • X is any amino acid
  • X 8 is selected from valine, leucine, isoleucine, and aspartate
  • X ⁇ is selected from valine, leucine, isoleucine, phenylalanine, and methionine
  • the variant of the bacterial protein includes an amino acid change at one or more of L G 4 , X 8 , X ⁇ of SEQ DD NO: _.
  • the invention features a bacterium including' two or more of the following: (a) a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O-acetyltransferase polypeptide is a C. glutamicum homoserine O-acetyltransferase polypeptide including an amino acid change in one or more of the following residues of SEQ ED NO: : Glycine 231, Lysine 233, Phenylalanine 251, and
  • Valine 253 (b) a nucleic acid encoding a variant bacterial homoserine O-acetylfransferase polypeptide, wherein the variant homoserine O-acetylfransferase polypeptide is a T.
  • variant homoserine O-acetyltransferase polypeptide is a mycobacterial homoserine O-acetyltransferase polypeptide including an amino acid change in a residue corresponding to one or more of the following residues of M. leprae homoserine O- acetyltransferase polypeptide set forth in SEQ DD NO: : Glycine 73, Aspartate 278, and
  • Tyrosine 260 (e) a nucleic acid encoding a variant bacterial homoserine O-acetyltransferase polypeptide, wherein the variant homoserine O-acetylfransferase polypeptide is an M. tuberculosis homoserine O-acetyltransferase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 73, Tyrosine 260, and Aspartate
  • a nucleic acid encoding a variant bacterial O-acetylhomoserine sulfhydrylase polypeptide, wherein the variant O-acetylhomoserine sulfhydrylase polypeptide is a C. glutamicum O-acetylhomoserine sulfhydrylase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 227, Leucine 229, Aspartate 231 , Glycine 232, Glycine 233, Phenylalanine 235, Aspartate 236, Valine 239, Phenylalanine
  • variant O-acetylhomoserine sulfhydrylase polypeptide is a T fusca O-acetylhomoserine sulfhydrylase polypeptide including an amino acid change in one or more of the following residues of SEQ DD NO: : Glycine 240, Aspartate 244, Phenylalanine 379, and Aspartate 394.
  • the invention features a bacterium including a nucleic acid encoding an episomal homoserine O-acetyltransferase polypeptide and an episomal O-acetylhomoserine sulfhydrylase polypeptide.
  • the bacterium is a Corynebacterium.
  • the episomal homoserine O-acetyltransferase polypeptide and the episomal O-acetylhomoserine sulfhydrylase polypeptide are of the same species as the bacterium (e.g., both are of C. glutamicum).
  • the episomal homoserine O- acetyltransferase polypeptide and the episomal O-acetylhomoserine sulfhydrylase polypeptide are of a different species than the bacterium, h various embodiments, the episomal homoserine O-acetyltransferase polypeptide is a variant of a bacterial homoserine O-acetyltransferase polypeptide with reduced feedback inhibition relative to a wild-type fonn of the homoserine O- acetyltransferase polypeptide.
  • the O-acetylhomoserine sulfhydrylase polypeptide is a variant of a bacterial O-acetylhomoserine sulfhydrylase polypeptide with reduced feedback inhibition relative to a wild-type form of the O-acetylhomoserine sulfhydrylase polypeptide.
  • Aspartic acid family of amino acids and related metabolites encompasses L-aspartate, ⁇ -aspartyl phosphate, L-aspartate- ⁇ -semialdehyde, L-2,3 -dihydrodipicolinate, L- ⁇ -piperideine- 2,6-dicarboxylate, N-succinyl-2-amino-6-keto-L-pimelate, N-succinyl-2, 6-L, L- diaminopimelate, L, L-diaminopimelate, D, L-diaminopimelate, L-lysine, homoserine, O-acetyl- L-homoserine, O-succinyl-L-homoserine, cystathionine, L-homocysteine, L-methionine, S- adenosyl-L-methionine, O-phospho-L-homoserine, threonine,
  • the aspartic acid family of amino acids and related metabolites encompasses aspartic acid, asparagine, lysine, tlireonine, methionine, isoleucine, and S-adenosyl-L- methionine.
  • a polypeptide or functional variant thereof with "reduced feedback inhibition” includes a polypeptide that is less inhibited by the presence of an inhibitory factor as compared to a wild-type form of the polypeptide or a polypeptide that is less inhibited by the presence of an inhibitory factor as compared to the corresponding endogenous polypeptide expressed in the organism into which the variant has been introduced.
  • a wild-type aspartokinase from E. coli or C.
  • glutamicum may have 10-fold less activity in the presence of a given concentration of lysine, or lysine plus threonine, respectively.
  • a variant with reduced feedback inhibition may have, for example, 5-fold less, 2-fold less, or wild-type levels of activity in the presence of the same concentration of lysine.
  • a "functional variant" protein is a protein that is capable of catalyzing the biosynthetic reaction catalyzed by the wild-type protein in the case where the protein is an enzyme, or providing the same biological function of the wild-type protein when that protein is not catalytic.
  • a functional variant of a protein that normally regulates the transcription of one or more genes would still regulate the transcription of one or more of the same genes when transformed into a bacterium, h certain embodiments, a functional variant protein is at least partially or entirely resistant to feedback inhibition by an amino acid.
  • the variant has fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 1 amino acid changes compared to the wild-type protein.
  • the amino acid changes are conservative changes.
  • a variant sequence is a nucleotide or amino acid sequence corresponding to a variant polypeptide, e.g., a functional variant polypeptide.
  • amino acid that is "corcesponding" to an amino acid in a reference sequence occupies a site that is homologous to the site in the reference sequence.
  • Corresponding amino acids can be identified by alignment of related sequences.
  • a heterologous nucleic acid or protein is meant to encompass a nucleic acid or protein, or functional variant of a nucleic acid or protein, of an organism (species) other than the host organism (species) used for the production of members of the aspartic acid family of amino acids and related metabolites, hi certain embodiments, when the host organism is a coryneform bacteria the heterologous gene will not be obtained from E. coli. In other specific embodiments, when the host organism is E. coli the heterologous gene will not be obtained from a coryneform bacteria.
  • Gene includes coding, promoter, operator, enhancer, terminator, co- transcribed (e.g., sequences from an operon), and other regulatory sequences associated with a particular coding sequence.
  • a "homologous" nucleic acid or protein is meant to encompass a nucleic acid or protein, or functional variant of a nucleic acid or protein, of an organism that is the same species as the host organism used for the production of members of the aspartic acid family of amino acids and related metabolites.
  • certain substitutions of one amino acid for another may be tolerated at one or more amino acid residues of a wild-type enzyme without eliminating the activity or function of the enzyme.
  • the term “conservative substitution” refers to the exchange of one amino acid for another in the same conservative substitution grouping in a protein sequence.
  • Conservative amino acid substitutions are known in the art and are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • conservative substitutions typically include substitutions within the following groups: Group 1: glycine, alanine, and proline; Group 2: valine, isoleucine, leucine, and methionine; Group 3: aspartic acid, glutamic acid, asparagine, glutamine; Group 4: serine, tlireonine, and cysteine; Group 5: lysine, arginine, and histidine; Group 6: phenylalanine, tyrosine, and tryptophan. Each group provides a listing of amino acids that may be substituted in a protein sequence for any one of the other amino acids in that particular group.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blosum 62 matrix and a gap weight of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes).
  • the length of a test sequence aligned for comparison purposes can be at least 30%, 40%, 50%, 60%, 70%, 80%,
  • nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the conesponding position in the second sequence, then the molecules are identical at that position (as used herein "identity” is equivalent to "homology").
  • the protein sequences described herein can be used as a "query sequence" to perform a search against a database of non-redundant sequences, for example.
  • Such searches can be performed using the BLASTP and TBLASTN programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • BLAST protein searches can be performed with the BLASTP program, using, for example, the Blosum 62 matrix, a wordlength of 3, and a gap existence cost of 11 and a gap extension penalty of 1.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology hifonnation, and default paramenter can be used.
  • Sequences described herein can also be used as query sequences in TBLASTN searches, using specific or default parameters.
  • the nucleic acid sequences described herein can be used as a "query sequence" to perform a search against a database of non-redundant sequences, for example.
  • Such searches can be performed using the BLASTN and BLASTX programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • Alignment of nucleotide sequences for comparison can also be conducted, e.g., by the local homology algorithm of Smith & Watennan, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I.
  • Nucleic acid sequences can be analyzed for hybridization properties.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology,
  • FIG 1. is a diagram of the biosynthesis of aspartate amino acid family.
  • FIG 2. is a diagram of the methionine biosynthetic pathway.
  • FIG 3. is a restriction map of plasmid MB3961 (vector backbone plasmid).
  • FIG 4. is a restriction map of plasmid MB4094 (vector backbone plasmid).
  • FIG 5. is a restriction map of plasmid MB4083 (hom-thrB deletion construct).
  • FIG 6. is a restriction map of plasmid MB4084 (thrB deletion construct).
  • FIG 7. is a restriction map of plasmid MB4165 (mcbR deletion construct).
  • FIG 8. is a restriction map of plasmid MB4169 (hom-thrB deletion/ gpd-M. smegmatis lysC(T31 ll)-asd replacement construct).
  • FIG 9. is a restriction map of plasmid MB4192 (hom-thrB deletion/ gpd-S. coelicolor hom(G362E) replacement construct.
  • FIG 10. is a restriction map of plasmid MB4276 (pck deletion/ gpd-M. smegmatis lysC(T311I)-asd replacement construct).
  • FIG 11. is a restriction map of plasmid MB4286 (mcbR deletion/ trcRBS-T fusca metA replacement construct).
  • FIG 12 A is a restriction map of plasmid MB4287 (mcbR deletion/ trcRBS-C. glutamicum metA (K233A)-metB replacement construct).
  • FIG 12B is a depiction of the nucleotide sequence of the DNA sequence in MB4278 (trcRBS-C. glutamicum rnetAYH) that spans from the trcRBS promoter to the stop of the metH gene.
  • FIG. 13 is a graph depicting the results of an assay to detennine in vitro O- acetyltransferase activity of C. glutamicum MetA from two C. glutamicum strains, MA-442 and MA-449, in the presence and absence of EPTG.
  • FIG. 14 is a graph depicting the results of an assay to determine sensitivity of MetA in C. glutamicum strain MA-442 to inhibition by methionine and S-AM1
  • FIG. 15 is a graph depicting the results of an assay to determine the in vitro O- acetyltransferase activity of T. fusca MetA expressed in C. glutamicum strains MA-456, MA570, MA-578, and MA-479. Rate is a measure of the change in OD412 divided by time per nanograms of protein.
  • FIG. 16 is a graph depicting the results of an assay to determine in vitro MetY activity of T fusca MetY expressed in C. glutamicum strains MA-456 and MA-570. Rate is defined as the change in OD412 divided by time per nanograms of protein.
  • FIG 17. is a graph depicting the results of an assay to detennine lysine production in C. glutamicum and B. lactofermentum strains expressing heterologous wild-type and mutant lysC variants.
  • FIG. 18 is a graph depicting results from an assay to determine lysine and homoserine production in C. glutamicum strain, MA-0331 in the presence and absence of the S. coelicolor horn G362E variant.
  • FIG 19. is a graph depicting results from any assay to determine asparate concentrations in C. glutamicum strains MA-0331 and MA-0463 in the presence and absence of E chrysanthemi ppc.
  • FIG. 20 is a graph depicting results from an assay to detennine lysine production in C. glutamicum strains MA-0331 and MA-0463 transformed with heterologous wild-type dapA genes.
  • FIG. 21 is a graph depicting results from an assay to determine metabolite levels in C. glutamicum strain MA-1378 and its parent strains.
  • FIG. 22 is a graph depicting results from an assay to determine homoserine and O- acetylhomoserine levels in C. glutamicum strains MA-0428, MA-0579, MA-1351, MA-1559 grown in the presence or absence of ⁇ PTG.
  • ⁇ PTG induces expression of the episomal plasmid borne T fusca metA gene.
  • FIG 23. is a graph depicting results from an assay to determine metabolite levels in C. glutamicum strain MA-1559 and its parent strains.
  • FIG. 24 is a graph depicting methionine concentrations in broths from fermentations of two C. glutamicum strains, MA-622, and MA-699, which express a MetA K233A mutant polypeptide. Production by cells cultured in the presence and absence of ⁇ PTG is depicted.
  • FIG. 25 is a graph depicting methionine concentrations in broths from fermentations of two C. glutamicum strains, MA-622 and MA-699, expressing a MetY D231A mutant polypeptide. Production by cells cultured in the presence and absence of ⁇ PTG is depicted.
  • FIG. 26 is a graph depicting methionine concentrations in broths from fermentations of two C. glutamicum strains, MA-622 and MA-699, expressing a C. glutamicum MetY G232A mutant polypeptide. Production by cells cultured in the presence and absence of ⁇ PTG is depicted.
  • FIG. 27 is a graph depicting results from an assay to detennine metabolite levels in C. glutamicum strains MA-1906, MA-2028, MA-1907, and MA-2025. Strains were grown in the presence and absence of ⁇ PTG.
  • FIG. 28 is a graph depicting results from an assay to determine metabolite levels in C. glutamicum strains MA-1667 and MA-1743. Strains were grown in the presence and absence of IPTG. ⁇
  • FIG. 29 is a graph depicting results from an assay to determine metabolite levels in C. glutamicum strains MA-0569, MA-1688, MA-1421, and MA-1790. Strains were grown in the absence and/or presence of IPTG.
  • FIG. 30 is a graph depicting results from an assay to determine metabolite levels in C. glutamicum strain MA-1668 and its parent strains.
  • the invention provides nucleic acids and modified bacteria that comprise nucleic acids encoding proteins that improve fermentative production of aspartate-derived amino acids and intermediate compounds.
  • nucleic acids and bacteria relevant to the production of L-aspartate, L-lysine, L-methionine, S-adenosyl-L-methionine, threonine, L-isoleucine, homoserine, O-acetyl homoserine, homocysteine, and cystathionine are disclosed.
  • the nucleic acids include genes that encode metabolic pathway proteins that modulate the biosynthesis of these amino acids, intermediates, and related metabolites either directly (e.g., via enzymatic conversion of intermediates) or indirectly (e.g., via transcriptional regulation of enzyme expression or regulation of amino acid export).
  • the nucleic acid sequences encoding the proteins can be derived from bacterial species other than the host organism (species) used for the production of members of the aspartic acid family of amino acids and related metabolites.
  • the invention also provides methods for producing the bacteria and the amino acids, including the production of amino acids for use in animal feed additives.
  • Modification of the sequences of certain bacterial proteins involved in amino acid production can lead to increased yields of amino acids.
  • Regulated (e.g., reduced or increased) expression of modified or unmodified (e.g., wild type) bacterial enzymes can likewise enhance amino acid production.
  • the methods and compositions described herein apply to bacterial proteins that regulate the production of amino acids and related metabolites, (e.g., proteins involved in the metabolism of methionine, threonine, isoleucine, aspartate, lysine, cysteine and sulfur), and nucleic acids encoding these proteins.
  • proteins include enzymes that catalyze the conversion of intermediates of amino acid biosynthetic pathways to other intermediates and/or end product, and proteins that directly regulate the expression and/or function of such enzymes.
  • Target proteins for manipulation include those enzymes that are subject to various types of regulation such as repression, attenuation, or feedback-inhibition.
  • Amino acid biosynthetic pathways in bacterial species, information regarding the proteins involved in these pathway, links to sequences of these proteins, and other related resources for identifying proteins for manipulation and/or expression as described herein can be accessed through linked databases described by Error! Hyperlink reference not valid.Bono et al, Genome Research, 8:203-210, 1998.
  • S-AM S-adenosylmethionine
  • target genes for manipulation are bacterial dapA, horn, thrB, ppc, pyc, pck, metE, glyA, metA, rnetY, mcbR, lysC, asd, rnetB, metC, rnetH, and metK genes. These target genes can be manipulated individually or in various combinations.
  • strains such that the activity of particular genes is reduced (e.g., by mutation or deletion of an endogenous gene).
  • stains with reduced activity of one or more of horn, thrB, pck, or mcbR gene products can exhibit enhanced production of amino acids and related intermediates.
  • Two central carbon metabolism enzymes that direct carbon flow towards the aspartic acid- family of amino acids and related metabolites include phosphoenolpyruvate carboxylase (Ppc) and pyruvate carboxylase (Pyc).
  • Ppc phosphoenolpyruvate carboxylase
  • Pyc pyruvate carboxylase
  • the initial steps of biosynthesis of aspartatic acid family amino acids are diagrammed in Figure 1.
  • Both enzymes catalyze the formation of oxaloacetate, a tricarboxylic acid (TCA) cycle component that is transaminated to aspartic acid.
  • Aspartokinase (which is encoded by lysC in corynefonn bacteria) catalyzes the first enzyme reaction in the aspartic acid family of amino acids, and is known to be regulated by both feedback-inhibition and repression.
  • deregulation of this enzyme is critical for the production of any of the commercially important amino acids and related metabolites of the aspartic acid amino acid pathway (e.g. aspartic acid, asparagine, lysine, methionine, S-adenosyl-L-methionine, threonine, and isoleucine).
  • aspartic acid asparagine, lysine, methionine, S-adenosyl-L-methionine, threonine, and isoleucine.
  • overexpression by increasing copy number and/or the use of strong promoters
  • deregulation of each or both of these enzymes can enhance production of the amino acids listed above.
  • biosynthetic enzymes can be employed to enhance production of specific amino acids.
  • enzymes involved in L-lysine biosynthesis include: dihydrodipicolinate synthase (DapA), dihydrodipicolinate reductase (DapB), diaminopimelate dehydrogenase (Ddh), and diaminopimelate decarboxylase (Lys A).
  • DapA dihydrodipicolinate synthase
  • DapB dihydrodipicolinate reductase
  • Ddh diaminopimelate dehydrogenase
  • Lis A diaminopimelate decarboxylase
  • Table 1 A list of enzymes involved in lysine biosynthesis is provided in Table 1. Overexpression and/or deregulation of each of these enzymes can enhance production of lysine. Overexpression of biosynthetic enzymes can be achieved by increasing copy number of the gene of interest and/or operably linking the gene to apromoter optimal for expression
  • Lysine productivity can be enhanced in strains overexpressing general and specific regulatory enzymes. Specific amino acid substitutions in aspartokinase and dihydrodipicolinate synthase in E. coli can lead to increased lysine production by reducing feedback inhibition. Enhanced expression of lysC and/or dapA (either wild-type or feedback-insensitive alleles) can increase lysine production. Similarly, deregulated alleles of heterologous lysC and dap A genes can be expressed in a sfrain of coryneform bacteria such as Corynebacterium glutamicum. Likewise, overexpression of either pyc ox ppc can enhance lysine production.
  • Steps in the biosynthesis of methionine are diagrammed in Figure 2.
  • enzymes that regulate methionine biosynthesis include: Homoserine dehydrogenase (Hom), O- homoserine acetyltransferase (MetA), and O-acetylhomoserine sulfhydrylase (MetY). Overexpression (by increasing copy number of the gene of interest and/or through the use of strong promoters) and/or deregulation of each of these enzymes can enhance production of methionine.
  • Homoserine dehydrogenase Homoserine dehydrogenase
  • MetalA O- homoserine acetyltransferase
  • MetalY O-acetylhomoserine sulfhydrylase
  • Methionine adenosyltransferase catalyzes the production of S-adenosyl-L- methionine from methionine. Reduction of ⁇ neti -expressed enzyme activity can prevent the conversion of methionine to S-adenosyl-L-methionine, thus enhancing the yield of methionine from bacterial strains. Conversely, if one wanted to enhance carbon flow from metliionine to S- adenosyl-L-methionine, the metK gene could be overexpressed or desensitized to feedback inhibition.
  • Suitable host species for the production of amino acids include bacteria of the family
  • Enterobacteriaceae such as an Escherichia coli bacteria and strains of the genus Corynebacterium.
  • the list below contains examples of species and strains that can be used as host sfrains for the expression of heterologous genes and the production of amino acids.
  • Corynebacterium glutamicum ATCC (American Type Culture Collection) 13032 Corynebacterium glutamicum ATCC 21526
  • Suitable species and strains for heterologous bacterial genes include, but are not limited to, these listed below.
  • Amino acid sequences of exemplary proteins which can be used to enhance amino acid production, are provided in Table 16.
  • Nucleotide sequences encoding these proteins are provided in Table 17.
  • the sequences that can be expressed in a host strain are not limited to those sequences provided by the Tables.
  • Aspartokinases are enzymes that catalyze the first committed step in the biosynthesis of aspartic acid family amino acids.
  • the level and activity of aspartokinases are typically regulated by one or more end products of the pathway (lysine or lysine plus threonine depending upon the bacterial species), both through feedback inhibition (also referred to as allosteric regulation) and transcriptional control (also called repression).
  • Bacterial homologs of coryneform and E. coli aspartokinases can be used to enhance amino acid production.
  • Coryneform and E. coli aspartokinases can be expressed in heterologous organisms to enhance amino acid production.
  • lysC locus contains two overlapping genes, lysC alpha and lysC beta. LysC alpha and lysC beta code for the 47- and 18-kD subunits of aspartokinase, respectively.
  • a third open-reading frame is adjacent to the lysC locus, and encodes aspartate semialdehyde dehydrogenase (asd). The asd start codon begins 24 base-pairs downstream from the end of the lysC open-reading frame, is expressed as part of the lysC operon.
  • the primary sequence of aspartokinase proteins and the structure of the lysC loci are conserved across several members of the order Actinomycetales.
  • Examples of organisms that encode both an aspartokinase and an aspartate semialdehyde dehydrogenase that are highly related to the proteins from coryneform bacteria include Mycobacterium smegmatis, Amycolatopsis mediterranei, Streptomyces coelicolor A3 (2), and ITiermobifida fusca. In some instances these organisms contain the lysC and asd genes arranged as in coryneform bacteria. Table 2 displays the percent identity of proteins from these Actinomycetes to the C. glutamicum aspartokinase and aspartate semialdehyde dehydrogenase proteins.
  • Isolates of source strains such as Mycobacterium smegmatis, Amycolatopsis mediterranei, Streptomyces coelicolor, and Tl ermobifida fusca are available.
  • the lysC operons can be amplified from genomic DNA prepared from each source strain, and the resulting PCR product can be ligated into an E. coli / C. glutamicum shuttle vector.
  • the homolog of the aspartokinase enzyme from the source strain can then be introduced into a host sfrain and expressed.
  • E. coli Asyartol ⁇ nase III homoloss hi coryneform bacteria there is concerted feedback inhibition of aspartokinase by lysine and threonine. This is in contrast to E. coli, where there are three distinct aspartokinases that are independently allosterically regulated by lysine, threonine, or methionine. Homologs of the E. coli aspartokinase III (and other isoenzymes) can be used as an alternative source of deregulated aspartokinase proteins. Expression of these enzymes in coryneform bacteria may decrease the complexity of pathway regulation.
  • the aspartokinase III genes are feedback- inhibited only by lysine instead of lysine and threonine. Therefore, the advantages of expressing feedback-resistant alleles of aspartokinase III alleles include: (1) the increased likelihood of complete deregulation; and (2) the possible removal of the need for constructing either "leaky” mutations in horn or threonine auxotrophs that need to be supplemented. These features can result in decreased feedback inhibition by lysine.
  • Genes encoding aspartokinase III isoenzymes can be isolated from bacteria that are more distantly related to Corynebacteria than the Actinomycetes described above.
  • the E. chysanthemi and S. oneidensis gene products are 77% and 60% identical to the E. coli lysC protein, respectively (and 26% and 35% identical to C. glutamicum LysC).
  • the genes coding for aspartokinase III, or functional variants therof, from the non- ⁇ scherichia bacteria, Erwinia chrysanthemi and Shewanella oneidensis can be amplified and ligated into the appropriate shuttle vector for expression in C. glutamicum.
  • Lysine analogs e.g. S-(2-aminoethyl)cysteine (A ⁇ C)
  • a ⁇ C 2-aminoethylcysteine
  • threonine can be used to identify strains with enhanced production of lysine.
  • A- significant portion of the known lysine-resistant strains from both C. glutamicum and E. coli contain mutations at the lysC locus.
  • specific amino acid substitutions that confer increased resistance to A ⁇ C have been identified, and these substitutions map to well-conserved residues.
  • Specific amino acid substitutions that result in increased lysine productivity, at least in wild-type strains include, but are not limited to, those listed in Table 3. i many instances, several useful substitutions have been identified at a particular residue.
  • strains have been identified that contain more than one lysC mutation. Sequence alignment confirms that the residues previously associated with feedback-resistance (i.e. A ⁇ C- resistance) are conserved in a variety of aspartokinase proteins from distantly related bacteria.
  • Standard site-directed mutagenesis techniques can be used to construct aspartokinase variants that are not subject to allosteric regulation. After cloning PCR-amplified lysC or aspartokinase III genes into appropriate shuttle vectors, oligonucleotide-mediated site-directed mutagenesis is use to provide modified alleles that encode substitutions such as those listed in Table 3. Vectors containing either wild-type genes or modified alleles can be be transformed into C. glutamicum alongside control vectors. The resulting transformants can be screened, for example, for lysine productivity, increased resistance to AEC, relative cross-feeding of lysine auxotrophs, or other methods known to those skilled in the art to identify the mutant alleles of most interest.
  • Assays to measure lysine productivity and/or enzyme activity can be used to confirm the screening results and select useful mutant alleles.
  • Teclmiques such as high pressure liquid chromatography (HPLC) and HPLC-mass specfromefry (MS) assays to quantify levels of members of the aspartic acid family of amino acids and related metabolites are known to those skilled in the art.
  • Methods for random generating amino acid substitutions within the lysC coding sequence, through methods such as mutagenenic PCR, can be used. These methods are familiar to those skilled in the art; for example, PCR can be performed using the GeneMorph PCR mutagenesis kit (Stratagene, La Jolla, Ca) according to manufacturer's instructions to achieve medium and high range mutation frequencies. Evaluation of the heterologous enzymes can be carried out in the presence of the LysC,
  • DapA, Pyc, and Ppc proteins that are endogenous to the host strain.
  • Phenotypic assays for AEC resistance or enzyme assays can be used to confirm function of wild-type and modified variants of heterologous aspartokinases.
  • the function of cloned heterologous genes can be confirmed by complementation of genetically characterized mutants of E. coli or C. glutamicum.
  • E. coli strains are publicly available from the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/top.html).
  • C. glutamicum mutants have also been described.
  • Dihydrodipicolinate synthase encoded by dapA, is the branch point enzyme that commits carbon to lysine biosynthesis rather than threonine/methionine production. DapA converts aspartate- ⁇ -semialdehyde to 2,3-dihydrodipicolinate. DapA overexpression has been shown to result in increased lysine production in both E. coli and coryneform bacteria, hi E. coli, DapA is allosterically regulated by lysine, whereas existing evidence suggests that C. glutamicum regulation occurs at the level of gene expression. Dihydrodipicolinate synthase proteins are not as well conserved amongst Actinomycetes as compared to LysC proteins.
  • Both wild-type and deregulated DapA proteins that are homologous to the C. glutamicum protein or the E. coli DapA protein can be expressed to enhance lysine production.
  • Candidate organisms that can be sources of dapA genes are shown in Table 4. The known sequence from M. tuberculosis or M. leprae can be used to identify homologous genes from M. smegmatis.
  • DapA isolates can be tested for increased lysine production using methods described above. For instance, one could distribute a culture of a lysine-requiring bacterium on a growth medium lacking lysine. A population of dapA mutants obtained by site-directed mutagenesis could then be introduced (through transformation or conjugation) into a wild-type coryneform sfrain, and subsequently spread onto the agar plate containing the distributed lysine auxofroph. A feedback-resistant dapA mutant would overproduce lysine which would be excreted into the growth medium and satisfy the growth requirement of the auxofroph previously distributed on the agar plate. Therefore a halo of growth of the lysine auxofroph around a dap A mutation- containing colony would indicate the presence of the desired feedback-resistant mutation.
  • Pyruvate carboxylase (Pyc) and phosphoenolpyruvate carboxylase (Ppc) catalyze the synthesis of oxaloacetic acid (OAA), the citric acid cycle intermediate that feeds directly into lysine biosynthesis.
  • OAA oxaloacetic acid
  • These anaplerotic reactions have been associated with improved yields of several amino acids, including lysine, and are obviously important to maximize OAA formation.
  • a variant of the C. glutamicum Pyc protein containing a P458S substitution has been shown to have increased activity, as demonstrated by increased lysine production.
  • Proline 458 is a highly conserved amino acid position across a broad range of pyruvate carboxylases, including proteins from the Actinomycetes S.
  • PEP carboxykinase expresses an enzyme that catalyzes the formation of phosphoenolpyruvate from OAA (for gluconeogenesis), and thus functionally competes it , pyc and ppc. Enhancing expression of pyc and ppc can maximize OAA formation. Reducing or eliminating pck activity can also improve OAA formation.
  • Homoserine dehydrogenase catalyzes the conversion of aspartate semialdehyde to homoserine. Hom is feedback-inhibited by threonine and repressed by methionine in coryneform bacteria. It is thought that this enzyme has greater affinity for aspartate semialdehyde than does the competing dihydrodipicolinate synthase (DapA) reaction in the lysine branch, but slight carbon "spillage" down the threonine pathway may still block Hom activity.
  • DapA dihydrodipicolinate synthase
  • Feedback-resistant variants of Hom, overexpression of hom, and/or deregulated transcription of horn, or a combination of any of these approaches, can enhance methionine, threonine, isoleucine, or S- adenosyl-L-methionine production.
  • Decreased Hom activity can enhance lysine production.
  • Bifunctional enzymes with homoserine dehydrogenase activity such as enzymes encoded by E. coli metL (aspartokinase Il-homoserine dehydrogenase II) and thrA (aspartokinase I-homoserine dehydrogenase I), can also be used to enhance amino acid production.
  • Targeted amino acid substitutions can be generated either to decrease, but not eliminate, Hom activity or to relieve Hom from feedback inhibition by tlireonine. Mutations that result in decreased Hom activity are referred to as "leaky" Hom mutations, hi the C. glutamicum homoserine dehydrogenase, amino acid residues have been identified that can be mutated to either enhance or decrease Hom activity. Several of these specific amino acids are well- conserved in Hom proteins in other Actinomycetes (see Table 6).
  • the hom dr mutation is described on page 11 of WO 93/09225. This mutation is a single base pair deletion at 1964 bp that disrupts the hom dr reading frame at codon 429. This results in a frame shift mutation that induces approximately ten amino acid changes and a premature termination, or truncation, i.e., deletion of approximately the last seven amino acid residues of the polypeptide.
  • MetA Homoserine O-acetyltransferase acts at the first committed step in methionine biosynthesis (Park, S. et al., Mol. Cells 8:286-294, 1998).
  • the MetA enzyme catalyzes the conversion of homoserine to O-acetyl-homoserine.
  • MetA is sfrongly regulated by end products of the methionine biosynthetic pathway, hi E. coli, allosteric regulation occurs by both S-AM and methionine, apparently at two separate allosteric sites.
  • MetJ and S-AM cause transcriptional repression of metA.
  • MetA may be allosterically inhibited by methionine and S-AM, similarly to E. coli. MetA synthesis can be repressed by methionine alone, h addition, trifluoromethionine-resistance has been associated with metA in early studies. Reduction of negative regulation by S-AM and methionine can enhance methionine or S- adenosyl-L-methionine production. Increased MetA activity can enhance production of aspartate-derived amino acids such as methionine and S-AM, whereas decreased MetA activity can promote the formation of amino acids such as threonine and isoleucine.
  • O-Acetylhomoserine sulfhydrylase catalyzes the conversion of O-acetyl homoserine to homocysteine.
  • MetY may be repressed by methionine in coryneform bacteria, with a 99% reduction in enzyme activity in the presence of 0.5 mM methionine. It is likely that this inhibition represents the combined effect of allosteric regulation and repression of gene expression.
  • enzyme activity is inhibited by methionine, homoserine, and O- acetylserine. It is possible that S-AM also modulates MetY activity. Deregulated MetY can enhance methionine or S-AM production.
  • Homoserine kinase is encoded by thrB gene, which is part of the hom-thrB operon. ThrB phosphorylates homoserine. Threonine inhibition of homoserine kinase has been observed in several species. Some studies suggest that phosphorylation of homoserine by homoserine kinase may limit threonine biosynthesis under some conditions. Increased ThrB activity can enhance production of aspartate-derived amino acids such as isoleucine and threonine, whereas decreased ThrB activity can promote the formation of amino acids including, but not limited to, lysine and methionine.
  • Methionine adenosyltransferase converts methionine to S-adenosyl-L-methionine (S- AM).
  • S- AM S-adenosyl-L-methionine
  • Methionine adenosyltransferase converts methionine to S-adenosyl-L-methionine (S- AM).
  • MethodK Down-regulating methionine adenosyltransferase (MetK) can enhance production of methionine by inhibiting conversion to S-AM. Enhancing expression of metK or activity of MetK can maximize production of S-AM.
  • O-Succinylhomoserine (thio)-lyase (MetB; also known as cystatliionine gamma- synthase) catalyzes the conversion of O-succinyl homoserine or O-acetyl homoserine to cystathionine. Increasing expression or activity of MetB can lead to increased methionine or S- AM.
  • Cystathionine beta-lyase can convert cystathionine to homocysteine. Increasing production of homocysteine can lead to increased production of methionine. Thus, increased MetC expression or activity can increase methionine or S-adenosyl-L-methionine production.
  • glutamate dehydrogenase encoded by the gdh gene, catalyses the reductive amination of ⁇ -ketoglutarate to yield glutamic acid.
  • Increasing expression or activity of glutamate dehydrogenase can lead to increased lysine, threonine, isoleucine, valine, proline, or tryptophan.
  • Diaminopimelate dehydrogenase encoded by the ddh gene in coryneform bacteria, catalyzes the the NADPH-dependent reduction of ammonia and L-2-amino-6-oxopimelate to form meso-2,6-diaminopimelate, the direct precursor of L-lysine in the alternative pathway of lysine biosynthesis.
  • Overexpression of diaminopimelate dehydrogenase can increase lysine production.
  • Detergent sensitivity rescuer encoding a protein related to the alpha subunit of acetyl CoA carboxylase, is a surfactant resistance gene. Increasing expression or activity of DtsRl can lead to increased production of lysine.
  • MetalE 5-Methyltetrahydropteroyltriglutamate-homocysteine methyltransferase
  • MetE also catalyzes the conversion of homocysteine to methionine.
  • Increasing MetE expression or activity can lead to increased production of methionine or S-adenosyl-L-methionine.
  • GlyA serine hydroxymethyltransferase
  • MetF 5,10-Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetefrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine.
  • Increasing expression or activity of MetF can lead to increased methionine or S- adenosyl-L-methionine production.
  • CysE Serine O-acetyltransferase catalyzes the conversion of serine to O-acetylserine. Increasing expression or activity of CysE can lead to increased expression of methionine or S- adenosyl-L-methionine.
  • SerA D-3-phosphoglycerate dehydrogenase catalyzes the first step in serine biosynthesis, and is allosterically inhibited by serine. Increasing expression or activity of SerA can lead to increased production of methionine or S-adenosyl-L-methionine.
  • McbR Gene Product The mcbR gene product of C. glutamicum was identified as a putative transcriptional repressor of the TetR-family and may be involved in the regulation of the metabolic network directing the synthesis of methionine in C. glutamicum (Rey et al, J Biotechnol. 103(l):51-65, 2003).
  • the mcbR gene product represses expression of metY, metK, cysK, cysl, hom, pyk, ssuD, and possibly other genes. It is possible that McbR represses expression in combination with small molecules such as S-AM or methionine.
  • McbR is involved in the regulation of sulfur containing amino acids (e.g., cysteine, methionine).
  • Reduced McbR expression or activity can also enhance production of any of the aspartate family of amino acids that are derived from homoserine (e.g., homoserine, O-acetyl-L- homoserine, O-succinyl-L-homoserine, cystathionine, L-homocysteine, L-methionine, S- adenosyl-L-methionine (S-AM), O-phospho-L-homoserin'e, threonine, 2-oxobutanoate, (S)-2- aceto-2-hydroxybutanoate, (S)-2-hydroxy-3-methyl-3-oxopentanoate, (R)-2,3-Dihydroxy-3- methylpentanoate, (R)-2-oxo-3-methylpentanoate, and L-isoleucine).
  • homoserine e.g., homoserine, O-acetyl-L- homoserine
  • Lysine exporter protein is a specific lysine translocator that mediates efflux of lysine from the cell, hi C. glutamicum with a deletion in the lysE gene, L-lysine can reach an intracellular concentration of more than 1M. (Erdmann, A., et al. J Gen Microbiol 139,:3115- 3122, 1993). Overexpression or increased activity of this exporter protein can enhance lysine production.
  • a substantial number of bacterial genes encode membrane transport proteins.
  • a subset of these membrane transport protein mediate efflux of amino acids from the cell.
  • Corynebacterium glutamicum express a threonine efflux protein. Loss of activity of this protein leads to a high intracellular accumulation of threonine (Simic et al., JBacteriol 183(18):5317- 5324, 2001).
  • Increasing expression or activity of efflux proteins can lead to increased production of various amino acids.
  • Useful efflux proteins include proteins of the drug/metabolite fransporter family.
  • the C. glutamicum proteins listed in Table 16 or homologs thereof can be used to increase amino acid production.
  • Bacterial genes for expression in host strains can be isolated by methods known in the art.
  • Genomic DNA from source sfrains can be prepared using known methods (see, e.g., Saito, H. and, Miura, K. Biochim Biophys Ada. 72:619-629, 1963) and genes can be amplified from genomic DNA using PCR (U.S. Pats. 4,683,195 and 4,683,202, Saiki, et al. Science 230:350-1354, 1985).
  • DNA primers to be used for the amplification reaction are those complemental to both 3'- terminals of a double stranded DNA containing an entire region or a partial region of a gene of interest.
  • DNA fragments When only a partial region of a gene is amplified, it is necessary to use such DNA fragments as primers to perform screening of a DNA fragment containing the entire region from a chromosomal DNA library.
  • a PCR reaction solution including DNA fragments containing the amplified gene is subjected to agarose gel electrophoresis, and then a DNA fragment is extracted and cloned into a vector appropriate for expression in bacterial systems.
  • DNA primers for PCR may be adequately prepared on the basis of, for example, a sequence known in the source strain (Richaud, F. et al., J. Bacteriol 297,1986).
  • primers that can amplify a region comprising the nucleotide bases coding for the heterologous gene of interest can be used.
  • Synthesis of the primers can be performed by an ordinary method such as a phosphoamidite method (see Tetrahed Lett. 22:1859,1981) by using a commercially available DNA synthesizer (for example, DNA Synthesizer Model 380B produced by Applied Biosystems Inc.).
  • the PCR can be performed by using a commercially available PCR apparatus and Taq DNA polymerase, or other polymerases that display higher fidelity, in accordance with a method designated by the supplier.
  • enzymes that regulate amino acid production are subject to allosteric feedback inhibition by biosynthetic pathway intermediates or end products.
  • Useful variants of these enzymes can be generated by substitution of residues responsible for feedback inhibition.
  • enzymes such as homoserine O-acetyltransferase (encoded by metA) axe feedback- inhibited by S-AM.
  • Additional putative S-AM binding residues in various enzymes include, but are not limited to, those listed in Tables 9 and 10.
  • One or more of the residues in Tables 9 and 10 can be substituted with a non-conservative residue, or with an alanine (e.g., where the wild type residue is other than an alanine).
  • Sequence alignment confirms that the residues potentially associated with feedback-sensitivity to S-AM are conserved in a variety of MetA and MetY proteins from distantly related bacteria.
  • Standard site-directed mutagenesis techniques can be used to construct variants that are less sensitive to allosteric regulation. After cloning a PCR-amplified gene or genes into appropriate shuttle vectors, oligonucleotide-mediated site-directed mutagenesis is use to provide modified alleles that encode specific amino acid substitutions.
  • Vectors containing either wild- type genes or modified alleles can be transformed into C. glutamicum, or another suitable host strain, alongside control vectors. The resulting transformants can be screened, for example, for amino acid productivity, increased resistance to feedback inhibition by S-AM, activity of the enzyme of interest, or other methods known to those skilled in the art to identify the variant alleles of most interest.
  • Assays to measure amino acid productivity and/or enzyme activity can be used to confirm the screening results and select useful variant alleles.
  • Techniques such as high pressure liquid chromatography (HPLC) and HPLC-mass spectrometry (MS) assays to quantify levels of amino acids and related metabolites are known to those skilled in the art.
  • Methods for generating random amino acid substitutions within a coding sequence can be used (e.g., to generate variants for screening for reduced feedback inhibition, or for introducing further variation into enhanced variant sequences).
  • PCR can be performed using the GeneMorph ® PCR mutagenesis kit (Stratagene, La Jolla, Ca) according to manufacturer's instructions to achieve medium and high range mutation frequencies. Other methods are also known in the art.
  • Evaluation of enzymes can be carried out in the presence of additional enzymes that are endogenous to the host strain, hi certain instances, it will be helpful to have reagents to specifically assess the functionality of a biosynthetic protein that is not endogenous to the organism (e.g., an episomally expressed protein).
  • Phenotypic assays for feedback inhibition or enzyme assays can be used to confirm function of wild-type and variants of biosynthetic enzymes.
  • the function of cloned genes can be confirmed by complementation of genetically characterized mutants of the host organism (e.g., the host E. coli or C. glutamicum bacterium). Many of the E. coli strains are publicly available from the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/top.html). C. glutamicum mutants have also been described.
  • Bacterial genes can be expressed in host bacterial strains using methods known in the art. hi some cases, overexpression of a bacterial gene (e.g., a heterologous and/or variant gene) will enhance amino acid production by the host strain. Overexpression of a gene can be achieved in a variety of ways. For example, multiple copies of the gene can be expressed, or the promoter, regulatory elements, and/or ribosome binding site upstream of a gene (e.g., a variant allele of a gene, or an endogenous gene) can be modified for optimal expression in the host strain. In addition, the presence of even one additional copy of the gene can achieve increased expression, even where the host strain already harbors one or more copies of the conesponding gene native to the host species.
  • a bacterial gene e.g., a heterologous and/or variant gene
  • Overexpression of a gene can be achieved in a variety of ways. For example, multiple copies of the gene can be expressed, or the promoter, regulatory elements, and/or rib
  • the gene can be operably linked to a strong constitutive promoter or an inducible promoter (e.g., trc, lac) and induced under conditions that facilitate maximal amino acid production.
  • a strong constitutive promoter or an inducible promoter e.g., trc, lac
  • Methods to enhance stability of the mRNA are known to those skilled in the art and can be used to ensure consistently high levels of expressed proteins. See, for example, Keasling, J., Trends in Biotechnology 17:452-460, 1999. Optimization of media and culture conditions may also enhance expression of the gene.
  • a gene of interest (e.g., a heterologous or variant gene) should be operably linked to an appropriate promoter, such as a native or host strain-derived promoter, a phage promoter, one of the well-characterized E. coli promoters (e.g. tac, trp, phoA, araBAD, or variants thereof etc.). Other suitable promoters are also available.
  • the heterologous gene is operably linked to a promoter that permits expression of the heterologous gene at levels at least 2-fold, 5-fold, or 10-fold higher than levels of the endogenous homolog in the host sfrain. Plasmid vectors that aid the process of gene amplification by integration into the chromosome can be used. See, for example, by Reinscheid et al. (Appl. Environ Microbiol. 60: 126-
  • the complete gene is cloned in a plasmid vector that can replicate in a host (typically E. coli), but not in C. glutamicum.
  • plasmid vectors include, for example, pSUP301 (Simon et al., Bio/Technol. 1, 784-79,1983), pKl ⁇ mob or pK19mob (Schfer et al., Gene 145:69- 73, 1994), PG ⁇ M-T (Promega Corp., Madison, Wise, USA), pCR2.1-TOPO (Shuman JBiol Chem. 269:32678-84, 1994; U.S. Pat. 5,487,993), pCR.RTM.Blunt (Invitrogen, Groningen,
  • the plasmid vector that contains the gene to be amplified is then transferred into the desired sfrain of C. glutamicum by conjugation or transformation.
  • the method of conjugation is described, for example, by Schfer et al. (Appl Environ Microbiol. 60:756-759,1994). Methods for transformation are described, for example, by Thierbach et al. (Appl Microbiol Biotechnol.
  • An appropriate expression plasmid can also contain at least one selectable marker.
  • a selectable marker can be a nucleotide sequence that confers antibiotic resistance in a host cell.
  • selectable markers include ampicillin, cefazolin, augmentin, cefoxitin, ceftazidime, ceftiofur, cephalothin, enrofloxicin, kanamycin, spectinomycin, streptomycin, tefracycline, ticarcillin, tilmicosin, or chloramphemcol resistance genes.
  • Additional selectable markers include genes that can complement nutritional auxotrophies present in a particular host strain (e.g. leucine, alanine, or homoserine auxotrophies).
  • a replicative vector is used for expression of the heterologous gene.
  • An exemplary replicative vector can include the following: a) a selectable marker, e.g., an antibiotic marker, such as kanR (from pACYC184); b) an origin of replication in E. coli, such as the P15a ori (from pACYC184); c) an origin of replication in C. glutamicum such as that found in pBLl; d) a promoter segment, with or without an accompanying repressor gene; and e) a terminator segment.
  • the promoter segment can be a lac, trc, trcRBS, tac, or XP ⁇ / ⁇ P R (from E.
  • the repressor gene can be lad ox ci857, for lac, trc, trcRBS, tac and P I / P R , respectively.
  • the tenninator segment can be from E. coli rrnB (from pfrc99a), the T7 terminator (from p ⁇ T26), or a terminator segment from C. glutamicum.
  • an integrative vector is used for expression of the heterologous gene.
  • An exemplary integrative vector can include: a selectable marker, e.g., an antibiotic marker, such as kanR (from pACYC184); b) an origin of replication in E. coli, such as the P15a ori (from pACYC184); c) and d) two segments of the C. glutamicum genome that flank the segment to be replaced, such as the pck or hom genes; e) the sacB gene from B. subtilis; f) a promoter segment to control expression of the heterologous gene, with or without an accompanying repressor gene; and g) a terminator segment.
  • a selectable marker e.g., an antibiotic marker, such as kanR (from pACYC184)
  • an origin of replication in E. coli such as the P15a ori (from pACYC184)
  • the promoter segment can be lac, trc, trcRBS, tac, or ⁇ P ⁇ /2P R (from E. coli), oxphoA, gpd, rplM, rpsJ (fxoxi C. glutamicum).
  • the repressor genes can be lad or ci, for lac, trc, trcRBS, tac and ⁇ Pi/ ⁇ Pn, respectively.
  • the terminator segment can be from E. coli rrnB (from pfrc99a), the T7 tenninator (from p ⁇ T26), or a terminator segment from C. glutamicum.
  • the possible integrative or replicative plasmids, or reagents used to construct these plasmids, are not limited to those described herein. Other plasmids are familiar to those in the art.
  • the terminator and flanking sequences can be supplied by a single gene segment, hi this case, the above elements will be ananged in the following sequence on the plasmid: marker; origin of replication; a segment of the C. glutamicum genome that flanks the segment to be replaced; promoter; C. glutamicum terminator; sacB gene.
  • the sacB gene can also be placed between the origin of replication and the C. glutamicum flanking segment. Integration and excision results in the insertion of only the promoter, terminator, and the gene of interest.
  • a multiple cloning site can be positioned in one of several possible locations between the plasmid elements described above in order to facilitate insertion of the particular genes of interest (e.g., lysC, etc.) into the plasmid.
  • an origin of conjugative transfer such as RP4 mob
  • a bacterial gene is expressed in a host strain with an episomal plasmid.
  • Suitable plasmids include those that replicate in the chosen host sfrain, such as a coryneform bacterium.
  • Many known plasmid vectors such as e.g.
  • pZl (Menkel et al., Applied Environ Microbiol. 64:549-554, 1989), p ⁇ K ⁇ xl ( ⁇ ikmanns et al., Gene 102:93-98,1991) or pHS2-l (Sonnen et al., Gene 107:69-74, 1991) are based on the cryptic plasmids pHM1519, pBLl or pGAl.
  • Other plasmid vectors that can be used include those based on pCG4 (U.S. Pat. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiol Lett.
  • the gene or genes maybe integrated into chromosome of a host microorganism by a method using transduction, transposon (Berg, D. ⁇ . and Berg, C. M., Bio/Technol. 1 :417,1983), Mu phage (Japanese Patent Application Laid-open No. 2-109985) or homologous or non-homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab., 1972):
  • amino acids it maybe advantageous for the production of amino acids to enhance one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, or of amino acid export, using more than one gene or using a gene in combination with other biosynthetic pathway genes.
  • Attenuation of metK expression or MetK activity can enhance methionine production by prevention conversion of methionine to S-AM.
  • the bacteria containing gene(s) of interest can be cultured continuously or by a batch fermentation process (batch culture).
  • Other commercially used process variations known to those skilled in the art include fed batch (feed process) or repeated fed batch process (repetitive feed process).
  • the culture medium to be used fulfills the requirements of the particular host sfrains.
  • General descriptions of culture media suitable for various microorganisms can be found in the book "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981), although those skilled in the art will recognize that the composition of the culture medium is often modified beyond simple growth requirements in order to maximize product formation.
  • Sugars and carbohydrates such as e.g., glucose, sucrose, lactose, fructose, maltose, starch and cellulose; oils and fats, such as e.g. soy oil, sunflower oil, groundnut oil and coconut fat; fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid; alcohols, such as e.g. glycerol and ethanol; and organic acids, such as e.g. acetic acid, can be used as the source of carbon, either individually or as a mixture.
  • oils and fats such as e.g. soy oil, sunflower oil, groundnut oil and coconut fat
  • fatty acids such as e.g. palmitic acid, stearic acid and linoleic acid
  • alcohols such as e.g. glycerol and ethanol
  • organic acids such as e.g. acetic acid
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy protein hydrolysate, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen.
  • the sources of nitrogen can be used individually or as a mixture.
  • Phosphoric acid potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salts can be used as the source of phosphorus.
  • Organic and inorganic sulfur-containing compounds such as, for example, sulfates, thiosulfates, sulfites, reduced sources such as H 2 S, sulfides, derivatives of sulfides, methyl mercaptan, thioglycolytes, thiocyanates, and thiourea, can be used as sulfur sources for the preparation of sulfur-containing amino acids.
  • the culture medium can also include salts of metals, e.g., magnesium sulfate or iron sulfate, which are necessary for growth.
  • Essential growth substances such as amino acids and vitamins (e.g. cobalamin), can be employed in addition to the above-mentioned substances.
  • Suitable precursors can moreover be added to the culture medium.
  • the starting substances mentioned can be added to the culture as a single batch, or can be fed in during the culture at multiple points in time.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, calcium carbonate, aimnonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH.
  • Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
  • Suitable substances having a selective action such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids.
  • oxygen or oxygen-containing gas mixtures such as e.g. air, are introduced into the culture.
  • the temperature of the culture is typically between 20-45°C and preferably 25-40°C. Culturing is continued until a maximum of the desired product has formed, usually within 10 hours to 160 hours.
  • the fermentation broths obtained in this way can contain a dry weight of 2.5 to 25 wt. % of the amino acid of interest. It also can be advantageous ifthe fermentation is conducted in such that the growth and metabolism of the production microorganism is limited by the rate of carbohydrate addtion for some portion of the fennentation cycle, preferably at least for 30% of the duration of the fermentation. For example, the concentration of utilizable sugar in the fermentation medium is maintained at ⁇ 3 g/1 during this period.
  • the fermentation broth can then be further processed. All or some of the biomass can be removed from the fermentation broth by any solid-liquid separation method, such as centrifugation, filtration, decanting or a combination thereof, or it can be left completely in the broth. Water is then removed from the broth by known methods, such as with the aid of a multiple-effect evaporator, thin film evaporator, falling film evaporator, or by reverse osmosis.
  • the concentrated fermentation broth can then be worked up by methods of freeze drying, spray drying, fluidized bed drying, or by other processes to give a preferably free-flowing, finely divided powder.
  • the free-flowing, finely divided powder can then in turn by converted by suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • organic or inorganic auxiliary substances or carriers such as starch, gelatin, cellulose derivatives or similar substances, such as are conventionally used as binders, gelling agents or thickeners in foodstuffs or feedstuffs processing, or further substances, such as, for example, silicas, silicates or stearates.
  • the product can be absorbed on to an organic or inorganic carrier substance which is known and conventional in feedstuffs processing, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • an organic or inorganic carrier substance which is known and conventional in feedstuffs processing, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • the product can be brought into a state in which it is stable to digestion by animal stomachs, in particular the stomach of ruminants, by coating processes using film-forming agents, such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920.
  • film-forming agents such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920.
  • biomass is separated off during the process, further inorganic solids, for example, those added during the fermentation, are generally removed.
  • the biomass can be separated off to the extent of up to 70%, preferably up to 80%, preferably up to 90%, preferably up to 95%, and particularly preferably up to 100%.
  • up to 20% of the biomass preferably up to 15%, preferably up to 10%, preferably up to 5%, particularly preferably no biomass is separated off.
  • Organic substances which are formed or added and are present in the solution of the fennentation broth can be retained or separated by suitable processes. These organic substances include organic by-products that are optionally produced, in addition to the desired L-amino acid, and optionally discharged by the microorganisms employed in the fermentation. These include L-amino acids chosen from the group consisting of L-lysine, L- valine, L-threonine, L- alanine, L-methionine, L-isoleucine, or L-tryptophan.
  • vitamins chosen from the group consisting of vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B 12 (cyanocobalamin), nicotinic acid/nicotinamide and vitamin E (tocopherol).
  • vitamins chosen from the group consisting of vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B 12 (cyanocobalamin), nicotinic acid/nicotinamide and vitamin E (tocopherol).
  • vitamins chosen from the group consisting of vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B 12 (cyanocobalamin), nicotinic acid/nicotinamide and vitamin E (tocopherol).
  • organic acids that carry one to three carboxyl groups, such as, acetic acid, lactic acid
  • organic substances including L- and/or D-amino acid and/or the racemic mixture D,L-amino acid, can also be added, depending on requirements, as a concentrate or pure substance in solid or liquid form during a suitable process step.
  • These organic substances mentioned can be added individually or as mixtures to the resulting or concentrated fermentation broth, or also during the drying or granulation process. It is likewise possible to add an organic substance or a mixture of several organic substances to the fermentation broth and a further organic substance or a further mixture of several organic substances during a later process step, for example granulation.
  • the product described above can be used as a feed additive, i.e. feed additive, for animal nutrition.
  • feed additive i.e. feed additive
  • Example 1 Construction of vectors for expression of genes for enhancing production of aspartate-derived amino acids
  • Plasmids were generated for expression of genes relevant to the production of aspartate- derived amino acids. Many of the target genes are shown in Figure 1 and 2, which depicts most of the biosynthetic genes directly involved in producing aspartate-derived amino acids. These plasmids, which may either replicate autonomously or integrate into the host C. glutamicum chromosome, were introduced into strains of corynebacteria by electroporation as described (see Follettie, M.T., et al. J. Bacteriol 167:695-702, 1993). All plasmids contain the kanR gene that confers resistance to the antibiotic kanamycin. Transformants were selected on media containing kanamycin (25mg/L).
  • episomal plasmids For expression from episomal plasmids, vectors were constructed using derivatives of the cryptic C. glutamicum low-copy pBLl plasmid (see Santamaria et al. J. Gen. Microbiol. 130:2237-2246, 1984). Episomal plasmids contain sequences that encode a replicase, which enables replication of the plasmid within C. glutamicum; therefore, these plasmids can be propagated without integration into the chromosome. Plasmids MB3961 and MB4094 were the vector backbones used to construct episomal expression plasmids described herein (see Figures 3 and 4).
  • Plasmid MB4094 contains an improved origin of replication, relative to MB3961, for use in corynebacteria; therefore, this backbone was used for most studies. Both MB3961 and MB4094 contain regulatory sequences from pTrc99A (see Amann et al., Gene 69:301-315, 1988). The 3' portion of the laclq-trc IPTG-inducible promoter cassette resides within the polylinker in such a way that genes of interest can be inserted as fragments containing Ncol-Notl compatible overhangs, with the Ncol site adjacent to the start site of the gene of interest
  • trcRBS modified trc promoter
  • C. glutamicum gpd, rplM, and rpsJ promoters can be inserted into the MB3961 and MB4094 backbones on convenient restriction fragments, including Nhe ⁇ -Ncol fragments.
  • the trcRBS promoter contains a modified ribosomal-binding site that was shown to enhance levels of expressed proteins. The sequences of promoters employed in these studies for expression of genes are found in Table 7.
  • Plasmids were also designed to inactivate native C, glutamicum genes by gene deletion. In some instances, these constructs both delete native genes and insert heterologous genes into the host chromosome at the locus of the deletion event. Table 8 lists the endogenous gene that was deleted and the heterologous genes that were introduced, if any.
  • Deletion plasmids contain nucleotide sequences homologous to regions upstream and downstream of the gene that is the target for the deletion event; in some instances these sequences include small amoimts of coding sequence of the gene that is to be inactivated. These flanking sequences are used to facilitate homologous recombination.
  • Deletion plasmids also contain the sacB gene, encoding the levansucrase gene from Bacillus subtilis. Transformants containing integrated plasmids were streaked to BHI medium lacking kanamycin. After 1 day, colonies were streaked onto BHI medium containing 10% sucrose. This protocol selects for strains in which the sacB gene has been excised, since it polymerizes sucrose to form levan that is toxic to C. glutamicum (see Jager, W., et al. J. Bacteriol 174:5462-5465, 1992).
  • Genomic DNA is isolated from M. smegmatis grown in BHI medium for 72 h at 37°C using QIAGEN Genomic-tips according to the recommendations of the manufacturer kits (Qiagen, Valencia, CA).
  • the Salting Out Procedure (as described in Practical Streptomyces Genetics, pp. 169-170, Kieser, T., et. al., John huies Foundation, Norwich, England 2000) is used on cells grown in TYE media (ATCC medium 1877 ISP Medium 1) for 7 days at 25°C.
  • TYG media ATCC medium 741
  • the 100 ml culture is spun down (5000 rpm for 10 min at 4°C) and washed twice with 40 ml lOmM Tris, 20mM EDTA pH 8.0.
  • the cell pellet is brought up in a final volume of 40 ml of lOmMTris, 20mM EDTA pH 8.0.
  • This suspension is passed through a Microfluidizer (Microfluidics Corporation, Newton MA) for 10 cycles and collected.
  • the apparatus is rinsed with an additional 20 ml of buffer and collected.
  • the final volume of lysed cells is 60 ml.
  • DNA is precipitated from the suspension of lysed cells by isopropanol precipitation, and the pellet is resuspended in 2 ml TE pH 8.0. The sample is extracted with phenol/chloroform and the DNA precipitated once again with isopropanol.
  • genomic DNA was prepared as described for E. coli (Qiagen genomic protocol) using a Genomic Tip 500/G.
  • primers are designed according to sequence upstream of the lysC gene and sequence near the stop of asd.
  • the upstream primer is 5'- CCGTGAGCTGCTCGGATGTGACG-3' (SEQ ID NO:_)
  • the downstream primer is 5'- TCAGAGGTCGGCGGCCAACAGTTCTGC-3' (SEQ DD NO:_ .
  • the genes are amplified using Pfu Turbo (Stratagene, La Jolla, CA) in a reaction mixture containing 10 ⁇ l 10X Cloned Pfu buffer, 8 ⁇ l dNTP mix (2.5mM each), 2 ⁇ l each primer (20uM), 1 ⁇ l Pfu Turbo, 10 ng genomic DNA and water in a final reaction volume of 100 ⁇ l.
  • the reaction conditions are 94°C for 2 min, followed by 28 cycles of 94°C for 30 sec, 60°C for 30sec, 72°C for 9 min.
  • the reaction is completed with a final extension at 72°C for 4 min, and the reaction is then cooled to 4°C.
  • the resulting product is purified by the Qiagen gel extraction protocol followed by blunt end ligation into the Smal site of pBluescript SK II-.
  • Ligations are fransformed into E. coli DH5 ⁇ and selected by blue/white screening. Positive fransformants are treated to isolate plasmid DNA by Qiagen methods and sequenced.
  • MB3902 is the resulting plasmid containing the expected insert.
  • Primer pairs for amplifying S. coelicolor genes are: 5'- ACCGCACTTTCCCGAGTGAC-3' (SEQ DD NO:_ and 5'- TCATCGTCCGCTCTTCCCCT- 3' (lysC-asd) (SEQ DD NO:_); 5'- ATGGCTCCGACCTCCACTCC-3' (SEQ DD NO:_) and 5'- CGTGCAGAAGCAGTTGTCGT-3' (d ⁇ pA) (SEQ DD NO:_); and 5'- TGAGGTCCGAGGGAGGGAAA-3' (SEQ DD NO:_) and 5'-
  • TTACTCTCCTTCAACCCGCA-3' (horn)
  • the primer pair for amplifying the metYA operon from T.fusc ⁇ is 5'- CATCGACTACGCCCGTGTGA-3' (SEQ DD NO:_ and 5'- TGGCTGTTCTTCACCGCACC-3' (SEQ DD NO:_).
  • chrysanthemi genes are: 5'- TTGACCTGACGCTTATAGCG-3' (SEQ DD NO:_) and 5'- CCTGTACAAAATGTTGGGAG-3' (dapA) (SEQ ID NO:_); and 5'- ATGAATGAACAATATTCCGCCA-3' (SEQ ID NO:_ and 5'- TTAGCCGGTATTGCGCATCC-3' (ppc) (SEQ ID NO:_).
  • Amplification of genes was done by similar methods as above or by using the TripleMaster PCR System from Eppendorf (Eppendorf, Hamburg, Germany). Blunt end ligations were performed to clone amplicons into the Smal site of pBluescript SK D-.
  • the resulting plasmids were MB3947 (S. coelicolor lysC-asd), MB3950 (S. coelicolor dapA), MB4066 (S. coelicolor hom), MB4062 (T. fusca metYA), MB3995 (E. chrysanthemi dap A), and MB4077 (E. chrysanthemi ppc).
  • Site-directed mutagenesis was performed on several of the pBluescript SK II- plasmids containing the heterologous genes described in Example 2. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit from Stratagene.
  • lysC/ask heterologous aspartokinase
  • substitution mutations were constructed that correspond to the T31 II, S301Y, A279P, and G345D amino acid substitutions in the C. glutamicum protein. These substitutions may decrease feedback inhibition by the combination of lysine and threonine.
  • the mutated lysC/ask alleles were expressed in an operon with the heterologous asd gene.
  • Oligonucleotides employed to construct M. smegmatis feedback resistant lysC alleles were: 5'-GGCAAGACCGACATCATATTCACGTGTGCGCGTG-3' (SEQ DD NO:_) and 5'-CACGCGCACACGTGAATATGATGTCGGTCTTGCC-3' (T311I) (SEQ DD NO:_); 5'-GGTGCTGCAGAACATCTACAAGATCGAGGACGGCAA-3' (SEQ DD NO:_) and 5'-TTGCCGTCCTCGATCTTGTAGATGTTCTGCAGCACC-3' (S301Y) (SEQ DD NO:_ ; 5'-GACGTTCCCGGCTACGCCGCCAAGGTGTTCCGC-3'(SEQ DD NO:_) and 5'- GCGGAACACCTTGGCGGCGTAGCCGGGAACGTC-3' (A279P) (SEQ DD NO:_J; and 5'- GTACGACGACCACATCGACAAGGTGTCGCT
  • Oligonucleotides employed to construct S. coelicolor feedback resistant lysC alleles were: 5'- CGGGCCTGACGGACATCRTCTTCACGCTCCCCAAG-3' (SEQ DD NO:_) and 5'- CTTGGGGAGCGTGAAGAYGATGTCCGTCAGGCCCG-3' (S314I/S314V) (SEQ DD NO:_ ; and 5'- GTCGTGCAGAACGTGTACGCCGCCTCCACGGGC-3' (SEQ D NO:_ and 5 '- GCCCGTGGAGGCGGCGTACACGTTCTGCACGAC-3 ' (S304Y) (SEQ DD NO:_J.
  • Site-directed mutagenesis can be performed to generate deregulated alleles of additional proteins relevant to the production of aspartate-derived amino acids. For example, mutations can be generated that correspond to the V59A, G378E, or carboxy-terminal truncations of the C. glutamicum hom gene.
  • the Transformer Site-Directed Mutagenesis Kit (BD Biosciences Clontech) was used to generate the S. coelicolor horn (G362E) substitution.
  • TTCATCGAACAGCGCTCGCACCTGCTGACCGCC-3' (SEQ DD NO:_) and 5'- GGCGGTCAGCAGGTGCGAGCGCTGTTCGATGAA-3' (SEQ DD NO:_)can be used to generate a substitution in the S. coelicolor pyc gene that conesponds to the C. glutamicum pyc P458S mutation.
  • Site-directed mutagenesis can also be utilized to introduce substitutions that correspond to deregulated dapA alleles described above.
  • Wild-type and deregulated alleles of heterologous (and C. glutamicum) genes were then cloned into vectors suitable for expression.
  • PCR was employed using oligonucleotides to facilitate cloning of genes as a Ncol-Notl fragment.
  • DNA sequence analysis was performed to verify that mutations were not introduced during rounds of amplification.
  • synthetic operons were constructed in order to express two or more genes, heterologous or endogenous, from the same promoter.
  • plasmid MB4278 was generated to express the C. glutamicum metA, metY, and metH genes from the trcRBS promoter.
  • Figure 12B displays the DNA sequence in MB4278 that spans from the trcRBS promoter to the stop of the metH gene; the gene order in this construct is rnetAYH.
  • the open reading frames in Figure 12B are shown in uppercase. Note that the construct was engineered such that each open reading frame is preceded by an identical stretch of DNA. This conserved sequence serves as a ribosomal-binding sequence that promotes efficient translation of C. glutamicum proteins. Similar intergenic sequences were used to construct additional synthetic operons.
  • Example 4 Isolation of additional threonine-insensitive mutants of homoserine dehydrogenase
  • oligonucleotide primers 5'- CACACGAAGACACCATGATGCGTACGCGTCCGCT -3' (contains aBbsl site and cleavage yields a Ncol compatible overhang) (SEQ DD NO: ) and 5'-
  • ATAAGAATGCGGCCGCTTACTCTCCTTCAACCCGCA -3' (contains aNotl site) (SEQ DD NO: ) are used to amplify the horn gene from plasmid MB4066.
  • the resulting mutant population is digested with Bbsl and Notl, ligated into NcoVNotl digested episomal plasmid containing the trcRBS promoter in the MB4094 plasmid backbone, and transformed into C. glutamicum ATCC 13032.
  • the transformed cells are plated on agar plates containing a defined medium for corynebacteria (see Guillouet, S., et al. Appl. Environ. Microbiol.
  • kanamycin 25 mg/L
  • 20 mg/L of AHV (alpha-amino, beta-hydroxyvaleric acid; a threonine analog)
  • O.OlmM IPTG O.OlmM IPTG
  • the resulting transformants are subsequently screened for homoserine excretion by replica plating to a defined medium agar plate supplemented with threonine, which was previously spread with ⁇ 10 6 cells of indicator C. glutamicum sfrain MA-331 (hom-thrB ⁇ ).
  • Putative feedback-resistant mutants are identified by a halo of growth of the indicator strain smiOunding the replica-plated fransformants.
  • the hom gene is PCR amplified using the above primer pair, the amplicon is digested as above, and ligated into the episomal plasmid described above.
  • Each of these putative horn mutants is subsequently re-transformed into C. glutamicum ATCC 13032 and plated on minimal medium agar plates containing 25 mg/L kanamycin and O.OlmM IPTG.
  • One colony from each transformation is replica plated to defined medium for corynebacteria containing 10, 20, 50, and 100 mg/L of AHV, and sorted based on the highest level of resistance to the threonine analog.
  • the heterologous metA gene cloned from T. fusca is subjected to enor prone PCR using the GeneMorph ® Random Mutagenesis kit obtained from Sfratagene. Under the conditions specified in this kit, oligonucleotide primers 5'- CACACACCTGCCACACATGAGTCACGACACCACCCCTCC -3' (contains aBspMl site and cleavage yields a Ncol compatible overhang) (SEQ DD NO: ) and 5'-
  • ATAAGAATGCGGCCGCTTACTGCGCCAGCAGTTCTT -3' (contains aNotl site) (SEQ DD NO: ) are used to amplify the metA gene from plasmid MB4062.
  • the resulting mutant amplicon is digested and ligated into the NcollNotl digested episomal plasmid described in Example 4, and then transformed into C. glutamicum sfrain MA-428.
  • MA-428 is a derivative of ATCC 13032 that has been transformed with integrating plasmid MB4192. After selection for recombination events, the resulting strain MA-428 is deleted for hom-thrB in a manner that results in insertion of a deregulated S.
  • coelicolor hom gene The fransformed MA-428 cells described are plated on minimal medium agar plates containing kanamycin (25 mg/L), 0.01 mM IPTG, and 100 ⁇ g/ml or 500 ⁇ g/ml of trifluoromethionine (TFM; a methionine analog). After 72 h at 30°C, the resulting fransformants are subsequently screened for O-acetylhomoserine excretion by replica plating to a minimal agar plate which was previously spread with ⁇ 10 6 cells of an indicator strain, S.
  • TMF trifluoromethionine
  • OAH O-acetylhomoserine
  • the metA gene is PCR amplified using the above primer pair, digested with BspMl and Notl, and ligated into the NotllNcoI digested episomal plasmid described in example 4.
  • Each of these putative metA mutant alleles is subsequently re-transformed into C. glutamicum ATCC 13032 and plated on minimal medium agar plates containing 25 mg/L kanamycin.
  • One colony from each transformation is replica plated to minimal medium containing 100, 200, 500, and 1000 ⁇ g/ml of TFM plus 0.01 mM IPTG, and sorted based on the highest level of resistance to the methionine analog.
  • Representatives from each group are grown in minimal medium to an OD of 2.0, the cells harvested by centrifugation, and homoserine O-acetyltransferase activity is determined by the methods described by Kredich and Tomkins (J Biol. Chem. 241 :4955-4965,1966) in the presence and absence of 20 mM methionine or S-AM.
  • the metA gene is PCR amplified from those cultures showing feedback-resistance and sequenced.
  • the resulting plasmids are used to generate expression plasmids to enhance amino acid production.
  • Oligonucleotide primers 5'- CACAGGTCTCCCATGGCACTGCGTCCTGACAGGAG-3' (contains a Bsal site and cleavage yields a Ncol compatible overhang) (SEQ DD NO: ) and 5'-
  • ATAAGAATGCGGCCGCTCACTGGTATGCCTTGGCTG -3' (contains aNotl site) (SEQ ID NO: ) are used for cloning into the episomal plasmid, as described above, and for canying out the mutagenesis reaction per the specifications of the GeneMorph ® Random Mutagenesis kit obtained from Sfratagene.
  • the major difference is that the mutated metY population is fransformed into a C. glutamicum sfrain that already produces high levels of O- acetylhomoserine.
  • This strain, MICmet2 is constructed by transforming MA-428 with a modified version of plasmid MB4286 that contains a deregulated T. fusca metA allele described above under the control of the trcRBS promoter. After transformation the sacB selection system enables the deletion of the endogenous mcbR locus and replacement with the deregulated heterologous metA allele.
  • the T. fusca metY variant fransformed MICmet2 strain is spread onto minimal agar plates containing 25 mg/L of kanamycin, 0.25mM IPTG, and an inhibiting concentration of toxic methionine analog(s) (e.g., ethionine, selenomethionine, TFM); the fransfonnants can be grown on these 3 different methionine analogs either individually or in double or triple combination).
  • the metY gene is amplified from those colonies growing on the selection plates, the amplicons are digested and ligated into the episomal plasmid described in example 4, and the resulting plasmids are fransformed into MICmet2.
  • the fransfonnants are grown on minimal medium agar plates containing 25 mg/L of kanamycin.
  • the resulting colonies are replica-plated to agar plates containing a 10-fold range of the toxic methionine analogs ethionine, TFM, and selenomethionine (plus 0.01 mM IPTG), and sorted on the basis of analog sensitivity.
  • Representatives from each group are grown in minimal medium to an OD of 2.0, the cells are harvested by centrifugation, and O-acetylhomoserine sulfhydrylase enzyme activity is determined by a modified version of the methods of Kredich and Tomkins (J. Biol. Chem.
  • An expression plasmid containing the feedback resistant metY and metA variants from T. fusca is constructed as follows. The T.
  • fusca metYA operon is amplified using oligonucleotides 5'- CACACACATGTCACTGCGTCCTGACAGGAGC-3' (contains a Aril site and cleavage yields a Ncol compatible overhang (also changes second codon from Ala>Ser)) (SEQ DD ⁇ O:_J and 5'-ATAAGAATGCGGCCGCTTACTGCGCCAGCAGTTCTT -3' (contains a Notl site) (SEQ DD NO: ).
  • the amplicon is digested with Pcil and Notl, and the fragment is ligated into the above episomal plasmid that has been treated sequentially treated with Notl, H ⁇ elll methylase, and Ncol.
  • Site directed mutagenesis performed using the QuikChange Site-Directed Mutagenesis Kit from Sfratagene, is used to incorporate the described substitution mutations in T. fusca metA and metY nto a single plasmid that expresses the deregulated alleles as an operon. The resulting plasmid is used to enhance amino acid production.
  • Trace elements solution comprises: 88 mg Na 2 B 4 O 7 -10H 2 O, 37 mg (NH 4 ) 6 Mo 7 O 27 -4H 2 O, 8.8 mg ZnSO 4 -7H 2 O, 270 mg CuSO 4 -5H 2 O, 7.2 mg MnCl 2 -4H 2 O, and 970 mg FeCl 3 -6H 2 O per liter of deionized water. (When needed to support auxotrophic requirements, amino acids and purines are supplemented to 30 mg/L final concentration.)
  • S-AM binding motifs have been identified in bacterial DNA methylfransferases (Roth et al, J. Biol. Chem., 273:17333-17342, 1998). Roth et al. identified a highly conserved amino acid motif in EcoRN -adenine- ⁇ 6 -D ⁇ A methyltransferase which appeared to be critical for S-AM binding by the enzyme. We searched for related motifs in the amino acid sequences of the following proteins of C.
  • MetA and MetY genes were cloned from C. glutamicum and T fusca as described in Example 2.
  • Table 11 lists the plasmids and strains used for the expression of wild-type and mutated alleles of MetA and MetY genes.
  • Tables 12 and 13 list the plasmids used for expression and the oligonucleotides employed for site-directed mutagenesis to generate MetA and MetY variants.
  • a single C. glutamicum colony was inoculated into seed culture media (see example 10 below) and grown for 24 hour with agitation at 33 °C.
  • the seed culture was diluted 1:20 in production soy media (40 mL) (example 10) and grown 8 hours. Following harvest by centrifugation, the pellet was washed lx in 1 volume of water.
  • the pellet was resuspended in 250 ⁇ l lysis buffer (1ml HEPES buffer, pH 7.5, 0.5ml 1M KOH, 1 O ⁇ l 0.5M EDTA, water to 5ml), 30 ⁇ l protease inhibitor cocktail, and 1 volume of 0.1 mm acid washed glass beads.
  • the mixture was alternately vortexed and held on ice for 15 seconds each for 8 reptitions. After centrifugation for 5' at 4,000 rpm, the supernatant was removed and re-spun for 20' at 10,000 rpm. The Bradford assay was used to determine protein concentration in the cleared supernatant.
  • Example 8 Quantifying MetA activity in C. glutamicum strains containing episomal plasmids
  • MetA activity in C. glutamicum expressing endogenous and episomal metA genes was determined. MetA activity was assayed in crude protein extracts using a protocol described by Kredich and Tomkins (J. Biol. Chem.24 ⁇ (2 ⁇ ):4955-4965, 1966). Preparation of protein extracts is described in the Example 7. Briefly, 1 ⁇ g of protein extract was added to a microtiter plate.
  • Reaction mix 250 ⁇ l; lOOmM tris-HCl pH 7.5, 2mM 5,5'-Dithiobis(2-nitrobenzoic acid) (DTN), 2mM sodium EDTA, 2mM acetyl Co A, 2mM homoserine
  • DTN 5,5'-Dithiobis(2-nitrobenzoic acid)
  • MetA activity liberates Co A from acetyl-CoA.
  • a disulfide interchange occurs between the CoA and DTN to produce thionifrobenzoic acid.
  • the production of thionifrobenzoic acid is followed spectrophotometrically. Absorbance at 412 mn was measured every 5 minutes over a period of 30 minutes. A well without protein extract was included as a control.
  • Inhibition of MetA activity was determined by addition of S-adenosyl methionine (S-AM; .02 mM, .2 mM, 2 mM) and methionine (.5mM, 5 mM, 50 mM). Inhibitors were added directly to the reaction mix before it was added to the protein extract, h vitro O-acetyltransferase activity was measured in crude protein extracts derived from C. glutamicum strains MA-442 and MA-449 which contain both endogenous and episomal C. glutamicum MetA and MetY genes.
  • Episomal metA and metY genes were expressed as a synthetic operon; the nucleic acid sequence of the metAY operon is as shown in the rnetAYH operon of Figure 12B, only lacking metH sequence.
  • the trcRBS promoter was employed in these episomal plasmids.
  • MA-442 expresses the episomal genes in the order metA-metY.
  • MA-449 expresses the episomal genes in the order metY-metA. Experiments were performed in the presence and absence of IPTG that induces expression of the plasmid borne MetA and MetY genes.
  • Figure 13 shows a time course of MetA activity. MetA activity was observed only when the genes were in the MetA-MetY (MA-442) configuration in samples from 8 hour and 20 hour cultures.
  • MetA activity in extracts from strain MA-449 was not significantly elevated relative to a control sample lacking protein at both 8 hour and 20 hour time points, with and without induction. This data is consistent with Northern blot analysis that showed low expression of metA when the two genes were in the metY-metA orientation.
  • MA- 442 extracts were assayed in the presence of 5 mM methionine, 0.2 mM S-AM, or in the absence of additional methionine or S-AM, and MetA activity was assayed as described above. As shown in Figure 14, MetA activity was reduced in the presence of 5 mM methionine and 0.2 mM S-AM. Thus, reducing allosteric repression of MetA may enhance MetA activity, allowing production of higher levels of methionine. It is possible that allosteric repression would also be observed at much lower levels of methionine or S-AM.
  • the levels tested are physiologically relevant levels in strains engineered for the production of amino acids such as methionine.
  • C. glutamicum sfrains expressing episomal T. fusca MetA (strains MA-578 and MA-579), or both episomal T fusca MetA and MetY (strains MA-456 and MA-570) were constructed and extracts were prepared from these sfrains and assayed for MetA activity.
  • the regulatory elements associated with each episomal gene are listed in Table 12. The rate of MetA activity in extracts of each sfrain was determined by calculating the change in OD 12 divided by time per ng of protein.
  • strain MA-578 exhibited a rate of approximately 2.75 units (change in OD 412 / time/ng protein) under inducing conditions, whereas the rate under non-inducing conditions was approximately 1.
  • Strain MA- 579 exhibited a rate of approximately 2.5 under inducing conditions and a rate of approximately 0.4 under non-inducing conditions.
  • Sfrain MA-456 which expresses metA and metY under the control of a constitutive promoter, exhibited a rate of approximately 2.2.
  • Sfrain MA-570 exhibited a rate of approximately 1 under inducing conditions and a rate of 0.3 under non- inducing conditions.
  • the negative control sample (no protein) exhibited a rate of approximately 0.1.
  • Example 9 Quantifying MetY activity in C. glutamicum strains containing episomal plasmids
  • EDTA lmM sodium sulfide nonahydrate (Na 2 S), 0.2mM pyridoxal-5-phosphoric acid (PLP) was mixed with 45 ⁇ g of protein extract.
  • O-acetyl homoserine OAH; Toronto Research Chemicals hie was added to a final concentration of 0.625mM. 200 ⁇ l of the reaction was removed immediately for the zero time point. The remainder of the reaction was incubated at 30°C. Three 200 ⁇ l samples were removed at 10 minute intervals.
  • Strain MA-456 which expresses episomal wild type T. fusca metA and metY alleles under the control of a constitutive promoter, exhibited a rate of 0.04.
  • Strain MA-570 which expresses episomal wild type T. fusca metA and metY alleles under the confrol of an inducible promoter, exhibited a rate of approximately 0.038 under inducing conditions, and a rate of less than 0.01 under non-inducing conditions.
  • expression of heterologous MetY results in enzyme activity that is significantly elevated over that of the endogenous MetY.
  • TrcRBS (see above) (lacIQ-Trc regulatory sequence from pTrc99A (Amann et al.,
  • gpd C. glutamicum gpd promoter
  • MO4040 5 CAGGTACGACTCGACAGCGGCCCGGCCCCCGGCCATCGG 3 '
  • MO4041 5 AAACTCGCCCGCCGGTTCGCCGCGGGCAGCTACGTCGTG 3 '
  • MO4042 5 CACGACGTAGCTGCCCGCGGCGAACCGGCGGGCGAGTTT 3 '
  • MO4043 5 CACGGCACCACGATCGCGGCCATCGTGGTGGACGCCGGC 3 '
  • MO4045 5 ATCGCGGGCATCGTGGTGGCCGCCGGCACCTTCGACTTC 3 '
  • MO4047 5 ATCGAGGCCGGACGCGCCGCCGTGGACGGCACCGAACTG 3 '
  • MO4048 5 CAGTTCGGTGCCGTCCACGGCGGCGCGTCCGGCCTCGAT 3 '
  • MO4050 5 GACGATGAGGCTGCGCACGGCACCGATGTTGACGAGCTG 3 '
  • MO4052 5 GTTTTCGTTCTTTTGGGCTGCGGTGCCGAAGCGTTCGTC 3 '
  • MO4059 5 GGCGGCGTGCTTATCGACGCCGGAAAGTTCGATTGGACT 3 '
  • MO4060 5 AGTCCAATCGAACTTTCCGGCGTCGATAAGCACGCCGCC 3 '
  • Example 10 Methods for producing and detecting aspartate-derived amino acids
  • each sfrain was inoculated from an agar plate into 10 ml of Seed Culture Medium in a 125 ml Erlenmeyer flask.
  • the seed culture was incubated at 250 rpm on a shaker for 16 h at 31°C.
  • a culture for monitoring amino acid production was prepared by performing a 1 :20 dilution of the seed culture into 10 ml of Batch Production Medium in 125 ml Erlenmeyer flasks.
  • IPTG was added to a set of the cultures to induce expression of the IPTG regulated genes (final concentration 0.25 mM).
  • Methionine fermentations were carried out for 60-66 h at 31°C with agitation (250 rpm).
  • agitation 250 rpm
  • multiple fransformants were fermented in parallel, and each fransformant was often grown in duplicate.
  • Most reported data points reflect the average of at least two fermentations with a representative transfonnant, together with confrol strains that were grown at the same time.
  • LCMS liquid chromatography-mass spectrometry
  • the instrument was operated in MRM mode to detect amino acids (lysine: 147 - ⁇ 84 (15 eV); methionine: 150 - 104 (12 eV); threonine/homoserine: 120 - 74 (10 eV); aspartic acid: 134 - 88 (15 eV); glutamic acid: 148 -» 84 (15 eV); O-acetylhomoserine: 162 -» 102 (12 eV); and homocysteine: 136 - 90 (15 eV)).
  • additional amino acids were quantified using similar methods (e.g. homocystine, glycine, S-adenosylmethionine).
  • Example 11 Heterologous wild-type and mutant lysC variants increase lysine production in C. glutamicum and B. lactofermentum.
  • Aspartokinase is often the rate-limiting activity for lysine production in corynebacteria.
  • the primary mechanism for regulating aspartokinase activity is allosteric regulation by the combination of lysine and threonine.
  • Heterologous operons encoding aspartokinases and aspartate semi-aldehyde dehydrogenases were cloned from M. smegmatis and S. coelicolor as described in Example 2.
  • Site-directed mutagenesis was used to generate deregulated alleles (see Example 3), and these modified genes were inserted into vectors suitable for expression in corynebacteria (Example 1).
  • the resulting plasmids, and the wild-type counterparts, were transformed into sfrains, including wild-type C. glutamicum sfrain ATCC 13032 and wild-type B. lactofermentum strain ATCC 13869, which were analyzed for lysine production (Figure 17).
  • Strains MA-0014, MA-0025, MA-0022, MA-0016, MA-0008 and MA-0019 contain plasmids with the MB3961 backbone (see Example 1). Increased expression, via addition of IPTG to the production medium, of either wild-type or deregulated heterologous lysC-asd operons promoted lysine production. Sfrain ATCC 13869 is the untransfonned control for these strains.
  • the plasmids containing M. smegmatis S301Y, T311I, and G345D alleles were most effective at enhancing lysine production; these alleles were chosen for expression for expression from improved vectors. Improved vectors containing deregulated M.
  • smegmatis alleles were transformed into C. glutamicum (ATCC 13032) to generate sfrains MA-0333, MA-0334, MA- 0336, MA-0361, and MA-0362 (plasmids contain either trcRBS or gpd promoter, MB4094 backbone; see Example 1).
  • Sfrain ATCC 13032 (A) is the unfransformed control for strains MA- 0333, MA-0334 and MA-0336.
  • Strain ATCC 13032 (B) is the unfransformed control for sfrains MA-0361 and MA-0362. Strains MA-0333, MA-0334, MA-0336, MA-0361, and MA-0362 all displayed improvement in lysine production.
  • strain MA-0334 produced in excess of 20 g/L lysine from 50 g/L glucose, hi addition, the T31 II and G345D alleles were shown to be effective when expressed from either the trcRBS or gpd promoter.
  • Example 12 S. coelicolor hom G362E variant increases carbon flow to homoserine in C. glutamicum strain, MA-0331
  • deregulation of aspartokinase increased carbon flow to aspartate-derived amino acids.
  • aspartokinase activity could be increased by the use of deregulated lysC alleles and/or by elimination of the small molecules that mediate the allosteric regulation (lysine or threonine).
  • Figure 18 shows that high levels of lysine accumulated in the broth when the hom-thrB locus was inactivated.
  • Hom and thrB encode for homoserine dehydrogenase and homoserine kinase, respectively, two proteins required for the production of threonine.
  • Lysine accumulation was also observed when only the thrB gene was deleted (see strain MA-0933 in Figure 21 (MA-0933 is one example, though it is not appropriate to directly compare MA-0933 to MA-0331, as these sfrains are from different genetic backgrounds).
  • Figure 18 shows the results from shake flask experiments performed using strains MA-0331, MA-0384, MA-0386, and MA-0389, in whichbroths were analyzed for aspartate-derived amino acids, including lysine and homoserine.
  • Strains expressing the S. coelicolor homG362 ⁇ gene display a dramatic decrease in lysine production as well as a significant increase in homoserine levels. Broth levels of homoserine were in excess of 5 g/L in strains such as MA-0389. It is possible that significant levels of homoserine still remain within the cell or that some homoserine has been converted to additional products.
  • Overexpression of deregulated lysC and other genes downstream of hom, together with hom may increase production of homoserine-based amino acids, including methionine (see below).
  • Example 13 Heterologous phosphoenolpyruvate carboxylase (Ppc) enzymes increase carbon flow to aspartate-derived amino acids.
  • Ppc Heterologous phosphoenolpyruvate carboxylase
  • Phosphoenolpyruvate carboxylase Phosphoenolpyruvate carboxylase (Ppc), together with pyruvate carboxylase (Pyc), catalyze the synthesis of oxaloacetic acid (OAA), the citric acid cycle intermediate that feeds directly into the production of aspartate-derived amino acids.
  • OAA oxaloacetic acid
  • the wild-type E. chrysanthemi ppc gene was cloned into expression vectors under confrol of the IPTG inducible trcRBS promoter. This plasmid was transfonned into high lysine strains MA-0331 and MA-0463 ( Figure 19). Strains were grown in the absence or presence of IPTG and analyzed for production of aspartate-derived amino acids, including aspartate.
  • Strain MA-0331 contains the hom-thrB A mutation, whereas MA-0463 contains the M. smegmatis lysC (T31 l ⁇ )-asd operon integrated at the deleted hom-thrB locus; the lysC-asd operon is under confrol of the C. glutamicum gpd promoter.
  • Figure 19 shows that the E. chrysanthemi ppc gene increased the accumulation of aspartate. This difference was even detectable in strains that converted most of the available aspartate into lysine.
  • Example 14 Heterologous dihydrodipicolinate synthases (dapA) enzymes increase lysine production.
  • Dihydrodipicolinate synthase is the branch point enzyme that commits carbon to lysine biosynthesis rather than to the production of homoserine-based amino acids.
  • DapA converts aspartate-B-semialdehyde to 2,3-dihydrodipicolinate.
  • the wild-type E. chrysanthemi and S. coelicolor dap A genes were cloned into expression vectors under the control of the trcRBS and gpd promoters. The resulting plasmids were transformed into strains MA-0331 and MA-0463, two strains that had already been engineered to produce high levels of lysine (see Example 13). MA-0463 was engineered for increased expression of the M.
  • E. chrysanthemi or S. coelicolor dapA gene increases lysine production in the MA-0331 and MA-0463 backgrounds.
  • Sfrain MA-0502 produced nearly 35 g/L lysine in a 50 g/L glucose process. It may be possible to engineer further lysine improvements by constructing deregulated variants of these heterologous dapA genes.
  • Example 15 Constructing strains that produce high levels of homoserine.
  • Strains that produce high levels of homoserine-based amino acids can be generated through a combination of genetic engineering and mutagenesis strategies.
  • five distinct genetic manipulations were performed to construct MA-1378, a strain that produces >10 g/L homoserine ( Figure 21).
  • wild-type C. glutamicum was first mutated using nifrosoguanidine (NTG) mutagenesis (based on the protocol described in "A short course in bacterial genetics.” J. H. Miller. Cold Spring Harbor Laboratory Press. 1992, page 143) followed by selection of colonies that grew on minimal plates containing high levels of ethionine, a toxic methionine analog.
  • McbR is a transcriptional repressor that regulates the expression of several genes required for the production of sulfur-containing amino acids such as methionine (see Rey, D.A., Puhler, A., and Kalinowsld, J., J. Biotechnology 103:51-65, 2003). In several instances we observed that inactivation of McbR generated strains with increased levels of homoserine-based amino acids.
  • Plasmid MB4084 was utilized to delete the t ⁇ rR locus in MA-0622, causing the accumulation of lysine and homoserine; methionine and methionine pathway intermediates also accumulated to a lesser degree.
  • MA-0933 resulted from this manipulation. As described above, it is believed that the lysine and homoserine accumulation was a result of deregulation of lysC, via the lack of threonine production, hi order to further optimize carbon flow to aspartate-B- semialdehyde and downstream amino acids, MA-0933 was fransformed with an episomal plasmid expressing the M.
  • smegmatis lysC T31 ⁇ l-asd operon (sfrain MA-1162).
  • High homoserine producing strain MA-1162 was then mutagenized with NTG, and colonies were selected on minimal medium plates containing a level of methionine methylsulfonium chloride (MMSC) that is normally inhibitory to growth.
  • MMSC methionine methylsulfonium chloride
  • Example 16 Heterologous homoserine acetyltransferases (MetA) enzymes increase carbon flow to homoserine-based amino acids.
  • MetA is the commitment step to methionine biosynthesis.
  • the wild-type T. fusca metA gene was cloned into an expression vector under the control of the trcRBS promoter. This plasmid was fransformed into high homoserine producing sfrains to test for elevated MetA activity ( Figures 22 and 23).
  • MA-0428, MA-0933, and MA-1514 were example high homoserine producing strains.
  • MA-0428 is a wild-type ATCC 13032 derivative that has been engineered with plasmid MB4192 (see Example 1) to delete the hom-thrB locus and integrate the gpd- S. coelicolor hom(G362 ⁇ ) expression cassette.
  • MA-1514 was constructed by using novobiocin to allow for loss of the M. smegmatis lysC(T3l lT)-asd operon plasmid from sfrain MA-1378. This manipulation was performed to allow for transformation with the episomal plasmid containing the T fusca metA gene and the kanR selectable marker. Sfrain MA-1559 resulted from the transformation of sfrain MA-1514 with the T. fusca metA gene under control of the trcRBS promoter.
  • MA-0933 is as described in Example 15. Induction of T.
  • fusca metA expression in each of these high homoserine sfrains resulted in accumulation of O- acetylhomoserine in culture broths.
  • strain MA-1559 displayed OAH levels in excess of 9 g/L. Additional manipulations can be perfonned to elicit conversion of OAH to other products, including methionine.
  • Example 17 Effects of metA variants on methionine production in C. glutamicum.
  • C. glutamicum homoserine acetyltransferase (MetA) variants were generated by site- directed mutagenesis of MetA-encoding DNA (Example 6).
  • C. glutamicum strains MA-0622 and MA-0699 were transformed with a high copy plasmid, MB4236, that encodes MetA with a lysine to alanine mutation at position 233 (MetA (K233A)). This plasmid also contains a wild- type copy of the C. glutamicum metY gene.
  • Sfrain MA-0699 was constructed by transforming MA-0622 with plasmid MB4192 to delete the hom-thrB locus and integrate the gpd- S.
  • coelicolor hom(G362E) expression cassette metA and metY axe expressed in a synthetic metAY operon under control of a modified version of the trc promoter.
  • the strains were cultured in the presence and absence of IPTG induction, and methionine productivity was assayed. Methionine production from each sfrain is plotted in Figure 24. As shown, individual transformants of MA- 622 and MA-699, when cultured under inducing conditions, each produced over 3000 ⁇ M methionine.
  • Example 17 Effects of metY variants on methionine production in C. glutamicum
  • C. glutamicum O-acetylhomoserine sulfhydrylase (MetY) variants were generated by site-directed mutagenesis of MetY-encoding DNA (Example 6).
  • C. glutamicum strain MA-622 and strain MA-699 were fransformed with a high copy plasmid, MB4238, that encodes MetY with an aspartate to alanine mutation at position 231 (MetY (D231 A)). This plasmid also contains the wild-type copy of the C. glutamicum metA gene, expressed as in Example 16.
  • the strains were cultured in the presence and absence of IPTG induction, and methionine productivity was assayed.
  • the methionine production from each strain is plotted in Figure 25.
  • individual fransfonnants of MA-622 when cultured under conditions in which expression of MetY (D231A) was induced, each produced over 1800 ⁇ M methionine.
  • MA-622 strains showed variation in the levels of methionine produced by individual transformants (i.e., fransformants 1 and 2 produced approx. 1800 ⁇ M methionine when induced, whereas transformants 3 and 4 produced over 4000 ⁇ M methionine when induced).
  • MA-699 strains which express an S.
  • coelicolor Hom variant produced approximately 3000 ⁇ M methionine in the absence of IPTG.
  • IPTG induction increased methionine production by 1500-2000 ⁇ M.
  • C. glutamicum sfrain MA-622 and strain MA-699 were fransformed with a high copy plasmid, MB4239, that encodes MetY with a glycine to alanine mutation at position 232 (MetY (G232A)).
  • the sfrains were cultured in the presence and absence of IPTG induction, and methionine productivity was assayed. The methionine production from each sfrain is plotted in Figure 26.
  • Methionine production was assayed in five different C. glutamicum strains. Four of these strains express a unique combination of episomal C. glutamicum metA and metY alleles, as listed in Table 14. A fifth strain, MA-622, does not contain episomal metA or metY alleles. The amount of methionine produced by each strain (g/L) is listed in Table 14.
  • Example 19 Combinations of genetic manipulations, using both heterologous and native genes, elicits production of aspartate-derived amino acids
  • FIG. 27 shows the production of several aspartate- derived amino acids by strains MA-2028 and MA-2025 along with titers from their parent strains MA-1906 and MA-1907, respectively.
  • MA-1906 was constructed by using plasmid MB4276 to delete the native pck locus in MA-0622 and replace pck with a cassette for constitutive expression of the M. smegmatis lysC(T31l ⁇ )-asd operon.
  • MA-1907 was generated by similar transformation of MB4276 into MA-0933.
  • MA-2028 and MA-2025 were constructed by transformation of the respective parents with MB4278, an episomal plasmid for inducible expression of a synthetic C. glutamicum metAYH operon (see Example 3).
  • Parent sfrains MA- 1906 and MA-1907 produce lysine or lysine and homoserine, respectively; methionine and methionine pathway intermediates are also produced by these strains.
  • the scale for lysine and homoserine is on the left y-axis; the scale for methionine and O-acetylhomoserine is on the right y-axis.
  • IP TG induction MA-2028 showed a decrease in lysine levels and an increase in methionine levels.
  • MA-2025 also displayed an IPTG-dependent decrease in lysine production, together with increased production of methionine and O-acetylhomoserine.
  • Strain MA- 1743 is another example of how combinatorial engineering can be employed to generate strains that produce methionine.
  • MA- 1743 was generated by transformation of MA-
  • MA- 1667 with metAYH expression plasmid MB4278 was constructed by first engineering strain MA-0422 (see Example 15) with plasmid MB4084 to delete thrB, and next using plasmid MB4286 to both delete the mcbR locus and replace mcbR with an expression cassette containing trcRBS-T. fusca metA.
  • expression does not appear to be as tightly regulated as seen with the episomal plasmids (as judged by amino acid production). Thismay be due to decreased levels of the laclq inhibitor protein.
  • IPTG induction of strain MA- 1743 elicits production of methiomne and pathway intermediates, including O-acetylhomoserine ( Figure 28; the scale for lysine and homoserine is on the left y-axis; the scale for methionine and O-acetylhomoserine is on the right y-axis).
  • MA-1688 and MA-1790 are two additional strains that were engineered with multiple genes, including the MB4278 metAYH expression plasmid (see Figure 29; the scale for lysine and homoserine is on the left y-axis; the scale for methionine and O-acetylhomoserine is on the right y-axis).
  • MA-0569 was constructed by sequentially using MB4192 and MB4165 to first delete the hom-thrB locus and integrate the gpd- S. coelicolor hom(G362E) expression cassette and then delete mcbR.
  • MA- 1790 construction required several steps.
  • a NTG mutant derivative of MA-0428 was identified based on its ability to allow for growth of a Salmonella metE mutant.
  • a population of mutagenized MA-0428 cells was plated onto a minimal medium containing tlireonine and a lawn (>10 6 cells of the Salmonella metE mutant).
  • the Salmonella metE mutant requires methionine for growth.
  • the corynebacteria colonies e.g. MA- 0600
  • surrounded by a halo of Salmonella growth were isolated and subjected to shake flask analysis.
  • Strain MA-600 was next mutagenized to ethionine resistance as described above, and one resulting sfrain was designated MA-0993.
  • Figure 30 shows the metabolite levels of sfrain MA-1668 and its parent sfrains.
  • the scale for lysine and homoserine is on the left y-axis; the scale for methionine and O-acetylhomoserine is on the right y-axis.
  • Strain MA-1668 was generated by transformation of MA-0993 with plasmid MB4287. Manipulation with MB4287 results in deletion of the mcbR locus and replacement with C. glutamicum metA(K233A)-metB.
  • Strain MA-1668 produces approximately 2 g/L methionine, with decreased levels of lysine and homoserine relative to its progenitor sfrains. Strain MA-1668 is still amenable to further rounds of molecular manipulation.
  • EthR6 and EthRlO represent independently isolated ethionine resistant mutants.
  • the Mcf3 mutation confers the ability to enable a Salmonella metE mutant to grow (see example 19).
  • the Mmsl3 mutation confers methionine methylsulfonium chloride resistance (see example 15).
  • Table 16 Amino acid sequences of exemplary heterologous proteins for amino acid production in Escherichia coli and corynefonn bacteria.
  • the NC number under the Gene column corresponds to the Genbank® protein record for the conesponding Corynebacterium glutamicum gene.
  • Table 17 Nucleotide sequences of exemplary heterologous proteins for amino acid production in Escherichia coli and coryneform bacteria. Note: This table provides coding sequences of each gene. Some GenBank® entries contain additional non-coding sequence associated with the gene.
  • CoryneX57226 ATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGGT 240 bacterium CGGCCAGGTTATGCGCACCCTTTTGGAAGAGCGCAATT glutamicum TCCCAGCTGACACTGTTCGTTTCTTTGCTTCCCCACGT TCCGCAGGCCGTAAGATTGAATTCCGTGGCACGGAAAT CGAGGTAGAAGACATTACTCAGGCAACCGAGGAGTCCC TCAAGGACATCGACGTTGCGTTGTTCTCCGCTGGAGGC ACCGCTTCCAAGCAGTACGCTCCACTGTTCGCTGCTGC AGGCGCGACTGTTGTGGATAACTCTTCTGCTTGGCGCA AGGACGACGAGGTTCCACTAATCGTCTCTGAGGTGAAC CCTTCCGACAAGGATTCCCTGGTCAAGGGCATTATTGC GAACCCTAACTGCACCACCATGGCTGCGATGCCAGTGC TGAAGCCACTTCACGATGCCGCTGGTCTTGTAAAGCTT CACGTTTCCTCTTACCAGGCTGTTTCCG
  • Escherichia NC 000913 ATGAAAAATGTTGGTTTTATCGGCTGGCGCGGTATGGT 241 coll CGGCTCCGTTCTCATGCAACGCATGGTTGAAGAGCGCG ACTTCGACGCCATTCGCCCTGTCTTCTTTTCTACTTCT CAGCTTGGCCAGGCTGCGCCGTCTTTTGGCGGAACCAC TGGCACACTTCAGGATGCCTTTGATCTGGAGGCGCTAA AGGCCCTCGATATCATTGTGACCTGTCAGGGCGGCGAT TATACCAACGAAATCTATCCAAAGCTTCGTGAAAGCGG ATGGCAAGGTTACTGGATTGACGCAGCATCGTCTCTGC GCATGAAAGATGACGCCATCATCATTCTTGACCCCGTC AATCAGGACGTCATTACCGACGGATTAAATAATGGCAT CAGGACTTTTGTTGGCGGTAACTGTACCGTAAGCCTGA TGTTGATGTCGTTGGGTGGTTTATTCGCCAATGATCTT GTTGATTGGGTGTCCGTTGCAACCTACCAGGC

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BRPI0614716A2 (pt) * 2005-08-05 2011-04-12 Univ Michigan State método para produção de um produto quìmico a partir da via c4 de a. succinogenes
JP2007068437A (ja) * 2005-09-05 2007-03-22 Chisso Corp 変異型アスパルトキナーゼとその利用方法
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EP2520645B1 (de) 2007-04-11 2015-01-14 CJ CheilJedang Corporation Zusammensetzungen und Verfahren zur Herstellung von Methionin
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KR101485222B1 (ko) * 2012-10-05 2015-01-22 상지대학교산학협력단 ｄａｐＡ 활성 조절을 통해 Ｌ-쓰레오닌 생산능이 향상된 코리네박테리움 속 미생물 및 이를 이용한 Ｌ-쓰레오닌 생산방법
KR101947959B1 (ko) 2018-05-28 2019-02-13 씨제이제일제당 (주) 변이형 호모세린 디하이드로게나제 및 이를 이용한 호모세린 또는 호모세린 유래 l-아미노산의 생산 방법
KR101996769B1 (ko) 2018-12-21 2019-10-01 씨제이제일제당 (주) 변이형 호모세린 디하이드로게나제 및 이를 이용한 호모세린 또는 호모세린 유래 l-아미노산의 생산 방법
AU2020292062B2 (en) * 2019-06-14 2024-01-18 Cj Cheiljedang Corporation Composition for preventing, treating, or improving gastrointestinal diseases comprising strain of genus Corynebacterium and culture thereof
KR102472559B1 (ko) * 2019-06-28 2022-12-01 씨제이제일제당 주식회사 황 함유 아미노산 또는 그 유도체의 제조방법
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