WO2017194696A1 - Cellules bactériennes à tolérance améliorée pour l'acide isobutyrique - Google Patents

Cellules bactériennes à tolérance améliorée pour l'acide isobutyrique Download PDF

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WO2017194696A1
WO2017194696A1 PCT/EP2017/061379 EP2017061379W WO2017194696A1 WO 2017194696 A1 WO2017194696 A1 WO 2017194696A1 EP 2017061379 W EP2017061379 W EP 2017061379W WO 2017194696 A1 WO2017194696 A1 WO 2017194696A1
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rpob
acid
mutation
ilvh
ilvn
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Rebecca Lennen
Alex Toftgaard Nielsen
Markus HERRGÅRD
Morten Sommer
Adam FEIST
Elsayed Tharwat Tolba MOHAMED
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Danmarks Tekniske Universitet
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
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    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01014—Glycine-tRNA ligase (6.1.1.14)

Definitions

  • the present invention relates to bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as isobutyric acid, and to methods of preparing and using such bacterial cells for production of isobutyric acid and other compounds.
  • Isobutyric acid or 2-methylpropanoic acid
  • is a high-production volume chemical >500 tons/year
  • isobutyrated products such as diisopropyl ketone and isopropyl esters (which are used in perfumes, oils for aircraft turbines, and solvents) (Riemenschneider et al. , 2012).
  • Isobutyrate salts are used to produce chemicals for the tanning and textile industries and for use as stabilizers, preservatives, and catalysts.
  • Isobutyric acid may also be used as a precursor for chemical conversion to methacrylic acid via oxidative dehydrogenation, followed by esterification with methanol to generate methyl methacrylate (MMA); the direct precursor for production of polymethylmethacrylate (PMMA) plastics (Bauer et ai, 2012).
  • isobutyric acid can be produced biologically from glucose via the same upstream pathway to
  • isobutyraldehyde that is employed for isobutanol production in E. coli (Zhang et ai , 2011), but instead employing, e.g. , an isobutyraldehyde dehydrogenase to oxidize isobutyraldehyde to isobutyric acid, and deleting the gene encoding a native alcohol dehydrogenase that ordinarily reduces isobutyraldehyde to isobutanol (Atsumi et a/., 2010; WO 2012/109534 A2).
  • Escherichia coli being a suitable host for industrial applications, there has been some interest in developing E. coli strains with improved tolerance to chemicals of interest for production, such as, e.g., n-butanol, ethanol and isobutanol, or to stress conditions present during fermentation (see, e.g. , Sandberg et al., 2014; Lennen and Herrgard, 2014; Tenaillon et al. , 2012; Minty et al. , 2011; Dragosits et al. , 2013; Winkler et al. , 2014; Wu et al., 2014;
  • the invention relates to bacterial cells with improved tolerance to at least isobutyric acid, as well as bacterial cells which are capable of producing isobutyric acid or another branched- or straight-chain aliphatic acid or alcohol, and have improved tolerance to certain chemicals, including isobutyric acid.
  • the invention also relates to compositions comprising such bacterial cells and isobutyric acid or a related compound, methods of preparing or screening for such bacterial cells, and methods of producing isobutyric acid or a related chemical compound using such bacterial cells.
  • various aspects of the invention provide for genetically modified bacterial host cells with a higher tolerance to isobutyric acid or other, related chemical compounds of interest.
  • the genetically modified bacterial host cells of the invention result in improved production of the compound from carbon feedstock, since they maintain robust metabolic activity in the presence of higher concentrations of the compound than the unmodified parent cells.
  • the bacterial cell comprises a recombinant biosynthetic pathway for producing isobutyric acid and at least one genetic modification which reduces expression of an endogenous gene selected from the group consisting of pykF, yobF, yjcF, rph, cheR and phoil, such as from the group consisting of pykF, yobF and phoU, or a combination of any thereof.
  • the bacterial cell comprises a genetic modification which reduces the expression of pykF, yobF or both.
  • the recombinant biosynthetic pathway provides for a production of more than 10 mg/L isobutyric acid within 48 hours of culture in the presence of a suitable carbon source.
  • the bacterial cell comprises a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of rpoS, yobF, phoU, or a combination thereof; (b) a mutant GlyQ (SEQ ID NO:9) comprising a mutation in residue E48; (c) a mutant RpoB (SEQ ID NO: 18) comprising at least one mutation that alters the structure of the rifampicin binding pocket, such as a mutation in at least one of residues H526, S531, R540, Q513, F514 and D516; (d) a mutant RpoB (SEQ ID NO: 18) comprising a mutation in residue A1183; (e) a mutant SapC (SEQ ID NO: 20) comprising a mutation in
  • mutations include, but are not limited to, GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB- A1183I, RpoB-A1183L, RpoB-A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB- H526T, RpoB-H526F, RpoB-H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L,
  • the invention in another aspect, relates to a bacterial cell comprising a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of yobF, rpoS or both; and/or (b) mutant GlyQ comprising a mutation in residue E48.
  • the bacterial cell also comprises a mutant regulatory subunit of acetohydroxybutanoate synthase/acetolactate synthase (AHAS) and a genetic modification which increases the expression of pyrE; the mutant regulatory subunit of AHAS providing for feedback-resistance to inhibition by L-valine, L-leucine or both.
  • AHAS acetohydroxybutanoate synthase/acetolactate synthase
  • the mutant regulatory subunit of AHAS may, for example, be a mutant IlvH or a mutant IlvN.
  • the bacterial cell comprises genetic modifications reducing the expression of pykF and one or both of yobF and rpoS.
  • the genetic modification can be a knock-down or a knock-out, e.g., a knock-out.
  • the bacterial cell may further comprise a recombinant biosynthetic pathway for producing a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid.
  • the recombinant biosynthetic pathway may produce a C3 to C8 branched-chain or straight-chain aliphatic acid, or a C3 to C5 branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid.
  • the recombinant biosynthetic pathway may produce a C3 to C8 branched-chain or straight-chain aliphatic acid, or a C3 to C5 branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid.
  • recombinant pathway is for producing a C4 to C8 branched-chain or straight-chain aliphatic acid.
  • a bacterial cell having improved tolerance may, for example, have an increased growth rate, reduced lag time, or both, in at least one of isobutyrate, butyric acid, valeric acid, 2- methylbutyric acid, isovaleric acid, 4-methylvaleric acid, 2-methylhexanoic acid, 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol and 1-pentanol, as compared to the parent bacterial cell.
  • the one or more genetic modifications or mutants increase the growth rate, reduces the lag time, or both, of the bacterial cell in about 1 g/L, about 6 g/L and/or about 12 g/L isobutyric acid or isobutyrate. In one embodiment, the bacterial cell has an increased growth rate, reduced lag time, or both, in at least about 6 g/L isobutyrate.
  • the invention relates to a bacterial cell comprising a recombinant biosynthetic pathway for producing isobutyric acid and a mutant regulatory subunit of AHAS, the mutant regulatory subunit of AHAS providing for feedback-resistance to inhibition by L-valine, L- leucine or both.
  • a process for preparing a recombinant E. coli cell for producing isobutyric acid or a related compound may comprise genetically modifying an E.
  • the process comprises comprising genetically modifying an E.
  • coli cell to knockdown or knock-out at least one endogenous gene selected from the group consisting of pykF, yobF, and phoU, and (a) further knocking-down or knocking-out the endogenous gene rpoS; (b) introducing into the E.
  • coli cell a recombinant biosynthetic pathway for producing isobutyric acid or a related compound, optionally wherein the recombinant biosynthetic pathway provides for a production of more than 10 mg/L isobutyric acid within 48 hours of culture in the presence of a carbon source; and/or (c) introducing at least one mutation selected from GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB-A1183I, RpoB-A1183L, RpoB- A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB-H526T, RpoB-H526F, RpoB- H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L, RpoB-D
  • a process for improving the tolerance of an E. coli cell to a C3 to C8 branched- chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid may comprise genetically modifying the E. coli cell to knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, rph, yjcF, yobF, cheR and phoU.
  • such processes may further comprise genetically modifying the E. coli cell to express a mutant GlyQ, express a mutant IlvH, express a mutant IlvN,
  • overexpress PyrE or a combination of any thereof.
  • a process for improving the tolerance of an E. coli cell to a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid comprises (a) genetically modifying an E. coli cell to knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, yobF, and phoU, optionally further knocking-down or knocking-out the endogenous gene rpoS; (b) preparing a population of the genetically modified E.
  • coli cell which population comprises one or more mutations in one or more endogenous genes selected from glyQ, rpoB, sapC, spsD, ilvN, ilvH, rph and the pyrE/rph intergenic region, and (c) selecting any E. coli cell which has an improved tolerance.
  • the invention relates to a method for producing a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid, comprising culturing the bacterial cell of any aspect or embodiment described herein, or a bacterial cell obtained or obtainable by the process of any aspect or embodiment described herein, in the presence of a carbon source.
  • the invention relates to a composition
  • a composition comprising isobutyric acid, butyric acid, valeric acid, 2-methylbutyric acid, isovaleric acid, 4-methylvaleric acid, 2-methylhexanoic acid, 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol and 1- pentanol at a concentration of at least about 1 g/L, at least 6 g/L or at least about 12 g/L, and a plurality of bacterial cells according to any aspect or embodiment herein.
  • the composition comprises isobutyric acid and a plurality of bacterial cells according to any aspect or embodiment described herein, a bacterial cell obtained or obtainable by the process of any aspect or embodiment described herein, wherein the isobutyric acid is present at a concentration of at least about 1 g/L.
  • Organic compounds may be described herein by their IUPAC name and/or other synonym known in the art.
  • isobutyric acid is also known as 2- methylpropanoic acid, 2-methylpropionic acid and isobutanoic acid.
  • a "related compound" to isobutyric acid includes, but is not limited to, other branched-chain aliphatic acids such as isovaleric acid (3-methylbutanoic acid), 2-methylbutanoic acid, 2- methylhexanoic acid, 4-methylvaleric acid, 3-methylhexanoic acid, 3-methyl-2-hexenoic acid, and other unsaturated derivatives and/or salts thereof; other straight-chain aliphatic acids such as butyric acid, valeric acid (pentanoic acid), hexanoic acid (caproic acid), octanoic acid (caprylic acid), propanoic acid, and unsaturated derivatives thereof and/or salts thereof; and branched- or straight-chain aliphatic alcohols including isobutanol, isopropanol, n-pentanol, n-hexanol, n-octanol, n-butanol, 1-propanol, 2-
  • the related compound comprises a 3- to 8-carbon (C3 to C8) branched or straight aliphatic chain. More preferably, the related compound comprises a C4 to C8 branched-chain or straight- chain aliphatic acid or a C3 to C5 branched-chain or straight-chain aliphatic alcohol. Most preferred are related compounds characterized by the presence of a carboxylic acid or carboxylate functionality and a branched or 4-carbon (C4) straight aliphatic carbon chain, such as e.g. , 2-methylbutanoic acid, isovaleric acid and butyric acid.
  • any embodiment pertaining to a carboxylic acid herein equally pertains to its anion and/or salt, formed by the deprotonation of the carboxylic acid group.
  • any embodiment pertaining to isobutyric acid includes isobutyrate (e.g., the predominant form in which isobutyric acid exists at a pH above the relevant pK a ).
  • a "recombinant biosynthetic pathway" for a compound of interest refers to an enzymatic pathway resulting in the production of a compound of interest in a host cell, wherein at least one of the enzymes is expressed from a transgene, i.e. , a gene added to the host cell genome by transformation.
  • the recombinant biosynthetic pathway also comprises a deletion of one or more native genes in the host cell.
  • the compound of interest is typically isobutyric acid or a related compound, and may be the actual end product or a precursor or intermediate in the production of another end product.
  • tolerant when used to describe a genetically modified bacterial cell of the invention or a strain derived therefrom, refers to a genetically modified bacterial cell or strain that shows a reduced lag time, an improved growth rate, or both, in the presence of isobutyric acid than the parent bacterial cell or strain from which it is derived, typically at concentrations of at least 1 g/L, such as at least 2.5 g/L, such as at least 4 g/L, such as at least 6 g/L, such as at least 6.3 g/L, such as at least 7.5 g/L, such as at least 10 g/L, such as at least 12.5 g/L, such as at least 15 g/L, such as at least 20 g/L.
  • concentrations of at least 1 g/L such as at least 2.5 g/L, such as at least 4 g/L, such as at least 6 g/L, such as at least 6.3 g/L, such as at least 7.5 g/L, such as at
  • An improved growth rate is at least 5%, such as at least 10%, such as at least 20%, such as at least 50%, such as at least 75% higher than that of a control, typically the parent cell or strain.
  • a reduced lag time is at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 90% shorter than that of a control, typically the parent cell or strain.
  • gene refers to a nucleic acid sequence that encodes a cellular function, such as a protein, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • An "endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • a “transgene” is a gene, native or heterologous, that has been introduced into the genome by a transformation procedure.
  • Gene names are herein set forth in italicised text with a lower-case first letter (e.g., pykF) whereas protein names are set forth in normal text with a capital first letter (e.g., PykF) .
  • coding sequence refers to a DNA sequence that encodes a specific amino acid sequence.
  • nucleic acid or amino acid sequence as found in the host cell.
  • heterologous when used to characterize a gene or protein with respect to a host cell, refers to a gene or protein which has a nucleic acid or amino acid sequence not normally found in the host cell.
  • transformation refers to the transfer of a nucleic acid fragment, such as a gene, into a host cell .
  • Host cells containing a gene introduced by transformation or a "transgene” are referred to as “transgenic” or “recombinant” or “transformed” cells.
  • a “genetic modification” refers to the introduction a genetically inherited change in the host cell genome. Examples of changes include mutations in genes and regulatory sequences, coding and non-coding DNA sequences. “Mutations” include deletions, substitutions and insertions of one or more nucleotides or nucleic acid sequences in the genome. Other genetic modifications include the introduction of heterologous genes or coding DNA sequences by recombinant techniques.
  • expression refers to the process in which a gene is transcribed into mRNA, and may optionally include the subsequent translation of the mRNA into an amino acid sequence, i. e. , a protein or polypeptide.
  • reduced expression or “downregulation" of an endogenous gene in a host cell means that the levels of the mRNA, protein and/or protein activity encoded by the gene are significantly reduced in the host cell, typically by at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control .
  • the control is the unmodified host cell.
  • the reduction of native mRNA and functional protein encoded by the gene is higher, such as 99% or greater.
  • “Increased expression”, “upregulation”, “overexpressing” or the like when used in the context of a protein or activity described herein, means increasing the protein level or activity within a bacterial cell.
  • An increase in protein level can be achieved by, e.g., a mutation in the promoter region or other neighbouring segment providing for increased expression of an endogenous gene, the expression of a transgene encoding the protein, or other techniques known in the art.
  • An up-regulation of an activity can occur through, e.g. , increased activity of a protein, increased potency of a protein or increased expression of a protein.
  • the protein with increased activity, potency or expression can be encoded by genes disclosed herein.
  • Genetic modifications resulting in a reduced expression of a target gene/protein can include, e.g. , knock-down of the gene (e.g. , a mutation in a promoter that results in decreased gene expression), a knock-out of the gene (e.g. , a mutation or deletion of the gene that results in 99 percent or greater decrease in gene expression), a mutation or deletion in the coding sequence which results in the expression of non-functional protein, and/or the introduction of a nucleic acid sequence that reduces the expression of the target gene, e.g. a repressor that inhibits expression of the target or inhibitory nucleic acids (e.g. CRISPR etc.) that reduces the expression of the target gene.
  • a nucleic acid sequence that reduces the expression of the target gene
  • a repressor that inhibits expression of the target or inhibitory nucleic acids (e.g. CRISPR etc.) that reduces the expression of the target gene.
  • a “conservative" amino acid substitution in a protein is one that does not negatively influence protein activity.
  • a conservative substitution can be made within groups of amino acids sharing physicochemical properties, such as, e.g. , basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagines), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, and threonine).
  • substitutions can be made between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • Other preferred substitutions are set out in Table 1 below.
  • lag times of native K-12 MG1655 cells began increasing already at isobutyric acid concentrations above 1 g/L, and at concentrations above 10 g/L, growth was almost completely inhibited.
  • bacterial cells comprising one or more mutations according to the invention exhibit a dramatically improved growth at high concentrations isobutyric acid, e.g., 1 g/L or more, typically reflected by an increased growth rate, a reduced lag time, or both.
  • the invention provides bacterial cells with improved tolerance to isobutyric acid and related compounds, as well as related processes and materials for producing and using such bacterial cells.
  • the genetic modifications according to the invention include those resulting in reduced expression of genes, e.g. , by gene knock-down or knock-out, herein referred to as "Group 1 modifications"; as well as silent mutations in coding or non-coding regions and non-silent
  • Group 2 modifications (I.e. , coding) mutations in coding regions, herein referred to as "Group 2 modifications"; and combinations thereof, as described below. a) Group 1 modifications
  • the bacterial cell has a genetic modification which reduces the expression of one or more endogenous genes listed in Table 4, including, but not limited to pykF, rph, yjcF, rpoS, yobF, cheR, and phoil.
  • the bacterial cell has a genetic modification which reduces the expression of one or more endogenous genes selected from the group consisting of pykF, yobF and phoU.
  • the one or more endogenous genes comprise pykF.
  • a bacterial cell which comprises genetic modifications reducing the expression of at least two endogenous genes selected from the group consisting of pykF, rph, yjcF, rpoS, yobF, cheR and phoU.
  • the bacterial cell comprises a first genetic modification which reduces the expression of a gene selected from pykF, rpoS, and yobF, and, optionally, a second genetic modification which reduces the expression of rpoS.
  • the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of a gene selected from rpoS, yobF, and phoil. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of rpoS. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of yobF. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF, a second genetic modification which reduces the expression of rpoS, and a third genetic modification which reduces the expression of yobF.
  • the bacterial cell comprises: - a first genetic modification which reduces the expression of rph, and a second genetic modification which reduces the expression of a gene selected from pykF, yjcF, rpoS, yobF, cheR and phoil;
  • a first genetic modification which reduces the expression of yjcF and a second genetic modification which reduces the expression of a gene selected from of pykF, rph, rpoS, yobF, cheR and phoil;
  • a first genetic modification which reduces the expression of cheR and a second genetic modification which reduces the expression of a gene selected from pykF, rph, yjcF, rpoS, yobF and phoU;
  • a first genetic modification which reduces the expression of phoU and a second genetic modification which reduces the expression of a gene selected from pykF, rph, yjcF, rpoS, yobF and cheR.
  • Knock-down or knock-out of a gene can be accomplished by any method known in the art for bacterial cells, and include, e.g. , lambda Red mediated recombination, PI phage
  • a knock-down of a gene can be accomplished by, for example, a mutation in the promoter region or transcriptional regulator binding sites resulting in decreased transcription, a mutation in the ribosome binding site resulting in decreased translation, a deletion or mutation in the coding region of the gene resulting in a reduced or fully or substantially eliminated activity of the protein, or by the presence of antisense sequences that interfere with transcription or translation of the gene, resulting in reduced expression of the protein.
  • the knocking-down of a gene results in at least 20% reduction in the expression level of the gene product in the bacterial cell, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% or higher.
  • a knock-out of a gene includes elimination of a gene's expression, such as by introducing a mutation in the coding sequence and/or promoter so that at least a portion (up to and including all) of the coding sequence and/or promoter is disrupted or deleted deletion, mutation, or insertion, resulting in loss of expression of the protein, or expression only of a non-functional mutant or non-functional fragment of the endogenous protein.
  • the symbol "DELTA" denotes a deletion of an endogenous gene.
  • a knock-out of a gene results in 1% or less of the native gene product being detectable, such as no detectable gene product.
  • either one or both of the first and second genetic modifications is a knock-out of the gene, optionally a deletion.
  • at least one of the first and second genetic modifications is a knock-down of the gene.
  • the genetic modification is a knock-down or knock-out of the one or more endogenous genes, resulting in at least 20%, such as at least 50%, such as at least 80%, such as at least 90%, such as at least 95%, reduction in the level of mRNA encoded by the gene.
  • a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-down or knock-out of the one or more endogenous genes, resulting in at least 20%, such as at least 50%, such as at least 80%, such as at least 90%, such as at least 95%, reduction in the level of protein encoded by the gene.
  • a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-out of the one or more endogenous genes.
  • the bacterial cell of any aspect or embodiment described herein comprises a mutation in at least one of GlyQ, RpoB, SapC, RpsD, IlvH and/or in IlvN which provides for improved tolerance to isobutyric acid.
  • the bacterial cell comprises a mutation in GlyQ.
  • the bacterial cell comprises a mutation in RpoB and SapC.
  • the bacterial cell comprises a mutation in RpoB and RpsD.
  • the bacterial cell of any one of the preceding embodiments comprises a mutation which increases the expression level of PyrE.
  • a native or mutant protein can be expressed from a mutated version of the endogenous gene, or from a transgene.
  • a mutant protein is expressed in the bacterial cell, e.g., from a mutated version of the endogenous gene, or from a transgene encoding the mutant protein.
  • the bacterial cell comprises a mutation in GlyQ which increases tolerance to isobutyric acid.
  • the GlyQ comprises a mutation, such as a deletion or amino acid substitution, in residue E48 or in the residue that aligns with residue E48 in E. coli GlyQ.
  • the mutation is an amino acid substitution of this residue into D or N.
  • the mutation is E48D or a conservative amino acid substitution thereof, such as E48N.
  • the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein.
  • the Group 1 modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF.
  • the Group 2 modification can be, for example, a mutation in one or more of RpoB, SapC, RpsD, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic
  • the bacterial cell comprises one or more mutations which provide for, or increase, feedback-resistance to inhibition by L-valine, L-leucine, or both.
  • mutations which provide for, or increase, feedback-resistance to inhibition by L-valine, L-leucine, or both.
  • the three AHAS isoforms catalyze the first common steps in the biosynthesis of the branched chain amino acids, L-valine, L-isoleucine, and L-leucine, through complex allosteric inhibition mediated by the small regulatory subunits of each isoform (IlvH, IlvN, and IlvO) which can be inhibited by valine and/or leucine.
  • the bacterial cell comprises a mutant regulatory subunit of AHAS, e.g., in IlvH and/or IlvN.
  • Non-limiting examples of mutations in IlvH include a deletion or amino acid substitution in the N-terminal region, the C-terminal region, or both, such as (when aligned with E.
  • IlvH coli IlvH
  • L9F, L9A, L9V, G14D S17F, N11A, N29H, A36V, T34I, N29K, as well as conservative substitutions thereof
  • Non-limiting examples of mutations in IlvN include N17H, N17K, G20D, A30P, V21D, F34L, I44R, and I44F, as well as conservative substitutions thereof (EP 1 942 183 Al; Elisakova et a/. , 2005; Kopecky et a/. , 1999; US Patent Application No.
  • Preferred mutations include IlvH-L9F and IlvN-N17H.
  • the bacterial cell comprises one or more mutations which increase(s) the expression level or activity of PyrE.
  • E. coli K-12 MG1655 and W3110, plus their common ancestor strain W1485, are known to exhibit pyrimidine starvation in minimal media due to the presence a frameshift mutation occurring in rph relative to other E. coli strains (Jensen et al., 1993). This mutation disrupts the transcriptional/translational coupling required for efficient translation of pyrE, encoding orotate phosphoribosyltransferase in the pyrimidine biosynthesis pathway. Compensatory mutations that correct this deficiency are well-known in the art.
  • One of these mutations is an 82 bp deletion near the 3' terminus of rph, due to presence of two homologous GCAGAAGGC sequences flanking this 82 bp region (Conrad et al., 2009).
  • This mutation precisely corresponds to the 82 bp deletion found in resequenced isolates from population IBUA8 (from NC_000913.3 coordinates 3815859 to 3815931; Table 4).
  • a 1 bp deletion at coordinate 3815809 in the pyrE/rph intrgenic region has previously been encountered in strains evolved for growth on a minimal glucose medium (LaCroix et al., 2015), and a wide array of other frameshift mutations, substitutions, and coding mutations near the 3' terminus of rph were encountered in a short- term selection/evolution of combinatorial mutant libraries in minimal medium at an elevated temperature of 42°C (Sandberg et al., 2014).
  • the same 1 bp deletion in the pyrE/rph intergenic region was also found to be present in evolved isolate IBUA6-7.
  • the bacterial cell comprises mutations in rph or the pyrE/rph intergenic region, such as, e.g., an 82 bp deletion near the 3' terminus of rph, or 1 or 82 bp deletions in the intergenic region between pyrE and rph.
  • the bacterial cell comprises one or more mutations in RpoB, which is the beta subunit of RNA polymerase.
  • the mutation alters the structure of the rifampicin-binding pocket of RpoB, and may, for example, alter rifampicin binding to RpoB, confer rifampicin resistance, or both.
  • Non-limiting residues in the rifampicin-binding pocket of RpoB that are suitable for such mutations include H526, S531, R540, Q513, F514, and D516 (when aligned with E. coli RpoB), such as, e.g.
  • Preferred mutations are those in H526 that are known to confer rifampicin-resistance, such as H526Y, H526D, H526N, H526R and H526L, as well as conservative amino acid substitutions of H526Y, such as H526Y, H526W, H526T, H526F and H526S, with H526Y being most preferred.
  • the mutation in RpoB is in residue A1183, such as A1183V, A1183I, A1183L, A1183M, or A1183F, preferably A1183V.
  • suitable RpoB mutations see, e.g. , Kapur et al. , 1994; Ramaswamy et al., 2004;
  • the bacterial cell comprises a mutation in SapC which increases tolerance to isobutyric acid.
  • the SapC comprises a mutation, such as a deletion or amino acid substitution, in residue S69 or in the residue that aligns with residue S69 in E. coli SapC.
  • the mutation is an amino acid substitution of this residue into P or A.
  • the mutation is S69P or a conservative amino acid substitution thereof, such as S69A.
  • the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein.
  • the Group 1 modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF.
  • the Group 2 modification can be, for example, a mutation in one or more of GlyQ, RpoB, RpsD, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic
  • the bacterial cell comprises a mutation in RpsD which increases tolerance to isobutyric acid.
  • the RpsD comprises a mutation, such as a deletion or amino acid substitution, in residue G87 or in the residue that aligns with residue G87 in E. coli SapC.
  • the mutation is an amino acid substitution of this residue into C, S or A.
  • the mutation is G87C or a conservative amino acid substitution thereof, such as G87S or G87A.
  • the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein.
  • the Group 1 is a deletion or amino acid substitution, in residue G87 or in the residue that aligns with residue G87 in E. coli SapC.
  • the mutation is an amino acid substitution of this residue into C, S or A.
  • the mutation is G87C or a conservative amino acid substitution thereof, such as G87S or G87A.
  • the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or
  • the modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF.
  • the Group 2 modification can be, for example, a mutation in one or more of GlyQ, RpoB, SapC, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic modifications are described herein.
  • Preferred Group 2 mutations include those in GlyQ residue E48; RpoB residue H526 or
  • RpsD residue G87 and SapC residue S69 as well as vertain residues in IlvH and IlvN, such as, e.g., GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB-A1183I, RpoB-A1183L, RpoB- A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB-H526T, RpoB-H526F, RpoB- H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L, RpoB-D516V, RpoB-D516Y, RpoB-S531L, RpoB-S531W, SapC-S69P, SapC-S69A, R
  • the bacterial cell also or alternatively comprises one or more other mutations listed in Table 4, selected from RpoB-Q618L, RpoB-E565A, SapD-G235S, RpsC- E166K, YijD-L66M, RpoC-T757K, SapD-G235S, RpsC with a duplication of residues 128-132 (VMFRR), RpoB-N357H, RpoC-D622V and RpoC-D622A, SapF-R158C, RpoB-H526Y, SapB- V262G, RpsD-G87C, RpoB-A1183V, SapC-S69P, RpsL-A23V, and ptsP with an IS5 element insertion after nucleotide 858, or a combination of any two or more thereof.
  • VFRR residues 128-132
  • the bacterial cell comprises a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of one or more of yobF, rpoS, phoU; and/or (b) mutant GlyQ comprising a mutation in residue E48; and/or (c) a mutant RpoB comprising a mutation in residue H526 or A1183; and/or (d) a mutant RpsD comprising a mutation in residue G87; and/or (e) a mutant SapC comprising a mutation in residue S69.
  • the bacterial cell comprises a genetic modification which reduces the expression of pykF, and
  • a mutant RpoB comprising an A1183V, A1183I, A1183L, A1183M, or A1183F mutation:
  • a mutant GlyQ comprising an E48D or E48N mutation, a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyrE, and a mutant RpoB comprising an A1183V, A1183I, A1183L, A1183M, or A1183F mutation;
  • the bacterial cell comprises a mutation in GlyQ-E48 selected from E48D and E48N, and a knock-out or knockdown of at least one of pykF, rph, yjcF, rpoS, yobF, cheR and phoil, such as a knock-out or knock-down of at least one of pykF, yobF and PhoU, such as pykF;
  • GlyQ-E48 selected from E48D and E48N, and a knock-out or knockdown of pykF and at least one of yobF, phoil, and rpoS, such as rpoS or yobF;
  • GlyQ-E48 selected from E48D and E48N, a knockout or knockdown of pykF, increased expression of pyrE, and a mutation in IlvH providing for increased feedback-resistance to inhibition by at least L-valine;
  • GlyQ-E48 selected from E48D and E48N, a knockout or knockdown of pykF, increased expression of pyrE, and a mutation in IlvN providing for increased feedback-resistance to inhibition by at least L-valine;
  • a mutation in IlvH providing for increased feedback-resistance to inhibition by at least L-valine, and a knock-down or knock-out of pykF and at least one of rpoS, yobF, and phoil, such as rpoS.
  • a mutation in IlvN providing for increased feedback-resistance to inhibition by at least L-valine, and a knock-down or knock-out of pykF and at least one of rpoS, yobF, and phoil, such as rpoS.
  • RpoB a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, and a knock-down or knock-out of pykF.
  • RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF. and at least one of rpoS, yobF, and phoil, such as rpoS.
  • RpsD-G87 selected from G87C, G87S, and G87A.
  • RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or
  • A1183F a knock-down or knock-out of pykF, and at least one of rpoS, yobF, and phoil, such as rpoS.
  • RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or
  • A1183F a knock-down or knock-out of pykF.
  • RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or
  • RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or
  • A1183F a knock-down or knock-out of pykF, and a mutation in GlyQ-E48 selected from E48D and E48N.
  • a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or
  • RpoB which alters the rifampicin binding pocket such as H526Y, and a knock-down or knock-out of pykF.
  • RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF, and at least one of rpoS, yobF, and phoU, such as rpoS.
  • RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF, and a mutation in SapC-S69 selected from S69P and S69A.
  • the bacterial cell comprises an upregulation of at least one of GlyQ, PyrE, SapC, RpsD, RpoB, IlvH and/or IlvN, e.g., by transforming the bacterial cell with a transgene expressing the endogenous protein or a mutant thereof as described herein, e.g.
  • the copy number of a gene or genes encoding the protein may be increased.
  • a strong and/or inducible promoter can be used to direct the expression of the gene, the gene being expressed either as a transient expression vehicle or homologously or heterologously incorporated into the bacterial genome.
  • the promoter, regulatory region and/or the ribosome binding site upstream of the gene can be altered to achieve the over-expression.
  • the expression can also be enhanced by increasing the relative half-life of the messenger or other forms of RNA. Any one or a combination of these approaches can be used to effect upregulation of a desired target protein as needed.
  • Some bacterial species such as, e.g., Bacteroides ruminicola, have a native capability to produce isobutyrate and/or related compounds from a suitable carbon source.
  • Allison (1978) reported that on the order of 1 g/L (11 ⁇ / ⁇ _) of combined C4 and C5 branched-chain fatty acids, including a small proportion of isobutyric acid ( ⁇ 0.1 g/L), was obtained when cells of wild-type Bacteroides ruminicola and Megasphera elsdenii were subjected to batch culturing at 38°C for 120 h in media containing 1 g/L glucose as carbon source.
  • higher production levels e.g.
  • a recombinant biosynthetic pathway is typically needed.
  • a "recombinant synthetic pathway" for isobutyric acid provides a higher production level of isobutyric acid than 0.1 g/L, 0.5 g/L, 1 g/L, 5 g/L, 6 g/L, 10 g/L, 12 g/L, 20 g/L, 50 g/L, or 100 g/L within 120h, typically within 48 h, from a supplied carbon source, e.g. , about 1 g/L glucose.
  • a bacterial cell with improved tolerance to at least isobutyric acid according to any aspect or embodiment described herein, wherein the bacterial cell further comprises a recombinant biosynthetic pathway for producing isobutyric acid or a related compound, such as, e.g. , valeric acid, isovaleric acid, 2-methylbutanoic acid, butyric acid or isobutanol.
  • a recombinant biosynthetic pathway for producing isobutyric acid or a related compound such as, e.g. , valeric acid, isovaleric acid, 2-methylbutanoic acid, butyric acid or isobutanol.
  • the bacterial cell comprises a recombinant biosynthetic pathway for producing at least one of the C4 to C7 branched-chain aliphatic acids, or a C4 to C5 straight-chain aliphatic acid, optionally isobutyric acid, butyric acid, valeric acid, 2-methylbutyric acid, isovaleric acid, 4-methylvaleric acid, or 2- methylhexanoic acid.
  • the bacterial cell comprises a recombinant biosynthetic pathway for producing at least one of the C3 to C5 branched-chain or straight- chain aliphatic alcohols, optionally 1-propanol, 2-propanol, isobutanol, 3-methyl-l-butanol, 2-methyl-l-butanol, or 1-pentanol.
  • any such recombinant biosynthetic pathway which is known in the art can be introduced into the cell by standard recombinant technologies.
  • Biosynthetic pathways suitable for production of isobutyric acid and related compounds in bacteria are well-known in the art and have been described by, e.g. , Atsumi et al. (2007), Atsumi et al. (2010), Saini et al. (2014), Dhande et al. (2012), Jawed et al. (2016), Volker et al. (2014), Yu et al. (2016) and Zhang et al. (2011).
  • Example 1 “Production of isobutyric acid in evolved isolates.” It is to be understood that, when a specific enzyme of these biosynthetic pathways is mentioned by name such as, e.g. , "acetolactate synthase", the enzyme may be any characterized and sequenced enzyme, from any species, that have been reported in the literature so long as it provides the desired activity. In some embodiments, the enzyme is an overexpressed gene which is native to the host cell used. In some embodiments, the enzyme is a functionally active fragment or variant of an enzyme which is heterologous or native to the host cell.
  • the recombinant biosynthetic pathway comprises a knock-down or a knock-out of one or more genes, typically for the purpose of avoiding competing reactions reducing the yield of the desired isobutyric acid or a related compound.
  • the biosynthetic pathway is for isobutyric acid from pyruvate, and comprises genes, optionally overexpressed, encoding :
  • - acetolactate synthase e.g., AlsS from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from
  • acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (2S)-hydroxy-2-methyl-3-oxobutanoic acid to (2R)-2,3- dihydroxy-3-methylbutanoic acid;
  • IlvD dihydroxy acid dehydratase
  • KDC 2-ketoacid decarboxylase
  • aldehyde dehydrogenases such as 3-hydroxypropionaldehyde
  • AldH dehydrogenase
  • native alcohol dehydrogenases which actively convert isobutyraldehyde to isobutanol such as (in E. coli), YqhD, can be deleted (Zhang et al. , 2011).
  • the biosynthetic pathway is for 2-methylbutyric acid from L- threonine, and comprises genes, optionally overexpressed, encoding :
  • threonine deaminase e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;
  • AlsS acetolactate synthase
  • acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (S)-2-aceto-2-hydroxybutanoic acid to (R)-2,3-dihydroxy-3- methylpentanoic acid;
  • IlvD dihydroxy acid dehydratase
  • KDC 2-ketoacid decarboxylase
  • S decarboxylation of (S)-3-methyl-2-oxopentanoic acid to 2-methylbutyraldehyde
  • aldehyde dehydrogenases such as 3-hydroxypropionaldehyde dehydrogenase (AldH) from, e.g., E. coli or promiscuous native enzymes, catalyzing the oxidization of 2-methylbutyraldehyde to 2-methylbutyric acid.
  • AldH 3-hydroxypropionaldehyde dehydrogenase
  • native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC).
  • native alcohol dehydrogenases which actively convert 2-methylbutyraldehyde to 2-methylbutanol such as (in E. coli), YqhD, can be deleted (Zhang et a/. , 2011).
  • the biosynthetic pathway is for 3-methylbutyric acid from pyruvate, and comprises genes, optionally overexpressed, encoding:
  • AlsS acetolactate synthase
  • IlvC acetohydroxy acid isomeroreductase
  • IlvD dihydroxy acid dehydratase
  • (2S)-2-isopropylmalate hydro-lyase (LeuCD), e.g., from E. coli, catalyzing the dehydration, isomerization, and hydration of (2S)-2-isopropylmalate to (2S,3R)-3- isopropylmalate;
  • KDC 2-ketoacid decarboxylase
  • Lactococcus lactis KIVD catalyzing the decarboxylation of 4-methyl-2-oxopentanoate to 3-methylbutyraldehyde
  • aldehyde dehydrogenases such as 3-hydroxypropionaldehyde
  • AldH dehydrogenase
  • native alcohol dehydrogenases which actively convert 2-methylbutyraldehyde to 2-methylbutanol such as (in E. coli), YqhD, can be deleted (Zhang et al. , 2011).
  • the biosynthetic pathway is for butyric acid from L-threonine, and comprises genes, optionally overexpressed, encoding:
  • threonine deaminase e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;
  • KDC 2- ketoacid decarboxylase
  • aldehyde dehydrogenases such as 3-hydroxypropionaldehyde
  • AldH dehydrogenase
  • native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC).
  • native alcohol dehydrogenases which actively convert butyraldehyde to n-butanol, such as (in E. coli), YqhD, can be deleted (Zhang et ai , 2011).
  • the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding :
  • acetyl-CoA carboxylase e.g., from E. coli, catalyzing the carboxylation of acetyl-CoA to malonyl-CoA;
  • FAD malonyl-CoA-acyl carrier protein transacylase
  • beta-ketoacyl-[acyl carrier protein] synthase III (FabH), e.g., from E. coli, catalyzing the condensation of malonyl-ACP and acetyl-CoA to acetoacetyl-ACP;
  • enoyl-[acyl carrier protein] reductase Fabl
  • E. coli catalyzing the reduction of a frans-2-enoyl-ACP to an acyl-ACP
  • beta-ketoacyl-[acyl carrier protein] synthase I and/or II (FabB or FabA), e.g., from E. coli, catalyzing the condensation of an acyl-ACP with malonyl-ACP to form a 3- oxoacyl-ACP with two additional carbons compared to the substrate acyl-ACP; and, an butyryl-ACP thioesterase (TesBT), e.g. from Bacteroides thetaiotaomicron, catalyzing the thioesterification of butyryl-ACP to butyric acid and free ACP.
  • fatty acid degradation via the beta-oxidation pathway can be deleted via knockouts such as (in E. coli), fadD, fadE, fadB, fadA, fadR, or fabR, singly or in any combination.
  • the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding :
  • beta-ketothiolase e.g., from Ralstonia eutropha, catalyzing the condensation of two acetyl-CoAs to acetoacetyl-CoA;
  • Hbd 3-hydroxybutyryl-CoA dehydrogenase
  • Prt crotonase
  • frans-enoyl-CoA reductase e.g., from Terponema denticola, catalyzing the reduction of crotonyl-CoA to butyryl-CoA; and,
  • TesBT butyryl-CoA thioesterase
  • Bacteroides thetaiotaomicron catalyzing the thioesterification of butyryl-CoA to butyric acid and free CoA.
  • additional deletions can be made that prevent the conversion of acetyl-CoA and its glycolytic precursors to fermentative products, such as (in E. coli), frdA, IdhA, adhE, ackA, and pta, singly or in any combination.
  • the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding:
  • beta-ketothiolase e.g., from Ralstonia eutropha, catalyzing the condensation of two acetyl-CoAs to acetoacetyl-CoA;
  • Hbd 3-hydroxybutyryl-CoA dehydrogenase
  • crotonase e.g., from Clostridium acetobutylicum, catalyzing the dehydration of
  • frans-enoyl-CoA reductase e.g., from Terponema denticola, catalyzing the reduction of crotonyl-CoA to butyryl-CoA; and,
  • acetoacetyl-CoA transferase e.g. from E. coli, catalyzing the conversion of butyryl-CoA and acetate to butyric acid and acetyl-CoA.
  • the biosynthetic pathway is for valeric acid from L-threonine, and comprises genes, optionally overexpressed, encoding :
  • threonine deaminase e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;
  • (2S)-2-isopropylmalate hydro-lyase (LeuCD), e.g., from E. coli, catalyzing the dehydration, isomerization, and hydration of (R)-2-ethyl-2-hydroxysuccinic acid to (2R,3S)-2-ethyl-3-hydroxysuccinic acid, and the hydration of (R)-2-hydroxy-2- propylsuccinic acid to (2S,3R)-2-hydroxy-3-propylsuccinic acid;
  • LeuCD (2S)-2-isopropylmalate hydro-lyase
  • KDC 2-ketoacid decarboxylase
  • aldehyde dehydrogenases such as alpha-ketoglutaric semialdehyde dehydrogenase (KDH) from, e.g., Burkholderia ambifaria, or promiscuous native enzymes, catalyzing the oxidization of valeraldehyde to valeric acid.
  • KDH alpha-ketoglutaric semialdehyde dehydrogenase
  • native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC).
  • the biosynthetic pathway is for isobutanol from pyruvate, and comprises genes, optionally overexpressed, encoding :
  • acetolactate synthase e.g., AlsS from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from E. coli, catalyzing the conversion of pyruvate to (2S)-hydroxy-2-methyl-3- oxobutanoic acid;
  • IlvC acetohydroxy acid isomeroreductase
  • IlvD dihydroxy acid dehydratase
  • KDC 2-ketoacid decarboxylase
  • KIVD from Lactococcus lactis
  • 2-ketovaline to isobutyraldehyde
  • alcohol dehydrogenases such as, e.g., AdhA from Lactococcus lactis or promiscuous native enzymes, catalyzing the reduction of isobutyraldehyde to isobutanol.
  • a process for preparing a recombinant bacterial cell e.g. , an E. coli cell.
  • a process for improving the tolerance of a bacterial cell, e.g. , an E. coli cell, to isobutyric acid is also provided.
  • a method of identifying a bacterial cell which is tolerant to at least isobutyric acid Also provided is a process for preparing a recombinant bacterial cell, e.g. , an E. coli cell, for producing isobutyric acid or a related compound such as, e.g., isovaleric acid, 2-methylbutanoic acid, butyric acid, and isobutanol.
  • These processes may comprise one or more steps of genetically modifying a bacterial cell to knock-down or knock-out one or more endogenous genes of any aspect or embodiment of the Group 1 modifications and/or introducing one or more mutations in the endogenous protein(s) or gene(s) of any Group 2 aspect or embodiment.
  • This can be achieved by, e.g. , transforming the bacterial cell with genetic constructs, e.g. , vectors, antisense nucleic acids or siRNA, which effect the knock-out or knock-down or which introduce the mutation into the endogenous gene or encode the mutated protein from a transgene.
  • the genetic constructs can also comprise suitable regulatory sequences, typically nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • suitable regulatory sequences typically nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • Regulatory sequences may include promoters (e.g. , constitutive promoters or inducible promoters), translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
  • bacterial cells can be exposed to selection pressure (as described in the
  • the Group 1 modification is a knock-down or knock-out of one or more endogenous genes selected from pykF, rph, yjcF, rpoS, yobF, cheR and phoU, or a combination thereof, such as a knock-down or knock-out of pykF; yobF; phoU; pykF and rpoS; pykF and yobF; pykF and phoU; or pykF, rpoS and yobF.
  • the Group 2 modification is a mutation in at least one endogenous protein or gene selected from GlyQ, RpoB, SapC, RpsD, IlvH and IlvN, such as, e.g., a GlyQ-E48D, RpoB-H526Y, RpoB-A1183V, RpoB-Q618L, RpoB-E565A, RpoB-N357H, SapC-S69P, RpsD-G87C, IlvH-L9F, or IlvN-N17H mutation and/or a mutation which increases the expression of PyrE, such as, e.g. a mutation in rph or the pyrE/rph intergenic region.
  • a mutation which increases the expression of PyrE such as, e.g. a mutation in rph or the pyrE/rph intergenic region.
  • the process may comprise genetically modifying the E. coli cell to express a mutant GlyQ, express a mutant IlvH, express a mutant IlvN, overexpress PyrE, or a combination of any thereof.
  • the process may comprise a knock-out of knock-down of rpoS. In one embodiment, the process may comprise genetically modifying the E. coli cell to express a mutant RpoB and a mutant SapC, or a combination of any thereof.
  • the process may comprise genetically modifying the E. coli cell to express a mutant GlyQ, overexpress PyrE, and express a mutant RpoB, or a combination of any thereof. In one embodiment, the process may further comprise genetically modifying the E. coli cell to express a mutant RpoB and a mutant RpsD, or a combination of any thereof.
  • the processes may further comprise a step of selecting any bacterial cell which has an improved tolerance to isobutyric acid at a predetermined concentration, such as at least 3 g/L or higher, such as at least 5 g/L or higher, such as at least about 6 g/L, 6.3 g/L or higher, such as at least
  • Also provided is a method of producing isobutyric acid comprising culturing the bacterial cell obtained by any one of these methods, or the bacterial cell of any preceding aspect or embodiment, under conditions where isobutyric acid is produced.
  • these conditions include the presence of a suitable carbon source or mixes of different suitable carbon sources.
  • suitable carbon sources include, e.g. , sucrose, D-glucose, D-xylose, L-arabinose, glycerol, pyruvate, as well as hydrolysates produced from cellulosic or lignocellulosic materials. For further details see, e.g., Zhang et ai, 2011 and Atsumi et ai, 2007.
  • compositions A bacterial cell which has an increased tolerance to isobutyric acid or a related compound can be useful for preparing producer cells for one or more such compound.
  • Bacterial cells according to the invention may have an increased growth rate, an decreased lag time, or both.
  • the bacterial cell may have Group 1 and/or Group 2 modifications providing for an increased growth rate, a reduced lag time, or both, of the cell in at least one of the C3 or C4 to C8 branched-chain aliphatic acids, or at least one of the C3 or C4 to C8 straight-chain aliphatic acid, optionally isobutyric acid, butyric acid, valeric acid, 2- methylbutyric acid, isovaleric acid, 4-methylvaleric acid, or 2-methylhexanoic acid.
  • composition of a plurality of bacterial cells according to any aspect or embodiment described herein, e.g., a culture of such bacterial cells, optionally in a suitable culture medium or a medium comprising a carbon source.
  • composition comprising
  • the isobutyric acid or the related compound is present at a concentration at which the genetic modification(s) and/or mutant(s) comprised in the bacterial cells results in an improved tolerance.
  • concentrations at which bacterial cells according to the invention have improved tolerance are shown in Example 1, e.g., in "Cross-compound tolerance testing" (see, e.g., Table 22).
  • the concentration of isobutyric acid or the related compound is at least 1 g/L, such as at least 2 g/L, such as at least 2.5 g/L, such as at least 4 g/L, such as at least 6 g/L, such as at least 6.3 g/L, such as at least 7.5 g/L, such as at least 10 g/L, such as at least 12 g/L, such as at least 12.5 g/L, such as at least 15 g/L, such as at least 20 g/L, such as in the range of 1 to 300 g/L, such as in the range of 2 to 100 g/L, such as in the range of 4 to 50 g/L, such as in the range of 6 to 20 g/L.
  • the composition comprises isobutyric acid. In one embodiment, the composition comprises butyric acid. In one embodiment, the composition comprises valeric acid. In one embodiment, the composition comprises 2-methylbutyric acid. In one embodiment, the composition comprises isovaleric acid. In one embodiment, the composition comprises 4-methylvaleric acid. In one
  • the composition comprises 2-methylhexanoic acid. In one embodiment, the composition comprises 1-propanol. In one embodiment, the composition comprises 2- propanol. In one embodiment, the composition comprises isobutanol. In one embodiment, the composition comprises 2-methyl-l-butanol. In one embodiment, the composition comprises 3-methyl-l-butanol. In one embodiment, the composition comprises 1-pentanol.
  • the bacterial cells are of the Escherichia, Bacillus, Pseudomonas, Lactobaccillus or Lactococcus family, such as, e.g. , E. coll cells, and comprise
  • pykF, rph, yjcF, rpoS, yobF, cheR and phoil or a combination of any thereof, such as a knock-down or knock-out of pykF; yobF; phoU; pykF and rpoS; pykF and yobF; pykF and phoU; or pykF, rpoS and yobF;
  • the PyrE or mutant GlyQ, RpoB, SapC, RpsD, IlvH and/or IlvN can be expressed from a transgene.
  • Assays for assessing the tolerance of a modified bacterial cell to isobyturic acid or a related compound typically evaluate the growth rate, lag time, or both, of the bacterial cell at one or more predetermined concentrations of the compound, typically as compared to a control (e.g., no compound).
  • a control e.g., no compound.
  • the predetermined concentrations(s) could be, for example, 1 g/L, 6.3 g/L and/or 12.5 g/L isobutyric acid or isobutyrate.
  • the control is the native or unmodified parent cell or strain, and an improved tolerance is identified as an improved growth rate, a reduced lag-time or both.
  • an improved growth rate can be at least 5%, such as at least 10%, such as at least 20%, such as at least 50%, such as at least 75% higher than that of the control, while a reduced lag time can be at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 90% shorter than that of the control.
  • Specific assays are described, in detail, in the Examples.
  • strains, clones and other progeny of the bacterial cells of these and other aspects and embodiments typically refers to a group of cells which are descendants of a initial single colony of parent cells whereas a "clone” is a group of cells which are the descendants of an initial genetically modified single parent cell.
  • Non-limiting examples of bacterial cells suitable for modification according to any one of the aspects and embodiments described herein include bacteria of the Enterobacteriaceae, Bacillaceae, Ralstoniaceae, Pseudomonadaceae, Lactobacillaceae or Streptococcaceae families, particularly the Escherichia, Bacillus, Ralstonia, Pseudomonas, Lactobacillus and Lactococcus genera.
  • the bacterial cell is an E.
  • the bacterial cell such as a cell of the commercially available and/or fully characterized strains K-12 MG1655, BW25113, BL21, BL21(DE3), K-12 W3110, W, JM109, or Crooks (ATCC 8739).
  • the bacterial cell is derived from an E. coli K12 strain.
  • the bacterial cell is a Lactobacillus cell, such as a cell of the commercially available and/or fully characterized strains Lactobacillus plantarum JDM1, Lactobacillus plantarum WCFS1, and Lactobacillus plantarum NCIMB 8826.
  • the bacterial cell is a Lactococcus cell, such as a cell of the commercially available and/or fully characterized strains Lactococcus lactis lactis CV56, Lactococcus lactis lactis NIZO B40, and Lactococcus lactis cremoris NZ9000.
  • the bacterial cell is a Bacillus cell, such as a cell of the commercially available and/or fully characterized strains Bacillus subtilis 168 and Bacillus subtilis PY79.
  • the bacterial cell is a Pseudomonas cell, such as a cell of the commercially available and/or fully characterized strain Pseudomonas putida KT2440.
  • the bacterial cell is a Ralstonia cell, such as a cell of the commercially available and/or fully characterized strains Ralstonia eutropha H16 and Ralstonia eutropha JMP134.
  • While aspect and embodiments relating to bacterial cells herein typically refer to genes or proteins according to their designation in E. coli, for bacterial cells of another family or species, it is within the level of skill in the art to identify the corresponding gene or protein, e.g. , the homolog, ortholog and/or paralog, in the other family or species, typically by identifying sequences having moderate (typically ⁇ 30%) or high (typically ⁇ 50%) homology to the E. coli sequence, preferably taking the function of the protein expressed by the gene and/or the locus of the gene in the genome into account.
  • Table 2A sets out the function of the protein encoded by each specific gene, the corresponding E.C. number (if applicable), its locus in the E. coli K-12 MG1655 genome and the SEQ ID number of the coding or non-coding sequence and, where applicable, the encoded amino acid sequence.
  • Table 2B sets out some examples of homologs in selected organisms, identified in a preliminary and non-limiting analysis. Indeed, homologs of these proteins exist also in other bacteria, and other homologs not identified in this preliminary search can exist in the species listed in Table 2B. The skilled person is well-familiar with different searching and/or screening methods for identifying homologs across different species. To briefly summarize some of the preliminary findings in Table 2B:
  • residue aligning with residue E48 in E. coli GlyQ may not always be conserved, and may be, for example, an A (Ala or alanine) or S (Ser or serine).
  • RpoS was found in at least Bacillus, Pseudomonas, and Ralstonia.
  • E. coli gene Protein function E.C. Locus ID SEQ ID NO:
  • oligopeptide binding- "peptide ABC (219-306 293 aa) "ABC transport protein- transporter aa) "peptide transporter system dependent permease” ABC permease” permease transport (YP 00306265 transporter (YP_725975.1, protein AppC” system inner 3.1) permease” YP_726565.1,
  • each recited gene is instead (i) a gene encoding the corresponding protein in Table 2A or 2B above, (ii) a gene located at the corresponding locus, or (iii) both.
  • improved tolerance toward and endogenous production of isobutyric acid can be achieved by genetic modifications which alleviate L- isoleucine starvation due to intracellular accumulation of L-valine and/or L-leucine via mutations that provide feedback-resistance to inhibition by L-valine, L-leucine or both. This can, e.g., be achieved by a mutation in IlvH, IlvN, or one or more other genetic modifications described herein.
  • improved tolerance towards isobutyric acid can also be achieved by genetic modifications which reduce the cellular metabolic flux through lower glycolysis, reduce pyruvate accumulation and acetate formation, increase metabolic flux through the pentose-phosphate pathway, decrease expression of the RpoS regulon, decrease the activity of glycyl-tRNA synthetase, and/or increase transcription of pyrE.
  • a decreased activity of glycyl-tRNA synthetase can be achieved by a mutant GlyQ, e.g., E48D.
  • the bacterial cell has a genetic modification which reduces the expression of one or more endogenous proteins selected from the group consisting of - a pyruvate kinase
  • the bacterial cell further comprises a recombinant biosynthetic pathway for producing isobutyric acid.
  • the bacterial cell is of the Lactobacillus or Lactococcus genera. In one particular embodiment, the bacterial cell is of the Escherichia genera. In one particular embodiment, the bacterial cell is of the
  • the bacterial cell is of the Bacillus genera. In one particular embodiment, the bacterial cell is of the Ralstonia genera. EXAMPLE 1 Methods
  • Escherichia coli K-12 MG1655 was grown overnight in M9 minimal medium + 1% glucose and subcultured the following morning to an initial OD 600 of 0.05 in M9 + 1% glucose.
  • Cells were grown to mid-exponential phase (OD 500 0.7-1.0) and were back-diluted with fresh medium to an OD 500 of 0.7.
  • the diluted cells were used to inoculate M9 + 1% glucose containing varying concentrations of isobutyric acid (first neutralized to pH 7.0 with sodium hydroxide), and growth was measured in FlowerPlates in a Biolector microbioreactor system (m2p-labs) at 37°C with 1000 rpm shaking.
  • the culture volume in each well was 1.4 mL.
  • E. coli K-12 MG1655 was grown overnight in M9 minimal medium and 150 ⁇ _ was transferred the next day into 8 tubes containing 15 mL of M9 + 1% glucose + 3 g/L isobutyric acid on a Tecan Evo robotic platform custom-designed for performing adaptive laboratory evolutions (ALE).
  • Cells were cultured on a 37°C heat block with stirring by magnetic stir bars.
  • Culture OD 600 was monitored at times determined by a predictive custom script, and when the OD 600 reached approximately 0.3, 150 ⁇ of culture was inoculated into a new tube with the same media concentration. Instrument downtime would occasionally result in cells overgrowing to saturation or an OD 500 greater than 0.3, and reinoculations were occasionally performed from cryogenic stocks of the population.
  • Illumina TruSeq Nano kit was used according to the manufacturers' directions using an input quantity of 200 ng of genomic DNA from each isolate. Sequencing was performed on an Illumina MiSeq sequencer, with a minimum 20X average genomic coverage ensured for each isolate based on the number of reads. Fastq output files were analyzed for variants compared to the K-12 MG1655 reference genome (accession number NC_000913.3) using breseq.
  • Probable important losses-of-function were determined by identifying genes across all isolates that harbored mutations, especially those occurring in multiple populations, and by the presence of at least one mutation that either generated a premature stop codon, a frameshift mutation, or the presence of an insertion element sequence within the gene.
  • the corresponding knockout strain from the Keio collection of single knockout mutants was used as a donor strain for Plvir phage transduction (Baba et a/. , 2006).
  • the Keio strain was grown to early exponential phase in LB + 5 mM CaCI 2 and 80 ⁇ of a Plvir stock raised on K-12 MG1655 was added. After significant lysis was observed after 1.5 to 2 hours, the lysate was filter-sterilized to remove cells and stored at 4°C. Strain K-12 MG1655 was grown overnight in LB + 5 mM CaCI 2 and 100 ⁇ of the overnight culture was mixed with 100 ⁇ of the Plvir lysate of the Keio collection mutant, and the mixture was incubated at 37°C without shaking for 20 minutes.
  • the entire mixture was then plated on LB agar containing 1.25 mM sodium pyrophosphate as a chelating agent and 25 ⁇ g/mL kanamycin.
  • One colony was then restruck on LB + 1.25 mM Na 2 4 0 7 + 25 ⁇ g/mL kanamycin plate and analyzed for presence of the Keio cassette in place of the wild- type gene by colony PCR.
  • the Keio cassette was flipped out to generate a scar sequence such that Kan R marker could be recycled.
  • Keio collection mutants were inoculated directly from a cryogenic stock of the Keio collection into 300 ⁇ LB medium containing 25 ⁇ g/mL kanamycin in 96 well deepwell plates and grown at 37°C with 300 rpm shaking overnight.
  • the Keio background strain, BW25113 was also inoculated into wells of this plate as a control.
  • a cryogenic stock was made from each plate, and the cryogenic stock was replica plated into another 96 well deepwell plate containing 300 ⁇ _ M9 + 1% glucose and grown overnight.
  • cells were inoculated 1 : 100 into clear bottomed 96 well half-deepwell plates containing M9 + 1% glucose plus 7.5 g/L and 12.5 g/L isobutyric acid (neutralized), and cultivated in a Growth Profiler as previously described for screening of ALE isolates.
  • Keio collection mutants were struck on LB + 25 pg/mL kanamycin from the cryogenic stock plate prepared during primary screening above and biological triplicate colonies were inoculated into a 96 well deepwell plate containing 300 pL M9 + 1% glucose. The next day, cells were inoculated into plates for cultivation on the Growth Profiler as described above.
  • the Keio collection strain containing yqhD: :kan, JW2978 was used as the donor strain for Plvir phage transduction into recipient strains K-12 MG1655, IBUA2-9, IBUA7-9, and IBUA8- 3 as described in 'Construction of gene knockouts.
  • Plasmid pCP20 which encodes a constitutively expressed yeast flippase recombinase (FLP) was transformed into JW2978, K- 12 MG1655 yqhDr.kan, IBUA2-9 yqhDr.kan IBUA7-9 yqhDr.kan, and IBUA8-3 yqhDr.kan to remove the kanamycin resistance marker, generating strains K-12 BW25113 AyqhD, K-12 MG1655 AyqhD, IBUA2-9 AyqhD, IBUA7-9 AyqhD and IBUA8-3 AyqhD. Loss of the kanamycin resistance marker was confirmed by
  • cells were grown overnight to saturation as described, and were inoculated the next morning into 2.5 mL M9 medium containing 4% glucose, 0.5 g calcium carbonate, 100 ⁇ g/mL ampicillin, 50 ⁇ g/mL kanamycin, 0.1 mM IPTG (no yeast extract), and either no added isobutyric acid, or sodium isobutyrate to a final concentration of 7.5 g/L, in a 24 well deepwell plate. The plate was incubated at 37°C and 1 mL samples of cell supernatants were harvested for isobutyric acid analysis after 24 and 50 hours.
  • the batch phase medium (400 mL) contained 2 g/L ammonium sulfate, 5.6 g/L potassium phosphate dibasic, 0.12 g/L Antifoam 204, 2% glucose, 0.1 g/L zinc chloride, 0.15 g/L iron(II) sulfate heptahydrate, 1.5 g/L trisodium citrate, 2 mM magnesium sulfate, 0.125 mM calcium chloride, and 1 mL/L of a trace element solution containing 2.0 g/L aluminum sulfate octadecahydrate, 0.75 g/L cobalt(II) sulfate hexahydrate, 2.5 g/L copper(II) sulfate
  • the feed solution contained 600 g/L glucose, 18 g/L magnesium sulfate heptahydrate, 3 mL/L of trace element solution, 0.1 g/L zinc chloride, 0.15 g/L iron(II) sulfate heptahydrate, 1.5 g/L trisodium citrate, 0.2 mM IPTG, 100 ⁇ g/mL ampicillin, and 50 ⁇ g/mL kanamycin.
  • the bioreactors were maintained at 30°C, pH 7.0 using 15% ammonium hydroxide, with air sparged at 1 vvm (1 L air per L liquid volume per minute), and the stirrer set to 800 rpm during the batch phase and 1000 rpm during the feed phase.
  • IPTG was added to a concentration of 0.2 mM after 22 hours and in the feeding phase at a concentration of 0.2 mM in the feeding solution, whereas in the second run IPTG was added 1 hour after the start of the feeding phase to a concentration of 0.1 mM, with a concentration of 0.1 mM in the feeding solution.
  • a screen of a larger selection of evolved isolates harboring deletions in yqhD and plasmids pIBAl and pIBA7 was performed using Feed-in-Time (FIT) medium (m2p-labs, Baesweiler, Germany) diluted 1 : 1 with 200 mM MOPS buffer, herein referred to as V2 FIT medium.
  • FIT Feed-in-Time
  • Genomic point mutants were generated using MAGE (Wang et ai , 2009), which involves multiple cycles of electroporation of cells expressing the ⁇ protein of ⁇ Red recombinase with single stranded DNA oligonucleotides.
  • the single-stranded oligonucleotides are believed to behave like Okazaki fragments during DNA replication, and their use enables a high enough efficiency of allelic replacement to preclude needing to select for cells that received the mutation.
  • K-12 MG1655 or knockout strains of K-12 MG1655 were transformed with pMA7SacB (Lennen et al. , NAR 2015), a plasmid that harbors the ⁇ subunit of ⁇ Red recombinase and Dam under control of an arabinose-inducible promoter, and SacB to enable removing the plasmid by sucrose counterselection following the identification of a desired mutant.
  • K-12 MG1655/pMA7SacB was grown in 15 mL of LB medium plus 100 ⁇ g/mL ampicillin to mid-exponential phase at 37°C, induced for 10 minutes with 0.2% L-arabinose, chilled in an ice water bath, and washed and concentrated 3 times with autoclaved chilled MilliQ water in a typical electrocompetent cell preparation.
  • 50 pmol of oligonucleotide was added to a 50 ⁇ aliquot of cells in a 1 mm gap electroporation cuvette, and cells were electroporated at 1.8 kV. Cells were immediately recovered in 1 mL LB and the entire volume of cells was used to inoculate the next 15 mL LB culture.
  • the mutant forward primer had the last base designed to be complementary to the mutated base and an additional mutation at the -3 position from the 3' end of the primer such that primer binding would be maximally destabilized with the wild-type base.
  • the wild-type primer typically had the -3 position from the 3' end of the primer mutated to offer additional destabilization with the mutant base. This allowed discrimination of the desired mutant or wild-type base for each screened isolate by qualitatively observing a reversal in the fluorescence vs. cycle threshold curves by qPCR with the two primer sets. Individual isolates were verified to have the desired mutant sequence in the genome with no adjacent off-target mutations by Sanger sequencing.
  • the updated version of the software discarded regions where growth curves were fit but the signal-to-noise ratio was less than 1, to eliminate automatic detection of false growth phases. While automatic detection succeeded in detecting and fitting the dominant growth phase more than 95% of the time, all data was additionally manually curated to ensure that the main growth phase was always selected and that false growth phases were not detected when growth was essentially absent.
  • E. coli K-12 MG1655 exhibited a steadily decreasing growth rate as a function of isobutyric acid concentration, with very little growth observed above 10 g/L (Table 3). Lag times began increasing above 1 g/L and steadily increased with increasing isobutyric acid concentration. Table 3. Growth of K-12 MG1655 in varying concentrations of isobutyric acid (neutralized). mean (1) std. error (1) mean (2) std. error (2) isobutyric acid
  • IBUA1-7 is an isobutyric acid-evolved strain isolated from population 1).
  • strains are arranged such that all that were isolated from the same population are presented in the same rows.
  • strains with an asterisk (*) following their name are hypermutator strains, and only the mutation identified that can be associated with generating the hypermutator phenotype (here only in IBUA3-2, with an insertion in the intergenic region between secA and mutT and those mutations that are shared with other mutations in the same gene in other strains are shown.
  • Each re-sequenced isolate was characterized using the Biolector system for growth at the screening concentration of chemical (12.5 g/L isobutyric acid) in biological triplicates. Tables showing the calculated average growth rates and lag times for each isolate are shown in Table 5. Standard errors are standard deviations about the mean of the growth rate and lag time for the three independent biological replicates.
  • IBUA1-7 0.450 9.0 0.037 0.7 0.421 8.5 0.008 0.3
  • Probable loss-of-function mutations were identified from re-sequencing results as described in methods. More obvious loss-of-function mutations were found (frameshift mutations and IS element insertions) in pykF in several populations, with the remainder of mutations in pykF being coding mutations or in the intergenic region upstream of pykF. It can therefore be inferred that these latter mutations likely also partially or fully inactivate PykF activity or abolish or reduce its expression via disruption of the promoter. Additional probable loss-of- function mutations were identified in two other genes, rpoS and yobF.
  • MG1655 ApykF yobF::kan 0.348 11.1 0.030 0.1 0.294 12.9 0.002 0.1
  • deletion of pykF primarily decreased lag time compared to the wild-type when grown with 6.3 g/L or 12.5 g/L isobutyric acid.
  • Deletion of rpoS by itself dramatically increased the lag time in 6.3 g/L isobutyric acid, and resulted in no growth in 12.5 g/L isobutyric acid.
  • Deletion of yobF did not result in a growth phenotype vs. K-12 MG1655 at either concentration.
  • the average growth rates and lag times for three biological replicates are shown in Table 7.
  • the growth rate of the pykF single deletion strain was more than double that of K-12 MG1655 with a significantly reduced lag time, and recuperated 57 to 73% of the growth rate observed in the four tested evolved strains.
  • the pykF rpoS double deletion strain exhibited the best overall features, with a growth rate that was 67 to 87% of the evolved strains and with the lowest lag time.
  • the pykF yobF double deletion strain also performed better than the pykF deletion alone, but with a lag time similar to the pykF single deletion strain.
  • Keio collection of gene knockouts is a commercial collection of knockouts in nearly all non-essential genes and ORFs in E. coll strain BW25113.
  • This strain is a K-12 derivative and possesses known mutations relative to the K-12 MG1655 background.
  • All Keio collection strains with knockouts in genes that were found to be mutated in Table 1 were screened for growth against the BW25113 control in M9 + 1% glucose + 6.3 g/L or 12.5 g/L isobutyric acid (neutralized) in the Growth Profiler screening format.
  • Initial (preliminary) qualitative observations based on averaged growth curves for 3 biological replicate cultures are shown in Table 9, with calculated growth rates shown in Table 10.
  • Gene deletion strains of rph and yjcF exhibited increased growth rates in both 6.3 g/L and 12.5 g/L isobutyrate.
  • Gene deletion strains of phoU exhibited an increased growth rate only at 6.3 g/L isobutyrate, while deletion of cheR increased the growth rate only in 12.5 g/L isobutyrate.
  • Knockout mutations from Keio strains have already been PI phage transduced into K-12 MG1655 and are being validated and screened for growth in the Biolector format. Knockouts will additionally be constructed in the best performing knockout strain(s) obtained from earlier efforts.
  • Table 9 Initial (preliminary) qualitative evaluation of growth rates of Keio collection knockout strains compared to the background strain BW25113 in 6.3 g/L and 12.5 g/L isobutyrate as measured in the Growth Profiler testing format.
  • BW25113 yjcFr.kan Increased increased Table 10. Growth rates of Keio collection knockouts in M9 + 12.5 g/L isobutyric acid (neutralized) as measured in the Growth Profiler testing format.
  • Isobutyric acid production has been demonstrated in E. coli from pyruvate via 5 steps (Zhang et a/. , 2011), the first 4 of which are common to isobutanol production as well (Atsumi et a/., 2007).
  • pyruvate can be converted to (2S)-hydroxy-2-methyl-3-oxobutanoic acid with AlsS (acetolactate synthase) heterologously expressed from Bacillus subtilis.
  • this product can be reduced and isomerized to (2R)-2,3-dihydroxy-3-methylbutanoic acid by native E. coli IlvC (acetohydroxy acid isomeroreductase).
  • this product can be dehydrated to 2-ketovaline by native E. coli IlvD (dihydroxy acid dehydratase).
  • this product can be decarboxylated to isobutyraldehyde by Lactococcus lactis KIVD (2-ketoacid decarboxylase).
  • this product can be oxidized to isobutyric acid promiscuously by a number of aldehyde dehydrogenases. The most active enzymes for this activity out of 7 tested were determined to be 3-hydroxypropionaldehyde dehydrogenase AldH from E. coli, and phenylacetaldehyde dehydrogenase PadA from E. coli (Zhang et a/. , 2011).
  • coli ilvD in an artificial operon under control of a Piac promoter (lactose or isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) inducible) and a kanamycin resistance gene.
  • pIBA7 contains the KIVD gene from L. lactis and E. coli pad A, also in an artificial operon under control of a P
  • isobutyric acid was measured after 24 hours and 48 hours growth (Table 12). No significantly increased titers of isobutyric acid could be observed under these conditions in any of the evolved strain backgrounds compared to the control strains (BW25113 and MG1655).
  • IBUA7-9 and IBUA8- 3 generated statistically equivalent titers of isobutyric acid, while IBUA2-9 produced a significantly lower titer than the control strains.
  • Yeast extract was added to these cultures, which is likely not commercially feasible due to its high expense relative to the value of isobutyric acid.
  • Table 12 Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured after 24 h and 48 h in M9 glucose medium containing yeast extract at 30°C.
  • strains were grown in M9 glucose without added yeast extract at 37°C, with or without the addition of 7.5 g/L isobutyric acid from inoculation. Calcium carbonate and IPTG were added as previously described. Blank media wells were included to allow determination of the isobutyric acid concentration when accounting for evaporation, and the values measured in blank wells were subtracted from measured concentrations in order to determine the amount that was produced on top of what was added to the cultures, for cultures with added isobutyric acid. Isobutyric acid was measured after 24 hours and 50 hours growth (Table 13).
  • Table 13 Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured after 24 h and 48 h in M9 glucose medium with or without added isobutyric acid at 37°C.
  • IBUA2-9 0.00 ⁇ 0.00 0.00 ⁇ 0.00 -0.05 ⁇ 0.05 -0.28 ⁇ 0.04
  • IBUA7-9 0.00 ⁇ 0.00 2.11 ⁇ 0.17 0.00 ⁇ 0.03 -0.24 ⁇ 0.05
  • IBUA8-3 1.30 ⁇ 0.04 1.62 ⁇ 0.09 1.45 ⁇ 0.12 2.12 ⁇ 0.15
  • strain IBUA8-3 could further produce additional isobutyric acid, and at titers similar to or even higher than in the absence of added isobutyric acid.
  • Table 14 Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from fed-batch fermentation runs after 45.5 hours in a minimal medium supplemented with glucose at 30°C. final titer
  • Table 15 Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from fed-batch fermentation runs after 47.5 hours in a minimal medium supplemented with glucose at 30°C, with altered induction conditions as described in the text.
  • IBUA3-2 and IBUA6-9 harbored different mutations on residue D622 of RpoC.
  • IBUA3-2 was however a mutator strain possessing numerous additional mutations that are likely responsible for its higher production levels than IBUA6-9.
  • the RpoC-D622A mutation has previously been studied and found to render RpoC insensitive to the strigent response alarmone ppGpp. Thus while this was effective as a mechanism of isobutyrate tolerance, this effect is counteractive toward endongenous production of isobutyrate.
  • Table 16 Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from screening in a minimal slow-release glucose medium after 48 at 30°C.
  • IBUA8-3 strain background resultsed in a closer investigation of mutations that could enable higher production levels.
  • IBUA8-3 and IBUA8-4 appear genetically identical and both have IlvH-L9F mutations, while IBUA8-10 harbors an IlvN-N17H mutation. All isolates also harbor a GlyQ-E48D mutation.
  • IlvH and IlvN are both small regulatory subunits of different active isoforms of acetohydroxybutanoate
  • AHAS synthase/acetolactate synthase
  • E. coli E. coli
  • AHAS III is composed of Ilvl and IlvH
  • AHAS I is composed of IlvB and IlvN
  • AHAS II is composed of IlvG and IlvM.
  • ilvG is a pseudogene due to the presence of an internal frameshift mutation and AHAS II activity is abolished.
  • the three AHAS isoforms catalyzes the first common steps in the biosynthesis of the branched chain amino acids, L-valine, L-isoleucine, and L-leucine, through complex allosteric inhibition mediated by the small regulatory subunits of each isoform (IlvH, IlvN, and IlvO). Both AHAS I and AHAS III are inhibited by valine, while AHAS III is nearly completely inhibited by leucine. Because both functional isoforms are inhibited by valine, E. coli K-12 is sensitive to the addition or accumulation of valine due to isoleucine starvation (De Felice et ai. , 1979), and has also been shown to undergo oscillations in isoleucine starvation under normal growth conditions (Andersen et ai , 2001).
  • the mutations found in evolved isolates in both IlvH and IlvN are in the N-terminal regions of these proteins. It has previously been shown that only the first 14-25 amino acids of IlvH are required for activation of AHAS III, and that N- and C-terminal truncated mutants do not exhibit valine inhibition (Zhao et a/. , 2013). L9A and L9V mutants of E. coli IlvH have been shown to reduce valine inhibition of AHAS III (Kaplun et ai , 2006), and specific feedback-resistant N-terminal mutations of E. coli IlvH (notably in residues 14 and 17) have been described for the purpose of producing L-valine (US Patent No. 6,737,255 B2).
  • the GlyQ-E48D mutation improved growth in the presence of 12.5 g/L isobutyrate only when it was together with the pykF deletion.
  • the combination of the GlyQ-E48D, IlvH-L9F, and ApykF mutations did not improve growth over the strain that only harbored GlyQ-E48D and ApykF.
  • the IlvH reversion mutant of IBUA8-3 exhibited unusual behavior, with an initial growth phase to a low cell density at a growth rate similar to IBUA8-3, then with an approximately 14 hour lag phase before growth resumed at a nearly equivalent rate.
  • IBUA8-10 also evolved a an AHAS feedback resistance mutation in parallel in the population, it can be concluded that the condition of isoleucine starvation or valine accumulation is particularly induced by the combination of the ApykF, GlyQ-E48D, and the increased expression of pyrE resulting from the 82 bp deletion within the 5' terminal region of rph (the increased growth rate imparted by these mutations likely necessitated a higher biosynthetic flux toward isoleucine than was otherwise possible without an AHAS feedback resistance mutation), as this was the only remaining common mutation between IBUA8-3 and IBUA8-10.
  • Table 17 Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyric acid (neutralized), as measured in the Biolector testing format.
  • Table 18 Growth rates and lag times of selected strains in M9 + 1 g/L L-valine or L-leucine, as measured in the Biolector testing format.
  • Table 19 Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyrate, measured in the Biolector testin format.
  • Table 20 Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyrate + 1 g/L L-isoleucine, as measured in the Biolector testing format.
  • IBUA8-3 ilvH-revert 0.398 8.0 0.018 0.1
  • AHAS feedback resistance mutations may improve native production of balanced levels of valine, leucine, and isoleucine during production.
  • the production pathway employed uses a heterologous AHAS (AlsS) with a higher activity for utilizing pyruvate as a substrate, as well as overexpressed E. coli IlvD, which is also used for biosynthesis of L-valine and L- isoleucine. This may lead to accumulation of L-valine which then feedback-inhibits flux through native E.
  • each evolved isolate was tested for cross-tolerance toward other straight short- chain and branched short-chain carboxylic acids and alcohols of potential biotechnological interest.
  • K-12 MG1655 was tested in the Growth Profiler screening format for growth in the presence of a range of concentrations of each compound : butyric acid, 2-methylbutyric acid, valeric acid, isovaleric acid (3-methylbutyric acid), 4-methylvaleric acid, 2- methylhexanoic acid, 1-propanol, 2-propanol (isopropanol), isobutanol, 3-methyl-l-butanol, 2-methyl-l-butanol, and 1-pentanol.
  • a large number of evolved isolates exhibit greatly improved growth rates and often-reduced lag times in all of these compounds compared with K-12 MG1655.
  • Large numbers of the isolates exhibited cross- tolerance toward isovalerate, 4-methylvalerate, and 2-methylbutyrate. More selected numbers of isolates exhibited cross-tolerance toward valerate, butyrate, and 2- methylhexanoate.
  • the only isolates that exhibited cross-tolerance toward the majority of alcohols were those derived from the IBUA8 population (Table 25).
  • Some additional isolates from the IBUA5 population, IBUA4-9, and IBUA6-7 also exhibited cross-tolerance toward 3- methyl-l-butanol (Table 26).
  • Table 22 Growth rates and lag times of K-12 MG1655 in varying concentrations of short branched- and straight-chain carboxylic acids and alcohols, as measured in the Growth Profiler testing format. mean (2) std. error (2) mean (2) std. error (2)
  • Table 23 Growth rates and lag times of K-12 MG1655 in specified inhibitory concentrations of butyrate, 2-methylbutyrate, and valerate, as measured in the Growth Profiler testing format.
  • IBUA1-7 0.230 19.3 0.039 4.5 0.273 12.6 0.007 0.8 0.206 14.8 0.011 0.2
  • IBUA1-9 0.100 14.8 0.145 13.0 0.240 19.3 0.082 4.5 0.140 19.6 0.006 2.1
  • IBUA2-6 0.000 0.0 0.000 0.0 0.137 19.7 0.119 20.2 0.000 0.0 0.000 0.0
  • Table 24 Growth rates and lag times of K-12 MG1655 in specified inhibitory concentrations of isovalerate, 4-methylvalerate, and 2-methylhexanoate, as measured in the Growth Profiler testing format.
  • IBUA1-7 0 315 10.2 0.021 0.1 0 328 12.3 0.012 0.2 0 245 12.2 0.006 0.3
  • IBUA2-6 0 315 14.0 0.014 1.3 0 381 13.1 0.026 0.5 0 206 17.3 0.092 3.7
  • IBUA4-1 0 315 10.2 0.004 0.3 0 392 10.0 0.024 0.0 0 324 10.2 0.022 0.1
  • IBUA7-6 0.310 10.0 0.017 0.4 0.332 11.6 0.049 0.3 0.152 11.3 0.021 0.5
  • Table 25 Growth rates and lag times of K-12 MG1655 and evolved isolates derived from population IBUA8 in specified inhibitory concentrations of 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, and 1-pentanol, as measured in the Growth Profiler testing format.
  • Table 26 Growth rates and lag times of K-12 MG1655 and selected evolved isolates in 5.5 g/L 3-methyl-l-butanol, as measured in the Growth Profiler testing format.
  • IBUA5-5 0.660 27.0 0.044 0.7 IBUA5-6 0.675 28.4 0.054 1.5
  • Dragosits M Mattanovich D. Adaptive laboratory evolution - principles and applications for biotechnology, Microbial Gell Factories 12: 64 (2013) . Dragosits M, Mozhayskiy V, Quinones-Soto S, Park J, Tagkopoulos I. Evolutionary potential, cross-stress behavior and the genetic basis of acquired stress resistance in Escherichia coli . Mol . Syst. Biol . 9: 643 (2013) .
  • Kaplun A Vyazmensky M, Zherdev Y, Belenky I, Slutzker A, Mendel S, Barak Z, Chipman DM, Shaanan B. Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli. J. Mol. Biol. 357:951-963 (2006).
  • Lennen RM Herrgard MJ. Combinatorial strategies for improving multiple-stress resistance in industrially relevant Escherichia coli strains. Appl. Environ. Microbiol. 80: 6223-6242 (2014).
  • Lennen RM Nilsson Wallin AI, Pedersen M, Bonde M, Luo H, Herrgard MJ, Sommer MO.
  • Acetohydroxyacid synthase a proposed structure for regulatory subunits supported by evidence from mutagenesis. J. Mol. Biol. 307:465-477 (2001) .

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Abstract

L'invention concerne des cellules bactériennes génétiquement modifiées pour améliorer leur tolérance à certains produits chimiques de base, tel que l'acide isobutyrique et des composés apparentés, et des procédés de préparation et d'utilisation de telles cellules bactériennes pour la production d'acide isobutyrique et de composés apparentés.
PCT/EP2017/061379 2016-05-12 2017-05-11 Cellules bactériennes à tolérance améliorée pour l'acide isobutyrique Ceased WO2017194696A1 (fr)

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WO2019139981A1 (fr) * 2018-01-09 2019-07-18 Lygos, Inc. Cellules hôtes recombinées et procédés de production d'acide isobutyrique
EP4484564A1 (fr) * 2023-06-27 2025-01-01 OQ Chemicals GmbH Procédé pour augmenter le rendement de biogaz de fermentations anaérobies et composition d'acides monocarboxyliques ramifiés à chaîne courte
CN121022705A (zh) * 2025-10-28 2025-11-28 天津科技大学 生产l-异亮氨酸的基因工程菌及其构建方法与应用
WO2025259581A3 (fr) * 2024-06-10 2026-01-29 Massachusetts Institute Of Technology Ingénierie microbienne pour la production d'acides 3-hydroxy alpha-substitués

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WO2025003293A3 (fr) * 2023-06-27 2025-09-18 Oq Chemicals Gmbh Procédé pour augmenter le rendement en biogaz de fermentations anaérobies et composition d'acides monocarboxyliques ramifiés à chaîne courte
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