WO2017211883A1 - Cellules bactériennes à tolérance améliorée aux polyols - Google Patents

Cellules bactériennes à tolérance améliorée aux polyols Download PDF

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WO2017211883A1
WO2017211883A1 PCT/EP2017/063821 EP2017063821W WO2017211883A1 WO 2017211883 A1 WO2017211883 A1 WO 2017211883A1 EP 2017063821 W EP2017063821 W EP 2017063821W WO 2017211883 A1 WO2017211883 A1 WO 2017211883A1
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rpoc
rpob
rpsa
seq
mutation
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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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Publication of WO2017211883A1 publication Critical patent/WO2017211883A1/fr

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/245—Escherichia (G)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1014—Hydroxymethyl-, formyl-transferases (2.1.2)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

  • the present invention relates to bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as diols and other polyols, and to methods of preparing and using such bacterial cells for production of polyols and other compounds.
  • Polyols such as diols are versatile water-miscible compounds used in diverse applications including use as polyester and polyurethane resin precursors, antifreezes, synthetic lubricants, plasticizers and polymer additives, intermediates in the production of
  • 2,3- butanediol Another diol, 2,3- butanediol, can be reduced to butadiene, a component of synthetic rubber, and the largest current usage of 2,3-butanediol is as a cross-linking agent for hard rubbers, as a precursor for insecticides, and as pharmaceutical intermediates (Grafje et a/. , 2012).
  • butadiene was primarily synthesized from butene obtained from cracked naptha.
  • Biological production of diols can be particularly advantageous when compared to chemical synthesis, in that it can readily allow the production of pure stereoisomers or racemic mixtures of stereoisomers, depending on the enzymes employed.
  • the production of diols in metabolically engineered microbial cells have been reviewed and described in several publications such as, e.g. , Sabra et a/. (2016), Clomburg et al. (2011), Jain et al. (2015), Li et al. (2015) and Xu et al. (2014).
  • high product titers are essential in order to minimize capital equipment and downstream separations costs for product purification.
  • At the high titers required for economical fermentation processes however, most chemicals exhibit significant toxicity that reduce yields and productivities by negatively affecting microbial growth (Van Dien, 2013; Zingaro et a/. , 2013).
  • 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. , Haft et al, 2014; 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; LaCroix et al. , 2015; Jensen et al., 2015 and 2016 ; Doukyu et al., 2012; Shenhar et al., 2012; and Rath and Jawali, 2006).
  • chemicals of interest for production such as, e.g. , n-butanol
  • the invention provides bacterial cells with improved tolerance to at least one aliphatic polyol, as well as bacterial cells which are capable of producing an aliphatic polyol and have improved tolerance to the aliphatic polyol.
  • aliphatic diols such as e.g.
  • compositions comprising such bacterial cells and one or more aliphatic polyols, methods of preparing or screening for such bacterial cells, and methods of producing aliphatic polyols using such bacterial cells.
  • the bacterial cell comprises a biosynthetic, optionally recombinant, pathway for producing an aliphatic polyol and at least one genetic modification which reduces expression of an endogenous gene selected from the group consisting of metJ, iscR, yhjA, gtrS, ycdU, rzpD, sspA and rph, or a combination of any thereof, optionally wherein the cell further comprises a genetic modification which increases the expression of PyrE and/or a mutation in one or more of NanK, RpsA, RpoB, RpoC, SpoT, NusG, Flu, Lon, and YgaH.
  • the bacterial cell comprises a biosynthetic, optionally recombinant, pathway for producing an aliphatic polyol and at least one genetic modification which increases one or more of (a) the biosynthesis of methionine in the bacterial cell; (b) the growth of the bacterial cell during polyol-induced methionine starvation; (c) intracellular iron levels during polyol-induced growth inhibition; (d) biosynthesis of iron siderophores during polyol-induced growth inhibition; and (e) the biosynthesis of iron-sulfur clusters during polyol-induced growth inhibition.
  • the bacterial cell comprises at least one genetic modification which reduces expression of metJ and/or iscR.
  • the bacterial cell may further comprise genetic modifications which reduce expression of relA and pur T; or genetic modifications which reduce the expression of acrB, acrA, or both, optionally in combination with a genetic modification which increases the expression of PyrE, or a mutation in one or more of NanK, RpsA, RpoB, RpoC, SpoT, NusG, Flu, Lon and YgaH.
  • the bacterial cell comprises genetic modifications which reduce expression of metJ, relA and pur T; metJ and acrB and/or acrA; iscR and relA; or fabR and ygfF, optionally in combination with a genetic modification which increases the expression of PyrE, and/or a mutation in one or more of NanK, RpsA, RpoB, RpoC, SpoT, NusG, Flu, Lon, and YgaH.
  • the genetic modification comprises a knock-down or knock-out of the endogenous gene.
  • the genetic modification is a knock-out.
  • Preferred, non-limiting polyols include diols, the genetic modification providing for an increased growth rate, a reduced lag time, or both, of the cell in at least one of, e.g., 2,3- butanediol and 1,2-propanediol, as compared to a control.
  • the control may be, for example, the parent bacterial cell.
  • the pathway is a recombinant pathway.
  • the bacterial cell may comprise a recombinant biosynthetic pathway for producing at least one of a propanediol, butanediol, pentanediol and a hexanediol.
  • the bacterial cell may be of any suitable genus or origin.
  • Preferred, non-limiting genera include Escherichia, Enterobacter, Klebsiella, Lactobacillus, Lactococcus, Bacillus,
  • Escherichia coll is particularly preferred.
  • a process for preparing a recombinant E. coll cell for producing an aliphatic polyol comprising genetically modifying an E. coll cell to
  • polyol and knock-down or knock-out at least one endogenous gene selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph; or
  • a process for improving the tolerance of a bacterial cell to an aliphatic diol comprising genetically modifying the bacterial cell to knock-down or knock-out (a) at least one endogenous gene selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph; or (b) a combination of endogenous genes selected from metJ, relA and purT; metJ and acrB and/or acrA; iscR and relA; and fabR and ygfF, optionally also introducing a genetic modification which increases the expression of PyrE, or a mutation in one or more of NanK, RpsA, RpoB, RpoC, SpoT, NusG, Flu, Lon, and YgaH.
  • a process for preparing a recombinant E. coli cell for producing an aliphatic polyol comprising genetically modifying an E. coli cell to introduce a recombinant biosynthetic pathway for producing an aliphatic polyol, and
  • nusG SEQ ID NO: 29, Flu (SEQ ID NO: 31), Lon (SEQ ID NO: 33), and YgaH (SEQ ID NO: 35), optionally wherein the one or more mutations are selected from RpoC-L268K, RpoC-L268N, RpoC-L268Q, RpoC-L268R, RpoC- N309F, RpoC-N309S, RpoC-N309T, RpoC-N309W, RpoC-N309Y, RpoC-Y75A, RpoC-Y75C, RpoC-Y75S, RpoC-ATPVIE(822-827), RpoB-D549A, RpoB-D549G,
  • a process for improving the tolerance of a bacterial cell to an aliphatic polyol comprising genetically modifying the bacterial cell to knock-down or knock-out at least one endogenous gene selected from the group consisting of metJ, iscR, yhjA, gtrS, ycdU, rzpD, sspA and rph; or a combination of endogenous genes selected from metJ, relA and purT; metJ and acrB, acrA or both; iscR and relA; and fabR and ygfF; optionally introducing one or more mutations in one or more endogenous genes selected from NanK (SEQ ID NO:19), psA (SEQ ID NO:37), RpoA (SEQ ID NO:21); RpoB (SEQ ID NO:23), RpoC (SEQ ID NO:25), SpoT (SEQ ID NO:27), NusG (SEQ ID NO:
  • a method for producing an aliphatic polyol comprising culturing the bacterial cell of any aspect or embodiment herein in the presence of a carbon source, and, optionally, isolating the aliphatic polyol.
  • composition comprising a propanediol or a butanediol at a concentration of at least 6% and a plurality of bacterial cells according to any aspect or embodiment herein.
  • the bacterial cells may be, e.g.
  • the Escherichia genus genetically modified to knock-down or knock-out at least one endogenous gene selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph; or a combination of endogenous genes selected from metJ, relA and purT; metJ and acrB and/or acrA; iscR and relA ; and fabR and ygfF.
  • endogenous gene selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph; or a combination of endogenous genes selected from metJ, relA and purT; metJ and acrB and/or acrA; iscR and relA ; and fabR and ygfF.
  • a method for producing an aliphatic diol comprising (a) culturing a plurality of bacterial cells capable of producing the aliphatic diol in a medium comprising a carbon source, and (b) adding methionine to the medium, wherein the concentration of the added methionine is from about 0.004 g L "1 gDCW 1 to about 0.2 g L "1 gDCW 1 , optionally wherein the bacterial cell is the bacterial cell of any preceding aspect or embodiment.
  • a "diol” as used herein is an aliphatic diol
  • a "polyol” is an aliphatic polyol
  • An “aliphatic polyol” herein refers to an organic compound comprising an aliphatic carbon chain to which two or more hydroxyl (-OH) groups are attached, and includes linear aliphatic diols and other linear aliphatic polyols, as well as derivatives thereof.
  • Aliphatic polyols suitable for production in bacteria typically comprise from 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and, most preferably, 3 to 6 carbon atoms, and, optionally comprises one or more heteroatoms.
  • Linear aliphatic polyols comprising 2, 3 or 4 hydroxyl groups are preferred and include, but are not limited to, 2,3-butanediol; 1,2-propanediol; 1,5 pentanediol; 1,2- pentanediol; 1,4-butanediol; 1,3-propanediol; 1,2-butanediol; 1,6-hexanediol; 1,8- octanediol; 1,10-decanediol and 1,12-dodecanediol.
  • Linear aliphatic diols such as, e.g. , 2,3-butanediol; 1,2-propanediol; l,5-pentanediol; l,2-pentanediol; 1,6-hexanediol; 1,4- butanediol and 1,3-propanediol are most preferred.
  • 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 a diol or other polyol, 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 a diol or other polyol than the parent bacterial cell or strain from which it is derived, typically at concentrations of at least 1% v/v, such as at least 1.5% v/v, such as at least 3% v/v, such as at least 5% v/v, such as at least 6% v/v, such as at least 7% v/v, such as at least 7.5% v/v, such as at least 8% v/v, such as at least 10% v/v.
  • 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. Genes names are herein set forth in italicised text with a lower-case first letter (e.g. , metJ) whereas protein names are set forth in normal text with a capital first letter (e.g. , MetJ).
  • 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 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 or other expression control sequence that results in decreased gene expression), a knock-out or disruption 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, e.g. a repressor that inhibits expression of the target or inhibitory nucleic acids ⁇ e.g. CRISPR etc.
  • 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. Table 1 - Examples of amino acid substitutions
  • the invention provides bacterial cells with improved tolerance to diols and other polyols, 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 ⁇ i.e. , coding) mutations in coding regions, herein referred to as “Group 2 modifications”; and combinations thereof.
  • the one or more genetic modifications provide for an increased growth rate, a reduced lag time, or both, of the bacterial cell in at least one of 2,3-butanediol and 1,2-propanediol, e.g. , at a concentration of at least 6% or at least 7% as compared to the wild-type bacterial cell.
  • the bacterial cell has a genetic modification which reduces expression of one or more endogenous genes selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdil, iscR, sspA and rph.
  • the endogenous gene is metJ.
  • a bacterial cell which comprises genetic modifications reducing the expression of at least two endogenous genes.
  • the genetic modifications reduce the expression of metJ and one or more other endogenous genes.
  • the other endogenous genes are relA and purT.
  • the other endogenous gene or genes is acrB, acrA or both.
  • the bacterial cell comprises genetic modifications which reduce expression of iscR and relA. In another embodiment, the bacterial cell comprises genetic modifications which reduce expression of fabR and ygfF.
  • the at least two endogenous genes bacterial cell comprises genetic modifications which reduce the expression of two or more of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph.
  • the bacterial cell comprises:
  • a first genetic modification which reduces the expression of metJ
  • a second genetic modification which reduces the expression of a gene selected from rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph;
  • a first genetic modification which reduces the expression of rzpD and a second genetic modification which reduces the expression of a gene selected from of metJ, yhjA, gtrS, ycdU, iscR, sspA and rph;
  • a first genetic modification which reduces the expression of yjhA and a second genetic modification which reduces the expression of a gene selected from of metJ, rzpD, gtrS, ycdU, iscR, sspA and rph;
  • a first genetic modification which reduces the expression of ycdU and a second genetic modification which reduces the expression of a gene selected from metJ, rzpD, yhjA, gtrS, iscR, sspA and rph;
  • a first genetic modification which reduces the expression of iscR and a second genetic modification which reduces the expression of a gene selected from metJ, rzpD, yhjA, gtrS, ycdU, sspA and rph,and, optionally, a third genetic modification which reduces the expression of relA ;
  • a first genetic modification which reduces the expression of rph and a second genetic modification which reduces the expression of a gene selected from metJ, rzpD, yhjA, gtrS, ycdU, iscR and sspA.
  • 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 of the one or more endogenous genes, resulting in at least 25%, such as at least 50%, such as at least 75%, 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 of the one or more endogenous genes, resulting in at least 25%, such as at least 50%, such as at least 75%, 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.
  • 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 resulting in decreased transcription, 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, shifted or deleted, 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.
  • a mutant protein is expressed in the bacterial cell, e.g., from a mutated version of an endogenous gene, or from a transgene encoding the mutant protein.
  • the bacterial cell may comprise one or more mutations in at least one protein selected from NanK (SEQ ID NO: 19), RpsA (SEQ ID NO: 37), RpoA (SEQ ID NO: 21); RpoB (SEQ ID NO: 23), RpoC (SEQ ID NO: 25), SpoT (SEQ ID NO: 27), NusG (SEQ ID NO: 29, Flu (SEQ ID NO: 31), Lon (SEQ ID NO: 33), and YgaH (SEQ ID NO: 35), e.g.
  • the one or more mutations are selected from RpoC-L268K, RpoC-L268N, RpoC-L268Q, RpoC-L268R, RpoC-N309F, RpoC-N309S, RpoC-N309T, RpoC-N309W, RpoC-N309Y, RpoC-Y75A, RpoC- Y75C, RpoC-Y75S, RpoC-ATPVIE(822-827), RpoB-D549A, RpoB-D549G, RpoB-H447F, RpoB- H447S, RpoB-H447T, RpoB-H447W, RpoB-H447Y, RpoB-I1112S, RpoB-I1112T, RpoB-V931A, RpoB-V931I, Rpo
  • the bacterial cell may further comprise a Group 1 modification as set out herein.
  • the bacterial cell comprises a Group 1 modification according to any aspect or embodiment herein as well as a mutation in one or more of NanK ⁇ e.g. , NanK- T128S), RpsA (e.g. , RpsA-G21V, RpsA-G21I, RpsA-G21L, RpsA-G21M, RpsA-G21F, RpsA- G21A), RpoB (e.g.
  • YgaH-V39A, YgaH-V39L, or YgaH-V39I and/or a mutation in rph or the pyrE/rph intergenic region which increases the expression of PyrE, wherein the one or more mutations improve tolerance to an aliphatic polyol such as, e.g. 2,3- butanediol.
  • the bacterial cell comprises a Group 1 modification according to any aspect or embodiment herein as well as a mutation in one or more of RpoA (e.g. , RpoA- D305G, RpoA-D305A, RpoA-G279V, RpoA-G279I, RpoA-G279L, RpoA-G279M, RpoA-G279F, and/or RpoA-G279A) and RpsA (e.g., RpoA- D305G, RpoA-D305A, RpoA-G279V, RpoA-G279I, RpoA-G279L, RpoA-G279M, RpoA-G279F, and/or RpoA-G279A) and RpsA (e.g.
  • RpoA e.g. , RpoA- D305G, RpoA-D305A,
  • the bacterial cell comprises a Group 1 modification according to any preceding aspect or embodiment as well as an upregulation of at least one of the endogenous genes NanK, RpsA, SpoT, NusG, PyrE, Flu, Lon, and YgaH, e.g. , by transforming the bacterial cell with a transgene expressing the endogenous protein.
  • 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.
  • the bacterial cell comprises one or more mutations which increase(s) the expression level or activity of PyrE, optionally in combination with a Group 1 modification.
  • 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 a/., 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).
  • a 1 bp deletion at coordinate 3815809 in the pyrE/rph intergenic region has previously been encountered in strains evolved for growth on a minimal glucose medium (LaCroix et a/.
  • the bacterial cell comprises mutations in rph or the pyrE/rph intergenic region, such as, e.g. , the 82 bp deletion near the 3' terminus of rph, the 1 bp deletion in the intergenic region between pyrE and rph, or both.
  • the bacterial cell comprises
  • - a mutation selected from NanK-T128S, RpsA-G21V, RpoB-H447Y, RpoC-L268R, RpoB-D549G, RpoB-V931A, RpoC-ATPVIE(822-827), RpoC-N309Y, SpoT-l213L, NusG-F144V, RpoC-Y75C, Flu-L642Q, RpoC-L268R, RpoB-llll2S, Lon-l716S, and YgaH-V39A, or a conservative substitution of any thereof, and a knock-out or knockdown of met J, relA, and purT ' m combination;
  • - a mutation selected from NanK-T128S, RpsA-G21V, RpoB-H447Y, RpoC-L268R, RpoB-D549G, RpoB-V931A, RpoC-ATPVIE(822-827), RpoC-N309Y, SpoT-l213L, NusG-F144V, RpoC-Y75C, Flu-L642Q, RpoC-L268R, RpoB-llll2S, Lon-l716S, and YgaH-V39A, or a conservative substitution of any thereof, and a knock-out or knockdown of iscR and relA in combination; or
  • the bacterial cell comprises
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q or RpoC-L268N
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q, or RpoC-L268N
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q or RpoC-L268N
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q, or RpoC-L268N
  • NanK-T128S mutation and a mutant NanK comprising a NanK-T128S mutation, and at least one genetic modification which reduces the expression of met J, relA, and purT, and acrB, acrA or both;
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q, or RpoC-L268N
  • NanK-T128S mutation and a mutant NanK comprising a NanK-T128S mutation, and at least one genetic modification which reduces the expression of metJ and acrB, acrA or both;
  • RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q, or RpoC-L268N
  • NanK comprising a NanK-T128S mutation, and at least one genetic modification which reduces the expression of met J, relA, purT, and acrB, acrA or both; - a mutant RpoC comprising a RpoC-L268R, RpoC-L268K, RpoC-L268Q or RpoC-L268N
  • a mutant NanK comprising a NanK-T128S mutation
  • a mutant Flu comprising a Flu-L642Q, Flu-L642N, or Flu-L642E mutation
  • at least one genetic modification which reduces the expression of metJ, relA, purl, elfD and acrB, acrA or both;
  • mutant poB comprising a RpoB-llll2S or RpoB-llll2T mutation and at least one genetic modification which reduces the expression of iscR, relA, and acrB, acrA or both;
  • RpoB comprising a RpoB-llll2S or RpoB-llll2T mutation and at least one genetic modification which reduces the expression of iscR, relA, and acrB, acrA or both;
  • mutant RpoB comprising a RpoB-llll2S or RpoB-llll2T mutation, and a mutant Lon comprising a Lon-l716S or Lon-l716T mutation, and at least one genetic modification which reduces the expression of iscR, relA, and acrB, acrA or both;
  • mutant RpoB comprising a RpoB-llll2S or RpoB-llll2T mutation
  • a mutant Lon comprising a Lon-l716S or Lon-l716T mutation
  • a mutant YgaH comprising a YgaH-V39A
  • YgaH-V39L, or YgaH-V39l mutation a genetic modification that increases the expression of PyrE, and at least one genetic modification which reduces the expression of iscR, relA, and acrB, acrA or both.
  • AcrB is part of a protein complex which includes AcrA (AcrAB-TolC), with TolC also serving as the outer membrane component of a number of other protein complexes. Accordingly, a knock-down or knock-out AcrA can result in the same phenotype as a knockdown or knockout of AcrB.
  • a knock-down or knock-out of acrB a knock-down or knock-out of acrA, or of acrA and acrB, can be used as an alternative.
  • the bacterial cell comprises a knockdown or knockout of metJ, acrB, relA, and purT; and one or more Group 2 modifications selected from the group consisting of: a mutation in NanK such as NanK-T128S or a conservative substitution thereof; a mutation in RpoC such as RpoC-L268R or a conservative substitution thereof; and/or a mutation in Flu such as Flu-L642Q or a conservative substitution thereof.
  • the bacterial cell also comprises a knockdown or knockout of elfD.
  • the bacterial cell comprises a knockdown or knockout of iscR and relA ; and one or more Group 2 modifications selected from the group consisting of: a mutation in RpoB such as RpoB-I1112S or a conservative substitution thereof; a mutation in Lon such as I716S or a conservative substitution thereof; a mutation in YgaH such as YgaH- V39A or a conservative substitution thereof; and/or a mutation increasing the expression of PyrE.
  • RpoB such as RpoB-I1112S or a conservative substitution thereof
  • Lon such as I716S or a conservative substitution thereof
  • YgaH such as YgaH- V39A or a conservative substitution thereof
  • a mutation increasing the expression of PyrE a mutation increasing the expression of PyrE.
  • Bacterial strains capable of producing diols and other polyols can be found, e.g. , in the genera Enterobacter, Klebsiella, Serratia, Lactobacillus, Bacillus, Paenibacillus, Clostridia, Thermoanaerobacterium, Bacteroides, Pantoea, and Citrobacter sp. (Sabra et al. , 2016; Jiang et al. , 2014).
  • the bacterial cell comprises a recombinant pathway for producing the diol or other polyol of interest.
  • a recombinant pathway can, for example, be added to introduce the capability to produce the diol or other polyol in a bacterial cell which does not have a native pathway to do so, typically by transforming the cell with one or more heterologous enzymes catalyzing the desired reaction(s).
  • a recombinant pathway can nonetheless be introduced in order to increase the production yield, e.g. , by overexpressing one or more native enzymes or transforming the cell with heterologous enzymes.
  • a bacterial cell with improved tolerance to at least one aliphatic polyol according to any aspect or embodiment described herein, wherein the bacterial cell further comprises a recombinant biosynthetic pathway for producing an aliphatic polyol of interest, such as, e.g. , 2,3-butanediol, 1,2-propanediol, 1,4-butanediol, 1,3- propanediol, 1,2-butanediol, 1,5-pentanediol and/or 1,2-pentanediol.
  • a recombinant biosynthetic pathway for producing an aliphatic polyol of interest such as, e.g. , 2,3-butanediol, 1,2-propanediol, 1,4-butanediol, 1,3- propanediol, 1,2-butanediol, 1,5-pentanediol and/or 1,2-pentan
  • the bacterial cell further comprises a recombinant biosynthetic pathway for producing 2,3-butanediol. In another particular embodiment, the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,2-propanediol. In another particular embodiment, the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,4-butanediol. In another particular embodiment, the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,3-propanediol. In another particular embodiment, the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,2-butanediol.
  • the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,5- pentanediol.
  • the bacterial cell further comprises a recombinant biosynthetic pathway for producing 1,2-pentanediol.
  • 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 diols in bacteria are well-known in the art and have been described by, e.g. , Xu et al. (2014), Jiang et a/. (2014), Sabra et a/. (2016), Saxena et a/.
  • 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 . Also, in some embodiments, 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 aliphatic polyol.
  • the biosynthetic pathway is for producing 2,3-butanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : an acetolactate synthase, e.g., BudB from Enterobacter cloacae, catalyzing the conversion of two pyruvate molecules to acetolactate;
  • an acetolactate synthase e.g., BudB from Enterobacter cloacae
  • an acetolactate decarboxylase e.g., BudA from Enterobacter cloacae, catalyzing the conversion of acetolactate to acetoin;
  • the native genes adhE, gloA, IdhA, tplA, and/or zwf are knocked-down or -out to reduce lactate production, ethanol production, and carbon flux into the pentose phosphate pathway.
  • the biosynthetic pathway is for producing 2,3-butanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : an acetolactate synthase, e.g., BudB from Enterobacter cloacae, catalyzing the conversion of two pyruvate molecules to acetolactate, which spontaneously decarboxylates to diacetyl; a diacetyl reductase, e.g., BudC from Klebsiella pneumoniae, catalyzing the conversion of diacetyl to acetoin; and
  • an acetolactate synthase e.g., BudB from Enterobacter cloacae
  • a diacetyl reductase e.g., BudC from Klebsiella pneumoniae
  • the biosynthetic pathway is for producing 2,3-butanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : an acetolactate synthase, e.g., BudB from Enterobacter cloacae, catalyzing the conversion of two pyruvate molecules to acetolactate;
  • an acetolactate synthase e.g., BudB from Enterobacter cloacae
  • an acetolactate decarboxylase e.g., BudA from Enterobacter cloacae, catalyzing the conversion of acetolactate to acetoin;
  • an acetoin dehydrogenase e.g., BudC from Klebiella pneumoniae, catalyzing the conversion of acetoin to diacetyl;
  • an acetylacetoin synthase e.g., from Bacillus licheniformis, catalyzing the conversion of two diacetyl molecules to acetylacetoin;
  • an acetylacetoin reductase e.g., from Bacillus licheniformis, catalyzing the conversion of acetylacetoin to acetylbutanediol;
  • an acetylbutanediol reductase e.g., from Bacillus licheniformis, catalyzing the conversion of acetylbutanediol to 2,3-butanediol.
  • the biosynthetic pathway does not constitute an acetolactate decarboxylase nor an acetoin dehydrogenase, and acetolactate is instead spontaneously converted to acetoin.
  • the biosynthetic pathway is for producing 1,2-propanediol from the cellular glycolytic intermediate dihydroxyacetone phosphate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a methylglyoxal synthase, e.g., MgsA from E. coli, catalyzing the conversion of
  • a methylglyoxal reductase or glycerol dehydrogenase e.g., GlyD and GlyH from E. coli, catalyzing the conversion of methylglyoxal to lactaldehyde;
  • lactaldehyde reductase or 1,2-propanediol reductase e.g., FucO from E. coli, catalyzing the conversion of lactaldehyde to 1,2-propanediol.
  • native lactate dehydrogenases which convert pyruvate to lactate such as (in E. coli), LdhA, can be deleted (Altaras and Cameron, 2000).
  • the biosynthetic pathway is for producing 1,2-propanediol from the cellular glycolytic intermediate dihydroxyacetone phosphate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a methylglyoxal synthase, e.g., MgsA from E. coli, catalyzing the conversion of
  • aldehyde oxidoreductase e.g. YqhD from E. coli, catalyzing the conversion of methylglyoxal to acetol
  • a glycerol reductase e.g., GlyD and GlyH from E. coli, catalyzing the conversion of acetol to 1,2-propanediol.
  • native lactate dehydrogenases which convert pyruvate to lactate such as (in E. coli), LdhA, can be deleted (Altaras and Cameron, 2000).
  • the biosynthetic pathway is for producing 1,2-propanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a lactate dehydrogenase, e.g., LdhA from E. coli, catalyzing the conversion of pyruvate to lactate;
  • a lactate dehydrogenase e.g., LdhA from E. coli
  • lactaldehyde dehydrogenase e.g. AldA from E. coli, catalyzing the conversion of lactate to lactaldehyde
  • the biosynthetic pathway is for producing 1,2-propanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a methylglyoxal synthase, e.g., MgsA from E. coli, catalyzing the conversion of
  • Type I glyoxylase e.g. GloA from E. coli, catalyzing the conversion of methylglyoxal and glutathione to (S)-lactoylglutathione;
  • Type II glyoxylase e.g. GloB from E. coli, catalyzing the conversion of (S)-lactoylglutathione to lactate and glutathione;
  • lactaldehyde dehydrogenase e.g. AldA from E. coli, catalyzing the conversion of lactate to lactaldehyde
  • lactaldehyde reductase or 1,2-propanediol reductase e.g.,FucO from E. coli, catalyzing the conversion of lactaldehyde to 1,2-propanediol.
  • native lactate dehydrogenases which convert pyruvate to lactate such as (in E. coli), LdhA, can be deleted (Altaras and Cameron, 2000).
  • the biosynthetic pathway is for producing 1,2-propanediol from the cellular glycolytic intermediate pyruvate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a lactate dehydrogenase, e.g., LdhA from E. coli, catalyzing the conversion of pyruvate to lactate;
  • a lactate dehydrogenase e.g., LdhA from E. coli
  • CoA transferase e.g., Pet from Clostridium propionicum DSM 1682, catalyzing the conversion of lactate and CoA to lactoyl-CoA;
  • aldehyde dehydrogenase e.g., a CoA-dependent succinate semialdehyde dehydrogenase (PdcD) from Yersinia enterocolitica subsp. enterocolitica 8081, catalyzing the conversion of lactoyl-CoA to lactaldehyde; and
  • PdcD CoA-dependent succinate semialdehyde dehydrogenase
  • an alcohol dehydrogenase e.g. a 3-hydroxypropionate dehydrogenase (MmsB) from Bacillus cereus ATCC 14579, catalyzing the conversion of lactaldehyde to 1,2-propanediol.
  • the biosynthetic pathway is for producing 1,4-butanediol from the cellular tricarboxylic acid intermediate succinate, and comprises genes, optionally
  • a succinyl-CoA synthetase e.g., SucCD from E. coli, catalyzing the conversion of succinate to succinyl-CoA;
  • a CoA-dependent succinate semialdehyde dehydrogenase e.g., SucD from E. coli, catalyzing the conversion of succinyl-CoA to succinyl semialdehyde;
  • a 4-hydroxybutyrate dehydrogenase e.g., 4HBd from Porphyromonas gingivalis, catalyzing the conversion of succinyl semialdehyde to 4-hydroxybutryrate;
  • 4-hydroxybutyryl-CoA transferase e.g., Cat2 from Porphyromonas gingivalis, catalyzing the conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA;
  • a 4-hydroxybutyryl-CoA reductase e.g., the bifunctional enzyme 025B from Clostridium beijerinckii, catalyzing the conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyrylaldehyde; and
  • an alcohol dehydrogenase e.g., the bifunctional enzyme 025B from Clostridium beijerinckii, catalyzing the conversion of 4-hydroxybutryrylaldehyde to 1,4-butanediol.
  • native malate dehydrogenase such as (in E. coli), Mdh
  • one or more subunits of a global regulator of gene expression under microaerobic and/or aerobic conditions such as (in E. coli), ArcAB
  • native lactate and/or alcohol dehydrogenases, and/or pyruvate formate lyase such as (in E. coli) LdhA, AdhE, and PfIB
  • pyruvate dehydrogenase can be modified by deleting the native lipoamide dehydrogenase (e.g., LpdA in E.
  • heterologously expressed LpdA such as from Klebsiella pneumoniae.
  • the heterologously expressed LpdA can optionally harbor a mutation reducing NADH sensitivity, such as D354K.
  • tricarboxylic acid cycle flux can increased by introducing a mutation to reduce NADH inhibition of citrate synthase, e.g., by introducing a GltA-R163L mutation to E. coli GItA.
  • an a-ketoglutarate decarboxylase can be overexpressed, e.g., SucA from E.
  • the biosynthetic pathway is for producing 1,3-propanediol from the cellular glycolytic intermediate dihydroxyacetone phosphate, and comprises genes, optionally overexpressed and/or heterologous, encoding : a glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, e.g., DAR1 and GPP2 from Saccharomyces cerevisiae, catalyzing the conversion of dihydroxyacetone phosphate to glycerol;
  • glycerol dehydratase e.g., DhaBl, DhaB2, and DhaB3 from Klebsiella pneumoniae, catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde
  • aldehyde oxidoreductase e.g., YqhD from E. coli, catalyzing the conversion of 3- hydroxypropionaldehyde to 1,3-propanediol.
  • PEP-dependent glucose transport is eliminated via deletion of one or more genes in glucose-specific PTS enzyme II, e.g. , PtsG in E. coli, and an ATP-dependent glucose transport system composed of galactose permease (e.g. , GalP in E. coli) and glucokinase (e.g. , Glk in E. coli) are overexpressed or heterologously expressed.
  • glucose-specific PTS enzyme II e.g. , PtsG in E. coli
  • an ATP-dependent glucose transport system composed of galactose permease (e.g. , GalP in E. coli) and glucokinase (e.g. , Glk in E. coli) are overexpressed or heterologously expressed.
  • galactose permease e.g. , GalP in E. coli
  • glucokinase e.g. , Glk in E. coli
  • glyceraldehyde-3-phosphate dehydrogenase (e.g. , Gap in E. coli), is downregulated
  • the biosynthetic pathway is for producing 1,3-butanediol from the cellular intermediate acetyl-CoA, and comprises genes, optionally overexpressed and/or heterologous, encoding : a 3-ketothiolase, e.g., PhaA from Ralstonia eutropha NBRC 102504, catalyzing the conversion of acetyl-CoA to acetoacetyl-CoA;
  • an acetoacetyl-CoA reductase e.g., PhaB from Ralstonia eutropha NBRC 102504, catalyzing the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA;
  • butyraldehyde dehydrogenase e.g., Bid from Clostridium saccharoperbutylacetonicum ATCC 27012, catalyzing the conversion of 3-hydroxybutyryrl-CoA to 3- hydroxybutyraldehyde;
  • an aldehyde-alcohol dehydrogenase e.g., AdhE from E. coli, catalyzing the conversion of 3- hydroxybutyraldehyde to 1,3-butanediol.
  • 1,5-pentanediol is produced from glutaric acid, optionally via glutaryl- CoA, via reduction of the 1- and 5-carboxylic acids to alcohols.
  • Pathways describing the production of glutaric acid from the intracellular amino acid L-lysine have been described (Adkins et a/. , 2013; Park et a/. , 2013) .
  • Biosynthesis of the glutaryl-CoA intermediate has been described by Cheong et a/. , 2016. 3) Processes
  • 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 a diol or other polyol also provided is a method of identifying a bacterial cell which is tolerant to at least one diol or other polyol.
  • 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 result, e.g. , in the knock-out or knock-down of a gene, introduce a mutation into an endogenous gene, or which 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 Examples) or to conditions which introduce random mutations in endogenous genes, and bacterial cells which comprise one or more Group 1 and/or Group 2 modifications according to any preceding aspects and embodiments can then be identified.
  • this involves preparing a population of the genetically modified bacterial cell, having different Group 1 and/or Group 2 modifications, and then selecting from this population any bacterial cell which has an improved tolerance to the diol or other polyol, e.g., an aliphatic diol or other polyol.
  • the Group 1 modification is a knock-down or knock-out of one or more endogenous genes selected from metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph or, e.g., a knock-down or knock-out of metJ in combination with relA and purT or with acrB and/or acrA, and/or a knock-down or knock-out of iscR and relA.
  • endogenous genes selected from metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph or, e.g., a knock-down or knock-out of metJ in combination with relA and purT or with acrB and/or acrA, and/or a knock-down or knock-out of iscR and relA.
  • the Group 2 modification is a mutation in at least one endogenous protein or gene selected from NanK, RpsA, RpoB, RpoC, SpoT, NusG, Flu, Lon, or YgaH, such as e.g.
  • NanK-T128S RpoA-D305G, RpoA-D305A, RpsA-D310V, RpsA-D310I, RpsA-D310L, RpsA-D310M, RpsA- D310F, RpsA-D310A, RpoA-G279V, RpoA-G279I, RpoA-G279L, RpoA-G279M, RpoA-G279F, RpoA-G279A, RpsA-G21V, RpsA-G21I, RpsA-G21L, RpsA-G21M, RpsA-G21F, RpsA-G21A, RpoB-H447Y, RpoB-H447F, RpoB-H447W, RpoB-H447T, RpoB-H447S, RpoC-L268R
  • the process comprises genetically modifying the bacterial cells, e.g. , the E. coli cells, to express a mutant NanK, RpoC, Flu, RpoB, Lon, YgaH, such as, e.g. , NanK- T128S, RpoC-L268R, Flu-L642Q, RpoB-I1112S, Lon-I716S, and/or YgaH-V39A mutation, or a conservative substitution of any thereof, and/or a mutation which increases the expression of PyrE.
  • a mutant NanK, RpoC, Flu, RpoB, Lon, YgaH such as, e.g. , NanK- T128S, RpoC-L268R, Flu-L642Q, RpoB-I1112S, Lon-I716S, and/or YgaH-V39A mutation, or a conservative substitution of any thereof, and/or
  • the processes may further comprise a step of selecting any bacterial cell which has an improved tolerance to a diol or other polyol at a predetermined concentration, such as at least 1% v/v or higher, such as at least
  • v/v or higher 1.5% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7% v/v or higher, such as at least 8% v/v or higher, such as at least 10% v/v or higher; - an optional step of introducing a recombinant biosynthetic pathway for producing the diol or other polyol; or both of the above steps, in any order.
  • the diol is 2,3-butanediol
  • the predetermined concentration is at least 1% v/v or higher, such as at least 1.5% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7% v/v or higher, such as at least 10% v/v or higher.
  • the diol is 1,2- propanediol
  • the predetermined concentration is at least 1% v/v or higher, such as at least 1.5% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7% v/v or higher, such as at least 7.5% v/v or higher, such as at least 8% v/v or higher, such as at least 10% v/v or higher.
  • the diol is 1,5-pentanediol
  • the predetermined concentration is at least 0.5% v/v or higher, such as at least 1% v/v or higher, such as at least 2% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7% v/v or higher, such as at least 7.5% v/v or higher, such as at least 8% v/v or higher, such as at least 10% v/v or higher.
  • the diol is 1,2-pentanediol
  • the predetermined concentration is at least 0.5% v/v or higher, such as at least 1% v/v or higher, such as at least 2% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7% v/v or higher, such as at least 7.5% v/v or higher, such as at least 8% v/v or higher, such as at least 10% v/v or higher.
  • the predetermined concentration is at most 7%, such as at most 8%, such as at most 9%, such as at most 10%, such as at most 15%, such as at most 20%.
  • Assays for assessing the tolerance of a modified bacterial cell to the diol or other polyol typically evaluate the growth rate, lag time, or both, of the bacterial cell at predetermined concentrations for the diol or other polyol in question, typically as compared to a control.
  • 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.
  • a method of producing a diol or other polyol 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 the diol or other polyol 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; raw carbon feedstocks such as crude glycerol and cane syrup; as well as hydrolysates produced from cellulosic or lignocellulosic materials.
  • methionine supplementation can improve endogenous production of diols in diol-overproducing strains during fermentation.
  • robust growth of K-12 MG1655 in 6% 2,3-butanediol or 8% 1,2-propanediol was significantly restored by the addition of methionine, with a growth rate approaching that of evolved strains in such media, whereas evolved strains did not have a significantly enhanced growth rate with the addition of methionine.
  • the added methionine concentration is at most 0.03 g L "1 gDCW “1 , such as at most 0.07 g L “1 gDCW “1 , such as at most 0.2 g L “1 gDCW “ ⁇
  • the added methionine concentration is in the range from about 0.0004 to about 0.2, such as from about 0.007 to about 0.2, such as from about 0.015 to about 0.2, such as from about 0.03 to about 0.2, such as from about 0.07 to about 0.2 g L "1 gDCW "1 .
  • the bacterial cell may comprise one or more genetic modifications according to any aspect or embodiment described herein.
  • the medium comprises no more than 10, such as no more than 8, such as no more than 6, such as no more than 5, such as no more than 4 other natural amino acids, e.g. , selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at a biologically relevant level, e.g. , at a concentration of at least 0.0002 g L "1 gDCW "1 .
  • the method further comprises isolating the aliphatic diol.
  • a bacterial cell which has an increased tolerance to a diol or other polyol can be useful for preparing producer cells for the production of the diol or other polyol.
  • 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 a propanediol, butanediol, pentanediol or a hexanediol, e.g.
  • 2,3-butanediol 1,2- propanediol; 1,4-butanediol, 1,3-propanediol, 1,2-butanediol, 1,5 pentanediol and/or 1,2- pentanediol, such as in 2,3-butanediol, 1,2-propanediol, or both.
  • compositions of a plurality of bacterial cells according to any aspect or embodiment described herein, e.g. , an in vitro culture of such bacterial cells, optionally in a suitable culture medium and/or a chemically-defined medium comprising a carbon source.
  • the composition is substantially homogenous with respect to the bacterial cells.
  • a composition comprising a plurality of bacterial cells according to any preceding aspect or embodiment and a diol or other polyol.
  • the diol or other polyol 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 as compared to the parent bacterial cells, e.g.
  • the concentration of the a diol or other polyol is at least 1% v/v or higher, such as at least 1.5% v/v or higher, such as at least 3% v/v or higher, such as at least 5% v/v or higher, such as at least 6% v/v or higher, such as at least 7.5% v/v or higher, such as at least 10% v/v or higher, such as at least 20% v/v or higher; such as at least 30% v/v or higher; such as in the range of 1% to 30% v/v, such as in the range of 2% to 20% v/v; such as in the range of 5% to 15% v/v or 5 to 10% v/v.
  • the composition comprises 2,3-butanediol. In one embodiment, the composition comprises 1,2-propanediol. In one embodiment, the composition comprises 1,5- pentanediol. In one embodiment, the composition comprises 1,2-pentanediol. In one embodiment, the composition comprises 1,4-butanediol. In one embodiment, the
  • composition comprises 1,3-propanediol.
  • the composition comprises 1,2- butanediol.
  • “Cross-compound tolerance testing” some of the genetic modifications according to the invention also confer tolerance to other chemicals, such as to other polyols or diols, to hexanoate and/or to p-coumarate.
  • a composition comprising hexanoate at a concentration of at least 0.1 g/L, such as at least 1 g/L, such as at least 5 g/L, such as at least 10 g/L, such as at least 20 g/L, or p-coumarate at a concentration of at least
  • 1 g/L such as at least 2.5 g/L, such as at least 5 g/L, such as at least 7.5 g/L, such as at least 15 g/L; and a plurality of bacterial cells according to any preceding aspect or embodiment.
  • the bacterial cells are of the Escherichia, Lactobaccillus, Lactococcus,
  • Corynebacterium, Bacillus, Ralstonia, or Pseudomonas genera such as, e.g. , E. coll cells, and comprise a) at least one genetic modification which reduces expression of an endogenous gene selected from the group consisting of metJ, rzpD, yhjA, gtrS, ycdU, iscR, sspA and rph, or a combination of any thereof;
  • Such bacterial cells may further comprise one or more Group 2 modifications as described in any aspect or embodiment herein.
  • Assays for assessing the tolerance of a modified bacterial cell to a diol or other polyol 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).
  • the predetermined concentrations(s) could be, for example, 6% v/v, 7% v/v or 8% v/v.
  • 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, as well as cell cultures of such bacterial cells or strains 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 Escherichia, Enterobacter, Klebsiella, Lactobaccillus, Lactococcus, Corynebacterium, Bacillus, Ralstonia, Paenibacillus, Clostridia, Citrobacter sp. or Pseudomonas genera, such as from the Escherichia,
  • the bacterial cell is an E. coli 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
  • 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 H 16 and Ralstonia eutropha JMP134.
  • the bacterial cell is a Corynebacterium cell, such as a cell of the commercially available and/or fully characterized strains 534 (ATCC 13032), K051, MBOOl, R, SCgGl, and SCgG2.
  • While aspect and embodiments relating to bacterial cells herein typically refer to genes or proteins according to their designation in E. coll, 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, i.e. , the ortholog and/or paralog, in the other family or species, typically by identifying sequences having moderate or high homology to the E. coll sequence, optionally 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.
  • Table 2B sets out some examples of homologs or orthologs in selected organisms, identified in a preliminary and non-limiting analysis. Indeed, homologs or orthologs of these proteins exist also in other bacteria, and other homologs or orthologs 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 or orthologs across different species. To briefly summarize some of the preliminary findings in Table 2B: elA, PurT, YfgF, and PyrE are widely conserved and were identified in all organisms.
  • FabR, RzpD, and YhjA had the longest alignments with Pseudomonas putida proteins, with other more partial alignments to FabR annotated as transcriptional regulators in all other organisms.
  • IscR was found in Gram-negative organisms and Bacillus subtilis, with other more partial alignments annotated as transcriptional regulators in other organisms.
  • SspA was found to be conserved in Gram-negative organisms.
  • RpoA, RpoB, RpoC, SpoT, RpsA and NusG were found to be widely conserved in all organisms.
  • E. coli gene Protein function E.C. Locus ID SEQ ID NO: designation number
  • each recited gene is instead (i) a gene encoding the corresponding (homolog or ortholog) protein in Table 2A or 2B above, (ii) a gene located at the corresponding locus, or (iii) both.
  • improved tolerance toward an aliphatic diol or other aliphatic polyol can be achieved by one or more genetic modifications which increase one or more of (a) the biosynthesis of methionine in the bacterial cell; (b) growth of the bacterial cell during polyol-induced methionine starvation, and (c) reduced efflux of precursors or intermediates required for methionine biosynthesis.
  • This can, e.g. , be achieved by a reduced expression of metJ, optionally also of relA and purT, and/or one or more other genetic modifications described herein.
  • the bacterial cell has a genetic modification which reduces the expression of one or more endogenous proteins selected from the group consisting of - A transcriptional repressor of a methionine regulon
  • a stringent starvation protein synthesized predominantly when cells are exposed to amino acid starvation
  • a permease subunit of a multidrug efflux system A DNA-binding transcriptional repressor
  • a cyclic di-GMP phosphodiesterase A cyclic di-GMP phosphodiesterase.
  • Escherichia coli K-12 MG1655 was grown overnight in M9 minimal medium + 1% glucose and subcultured the following morning to an initial OD 500 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 5 oo Of 0.7. The diluted cells were used to inoculate M9 + 1% glucose containing varying concentrations of diols, 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.
  • Culture OD 500 was monitored at times determined by a predictive custom script, and when the OD 500 reached approximately 0.3, 150 pL 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 600 greater than 0.3, and reinoculations were occasionally performed from cryogenic stocks of the population. When the growth rate was observed to substantially increase, the media concentration was changed. These concentration changes were to 5.5%, 6.5%, 7%, and 8% for 1,2- propanediol and to 6.5%, 7%, and 8% for 2,3-butanediol. Approximately 100 pL of each population (8 per chemical) were plated on LB agar and incubated at 37°C overnight. Primary screening of ALE isolates
  • cryogenic glycerol stocks of the overnight culture were saved in a 96 well plate format.
  • Half deepwell plates were incubated at 37°C with 225 rpm shaking in a Growth Profiler (Enzyscreen), with optical scans of the plates taken at 15 minute intervals.
  • Green pixel values integrated over a 1 mm diameter circular area in each well were converted to OD(5oo values using a previously determined calibration between OD 600 and green pixel values. Resulting growth curves were visually inspected for isolates exhibiting the most robust or unique growth patterns within each population. In general, it was attempted to select three isolates per population for further analysis, and all populations were represented in the resequenced isolates.
  • 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 Pl vir phage transduction (Baba et ai , 2006) .
  • the Keio strain was grown to early exponential phase in LB + 5 mM CaCI 2 and 80 ⁇ _ of a Plw ' r 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 ⁇ L of the Pl vir 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 6% and 7% 2,3-butanediol or 6% and 8% 1,2-propanediol, and cultivated in a Growth Profiler as previously described for screening of ALE isolates.
  • Keio collection mutants were struck on LB + 25 ⁇ g/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 ⁇ M9 + 1% glucose. The next day, cells were inoculated into plates for cultivation on the Growth Profiler as described above.
  • Plasmid pET-RABC was obtained from Dr. Cuiqing Ma and Dr. Chao Gao (Shandong).
  • the hsdR deletion was found to be necessary for transformation in K-12 MG1655 due to the presence of EcoKI (HsdM/HsdR/HsdS) restriction sites in the plasmid.
  • the plasmid was transformed into each AhsdR strain by adding the plasmid to cells resuspended in TSS buffer followed by heat shocking for 30 seconds at 42°C, placing the cells on ice, resuspending in LB, and outgrowing at 37°C for 1-2 hours. The outgrown cells were plated on LB agar plates containing 50 ⁇ g/mL kanamycin to select for transformants.
  • E. coli K-12 MG1655 exhibited a steadily decreasing growth rate as a function of diol concentration in general (Table 3). Toxicity appeared to depend on carbon chain length, with toxicity increasing in order of 1,2-propanediol, 2,3-butanediol, and the pentanediols. Toxicity was much greater for 1,2-pentanediol than for 1,5-pentanediol, with growth observed at maximum concentrations of 1% and 3.5%, respectively. Maximum concentrations for robust growth in 2,3-butanediol and 1,2-propanediol were 5% and 7.5%, respectively.
  • Mean std. error mean std. error diol % (v/v) ⁇ (h 1 ) t, auß (h) ⁇ (h 1 ) t, a redesign (h) ⁇ (h 1 ) 3 ⁇ 4 a manner (h) ⁇ (h 1 ) 3 ⁇ 4 a provoke (h)
  • strains with an asterisk (*) following their name are hypermutator strains, and only the mutation identified that can be associated with generating the hypermutator phenotype (mutations in mutS, mutY, or mutL) and those mutations that are shared with other mutations in the same gene in other strains are shown.
  • the majority of isolates were hypermutator strains, with the exception of 12PD4-6, 12PD6-3, and 12PD6-9. A large number of called missing coverage deletions in 12PD6-9 were likely a result of an adapter problem, and these are not considered.
  • mutator strains only mutations in genes (or surrounding intergenic regions) that were common between the mutator isolates and the non-mutator isolates are listed. Mutations that occur independently across multiple populations, or that appear fixed in a highly variable population, are likely causative and of highest interest. For 2,3-butanediol, these include mutations in metJ, relA, nanK, purT, rpoB, and rpoC. Mutations also occur in acrB in 2 populations.
  • those of metJ, relA, purT, and acrB are likely loss- of-function mutations, due to the presence of frameshift mutations, large deletions, or IS element insertions in at least one population of individual isolate that possesses mutations in that gene.
  • Other mutations are likely gain-of-function or weakening of function, for example coding mutations in genes encoding subunits of RNA polymerase (RpoB and RpoC), which are essential, and the T128S mutation in NanK, which is present in nearly every population.
  • mutations in metJ were all coding, however they are also assumed to be loss-of-function mutations due to the co-occurrence of probable loss-of-function mutations for 2,3-butanediol. Because most isolates were hypermutators, SNPs are expected to be more common than other types of mutations. There were additionally mutations in relA in most isolates, which are also presumed to be losses-of-function based on loss-of-function mutations found for 2,3-butanediol evolved isolates.
  • Each re-sequenced isolate was characterized using the Biolector system for growth in M9 media containing 7% (v/v) 2,3-butanediol or 8% (v/v) 1,2-propanediol in biological triplicates. Tables showing the calculated average growth rates and lag times for each isolate of each detected phase (using custom automated growth parameter determination software) are shown in Table 6 for 2,3-butanediol, and Table 7 for 1,2-propanediol. Standard errors are standard deviations about the mean of the growth rate and lag time for the three independent biological replicates. In the presence of diols, many strains exhibited diauxic or triauxic growth patterns, manifesting in the presence of multiple growth phases. A value is only shown for second and third phases if two or more replicates had a growth phase detected, and that value is the average of the parameters calculated for those determined growth phases.
  • Probable loss-of-function mutations were identified from re-sequencing results as described in methods and the section on resequencing of selected isolates. Initially, single gene knockouts of metJ, relA, purT, fabR, clsA, yfgF, treA, and acrB were constructed and tested with a selection of evolved isolates in 7% (v/v) 2,3-butanediol (Table 8) or 8% (v/v) 1,2- propanediol (Table 9). The wild-type strain and the majority of single knockout strains did not grow in 7% 2,3-butanediol.
  • K-12 AmetJ acrBr.kan did not exhibit improved growth relative to the single knockout K-12 metJr.kan, nor did any other knockout combination with metJ.
  • K-12 AfabR yfgFrkan exhibited an increased growth rate in the primary growth phase and reduced lag times relative to the fabR and yfgF single deletion strains, as well as an increased secondary growth phase (which was present but not automatically detected for at least 2 out of 3 replicates for the fabR and yfgF single deletion strains). It also had a reduced lag time relative to K-12 MG1655 and a higher secondary phase growth rate than K-12 MG1655.
  • triple gene deletions were also constructed and tested with 7% 2,3-butanediol (Table 12) and 8% 1,2-propanediol (Table 13), with the single knockout strain K-12 MG1655 acrBr.kan also added. Additional genes were also tested in combination with deletions in metJ and relA, including rnb (co-occurring mutations in population 23BD1 and 23BD4-3), treR (co-occurring mutations in 23BD7-4 and 23BD7-7), and yeaR (co-occurring mutations in 23BD4-3, 23BD4-4, and 23BD5-1).
  • K-12 metJ relA purTr. kan had an increased growth rate vs. K-12 metJr. kan, indicating a positive epistatis between these three loss-of-function mutations.
  • the acrB single knockout strain did not have a detectable growth phase, also demonstrating that metJ and acrB losses-of-function have a synergetic effect when combined. None of the other triple knockout strains exhibited a detectable growth phase in at least 2 out of 3 replicates, with substantially lower growth than K-12 metJr.
  • K-12 AmetJ ArelA purTrkan had a higher average growth rate than K- 12 metJr.kan alone although with higher variability (individual replicates had growth rates of 0.39, 0.64, and 0.32 h "1 ).
  • the K-12 AmetJ acrBrkan did not have an increased growth rate over K-12 metJr.kan alone (again), and no other triple knockout combination with metJ and relA exhibited a higher growth rate than K-12 metJr.kan.
  • Keio collection of gene knockouts is a commercially available 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 Tables 4 and 5 were screened for growth against the BW25113 control in M9 + 1% glucose + 6% (Table 14) or 7% (v/v) 2,3-butanediol, and 6% or 8% (v/v) 1,2-propanediol (Table 15).
  • BW25113 iscR: : kan 0.240 3.7 0.008 0.7
  • Methionine supplementation was also tested for its ability to restore growth in the presence of 8% (v/v) 1,2-propanediol. Wild-type and a selection of evolved strains were tested (Table 21), and methionine was again found to restore growth of the wild-type strain, with minimal effect on evolved strains.
  • Table 20 Growth rates and lag times of the wild-type strain, selected 2,3-butanediol evolved strains, and K-12 MG1655 metJr.kan in M9 supplemented with 0.3 g/L methionine, 6% (v/v) 2,3-butanediol, or 6% (v/v) 2,3-butanediol and 0.3 g/L methionine, as measured in the Biolector testing format.
  • Table 21 Growth rates and lag times of the wild-type strain and selected 1,2-propanediol evolved strains in M9 supplemented 8% (v/v) 1,2-propanediol, or 8% (v/v) 1,2-propanediol and 0.3 g/L methionine, as measured in the Biolector testing format.
  • (p)ppGpp is largely synthesized by RelA, which associates with with the ribosome and is activated by binding of uncharged tRNAs.
  • (p)ppGpp regulates numerous gene products required for cell growth, with the net effect being the induction of a growth arrest (stringent response) when (p)ppGpp
  • (p)ppGpp also accumulates in diol-stressed cells, and that this occurs via either the sensing of uncharged tRNAs in general by RelA (Hauryiuk et a/. , 2015), or detection of ribosome stalling by RelA due to lack of methionyl-tRNAs (Haft et a/. , 2014), or indirectly due to iron starvation (Miethke et a/. , 2006; Vinella et a/. , 2005) induced by toxic concentrations of diols, as elaborated on below.
  • PurT is one of two transformylases in purine biosynthesis, with the other being PurN. PurT utilizes the formyl group from formate, whereas PurN utilizes the formyl group from
  • formyltetrahydrofolate which is also the formyl donor for generating initiator formylmethionine-tRNA (tRNA Met ) that is required for initiating translation of AUG start codons.
  • tRNA Met initiator formylmethionine-tRNA
  • This explanation provides a functional linkage between the metJ, relA, and pur T genes that all involve coping strategies for methionine starvation, and could explain the negative epistasis in the metJ relA double knockout and the positive epistasis in the metJ relA pur T triple knockout.
  • Methionine supplementation can thus be a strategy for improving endogenous production of diols in diol-overproducing strains during fermentation, since it is expected that growth would be inhibited by secreted diols at high concentrations due to the same mechanisms of toxicity observed here.
  • Iron starvation is known to trigger the stringent response and SpoT-dependent accumulation of (p)ppGpp in E. coli and other bacterial species (Miethke et a/. , 2006; Vinella et a/. , 2005), which is believed to help stimulate expression of iron uptake systems, thereby alleviating iron starvation conditions (Vinella et a/., 2005).
  • the loss-of-function in relA optionally in combination with a SpoT coding mutation such as SpoT-I213L or conservative substitutions thereof, may stimulate SpoT-dependent accumulation of (p)ppGpp and the increased expression of one or more iron uptake systems.
  • Iron starvation could potentially arise from either direct chelation of iron by diols, or from diols interfering with chelation of iron by siderophores such as enterobactin.
  • Derepression of iron-sulfur cluster biosynthesis and assembly enzymes via knockdown or knockout of iscR likely enables the more efficient use of cellular ferric iron for this critical function, as iron-sulfur clusters serve as catalytic cores of cytochromes involved in cellular respiration and in glutamate synthase.
  • McR isobactin
  • L-threonine is a precursor for production of a catecholic trilactone siderophore that is utilized for ferric iron uptake, and that the threonine, serine/glycine, and cysteine/methionine biosynthetic pathways are interdependent.
  • L-serine is a precursor for the production of enterobactin, another siderophore involved in ferric iron uptake.
  • enterobactin another siderophore involved in ferric iron uptake.
  • L- cysteine is synthesized from L-serine, a reduction in levels of L-serine could lead to L- cysteine starvation and thus also L-methionine starvation, as L-cysteine is also a precursor for biosynthesis of L-methionine.
  • Several 12PD isolates also exhibit cross-tolerance toward coumarate, however the only non-hypermutator strain is 12PD6-9.
  • This strain has non-coding mutations in ypjA, and mutations in ypjA thought to be inactivating were also found in p-coumarate evolved isolates.
  • the 2,3-butanediol evolved strain with the best overall tolerance toward the range of chemical stressors was 23BD8-7.
  • the majority of isolates being hypermutators was likely responsible for highly variable cross- tolerance between compounds in the 1,2-propanediol evolved strains, however the best- performing isolate was 12PD4-9.
  • Table 21 Normalized t 0 Di(evoived toDi(wiid-ty P e) values for 2,3-butanediol-evolved isolates grown in the presence of inhibitory concentrations of 12 different chemicals.
  • Table 22 Normalized t OD1(evo i ved) /t OD1(wl i d . f y pe) values for 1,2-propanediol-evolved isolates grown in the presence of inhibitory concentrations of 12 different chemicals.
  • each evolved isolate was tested for cross-tolerance toward other aliphatic diols 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 (note that this had been done in the Biolector format previously for 1,2-pentanediol and 1,5- pentanediol thus was not repeated here) : 1,3-propanediol and 1,4-butanediol. Variable concentrations of these compounds elicited growth inhibition in E. coli K-12 MG1655 (Table 23).
  • a screening concentration was selected for the evolved isolates for which wild-type cells could achieve at a growth rate of 0.15-0.3 h "1 (versus uninhibited growth at 0.7-0.9 h "1 in M9 glucose minimal medium). These concentrations were: 5.5% (v/v) 1,3-propanediol, 5.5% (v/v) 1,4-butanediol, 1.25% (v/v) 1,2-pentanediol, and 3.5% (v/v) 1,5-pentanediol. The results of 2,3-butanediol-evolved isolates grown in these concentrations of alternative diols are shown in Table 24.
  • diol mean std. error mean std. error
  • Table 24 Growth rates and lag times of K-12 MG1655 and 2,3-butanediol-evolved isolates in specified inhibitory concentrations of diols, as measured in the Growth Profiler testing format.
  • DHAP can be readily generated (e.g. glucose, glycerol, or xylose)
  • a methylglyoxal intermediate which depending on the choice of reducing enzyme, can generate either (S)- or (R)-lactaldehyde, or acetol. These can then be further reduced to (S)- or (R)-l,2- propanediol, or acetol can be reduced to a racemic mixture of both isomers.
  • S S- or (R)-l,2- propanediol
  • acetol can be reduced to a racemic mixture of both isomers.
  • coli is 5.6 g/L, and this was obtained using glycerol as a carbon source in a strain with inactivations of ackA-pta (acetate formation), replacement of the native PEP-dependent dihydroxyacetone kinase with an ATP-dependent enzyme from Citrobacter freundii, and overexpression of native methylglyoxal synthase (MgsA), L-1,2- propanediol dehydrogenase (GldA), and NADPH-dependent aldehyde reductase YqhD
  • acetoin reductases can then be utilized to generate 2,3-butanediol stereoisomers, or diacetyl can be used to generate acetylacetoin, from which different stereoisomers of 2,3- butanediol can ultimately derive.
  • Up to 119 g/L of 2,3-butanediol has been produced in Enterobacter cloacae subsp. dissolvens SDM utilizing lignocellulosic hydrolysates by simply deleting byproduct producing genes (Li et al. , 2015).
  • the best demonstrated production in recombinant E. coli from glucose is 73.8 g/L using E. coli BL21(DE3) containing a plasmid (pET-RABC) overexpressing a gene cluster from
  • yeast extract due to introduction of the heterologous acetolactate synthase, thus yeast extract was utilized in the screen. Results are shown in Table 25.
  • yeast extract which was not employed in the evolutions, the majority of evolved isolates did not exhibit improved endogenous production of 2,3-butanediol as compared with wild-type K-12 MG1655 harboring the same modifications.
  • 23BD7-5 is notable in possessing a loss-of-function mutation in acrB that the other isolates from population 23BD7 do not possess, however it also lacks mutations in to/C, treR, and yhjA that the other 23BD7 isolates possess.
  • 23BD8-2 is notable in being the only resequenced evolved isolate lacking a mutation in metJ. It instead harbors a probable loss- or reduction-of-function mutation in iscR (inferred by Keio screening results described above) as well as mutations in relA, rpoB, Ion, ygaH, and a mutation that increases the expression of PyrE.
  • Table 25 2,3-butanediol titers in background strains harboring the AhsdR mutation and plasmid pET-RABC, measured from screening in a minimal medium containing 5% (w/v) glucose and 1% (w/v) yeast extract after 48 hours at 30°C.
  • Datsenko KA Wanner BL.
  • Nakamura CE Whited GM . Metabolic engineering for the microbial production of 1,3- propanediol. Curr. Opin. Biotechnol. 14:454-459 (2003) .
  • Van Bogelen RA Kelley PM, Neidhardt FC. Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. 3. Bacteriol. 169:26-32 (1987) . Van Dien S. From the first drop to the first truckload : commercialization of microbial processes for renewable chemicals. Gurr. Opin. Biotechno. 24: 1-8 (2013) .
  • Zingaro KA Dissecting the assays to assess microbial tolerance to toxic chemicals in bioprocessing. Trends Biotechnol. 31 : 643-653 (2013) . US 2012/0282661 Al (Genomatica Inc.)
  • EP 2 580 315 A2 (Cobalt Technologies Inc.)

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Abstract

L'invention concerne des cellules bactériennes génétiquement modifiées pour améliorer leur tolérance à certains produits chimiques de base, tels que des diols et autres polyols, et des procédés de préparation et d'utilisation de telles cellules bactériennes pour la production de polyols et autres composés.
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CN110484466A (zh) * 2019-08-15 2019-11-22 华南理工大学 一种提高嗜热厌氧杆菌发酵性能的方法
CN110592109A (zh) * 2019-08-28 2019-12-20 黑龙江伊品生物科技有限公司 一种spoT基因改造的重组菌株及其构建方法与应用

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Publication number Priority date Publication date Assignee Title
CN108795963A (zh) * 2018-06-27 2018-11-13 青岛农业大学 对大肠杆菌gloA基因进行定点定向突变的方法
CN110484466A (zh) * 2019-08-15 2019-11-22 华南理工大学 一种提高嗜热厌氧杆菌发酵性能的方法
CN110484466B (zh) * 2019-08-15 2023-04-21 华南理工大学 一种提高嗜热厌氧杆菌发酵性能的方法
CN110592109A (zh) * 2019-08-28 2019-12-20 黑龙江伊品生物科技有限公司 一种spoT基因改造的重组菌株及其构建方法与应用

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