The present invention relates to bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as isobutyric acid, and to methods of preparing and using such bacterial cells for production of isobutyric acid and other compounds.

BACKGROUND OF THE INVENTION

Isobutyric acid, or 2-methylpropanoic acid, is a high-production volume chemical (>500 tons/year) currently manufacturered primarily as a solvent, herbicide, and as a precursor for isobutyrated products such as diisopropyl ketone and isopropyl esters (which are used in perfumes, oils for aircraft turbines, and solvents) (Riemenschneider et al. , 2012).

Isobutyrate salts are used to produce chemicals for the tanning and textile industries and for use as stabilizers, preservatives, and catalysts. Isobutyric acid may also be used as a precursor for chemical conversion to methacrylic acid via oxidative dehydrogenation, followed by esterification with methanol to generate methyl methacrylate (MMA); the direct precursor for production of polymethylmethacrylate (PMMA) plastics (Bauer et ai, 2012).

As an attractive alternative to the chemical route derived from petroleum products, isobutyric acid can be produced biologically from glucose via the same upstream pathway to

isobutyraldehyde that is employed for isobutanol production in E. coli (Zhang et ai , 2011), but instead employing, e.g. , an isobutyraldehyde dehydrogenase to oxidize isobutyraldehyde to isobutyric acid, and deleting the gene encoding a native alcohol dehydrogenase that ordinarily reduces isobutyraldehyde to isobutanol (Atsumi et a/., 2010; WO 2012/109534 A2).

For production of bulk chemicals from renewable plant-based carbon feedstocks, 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.

Escherichia coli being a suitable host for industrial applications, there has been some interest in developing E. coli strains with improved tolerance to chemicals of interest for production, such as, e.g., n-butanol, ethanol and isobutanol, or to stress conditions present during fermentation (see, e.g. , Sandberg et al., 2014; Lennen and Herrgard, 2014; Tenaillon et al. , 2012; Minty et al. , 2011; Dragosits et al. , 2013; Winkler et al. , 2014; Wu et al., 2014;

LaCroix et al. , 2015; Jensen et al. , 2015a and 2015b; Doukyu et al., 2012; Shenhar et al. , 2012; and Rath and Jawali, 2006).

Despite these and other advances in the art, there is still a need for bacterial cells with improved tolerance to chemicals of interest for bio-based production, such as isobutyric acid.

SUMMARY OF THE INVENTION

It has been found by the present inventors that certain genetic modifications unexpectedly improve the tolerance of bacterial cells, such as those of the Escherichia, Bacillus, Ralstonia, Pseudomonas, Lactobacillus or Lactococcus genera, to certain chemical compounds, particularly isobutyric acid and other branched- or straight-chain aliphatic acids, as well as branched-chain and straight-chain aliphatic alcohols.

Accordingly, the invention relates to bacterial cells with improved tolerance to at least isobutyric acid, as well as bacterial cells which are capable of producing isobutyric acid or another branched- or straight-chain aliphatic acid or alcohol, and have improved tolerance to certain chemicals, including isobutyric acid.

The invention also relates to compositions comprising such bacterial cells and isobutyric acid or a related compound, methods of preparing or screening for such bacterial cells, and methods of producing isobutyric acid or a related chemical compound using such bacterial cells. These and other aspects and embodiments are described further below. DETAILED DISCLOSURE OF THE INVENTION

As described herein, various aspects of the invention provide for genetically modified bacterial host cells with a higher tolerance to isobutyric acid or other, related chemical compounds of interest. When transformed with a recombinant biosynthetic pathway for producing the compound of interest from a carbon source, the genetically modified bacterial host cells of the invention result in improved production of the compound from carbon feedstock, since they maintain robust metabolic activity in the presence of higher concentrations of the compound than the unmodified parent cells. So, in one aspect, the bacterial cell comprises a recombinant biosynthetic pathway for producing isobutyric acid and at least one genetic modification which reduces expression of an endogenous gene selected from the group consisting of pykF, yobF, yjcF, rph, cheR and phoil, such as from the group consisting of pykF, yobF and phoU, or a combination of any thereof. In one embodiment, the bacterial cell comprises a genetic modification which reduces the expression of pykF, yobF or both. In one embodiment, the recombinant biosynthetic pathway provides for a production of more than 10 mg/L isobutyric acid within 48 hours of culture in the presence of a suitable carbon source. In one aspect, the bacterial cell comprises a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of rpoS, yobF, phoU, or a combination thereof; (b) a mutant GlyQ (SEQ ID NO:9) comprising a mutation in residue E48; (c) a mutant RpoB (SEQ ID NO: 18) comprising at least one mutation that alters the structure of the rifampicin binding pocket, such as a mutation in at least one of residues H526, S531, R540, Q513, F514 and D516; (d) a mutant RpoB (SEQ ID NO: 18) comprising a mutation in residue A1183; (e) a mutant SapC (SEQ ID NO: 20) comprising a mutation in residue S69; (f) a mutant RpsD (SEQ ID NO: 22) comprising a mutation in residue G87; (g) a mutant regulatory subunit of acetohydroxybutanoate synthase/acetolactate synthase (AHAS), the mutant regulatory subunit of AHAS providing for feedback-resistance to inhibition by L-valine, L-leucine or both; (h) a genetic modification which increases the expression of pyrE; (i) a genetic modification which reduces the expression of yqhD; or (j) a combination of any two or more of (a) to (i). Particular examples of mutations include, but are not limited to, GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB- A1183I, RpoB-A1183L, RpoB-A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB- H526T, RpoB-H526F, RpoB-H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L,

RpoB-D516V, RpoB-D516Y, RpoB-S531L, RpoB-S531W, SapC-S69P, SapC-S69A, RpsD-G87C, RpsD-G87S, RpsD-G87A, IlvH-L9F, IlvH-L9A, IlvH-L9V, IlvH-G14D, IlvH-S17F, IlvH-Nl lA, IlvH-N29H, IlvH-A36V, IlvH-T34I, IlvH-N29K, IlvN-N17H, IlvN-N17K, IlvN-G20D, IlvN-A30P, IlvN-V21D, IlvN-F34L, IlvN-I44R, and IlvN-I44F, as well as combinations thereof.

In another aspect, the invention relates to a bacterial cell comprising a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of yobF, rpoS or both; and/or (b) mutant GlyQ comprising a mutation in residue E48. In one embodiment, the bacterial cell also comprises a mutant regulatory subunit of acetohydroxybutanoate synthase/acetolactate synthase (AHAS) and a genetic modification which increases the expression of pyrE; the mutant regulatory subunit of AHAS providing for feedback-resistance to inhibition by L-valine, L-leucine or both. The mutant regulatory subunit of AHAS may, for example, be a mutant IlvH or a mutant IlvN. In a specific embodiment, the bacterial cell comprises genetic modifications reducing the expression of pykF and one or both of yobF and rpoS. The genetic modification can be a knock-down or a knock-out, e.g., a knock-out.

The bacterial cell may further comprise a recombinant biosynthetic pathway for producing a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid. For example, the recombinant biosynthetic pathway may produce a C3 to C8 branched-chain or straight-chain aliphatic acid, or a C3 to C5 branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid. In one embodiment, the

recombinant pathway is for producing a C4 to C8 branched-chain or straight-chain aliphatic acid.

A bacterial cell having improved tolerance may, for example, have an increased growth rate, reduced lag time, or both, in at least one of isobutyrate, butyric acid, valeric acid, 2- methylbutyric acid, isovaleric acid, 4-methylvaleric acid, 2-methylhexanoic acid, 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol and 1-pentanol, as compared to the parent bacterial cell. In one embodiment, the one or more genetic modifications or mutants increase the growth rate, reduces the lag time, or both, of the bacterial cell in about 1 g/L, about 6 g/L and/or about 12 g/L isobutyric acid or isobutyrate. In one embodiment, the bacterial cell has an increased growth rate, reduced lag time, or both, in at least about 6 g/L isobutyrate.

In one aspect, the invention relates to a bacterial cell comprising a recombinant biosynthetic pathway for producing isobutyric acid and a mutant regulatory subunit of AHAS, the mutant regulatory subunit of AHAS providing for feedback-resistance to inhibition by L-valine, L- leucine or both. In one aspect, a process for preparing a recombinant E. coli cell for producing isobutyric acid or a related compound, may comprise genetically modifying an E. coli cell to introduce a recombinant biosynthetic pathway for producing isobutyric acid or the related compound; and knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, rph, yjcF, yobF, cheR and phoil, such as a knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, phoU. In one embodiment, the process comprises comprising genetically modifying an E. coli cell to knockdown or knock-out at least one endogenous gene selected from the group consisting of pykF, yobF, and phoU, and (a) further knocking-down or knocking-out the endogenous gene rpoS; (b) introducing into the E. coli cell a recombinant biosynthetic pathway for producing isobutyric acid or a related compound, optionally wherein the recombinant biosynthetic pathway provides for a production of more than 10 mg/L isobutyric acid within 48 hours of culture in the presence of a carbon source; and/or (c) introducing at least one mutation selected from GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB-A1183I, RpoB-A1183L, RpoB- A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB-H526T, RpoB-H526F, RpoB- H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L, RpoB-D516V, RpoB-D516Y, RpoB-S531L, RpoB-S531W, SapC-S69P, SapC-S69A, RpsD-G87C, RpsD-G87S, RpsD-G87A, IlvH-L9F, IlvH-L9A, IlvH-L9V, IlvH-G14D, IlvH-S17F, IlvH-NllA, IlvH-N29H, IlvH-A36V, IlvH- T34I, IlvH-N29K, IlvN-N17H, IlvN-N17K, IlvN-G20D, IlvN-A30P, IlvN-V21D, IlvN-F34L, IlvN- I44R, and IlvN-I44F.

In one aspect, a process for improving the tolerance of an E. coli cell to a C3 to C8 branched- chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid, may comprise genetically modifying the E. coli cell to knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, rph, yjcF, yobF, cheR and phoU. In one embodiment, such processes may further comprise genetically modifying the E. coli cell to express a mutant GlyQ, express a mutant IlvH, express a mutant IlvN,

overexpress PyrE, or a combination of any thereof.

In one embodiment, a process for improving the tolerance of an E. coli cell to a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid, comprises (a) genetically modifying an E. coli cell to knock-down or knock-out at least one endogenous gene selected from the group consisting of pykF, yobF, and phoU, optionally further knocking-down or knocking-out the endogenous gene rpoS; (b) preparing a population of the genetically modified E. coli cell, which population comprises one or more mutations in one or more endogenous genes selected from glyQ, rpoB, sapC, spsD, ilvN, ilvH, rph and the pyrE/rph intergenic region, and (c) selecting any E. coli cell which has an improved tolerance.

In one aspect, the invention relates to a method for producing a C3 to C8 branched-chain aliphatic acid or branched-chain or straight-chain aliphatic alcohol, optionally isobutyric acid, comprising culturing the bacterial cell of any aspect or embodiment described herein, or a bacterial cell obtained or obtainable by the process of any aspect or embodiment described herein, in the presence of a carbon source.

In one aspect, the invention relates to a composition comprising isobutyric acid, butyric acid, valeric acid, 2-methylbutyric acid, isovaleric acid, 4-methylvaleric acid, 2-methylhexanoic acid, 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol and 1- pentanol at a concentration of at least about 1 g/L, at least 6 g/L or at least about 12 g/L, and a plurality of bacterial cells according to any aspect or embodiment herein. In one embodiment, the composition comprises isobutyric acid and a plurality of bacterial cells according to any aspect or embodiment described herein, a bacterial cell obtained or obtainable by the process of any aspect or embodiment described herein, wherein the isobutyric acid is present at a concentration of at least about 1 g/L.

Definitions Organic compounds may be described herein by their IUPAC name and/or other synonym known in the art. As a non-limiting example, "isobutyric acid" is also known as 2- methylpropanoic acid, 2-methylpropionic acid and isobutanoic acid.

A "related compound" to isobutyric acid includes, but is not limited to, other branched-chain aliphatic acids such as isovaleric acid (3-methylbutanoic acid), 2-methylbutanoic acid, 2- methylhexanoic acid, 4-methylvaleric acid, 3-methylhexanoic acid, 3-methyl-2-hexenoic acid, and other unsaturated derivatives and/or salts thereof; other straight-chain aliphatic acids such as butyric acid, valeric acid (pentanoic acid), hexanoic acid (caproic acid), octanoic acid (caprylic acid), propanoic acid, and unsaturated derivatives thereof and/or salts thereof; and branched- or straight-chain aliphatic alcohols including isobutanol, isopropanol, n-pentanol, n-hexanol, n-octanol, n-butanol, 1-propanol, 2-propanol, 2-methyl-l-butanol, 3-methyl-l- butanol, 2-methyl-l-hexanol, 3-methyl-l-hexanoland derivatives thereof. Preferably, the related compound comprises a 3- to 8-carbon (C3 to C8) branched or straight aliphatic chain. More preferably, the related compound comprises a C4 to C8 branched-chain or straight- chain aliphatic acid or a C3 to C5 branched-chain or straight-chain aliphatic alcohol. Most preferred are related compounds characterized by the presence of a carboxylic acid or carboxylate functionality and a branched or 4-carbon (C4) straight aliphatic carbon chain, such as e.g. , 2-methylbutanoic acid, isovaleric acid and butyric acid.

So long as it is not contradicted by context, any embodiment pertaining to a carboxylic acid herein equally pertains to its anion and/or salt, formed by the deprotonation of the carboxylic acid group. For example, any embodiment pertaining to isobutyric acid includes isobutyrate (e.g., the predominant form in which isobutyric acid exists at a pH above the relevant pK _a ).

As used herein, 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. In some cases, the recombinant biosynthetic pathway also comprises a deletion of one or more native genes in the host cell. The compound of interest is typically isobutyric acid or a related compound, and may be the actual end product or a precursor or intermediate in the production of another end product. The terms "tolerant" or "improved tolerance", when used to describe a genetically modified bacterial cell of the invention or a strain derived therefrom, refers to a genetically modified bacterial cell or strain that shows a reduced lag time, an improved growth rate, or both, in the presence of isobutyric acid than the parent bacterial cell or strain from which it is derived, typically at concentrations of at least 1 g/L, such as at least 2.5 g/L, such as at least 4 g/L, such as at least 6 g/L, such as at least 6.3 g/L, such as at least 7.5 g/L, such as at least 10 g/L, such as at least 12.5 g/L, such as at least 15 g/L, such as at least 20 g/L. 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.

The term "gene" refers to a nucleic acid sequence that encodes a cellular function, such as a protein, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "transgene" is a gene, native or heterologous, that has been introduced into the genome by a transformation procedure. Gene names are herein set forth in italicised text with a lower-case first letter (e.g., pykF) whereas protein names are set forth in normal text with a capital first letter (e.g., PykF) .

As used herein the term "coding sequence" refers to a DNA sequence that encodes a specific amino acid sequence.

The term "native", when used to characterize a gene or a protein herein with respect to a host cell, refers to a gene or protein having the nucleic acid or amino acid sequence as found in the host cell.

The term "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.

As used herein the term "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.

As used herein, 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. The term "expression", as used herein, 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.

As used herein, "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 . Typically, when the reduced expression is obtained by a genetic modification in the host cell, the control is the unmodified host cell. Sometimes, e.g. , in the case of gene knock-out, the reduction of native mRNA and functional protein encoded by the gene is higher, such as 99% or greater.

"Increased expression", "upregulation", "overexpressing" or the like, when used in the context of a protein or activity described herein, means increasing the protein level or activity within a bacterial cell. An increase in protein level can be achieved by, e.g., a mutation in the promoter region or other neighbouring segment providing for increased expression of an endogenous gene, the expression of a transgene encoding the protein, or other techniques known in the art. An up-regulation of an activity can occur through, e.g. , increased activity of a protein, increased potency of a protein or increased expression of a protein. The protein with increased activity, potency or expression can be encoded by genes disclosed herein.

Genetic modifications resulting in a reduced expression of a target gene/protein can include, e.g. , knock-down of the gene (e.g. , a mutation in a promoter that results in decreased gene expression), a knock-out of the gene (e.g. , a mutation or deletion of the gene that results in 99 percent or greater decrease in gene expression), a mutation or deletion in the coding sequence which results in the expression of non-functional protein, and/or the introduction of a nucleic acid sequence that reduces the expression of the target gene, e.g. a repressor that inhibits expression of the target or inhibitory nucleic acids (e.g. CRISPR etc.) that reduces the expression of the target gene.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J ., Fritsch, E. F. and Maniatis, T. Molecular Cloning : A Laboratory Manual, 4 ^th ed. ; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 2012; and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene

Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1984; and by Ausubel, F. M. et a/. , In Current Protocols in Molecular Biology, published by John Wiley & Sons (1995); and by Datsenko and Wanner, 2000; and by Baba et al., 2006; and by

Thomason et al. , 2007.

A "conservative" amino acid substitution in a protein is one that does not negatively influence protein activity. Typically, 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). Most commonly, 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

Specific embodiments of the invention

As described in the Examples, lag times of native K-12 MG1655 cells began increasing already at isobutyric acid concentrations above 1 g/L, and at concentrations above 10 g/L, growth was almost completely inhibited. By contrast, bacterial cells comprising one or more mutations according to the invention exhibit a dramatically improved growth at high concentrations isobutyric acid, e.g., 1 g/L or more, typically reflected by an increased growth rate, a reduced lag time, or both.

So, the invention provides bacterial cells with improved tolerance to isobutyric acid and related compounds, as well as related processes and materials for producing and using such bacterial cells.

1) Genetic modifications

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, as described below. a) Group 1 modifications

In one aspect, the bacterial cell has a genetic modification which reduces the expression of one or more endogenous genes listed in Table 4, including, but not limited to pykF, rph, yjcF, rpoS, yobF, cheR, and phoil. In one aspect, the bacterial cell has a genetic modification which reduces the expression of one or more endogenous genes selected from the group consisting of pykF, yobF and phoU. In one embodiment, the one or more endogenous genes comprise pykF.

In one aspect, there is provided a bacterial cell which comprises genetic modifications reducing the expression of at least two endogenous genes selected from the group consisting of pykF, rph, yjcF, rpoS, yobF, cheR and phoU. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of a gene selected from pykF, rpoS, and yobF, and, optionally, a second genetic modification which reduces the expression of rpoS.

In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of a gene selected from rpoS, yobF, and phoil. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of rpoS. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF and a second genetic modification which reduces the expression of yobF. In one embodiment, the bacterial cell comprises a first genetic modification which reduces the expression of pykF, a second genetic modification which reduces the expression of rpoS, and a third genetic modification which reduces the expression of yobF.

In other separate and specific embodiments, the bacterial cell comprises: - a first genetic modification which reduces the expression of rph, and a second genetic modification which reduces the expression of a gene selected from pykF, yjcF, rpoS, yobF, cheR and phoil;

a first genetic modification which reduces the expression of yjcF and a second genetic modification which reduces the expression of a gene selected from of pykF, rph, rpoS, yobF, cheR and phoil;

a first genetic modification which reduces the expression of rpoS and a second genetic modification which reduces the expression of a gene selected from of pykF, rph, yjcF, yobF, cheR and phoil; a first genetic modification which reduces the expression of yobF and a second genetic modification which reduces the expression of a gene selected from pykF, rph, yjcF, rpoS, cheR and phoU;

a first genetic modification which reduces the expression of cheR and a second genetic modification which reduces the expression of a gene selected from pykF, rph, yjcF, rpoS, yobF and phoU;

a first genetic modification which reduces the expression of phoU and a second genetic modification which reduces the expression of a gene selected from pykF, rph, yjcF, rpoS, yobF and cheR.

Knock-down or knock-out of a gene can be accomplished by any method known in the art for bacterial cells, and include, e.g. , lambda Red mediated recombination, PI phage

transduction, and single-stranded oligonucleotide recombineering/MAGE technologies (see, e.g. , Datsenko and Wanner, 2000; Thomason et ai , 2007; Wang et ai , 2009). Typically, a knock-down of a gene can be accomplished by, for example, a mutation in the promoter region or transcriptional regulator binding sites resulting in decreased transcription, a mutation in the ribosome binding site resulting in decreased translation, a deletion or mutation in the coding region of the gene resulting in a reduced or fully or substantially eliminated activity of the protein, or by the presence of antisense sequences that interfere with transcription or translation of the gene, resulting in reduced expression of the protein. Preferably, the knocking-down of a gene results in at least 20% reduction in the expression level of the gene product in the bacterial cell, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% or higher. A knock-out of a gene includes elimination of a gene's expression, such as by introducing a mutation in the coding sequence and/or promoter so that at least a portion (up to and including all) of the coding sequence and/or promoter is disrupted or deleted deletion, mutation, or insertion, resulting in loss of expression of the protein, or expression only of a non-functional mutant or non-functional fragment of the endogenous protein. As used herein, the symbol "DELTA" denotes a deletion of an endogenous gene. Preferably, a knock-out of a gene results in 1% or less of the native gene product being detectable, such as no detectable gene product.

In one specific embodiment, either one or both of the first and second genetic modifications is a knock-out of the gene, optionally a deletion. In an alternative embodiment at least one of the first and second genetic modifications is a knock-down of the gene. In one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-down or knock-out of the one or more endogenous genes, resulting in at least 20%, such as at least 50%, such as at least 80%, such as at least 90%, such as at least 95%, reduction in the level of mRNA encoded by the gene.

In one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-down or knock-out of the one or more endogenous genes, resulting in at least 20%, such as at least 50%, such as at least 80%, such as at least 90%, such as at least 95%, reduction in the level of protein encoded by the gene.

In one aspect, there is provided 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.

In one aspect, the bacterial cell of any aspect or embodiment described herein comprises a mutation in at least one of GlyQ, RpoB, SapC, RpsD, IlvH and/or in IlvN which provides for improved tolerance to isobutyric acid. In one embodiment, the bacterial cell comprises a mutation in GlyQ. In one embodiment, the bacterial cell comprises a mutation in RpoB and SapC. In one embodiment, the bacterial cell comprises a mutation in RpoB and RpsD. In one aspect, the bacterial cell of any one of the preceding embodiments comprises a mutation which increases the expression level of PyrE. A native or mutant protein can be expressed from a mutated version of the endogenous gene, or from a transgene. Advantageously, these mutations can be combined with each other and/or with one or more of the genetic modifications described in the preceding sections. b) Group 2 modifications In certain embodiments, a mutant protein is expressed in the bacterial cell, e.g., from a mutated version of the endogenous gene, or from a transgene encoding the mutant protein.

In one embodiment, the bacterial cell comprises a mutation in GlyQ which increases tolerance to isobutyric acid. In one particular embodiment, the GlyQ comprises a mutation, such as a deletion or amino acid substitution, in residue E48 or in the residue that aligns with residue E48 in E. coli GlyQ. In one embodiment, the mutation is an amino acid substitution of this residue into D or N. Preferably, the mutation is E48D or a conservative amino acid substitution thereof, such as E48N. In one particular embodiment, the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein. For example, the Group 1 modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF. The Group 2 modification can be, for example, a mutation in one or more of RpoB, SapC, RpsD, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic

modifications are described herein.

In one embodiment, the bacterial cell comprises one or more mutations which provide for, or increase, feedback-resistance to inhibition by L-valine, L-leucine, or both. As described in Example 1 in the section entitled "Investigation of mutations in IBUA8", several isolates harbored mutations in IlvH and IlvN, which are small regulatory subunits of different active isoforms of AHAS in E. coli. The three AHAS isoforms catalyze the first common steps in the biosynthesis of the branched chain amino acids, L-valine, L-isoleucine, and L-leucine, through complex allosteric inhibition mediated by the small regulatory subunits of each isoform (IlvH, IlvN, and IlvO) which can be inhibited by valine and/or leucine. Accordingly, in a preferred embodiment, the bacterial cell comprises a mutant regulatory subunit of AHAS, e.g., in IlvH and/or IlvN. Non-limiting examples of mutations in IlvH include a deletion or amino acid substitution in the N-terminal region, the C-terminal region, or both, such as (when aligned with E. coli IlvH), e.g., L9F, L9A, L9V, G14D, S17F, N11A, N29H, A36V, T34I, N29K, as well as conservative substitutions thereof (Kaplun et a/. , 2006; US Patent No. 6,737,255 B2; Mendel et al. , 2001). Non-limiting examples of mutations in IlvN include N17H, N17K, G20D, A30P, V21D, F34L, I44R, and I44F, as well as conservative substitutions thereof (EP 1 942 183 Al; Elisakova et a/. , 2005; Kopecky et a/. , 1999; US Patent Application No.

20070292914 Al). Preferred mutations include IlvH-L9F and IlvN-N17H.

In one embodiment, the bacterial cell comprises one or more mutations which increase(s) the expression level or activity of PyrE. E. coli K-12 MG1655 and W3110, plus their common ancestor strain W1485, are known to exhibit pyrimidine starvation in minimal media due to the presence a frameshift mutation occurring in rph relative to other E. coli strains (Jensen et al., 1993). This mutation disrupts the transcriptional/translational coupling required for efficient translation of pyrE, encoding orotate phosphoribosyltransferase in the pyrimidine biosynthesis pathway. Compensatory mutations that correct this deficiency are well-known in the art. One of these mutations is an 82 bp deletion near the 3' terminus of rph, due to presence of two homologous GCAGAAGGC sequences flanking this 82 bp region (Conrad et al., 2009). This mutation precisely corresponds to the 82 bp deletion found in resequenced isolates from population IBUA8 (from NC_000913.3 coordinates 3815859 to 3815931; Table 4). In addition to the 82 bp deletion, a 1 bp deletion at coordinate 3815809 in the pyrE/rph intrgenic region has previously been encountered in strains evolved for growth on a minimal glucose medium (LaCroix et al., 2015), and a wide array of other frameshift mutations, substitutions, and coding mutations near the 3' terminus of rph were encountered in a short- term selection/evolution of combinatorial mutant libraries in minimal medium at an elevated temperature of 42°C (Sandberg et al., 2014). The same 1 bp deletion in the pyrE/rph intergenic region was also found to be present in evolved isolate IBUA6-7. Without being limited to theory, all of these mutations can serve the same function of increasing expression of PyrE, with the selective pressure for these mutations being even stronger in minimal media with particular imposed stresses (certain chemicals or heat) than in minimal media alone. In one embodiment, the bacterial cell comprises mutations in rph or the pyrE/rph intergenic region, such as, e.g., an 82 bp deletion near the 3' terminus of rph, or 1 or 82 bp deletions in the intergenic region between pyrE and rph.

In one embodiment, the bacterial cell comprises one or more mutations in RpoB, which is the beta subunit of RNA polymerase. In one embodiment, the mutation alters the structure of the rifampicin-binding pocket of RpoB, and may, for example, alter rifampicin binding to RpoB, confer rifampicin resistance, or both. Non-limiting residues in the rifampicin-binding pocket of RpoB that are suitable for such mutations include H526, S531, R540, Q513, F514, and D516 (when aligned with E. coli RpoB), such as, e.g. , the amino acid substitutions H526Y, H526W, H526T, H526F, H526S, H526D, H526N, H526R, H526L, D516V, D516Y, S531L and S531W, as well as conservative substitutions thereof. Preferred mutations are those in H526 that are known to confer rifampicin-resistance, such as H526Y, H526D, H526N, H526R and H526L, as well as conservative amino acid substitutions of H526Y, such as H526Y, H526W, H526T, H526F and H526S, with H526Y being most preferred. As described in Example 1 in the section entitled "Resequencing of tolerant isolates", several isolates harbored the H526Y mutation in RpoB. In another embodiment, the mutation in RpoB is in residue A1183, such as A1183V, A1183I, A1183L, A1183M, or A1183F, preferably A1183V. For additional information on suitable RpoB mutations, see, e.g. , Kapur et al. , 1994; Ramaswamy et al., 2004;

Donnabella et al., 1994; Bodmer et al., 1995; Kim et al., 1997.

In one embodiment, the bacterial cell comprises a mutation in SapC which increases tolerance to isobutyric acid. In one particular embodiment, the SapC comprises a mutation, such as a deletion or amino acid substitution, in residue S69 or in the residue that aligns with residue S69 in E. coli SapC. In one embodiment, the mutation is an amino acid substitution of this residue into P or A. Preferably, the mutation is S69P or a conservative amino acid substitution thereof, such as S69A. In one particular embodiment, the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein. For example, the Group 1 modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF. The Group 2 modification can be, for example, a mutation in one or more of GlyQ, RpoB, RpsD, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic

modifications are described herein.

In one embodiment, the bacterial cell comprises a mutation in RpsD which increases tolerance to isobutyric acid. In one particular embodiment, the RpsD comprises a mutation, such as a deletion or amino acid substitution, in residue G87 or in the residue that aligns with residue G87 in E. coli SapC. In one embodiment, the mutation is an amino acid substitution of this residue into C, S or A. Preferably, the mutation is G87C or a conservative amino acid substitution thereof, such as G87S or G87A. In one particular embodiment, the bacterial cell further comprises at least one Group 1 modification, an additional Group 2 modification, or both, according to any aspects or embodiments herein. For example, the Group 1

modification can be a genetic modification which reduces the expression of one or more of pykF, yobF, phoU, rpoS or a combination thereof, such as pykF and rpoS, pykF and yobF, pykF and phoU, or yobF and rpoS, such as at least pykF. The Group 2 modification can be, for example, a mutation in one or more of GlyQ, RpoB, SapC, IlvH and IlvN and/or a genetic modification which increases the expression of PyrE. Examples of such mutations and genetic modifications are described herein. Preferred Group 2 mutations include those in GlyQ residue E48; RpoB residue H526 or

A1183; RpsD residue G87 and SapC residue S69 as well as vertain residues in IlvH and IlvN, such as, e.g., GlyQ-E48D, GlyQ-E48N, RpoB-A1183V, RpoB-A1183I, RpoB-A1183L, RpoB- A1183M, RpoB-A1183F, RpoB-H526Y, RpoB-H526W, RpoB-H526T, RpoB-H526F, RpoB- H526S, RpoB-H526D, RpoB-H526N, RpoB-H526R, RpoB-H526L, RpoB-D516V, RpoB-D516Y, RpoB-S531L, RpoB-S531W, SapC-S69P, SapC-S69A, RpsD-G87C, RpsD-G87S, RpsD-G87A, IlvH-L9F, IlvH-L9A, IlvH-L9V, IlvH-G14D, IlvH-S17F, IlvH-NllA, IlvH-N29H, IlvH-A36V, IlvH- T34I, IlvH-N29K, IlvN-N17H, IlvN-N17K, IlvN-G20D, IlvN-A30P, IlvN-V21D, IlvN-F34L, IlvN- I44R, and IlvN-I44F, and combinations thereof.

In one embodiment, the bacterial cell also or alternatively comprises one or more other mutations listed in Table 4, selected from RpoB-Q618L, RpoB-E565A, SapD-G235S, RpsC- E166K, YijD-L66M, RpoC-T757K, SapD-G235S, RpsC with a duplication of residues 128-132 (VMFRR), RpoB-N357H, RpoC-D622V and RpoC-D622A, SapF-R158C, RpoB-H526Y, SapB- V262G, RpsD-G87C, RpoB-A1183V, SapC-S69P, RpsL-A23V, and ptsP with an IS5 element insertion after nucleotide 858, or a combination of any two or more thereof. In one embodiment, the bacterial cell comprises a genetic modification which reduces the expression of pykF, and (a) at least one genetic modification which reduces the expression of one or more of yobF, rpoS, phoU; and/or (b) mutant GlyQ comprising a mutation in residue E48; and/or (c) a mutant RpoB comprising a mutation in residue H526 or A1183; and/or (d) a mutant RpsD comprising a mutation in residue G87; and/or (e) a mutant SapC comprising a mutation in residue S69.

In separate and specific embodiments, the bacterial cell comprises a genetic modification which reduces the expression of pykF, and

(a) a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyE, a mutant GlyQ comprising an E48D or E48N mutation, and one or both of a mutant

IlvH and IlvN, the mutant IlvH comprising a mutation in residue L9 and the mutant IlvN comprising a mutation in residue N17;

(b) a mutant RpoB comprising an H526Y, H526W, H526T, H526F, H526S, H526D, H526N, H526R, or H526L mutation, and a mutant SapC comprising an S69P or S69A mutation; (c) a genetic modification which reduces the expression of rpoS, a mutant GlyQ comprising an E48D or E48N mutation, a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyrE, and a mutant RpoB comprising an A1183V, A1183I, A1183L, A1183M, or A1183F mutation; or

(d) a mutant RpoB comprising an H526Y, H526W, H526T, H526F, or H526S mutation, and a mutant RpsD comprising a G87C, G87S, or G87A mutation.

(e) a mutant GlyQ comprising an E48D or E48N mutation, and one or both of a mutant IlvH and IlvN, the mutant IlvH comprising a mutation in residue L9 and the mutant IlvN comprising a mutation in residue N17;

(f) a mutant GlyQ comprising an E48D or E48N mutation;

(g) a mutant RpoB comprising an H526W, H526T, H526F, H526S, H526D, H526N, H526R or H526L mutation;

(h) a genetic modification which reduces the expression of rpoS, a mutant GlyQ comprising an E48D or E48N mutation, and a mutant RpoB comprising an A1183V, A1183I, A1183L,

A1183M, or A1183F mutation;

(i) a mutant RpoB comprising an A1183V, A1183I, A1183L, A1183M, or A1183F mutation: (j) a mutant GlyQ comprising an E48D or E48N mutation, a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyrE, and a mutant RpoB comprising an A1183V, A1183I, A1183L, A1183M, or A1183F mutation;

(k) a genetic modification which reduces the expression of rpoS, and a mutant GlyQ

comprising an E48D or E48N mutation; or

(I) a mutant GlyQ comprising an E48D or E48N mutation, and a mutant RpoB comprising an

A1183V, A1183I, A1183L, A1183M, or A1183F mutation. ther separate and specific embodiments, the bacterial cell comprises a mutation in GlyQ-E48 selected from E48D and E48N, and a knock-out or knockdown of at least one of pykF, rph, yjcF, rpoS, yobF, cheR and phoil, such as a knock-out or knock-down of at least one of pykF, yobF and PhoU, such as pykF;

a mutation in GlyQ-E48 selected from E48D and E48N, and a knock-out or knockdown of pykF and at least one of yobF, phoil, and rpoS, such as rpoS or yobF;

a mutation in GlyQ-E48 selected from E48D and E48N, a knockout or knockdown of pykF, increased expression of pyrE, and a mutation in IlvH providing for increased feedback-resistance to inhibition by at least L-valine;

a mutation in GlyQ-E48 selected from E48D and E48N, a knockout or knockdown of pykF, increased expression of pyrE, and a mutation in IlvN providing for increased feedback-resistance to inhibition by at least L-valine;

a mutation in IlvH providing for increased feedback-resistance to inhibition by at least L-valine, and a knock-down or knock-out of pykF and at least one of rpoS, yobF, and phoil, such as rpoS.

a mutation in IlvN providing for increased feedback-resistance to inhibition by at least L-valine, and a knock-down or knock-out of pykF and at least one of rpoS, yobF, and phoil, such as rpoS.

- a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, and a knock-down or knock-out of pykF.

a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF. and at least one of rpoS, yobF, and phoil, such as rpoS.

- a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, a

knock-down or knock-out of pykF, and a mutation in RpsD-G87 selected from G87C, G87S, and G87A.

- a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or

A1183F, a knock-down or knock-out of pykF, and at least one of rpoS, yobF, and phoil, such as rpoS.

- a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or

A1183F,a knock-down or knock-out of pykF.

- a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or

A1183F, a knock-down or knock-out of pykF, and a mutation in GlyQ-E48 selected from E48D and E48N.

- a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or

A1183F, a knock-down or knock-out of pykF, and a mutation in GlyQ-E48 selected from E48D and E48N. - a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or

A1183F, a knock-down or knock-out of pykF, a knock-down or knock-out of rpoS, and a mutation in GlyQ-E48 selected from E48D and E48N.a mutation in RpoB-A1183 selected from A1183V, A1183I, A1183L, A1183M, or A1183F, a knock-down or knock- out of pykF, a knock-down or knock-out of rpoS, a mutation in GlyQ-E48 selected from E48D and E48N, and increased expression of pyrE.

a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, and a knock-down or knock-out of pykF.

a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF, and at least one of rpoS, yobF, and phoU, such as rpoS.

a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, a knock-down or knock-out of pykF, and a mutation in SapC-S69 selected from S69P and S69A.

- a mutation in RpoB which alters the rifampicin binding pocket such as H526Y, a

knock-down or knock-out of pykF, a mutation in SapC-S69 selected from S69P and S69A, and a knock-down or knock-out of at least one of rpoS, yobF, and phoU, such as rpoS. In an alternative embodiment, the bacterial cell comprises an upregulation of at least one of GlyQ, PyrE, SapC, RpsD, RpoB, IlvH and/or IlvN, e.g., by transforming the bacterial cell with a transgene expressing the endogenous protein or a mutant thereof as described herein, e.g. , PyrE, GlyQ-E48D, IlvH-L9F, or IlvN-N17H. To cause an up-regulation through increased expression of a protein, the copy number of a gene or genes encoding the protein may be increased. Alternatively, 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. In another embodiment, 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.

2) Production pathways

Some bacterial species such as, e.g., Bacteroides ruminicola, have a native capability to produce isobutyrate and/or related compounds from a suitable carbon source. For example, Allison (1978) reported that on the order of 1 g/L (11 μιηοΙ/ιηΙ_) of combined C4 and C5 branched-chain fatty acids, including a small proportion of isobutyric acid (<0.1 g/L), was obtained when cells of wild-type Bacteroides ruminicola and Megasphera elsdenii were subjected to batch culturing at 38°C for 120 h in media containing 1 g/L glucose as carbon source. However, for higher production levels, e.g. , above 0.1 g/L, 0.5 g/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L, 50 g/L, 100 g/L or higher within 48h of culture in the presence of a carbon source, a recombinant biosynthetic pathway is typically needed. So, unless contradicted by context, as used herein, a "recombinant synthetic pathway" for isobutyric acid provides a higher production level of isobutyric acid than 0.1 g/L, 0.5 g/L, 1 g/L, 5 g/L, 6 g/L, 10 g/L, 12 g/L, 20 g/L, 50 g/L, or 100 g/L within 120h, typically within 48 h, from a supplied carbon source, e.g. , about 1 g/L glucose.

In one aspect, there is provided a bacterial cell with improved tolerance to at least isobutyric acid according to any aspect or embodiment described herein, wherein the bacterial cell further comprises a recombinant biosynthetic pathway for producing isobutyric acid or a related compound, such as, e.g. , valeric acid, isovaleric acid, 2-methylbutanoic acid, butyric acid or isobutanol. For example, in one embodiment, the bacterial cell comprises a recombinant biosynthetic pathway for producing at least one of the C4 to C7 branched-chain aliphatic acids, or a C4 to C5 straight-chain aliphatic acid, optionally isobutyric acid, butyric acid, valeric acid, 2-methylbutyric acid, isovaleric acid, 4-methylvaleric acid, or 2- methylhexanoic acid. In a further embodiment, the bacterial cell comprises a recombinant biosynthetic pathway for producing at least one of the C3 to C5 branched-chain or straight- chain aliphatic alcohols, optionally 1-propanol, 2-propanol, isobutanol, 3-methyl-l-butanol, 2-methyl-l-butanol, or 1-pentanol.

In principle, any such recombinant biosynthetic pathway which is known in the art can be introduced into the cell by standard recombinant technologies. Biosynthetic pathways suitable for production of isobutyric acid and related compounds in bacteria are well-known in the art and have been described by, e.g. , Atsumi et al. (2007), Atsumi et al. (2010), Saini et al. (2014), Dhande et al. (2012), Jawed et al. (2016), Volker et al. (2014), Yu et al. (2016) and Zhang et al. (2011). Some specific, preferred pathways are, however, exemplified below and in Example 1, "Production of isobutyric acid in evolved isolates." It is to be understood that, when a specific enzyme of these biosynthetic pathways is mentioned by name such as, e.g. , "acetolactate synthase", the enzyme may be any characterized and sequenced enzyme, from any species, that have been reported in the literature so long as it provides the desired activity. In some embodiments, the enzyme is an overexpressed gene which is native to the host cell used. In some embodiments, the enzyme is a functionally active fragment or variant of an enzyme which is heterologous or native to the host cell. 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 isobutyric acid or a related compound.

So, in one embodiment, the biosynthetic pathway is for isobutyric acid from pyruvate, and comprises genes, optionally overexpressed, encoding :

- acetolactate synthase, e.g., AlsS from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from

E. coll, catalyzing the conversion of pyruvate to (2S)-hydroxy-2-methyl-3- oxobutanoic acid;

acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (2S)-hydroxy-2-methyl-3-oxobutanoic acid to (2R)-2,3- dihydroxy-3-methylbutanoic acid;

dihydroxy acid dehydratase (IlvD), e.g., from E. coli, catalyzing the dehydration of (2R)-2,3-dihydroxy-3-methylbutanoic acid to 2-ketovaline;

2-ketoacid decarboxylase (KDC), e.g., KIVD from Lactococcus lactis, catalyzing the decarboxylation of 2-ketovaline to isobutyraldehyde; and,

- one or more aldehyde dehydrogenases such as 3-hydroxypropionaldehyde

dehydrogenase (AldH) from, e.g., E. coli or promiscuous native enzymes, catalyzing the oxidization of isobutyraldehyde to isobutyric acid.

Optionally, native alcohol dehydrogenases which actively convert isobutyraldehyde to isobutanol such as (in E. coli), YqhD, can be deleted (Zhang et al. , 2011). In another embodiment, the biosynthetic pathway is for 2-methylbutyric acid from L- threonine, and comprises genes, optionally overexpressed, encoding :

threonine deaminase (IlvA), e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;

acetolactate synthase (AlsS), e.g., from Bacillus subtilis, catalyzing the conversion of pyruvate and 2-oxobutanoic acid to (S)-2-aceto-2-hydroxybutanoic acid;

acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (S)-2-aceto-2-hydroxybutanoic acid to (R)-2,3-dihydroxy-3- methylpentanoic acid;

dihydroxy acid dehydratase (IlvD), e.g., from E. coli, catalyzing the dehydration of (R)-2,3-dihydroxy-3-methylpentanoic acid to (S)-3-methyl-2-oxopentanoic acid;

2-ketoacid decarboxylase (KDC), e.g., from Lactococcus lactis KIVD, catalyzing the decarboxylation of (S)-3-methyl-2-oxopentanoic acid to 2-methylbutyraldehyde; and, one or more aldehyde dehydrogenases such as 3-hydroxypropionaldehyde dehydrogenase (AldH) from, e.g., E. coli or promiscuous native enzymes, catalyzing the oxidization of 2-methylbutyraldehyde to 2-methylbutyric acid. Optionally, native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC). Optionally, native alcohol dehydrogenases which actively convert 2-methylbutyraldehyde to 2-methylbutanol such as (in E. coli), YqhD, can be deleted (Zhang et a/. , 2011).

In another embodiment, the biosynthetic pathway is for 3-methylbutyric acid from pyruvate, and comprises genes, optionally overexpressed, encoding:

acetolactate synthase (AlsS), e.g., from Bacillus subtilis, catalyzing the conversion of pyruvate to (2S)-hydroxy-2-methyl-3-oxobutanoic acid;

- acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (2S)-hydroxy-2-methyl-3-oxobutanoic acid to (2R)-2,3- dihydroxy-3-methylbutanoic acid;

dihydroxy acid dehydratase (IlvD), e.g., from E. coli, catalyzing the dehydration of (2R)-2,3-dihydroxy-3-methylbutanoic acid to 2-ketovaline;

- 2-isopropylmalate synthase (LeuA), e.g., from E. coli, catalyzing the acetylation of 2- ketovaline to (2S)-2-isopropylmalate;

(2S)-2-isopropylmalate hydro-lyase (LeuCD), e.g., from E. coli, catalyzing the dehydration, isomerization, and hydration of (2S)-2-isopropylmalate to (2S,3R)-3- isopropylmalate;

- 3-isopropylmalate dehydrogenase (LeuB), e.g., from E. coli, catalyzing the oxidation of (2S,3R)-3-isopropylmalate to (2S)-2-isopropyl-3-oxosuccinate, which subsequently spontaneously decarboxylates to 4-methyl-2-oxopentanoate;

2-ketoacid decarboxylase (KDC), e.g., from Lactococcus lactis KIVD, catalyzing the decarboxylation of 4-methyl-2-oxopentanoate to 3-methylbutyraldehyde; and, - one or more aldehyde dehydrogenases such as 3-hydroxypropionaldehyde

dehydrogenase (AldH) from, e.g., E. coli or promiscuous native enzymes, catalyzing the oxidization of 3-methylbutyraldehyde to 3-methylbutyric acid.

Optionally, native alcohol dehydrogenases which actively convert 2-methylbutyraldehyde to 2-methylbutanol such as (in E. coli), YqhD, can be deleted (Zhang et al. , 2011).

In another embodiment, the biosynthetic pathway is for butyric acid from L-threonine, and comprises genes, optionally overexpressed, encoding:

threonine deaminase (IlvA), e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;

2-isopropylmalate synthase (LeuA), e.g., from E. coli, catalyzing the acetylation of 2- oxobutanoic acid to (R)-2-ethyl-2-hydroxysuccinic acid; (2S)-2-isopropylmalate hydro-lyase (LeuCD), e.g., from E. coli, catalyzing the dehydration, isomerization, and hydration of (R)-2-ethyl-2-hydroxysuccinic acid to (2R,3S)-2-ethyl-3-hydroxysuccinic acid;

3-isopropylmalate dehydrogenase (LeuB), e.g., from E. coli, catalyzing the oxidation of (2R,3S)-2-ethyl-3-hydroxysuccinic acid to (R)-2-ethyl-3-oxosuccinic acid, which subsequently spontaneously decarboxylates to 2-oxopentanoic acid;

2- ketoacid decarboxylase (KDC), e.g., from Lactococcus lactis KIVD, catalyzing the decarboxylation of 2-oxopentanoic acid to butyraldehyde; and,

one or more aldehyde dehydrogenases such as 3-hydroxypropionaldehyde

dehydrogenase (AldH) from, e.g., E. coli or promiscuous native enzymes, catalyzing the oxidization of butyraldehyde to butyric acid.

Optionally, native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC). Optionally, native alcohol dehydrogenases which actively convert butyraldehyde to n-butanol, such as (in E. coli), YqhD, can be deleted (Zhang et ai , 2011).

In another embodiment, the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding :

acetyl-CoA carboxylase (AccABCD), e.g., from E. coli, catalyzing the carboxylation of acetyl-CoA to malonyl-CoA;

- malonyl-CoA-acyl carrier protein transacylase (FabD), e.g. from E. coli, catalyzing the transacylation of holo-acyl carrier protein and malonyl-CoA to malonyl-acyl carrier protein (ACP);

beta-ketoacyl-[acyl carrier protein] synthase III (FabH), e.g., from E. coli, catalyzing the condensation of malonyl-ACP and acetyl-CoA to acetoacetyl-ACP;

- 3-oxoacyl-[acyl carrier protein] reductase (FabG), e.g., from E. coli, catalyzing the reduction of a 3-oxoacyl-ACP to a 3-hydroxyacyl-ACP;

3- hydroxy-acyl-[acyl carrier protein] dehydratase (FabZ), e.g., from E. coli, catalyzing the dehydration of a 3-hydroxyacyl-ACP to a frans-2-enoyl-ACP;

enoyl-[acyl carrier protein] reductase (Fabl), e.g., from E. coli, catalyzing the reduction of a frans-2-enoyl-ACP to an acyl-ACP;

beta-ketoacyl-[acyl carrier protein] synthase I and/or II (FabB or FabA), e.g., from E. coli, catalyzing the condensation of an acyl-ACP with malonyl-ACP to form a 3- oxoacyl-ACP with two additional carbons compared to the substrate acyl-ACP; and, an butyryl-ACP thioesterase (TesBT), e.g. from Bacteroides thetaiotaomicron, catalyzing the thioesterification of butyryl-ACP to butyric acid and free ACP. Optionally, fatty acid degradation via the beta-oxidation pathway can be deleted via knockouts such as (in E. coli), fadD, fadE, fadB, fadA, fadR, or fabR, singly or in any combination.

In another embodiment, the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding :

beta-ketothiolase (PhaA), e.g., from Ralstonia eutropha, catalyzing the condensation of two acetyl-CoAs to acetoacetyl-CoA;

3-hydroxybutyryl-CoA dehydrogenase (Hbd), e.g., from Clostridium acetobutylicum, catalyzing the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA;

- crotonase (Crt), e.g., from Clostridium acetobutylicum, catalyzing the dehydration of

3-hydroxybutyryl-CoA to crotonyl-CoA;

frans-enoyl-CoA reductase (Ter), e.g., from Terponema denticola, catalyzing the reduction of crotonyl-CoA to butyryl-CoA; and,

a butyryl-CoA thioesterase (TesBT), e.g. from Bacteroides thetaiotaomicron, catalyzing the thioesterification of butyryl-CoA to butyric acid and free CoA.

Optionally, additional deletions can be made that prevent the conversion of acetyl-CoA and its glycolytic precursors to fermentative products, such as (in E. coli), frdA, IdhA, adhE, ackA, and pta, singly or in any combination.

In another embodiment, the biosynthetic pathway is for butyric acid from acetyl-CoA, and comprises genes, optionally overexpressed, encoding:

beta-ketothiolase (PhaA), e.g., from Ralstonia eutropha, catalyzing the condensation of two acetyl-CoAs to acetoacetyl-CoA;

- 3-hydroxybutyryl-CoA dehydrogenase (Hbd), e.g., from Clostridium acetobutylicum, catalyzing the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA;

crotonase (Crt), e.g., from Clostridium acetobutylicum, catalyzing the dehydration of

3-hydroxybutyryl-CoA to crotonyl-CoA;

frans-enoyl-CoA reductase (Ter), e.g., from Terponema denticola, catalyzing the reduction of crotonyl-CoA to butyryl-CoA; and,

acetoacetyl-CoA transferase (AtoDA), e.g. from E. coli, catalyzing the conversion of butyryl-CoA and acetate to butyric acid and acetyl-CoA.

Optionally, additional deletions can be made that prevent the conversion of acetyl-CoA and its glycolytic precursors to fermentative products, such as (in E. coli), frdA, IdhA, adhE, ackA, and pta, singly or in any combination. In another embodiment, the biosynthetic pathway is for valeric acid from L-threonine, and comprises genes, optionally overexpressed, encoding :

threonine deaminase (IlvA), e.g., from E. coli, catalyzing the deamination of L- threonine to 2-oxobutanoic acid;

- 2-isopropylmalate synthase (LeuA), e.g., from E. coli, catalyzing the acetylation of 2- oxobutanoic acid to (R)-2-ethyl-2-hydroxysuccinic acid, and the acetylation of 2- oxopentanoic acid to (R)-2-hydroxy-2-propylsuccinic acid;

(2S)-2-isopropylmalate hydro-lyase (LeuCD), e.g., from E. coli, catalyzing the dehydration, isomerization, and hydration of (R)-2-ethyl-2-hydroxysuccinic acid to (2R,3S)-2-ethyl-3-hydroxysuccinic acid, and the hydration of (R)-2-hydroxy-2- propylsuccinic acid to (2S,3R)-2-hydroxy-3-propylsuccinic acid;

3-isopropylmalate dehydrogenase (LeuB), e.g., from E. coli, catalyzing the oxidation of (2R,3S)-2-ethyl-3-hydroxysuccinic acid to (R)-2-ethyl-3-oxosuccinic acid, which subsequently spontaneously decarboxylates to 2-oxopentanoic acid, and the oxidation of (2S,3R)-2-hydroxy-3-propylsuccinic acid to (R)-2-oxo-3-propylsuccinic acid, which subsequently decarboxylates to 2-oxohexanoic acid;

2-ketoacid decarboxylase (KDC), e.g., KIVD from Lactococcus lactis, catalyzing the decarboxylation of 2-oxohexanoic acid to valeraldehyde; and,

one or more aldehyde dehydrogenases such as alpha-ketoglutaric semialdehyde dehydrogenase (KDH) from, e.g., Burkholderia ambifaria, or promiscuous native enzymes, catalyzing the oxidization of valeraldehyde to valeric acid.

Optionally, native threonine biosynthesis can be increased by overexpressing genes involved in its synthesis, such as homoserine dehydrogenase (ThrA), homoserine kinase (ThrB), and threonine synthase (ThrC). In another embodiment, the biosynthetic pathway is for isobutanol from pyruvate, and comprises genes, optionally overexpressed, encoding :

acetolactate synthase, e.g., AlsS from Bacillus subtilis, or IlvIH, IlvGM, or IlvBN from E. coli, catalyzing the conversion of pyruvate to (2S)-hydroxy-2-methyl-3- oxobutanoic acid;

- acetohydroxy acid isomeroreductase (IlvC), e.g., from E. coli, catalyzing the reduction and isomerization of (2S)-hydroxy-2-methyl-3-oxobutanoic acid to (2R)-2,3- dihydroxy-3-methylbutanoic acid;

dihydroxy acid dehydratase (IlvD), e.g., from E. coli, catalyzing the dehydration of (2R)-2,3-dihydroxy-3-methylbutanoic acid to 2-ketovaline;

- 2-ketoacid decarboxylase (KDC), e.g., KIVD from Lactococcus lactis, catalyzing the decarboxylation of 2-ketovaline to isobutyraldehyde; and, one or more alcohol dehydrogenases such as, e.g., AdhA from Lactococcus lactis or promiscuous native enzymes, catalyzing the reduction of isobutyraldehyde to isobutanol.

3) Processes In one aspect, there is provided a process for preparing a recombinant bacterial cell, e.g. , an E. coli cell. Also provided is a process for improving the tolerance of a bacterial cell, e.g. , an E. coli cell, to isobutyric acid. Also provided is a method of identifying a bacterial cell which is tolerant to at least isobutyric acid. Also provided is a process for preparing a recombinant bacterial cell, e.g. , an E. coli cell, for producing isobutyric acid or a related compound such as, e.g., isovaleric acid, 2-methylbutanoic acid, butyric acid, and isobutanol.

These processes may comprise one or more steps of genetically modifying a bacterial cell to knock-down or knock-out one or more endogenous genes of any aspect or embodiment of the Group 1 modifications and/or introducing one or more mutations in the endogenous protein(s) or gene(s) of any Group 2 aspect or embodiment. This can be achieved by, e.g. , transforming the bacterial cell with genetic constructs, e.g. , vectors, antisense nucleic acids or siRNA, which effect the knock-out or knock-down or which introduce the mutation into the endogenous gene or encode the mutated protein from a transgene.

The genetic constructs, particularly vectors, 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. 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. Alternatively, 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 be identified.

In one specific embodiment, the Group 1 modification is a knock-down or knock-out of one or more endogenous genes selected from pykF, rph, yjcF, rpoS, yobF, cheR and phoU, or a combination thereof, such as a knock-down or knock-out of pykF; yobF; phoU; pykF and rpoS; pykF and yobF; pykF and phoU; or pykF, rpoS and yobF. In one specific embodiment, the Group 2 modification is a mutation in at least one endogenous protein or gene selected from GlyQ, RpoB, SapC, RpsD, IlvH and IlvN, such as, e.g., a GlyQ-E48D, RpoB-H526Y, RpoB-A1183V, RpoB-Q618L, RpoB-E565A, RpoB-N357H, SapC-S69P, RpsD-G87C, IlvH-L9F, or IlvN-N17H mutation and/or a mutation which increases the expression of PyrE, such as, e.g. a mutation in rph or the pyrE/rph intergenic region.

In one embodiment, the process may comprise genetically modifying the E. coli cell to express a mutant GlyQ, express a mutant IlvH, express a mutant IlvN, overexpress PyrE, or a combination of any thereof.

In one embodiment, the process may comprise a knock-out of knock-down of rpoS. In one embodiment, the process may comprise genetically modifying the E. coli cell to express a mutant RpoB and a mutant SapC, or a combination of any thereof.

In one embodiment, the process may comprise genetically modifying the E. coli cell to express a mutant GlyQ, overexpress PyrE, and express a mutant RpoB, or a combination of any thereof. In one embodiment, the process may further comprise genetically modifying the E. coli cell to express a mutant RpoB and a mutant RpsD, or a combination of any thereof.

The processes may further comprise a step of selecting any bacterial cell which has an improved tolerance to isobutyric acid at a predetermined concentration, such as at least 3 g/L or higher, such as at least 5 g/L or higher, such as at least about 6 g/L, 6.3 g/L or higher, such as at least

12.5 g/L or higher; a step of introducing a recombinant biosynthetic pathway for producing isobutyric acid or a related compound; or both of the above steps, in any order.

Also provided is a method of producing isobutyric acid, comprising culturing the bacterial cell obtained by any one of these methods, or the bacterial cell of any preceding aspect or embodiment, under conditions where isobutyric acid is produced. Typically, these conditions include the presence of a suitable carbon source or mixes of different suitable carbon sources. Non-limiting examples of suitable carbon sources include, e.g. , sucrose, D-glucose, D-xylose, L-arabinose, glycerol, pyruvate, as well as hydrolysates produced from cellulosic or lignocellulosic materials. For further details see, e.g., Zhang et ai, 2011 and Atsumi et ai, 2007.

4) Compositions A bacterial cell which has an increased tolerance to isobutyric acid or a related compound can be useful for preparing producer cells for one or more such compound. Bacterial cells according to the invention may have an increased growth rate, an decreased lag time, or both. For example, the bacterial cell may have Group 1 and/or Group 2 modifications providing for an increased growth rate, a reduced lag time, or both, of the cell in at least one of the C3 or C4 to C8 branched-chain aliphatic acids, or at least one of the C3 or C4 to C8 straight-chain aliphatic acid, optionally isobutyric acid, butyric acid, valeric acid, 2- methylbutyric acid, isovaleric acid, 4-methylvaleric acid, or 2-methylhexanoic acid.

In one aspect, there is provided a composition of a plurality of bacterial cells according to any aspect or embodiment described herein, e.g., a culture of such bacterial cells, optionally in a suitable culture medium or a medium comprising a carbon source.

In one aspect, there is provided a composition comprising

a plurality of bacterial cells according to any preceding aspect or embodiment;

isobutyric acid or a related compound.

In one embodiment, the isobutyric acid or the related compound is present at a concentration at which the genetic modification(s) and/or mutant(s) comprised in the bacterial cells results in an improved tolerance. The concentrations at which bacterial cells according to the invention have improved tolerance are shown in Example 1, e.g., in "Cross-compound tolerance testing" (see, e.g., Table 22).

Typically, the concentration of isobutyric acid or the related compound is at least 1 g/L, such as at least 2 g/L, such as at least 2.5 g/L, such as at least 4 g/L, such as at least 6 g/L, such as at least 6.3 g/L, such as at least 7.5 g/L, such as at least 10 g/L, such as at least 12 g/L, such as at least 12.5 g/L, such as at least 15 g/L, such as at least 20 g/L, such as in the range of 1 to 300 g/L, such as in the range of 2 to 100 g/L, such as in the range of 4 to 50 g/L, such as in the range of 6 to 20 g/L. In one embodiment, the composition comprises isobutyric acid. In one embodiment, the composition comprises butyric acid. In one embodiment, the composition comprises valeric acid. In one embodiment, the composition comprises 2-methylbutyric acid. In one embodiment, the composition comprises isovaleric acid. In one embodiment, the composition comprises 4-methylvaleric acid. In one

embodiment, the composition comprises 2-methylhexanoic acid. In one embodiment, the composition comprises 1-propanol. In one embodiment, the composition comprises 2- propanol. In one embodiment, the composition comprises isobutanol. In one embodiment, the composition comprises 2-methyl-l-butanol. In one embodiment, the composition comprises 3-methyl-l-butanol. In one embodiment, the composition comprises 1-pentanol.

Preferably, the bacterial cells are of the Escherichia, Bacillus, Pseudomonas, Lactobaccillus or Lactococcus family, 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 pykF, rph, yjcF, rpoS, yobF, cheR and phoil or a combination of any thereof, such as a knock-down or knock-out of pykF; yobF; phoU; pykF and rpoS; pykF and yobF; pykF and phoU; or pykF, rpoS and yobF;

b) a mutation in at least one of glyQ, rpoB, sapC, rpsD, ilvH and ilvN, typically a coding mutation according to any aspect or embodiment herein, which improves the tolerance of the bacterial cell to isobutyric acid;

c) a mutation which increases the expression of pyrE; or

d) a combination of a) and b), a) and c) or a), b) and c).

In b), as an alternative to mutating an endogenous gene, the PyrE or mutant GlyQ, RpoB, SapC, RpsD, IlvH and/or IlvN can be expressed from a transgene.

Assays for assessing the tolerance of a modified bacterial cell to isobyturic acid or a related compound typically evaluate the growth rate, lag time, or both, of the bacterial cell at one or more predetermined concentrations of the compound, typically as compared to a control (e.g., no compound). For isobutyric acid, the predetermined concentrations(s) could be, for example, 1 g/L, 6.3 g/L and/or 12.5 g/L isobutyric acid or isobutyrate. Preferably, 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. For example, 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.

5) Bacterial cells

Also provided are strains, clones and other progeny of the bacterial cells of these and other aspects and embodiments. Typically, as used herein, a "strain" typically refers to a group of cells which are descendants of a initial single colony of parent cells whereas a "clone" is a group of cells which are the descendants of an initial genetically modified single parent cell.

Non-limiting examples of bacterial cells suitable for modification according to any one of the aspects and embodiments described herein include bacteria of the Enterobacteriaceae, Bacillaceae, Ralstoniaceae, Pseudomonadaceae, Lactobacillaceae or Streptococcaceae families, particularly the Escherichia, Bacillus, Ralstonia, Pseudomonas, Lactobacillus and Lactococcus genera. In one embodiment, 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). In a specific embodiment, the bacterial cell is derived from an E. coli K12 strain. In another embodiment, the bacterial cell is a Lactobacillus cell, such as a cell of the commercially available and/or fully characterized strains Lactobacillus plantarum JDM1, Lactobacillus plantarum WCFS1, and Lactobacillus plantarum NCIMB 8826. In another embodiment, 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. In another embodiment, 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. In one embodiment, the bacterial cell is a Pseudomonas cell, such as a cell of the commercially available and/or fully characterized strain Pseudomonas putida KT2440. In another embodiment, the bacterial cell is a Ralstonia cell, such as a cell of the commercially available and/or fully characterized strains Ralstonia eutropha H16 and Ralstonia eutropha JMP134.

While aspect and embodiments relating to bacterial cells herein typically refer to genes or proteins according to their designation in E. coli, for bacterial cells of another family or species, it is within the level of skill in the art to identify the corresponding gene or protein, e.g. , the homolog, ortholog and/or paralog, in the other family or species, typically by identifying sequences having moderate (typically≥30%) or high (typically≥50%) homology to the E. coli sequence, preferably taking the function of the protein expressed by the gene and/or the locus of the gene in the genome into account. Table 2A below sets out the function of the protein encoded by each specific gene, the corresponding E.C. number (if applicable), its locus in the E. coli K-12 MG1655 genome and the SEQ ID number of the coding or non-coding sequence and, where applicable, the encoded amino acid sequence.

Table 2B below sets out some examples of homologs in selected organisms, identified in a preliminary and non-limiting analysis. Indeed, homologs of these proteins exist also in other bacteria, and other homologs not identified in this preliminary search can exist in the species listed in Table 2B. The skilled person is well-familiar with different searching and/or screening methods for identifying homologs across different species. To briefly summarize some of the preliminary findings in Table 2B:

GlyQ is essential and was identified in all organisms. The residue aligning with residue E48 in E. coli GlyQ may not always be conserved, and may be, for example, an A (Ala or alanine) or S (Ser or serine).

IlvH was found in 4 out of 5 species and the L9 residue was conserved.

IlvN was found in 2 out of 5 species (the Gram-negatives) and in these N17 was conserved (although aligning as Ni l).

RpoS was found in at least Bacillus, Pseudomonas, and Ralstonia.

- PyrE was found in the Gram-negative species.

Table 2A - Protein function and Locus IDs

E. coli gene Protein function E.C. Locus ID SEQ ID NO:

designation number

pykF Pyruvate kinase I 2.7.1.40 bl676 1

rpoS RNA polymerase, sigma S N/A b2741 2

(sigma 38) factor

yobF Small protein involved in stress N/A bl824 3

response

cheR Chemotaxis protein 2.1.1.-, bl884 4

methyltransferase 2.1.1.80 phoU negative regulator of the Pho N/A b3724 5

regulon

Rph RNase PH 2.7.7.56 b3643 6

yjcF Conserved protein N/A b4066 7

glyQ Glycyl-tRNA synthetase, alpha 6.1.1.14 b3560 8 (DNA)

subunit

9 (protein) ilvH Small regulatory subunit of 2.2.1.6 b0078 10 (DNA)

acetohydroxybutanoate

11 (protein) synthase/acetolactate synthase

III

ilvN Small regulatory subunit of 2.2.1.6 b3670 12 (DNA)

acetohydroxybutanoate

13 (protein) synthase/acetolactate synthase I pyrE 0 rotate 2.4.2.10 b3642 14 (DNA) phosphoribosyltransferase

15 (protein) pyrE/rph - - - 16 (DNA) intergenic

region

rpoB RNA polymerase, β subunit 2.7.7.6 b3987 17 (DNA)

18 (protein) sapC putrescine ABC exporter 3.6.3.- bl292 19 (DNA)

membrane protein SapC

20 (protein) rpsD 30S ribosomal subunit protein N/A b3296 21 (DNA)

S4

22 (protein)

Table 2B - Homologs

Database references for the gene (or protein) sequences are indicated within parentheses.

Protein B. subtilis 168 P. putida L. plantarum L. lactis Ralstonia

KT2440 JDM1 KF147 eutropha H16

PykF 52% identity 38% identity 45% identity 39% identity 36-39%

"pyruvate "pyruvate "pyruvate "pyruvate identity (469- kinase" kinase" kinase" kinase" 478 aa)

(NP 390796. (NP_746417.1 (YP 00306317 (YP_003353 "pyruvate

1) 9.1) 857.1) kinase"

NP_743521.1) (YP_728028.1,

YP_725084.1)

RpoS 43% identity 76% identity 44% identity 40% identity 52% identity

"RNA

"RNA "RNA "RNA "RNA

polymerase polymerase polymerase polymerase polymerase

sigma factor sigma-43 sigma S sigma factor sigma

RpoS"

factor" factor" RpoD" factor"

(YP 726836.1

(NP 390399. (NP 743780.1 (YP 00306323 (YP 003352

2) ) 7.1) 999.1) )

YobF 42% identity

(31 aa)

"pre protein

translocase

subunit SecD"

(NP 742996.1

) CheR 32% identity 29-33%

"chemotaxis identity (208- protein 261 aa)

methyltransfe "chemotaxis

rase" protein CheR",

(NP 390153. "methyltransfe

2) rase, CheR- like"

(NP_746506.1

NP_743648.1,

NP_745890.1)

PhoU 49% identity 31% identity 29% identity 47% identity

(223 aa) (208 aa) (210 aa) (228 aa) "phosphate "phosphate "phosphate "phosphate transporter transport transport uptake PhoU" system system regulator

(NP 747426.1 protein" regulator" PhoU"

) (YP 00306220 (YP 003354 (YP 726897.1

5.1) 286.1) )

Rph 58% identity 69% identity 23-26% 62% identity "ribonuclease "ribonuclease identity (221 aa) PH" PH" (173-207 aa "ribonuclease

(NP 390715. (NP 747395.1 in stretches) PH"

1) ) "polyribonucl (YP 725462.1 eotide )

nucleotidyltr

ansferase"

(YP_003354

448.1)

YjcF 22% identity 22-29% 23% identity

"hypothetical identity (148- (181 aa)

protein 218 aa in "hypothetical

BSU10890" stretches) protein

(NP 388970. "penta peptide JDM1 2214"

1) repeat- (YP 00306379

containing 8.1)

protein"

(NP 744817.1

)

GlyQ 63% identity 76% identity 59% identity 59% identity 69% identity "glycine-tRNA "glycine-tRNA "glycine-tRNA "glycine- "glycine-tRNA ligase subunit ligase subunit ligase subunit tRNA ligase ligase subunit alpha" (E48 = alpha" (E48 = alpha" (E48 = subunit alpha" (E48 = S42) A50) A50) alpha" (E48 A43)

(NP 390405. (NP 742231.1 (YP 00306324 = S47) (YP 725041.1 1) ) 0.1) (YP_003353 )

574.1) PyrE 25-34% 67% identity 29% identity 30% identity 56% identity identity (in "orotate "orotate (131 aa) " "orotate stretches) phospho- phospho- orotate phospho-

"orotate ribosyl- ribosyl- phosphoribo ribosyl- phospho- transferase" transferase" syltransferas transferase" ribosyl- (NP 747392.1 (YP 00306374 e" (YP 724744.1 transferase" ) 6.1) (YP_003353 )

(NP 389439. 548.1)

1)

IlvH 37% identity 64% identity 35% identity 60% identity

"acetolactate "acetolactate "acetolactate "acetolactate synthase synthase synthase synthase small small subunit" small subunit" small subunit" (L9 =

(L9 = V9) (L9 = L9) subunit" (L9 L9)

(NP 390708. (NP 746788.1 = L9) (YP 725546.1

2) ) (YP 003353 )

709.1)

IlvN 36% identity 36% identity 33% identity "acetolactate "acetolactate "acetolactate synthase synthase synthase small small subunit" small subunit" subunit" (N 17 (NP 390708. (N 17 = N i l) = N i l) 2) (NP 746788.1 (YP 725546.1

) )

RpoB 59% identity 72% identity 47% identity 46% identity 66% identity

(533 aa) (1360 aa) (953 aa) (951 aa) (1370 aa)

"DNA-directed "DNA-directed "DNA-directed "DNA- "DNA-directed

RNA RNA RNA directed RNA RNA polymerase polymerase polymerase polymerase polymerase subunit beta" subunit beta" subunit beta" subunit subunit beta"

(NP 387988. (NP 742613.1 (YP 00306242 beta" (YP 727933.1

2) ) 6.1) (YP 003354 )

373.1)

SapC 29% identity 42% identity 25% identity 23-27% 28-37%

(288 aa) (281 aa) (302 aa) identity identity (216-

"oligopeptide "binding- "peptide ABC (219-306 293 aa) "ABC transport protein- transporter aa) "peptide transporter system dependent permease" ABC permease" permease transport (YP 00306265 transporter (YP_725975.1, protein AppC" system inner 3.1) permease" YP_726565.1,

(NP 389022. membrane (YP_003354 YP_726551.1,

1) protein" 430.1, YP_727395.1,

(NP 743041.1 YP 0033528 YP_725810.1,

) 73.1) YP_727738.1,

YP_726867.1)

RpsD 50% identity 73% identity 49% identity 47% identity 70% identity

(210 aa) "30S (206 aa) "30S (208 aa) "30S (209 aa) (207 aa) "30S ribosomal ribosomal ribosomal "30S ribosomal protein S4" protein S4" protein S4" ribosomal protein S4"

(NP 390844. (NP 742644.1 (YP 00306353 protein S4" (YP 727895.1

1) ) 3.1) (YP 003352 )

806.1) So, in one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein each recited gene is instead (i) a gene encoding the corresponding protein in Table 2A or 2B above, (ii) a gene located at the corresponding locus, or (iii) both. In particular, without being limited to theory, improved tolerance toward and endogenous production of isobutyric acid can be achieved by genetic modifications which alleviate L- isoleucine starvation due to intracellular accumulation of L-valine and/or L-leucine via mutations that provide feedback-resistance to inhibition by L-valine, L-leucine or both. This can, e.g., be achieved by a mutation in IlvH, IlvN, or one or more other genetic modifications described herein.

Additional and alternatively, without being limited to theory, improved tolerance towards isobutyric acid can also be achieved by genetic modifications which reduce the cellular metabolic flux through lower glycolysis, reduce pyruvate accumulation and acetate formation, increase metabolic flux through the pentose-phosphate pathway, decrease expression of the RpoS regulon, decrease the activity of glycyl-tRNA synthetase, and/or increase transcription of pyrE. Without being limited to theory, a decreased activity of glycyl-tRNA synthetase can be achieved by a mutant GlyQ, e.g., E48D.

In one embodiment, the bacterial cell has a genetic modification which reduces the expression of one or more endogenous proteins selected from the group consisting of - a pyruvate kinase

an RNA polymerase, sigma S factor

a protein involved in stress response

a chemotaxis protein methyltransferase

a negative regulator of the Pho regulon

- an RNase PH.

In one specific embodiment, the bacterial cell further comprises a recombinant biosynthetic pathway for producing isobutyric acid. In one particular embodiment, the bacterial cell is of the Lactobacillus or Lactococcus genera. In one particular embodiment, the bacterial cell is of the Escherichia genera. In one particular embodiment, the bacterial cell is of the

Pseudomonas genera. In one particular embodiment, the bacterial cell is of the Bacillus genera. In one particular embodiment, the bacterial cell is of the Ralstonia genera. EXAMPLE 1 Methods

Screening for tolerance in wild-type cells

Escherichia coli K-12 MG1655 was grown overnight in M9 minimal medium + 1% glucose and subcultured the following morning to an initial OD ₆₀₀ of 0.05 in M9 + 1% glucose. Cells were grown to mid-exponential phase (OD ₅₀₀ 0.7-1.0) and were back-diluted with fresh medium to an OD ₅₀₀ of 0.7. The diluted cells were used to inoculate M9 + 1% glucose containing varying concentrations of isobutyric acid (first neutralized to pH 7.0 with sodium hydroxide), and growth was measured in FlowerPlates in a Biolector microbioreactor system (m2p-labs) at 37°C with 1000 rpm shaking. The culture volume in each well was 1.4 mL.

Adaptive laboratory evolution of tolerant strains

Based on the screening results, E. coli K-12 MG1655 was grown overnight in M9 minimal medium and 150 μΙ_ was transferred the next day into 8 tubes containing 15 mL of M9 + 1% glucose + 3 g/L isobutyric acid on a Tecan Evo robotic platform custom-designed for performing adaptive laboratory evolutions (ALE). Cells were cultured on a 37°C heat block with stirring by magnetic stir bars. Culture OD ₆₀₀ was monitored at times determined by a predictive custom script, and when the OD ₆₀₀ reached approximately 0.3, 150 μί of culture was inoculated into a new tube with the same media concentration. Instrument downtime would occasionally result in cells overgrowing to saturation or an OD ₅₀₀ greater than 0.3, and reinoculations were occasionally performed from cryogenic stocks of the population. When the growth rate was observed to substantially increase, the media concentration was changed. These concentration changes were to 5 g/L, 7.5 g/L, 10 g/L, and 12.5 g/L isobutyric acid. Approximately 100 μί of each population (8 per chemical) were plated on LB agar and incubated at 37°C overnight. Primary screening of ALE isolates

Five colonies from wild-type K-12 MG1655 and 10 individual colonies deriving from each population were inoculated into 300 μί M9 + 1% glucose in 96 well deepwell plates and incubated in a 300 rpm plate shaker at 37°C. The next day, cells were diluted 10X in M9 + 1% glucose and 30 μί was transferred into clear-bottomed 96 well half-deepwell plates (with rectangular wells) containing M9 + 1% glucose and M9 + 1% glucose + 13.89 g/L isobutyric acid, such that the final concentration of isobutyric acid was 12.5 g/L. In addition, 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 ₅₀ o values using a previously determined calibration between OD ₅₀₀ 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.

Secondary screening of ALE isolates

Selected isolates from the primary screen were restruck onto LB agar from the cryogenic stock made from the overnight culture plate for the primary screen. Five K-12 MG1655 colonies and three individual colonies from each isolate were inoculated as biological replicates into a new 96 well deepwell plate containing 300 μί of M9 + 1% glucose, and grown overnight as for the primary screen. The next day, a cryogenic stock and half deepwell plates containing M9 + 1% glucose with or without isobutyric acid were inoculated using the plate of overnight cultures, and growth was measured as described for the primary screen. Resulting growth curves were visually inspected for isolates exhibiting robust and reproducible growth between replicates in high concentrations of isobutyric acid.

Re-sequencing of ALE isolates

A total of 20 isolates were selected from the secondary screen for whole-genome

resequencing. An individual colony was taken from the LB agar plates prepared following the primary screen, inoculated into 2 mL LB, and grown overnight at 37°C in a 250 rpm shaker. The following morning, 0.5 mL of cells were transferred to microcentrifuge tubes and centrifuged at 16000 x g for 2 minutes. The supernatant was removed and pellets were stored at -20°C until further processing. Genomic DNA was extracted from thawed cell pellets using a PureLink genomic DNA extraction kit, with further concentration and purification performed by ethanol precipitation. To generate libraries for sequencing, the

Illumina TruSeq Nano kit was used according to the manufacturers' directions using an input quantity of 200 ng of genomic DNA from each isolate. Sequencing was performed on an Illumina MiSeq sequencer, with a minimum 20X average genomic coverage ensured for each isolate based on the number of reads. Fastq output files were analyzed for variants compared to the K-12 MG1655 reference genome (accession number NC_000913.3) using breseq.

Construction of gene knockouts

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. For those genes, the corresponding knockout strain from the Keio collection of single knockout mutants (where each gene is replaced with a cassette consisting of a kanamycin resistance gene flanked by FRT sites) was used as a donor strain for Plvir phage transduction (Baba et a/. , 2006). Briefly, the Keio strain was grown to early exponential phase in LB + 5 mM CaCI ₂ and 80 μί of a Plvir stock raised on K-12 MG1655 was added. After significant lysis was observed after 1.5 to 2 hours, the lysate was filter-sterilized to remove cells and stored at 4°C. Strain K-12 MG1655 was grown overnight in LB + 5 mM CaCI ₂ and 100 μί of the overnight culture was mixed with 100 μί of the Plvir lysate of the Keio collection mutant, and the mixture was incubated at 37°C without shaking for 20 minutes. The entire mixture was then plated on LB agar containing 1.25 mM sodium pyrophosphate as a chelating agent and 25 μg/mL kanamycin. One colony was then restruck on LB + 1.25 mM Na _{2 4} 0 ₇ + 25 μg/mL kanamycin plate and analyzed for presence of the Keio cassette in place of the wild- type gene by colony PCR. When further knockouts were constructed in the same strain, the Keio cassette was flipped out to generate a scar sequence such that Kan ^R marker could be recycled. This was performed by transforming with pCP20, which constitutively expresses a flippase recombinase, and plating cells on LB agar + 100 μg/mL ampicillin and incubating at 30°C. The next day, one or more colonies was tested by colony PCR for loss of the Keio cassette, and successful mutants were then cured of pCP20 by elevated temperature curing at 40°C. Strains were verified to be cured of plasmid by plating on LB agar + 100 μg/mL ampicillin and incubation at 30°C. Plvir transductions were then performed using these mutant strains as recipients.

Biolector growth screening of evolved isolates and reconstructed mutants

Biological triplicate cultures of each strain were grown to saturation overnight in 96 well deepwell plates containing 300 μί M9 + 1% glucose. The next day, cells were diluted 1 : 10 in deionized water in a clear 96 well plate and the OD ₆₀₀ was measured on a BioTek plate reader. 48 well FlowerPlates containing a final volume of 1.4 mL of M9 + 1% glucose + 12.5 g/L isobutyric acid (neutralized to pH 7.0 with sodium hydroxide) were inoculated to OD ₅₀₀ 0.03 (with plate reader pathlength, 200 μί volume) with the overnight culture and sealed with Breathseal film. Light backscatter intensity was monitored in a Biolector microbioreactor system at 37°C with 1000 rpm shaking.

Keio collection screening for loss-of-function mutations

For primary screening, Keio collection mutants were inoculated directly from a cryogenic stock of the Keio collection into 300 μί LB medium containing 25 μg/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 μΙ_ M9 + 1% glucose and grown overnight. The next day, cells were inoculated 1 : 100 into clear bottomed 96 well half-deepwell plates containing M9 + 1% glucose plus 7.5 g/L and 12.5 g/L isobutyric acid (neutralized), and cultivated in a Growth Profiler as previously described for screening of ALE isolates.

As a secondary screen, promising Keio collection mutants were struck on LB + 25 pg/mL kanamycin from the cryogenic stock plate prepared during primary screening above and biological triplicate colonies were inoculated into a 96 well deepwell plate containing 300 pL M9 + 1% glucose. The next day, cells were inoculated into plates for cultivation on the Growth Profiler as described above.

Generation of isobut rate production strains

The Keio collection strain containing yqhD: :kan, JW2978, was used as the donor strain for Plvir phage transduction into recipient strains K-12 MG1655, IBUA2-9, IBUA7-9, and IBUA8- 3 as described in 'Construction of gene knockouts.' Plasmid pCP20, which encodes a constitutively expressed yeast flippase recombinase (FLP), was transformed into JW2978, K- 12 MG1655 yqhDr.kan, IBUA2-9 yqhDr.kan IBUA7-9 yqhDr.kan, and IBUA8-3 yqhDr.kan to remove the kanamycin resistance marker, generating strains K-12 BW25113 AyqhD, K-12 MG1655 AyqhD, IBUA2-9 AyqhD, IBUA7-9 AyqhD and IBUA8-3 AyqhD. Loss of the kanamycin resistance marker was confirmed by colony PCR of a region flanking the yqhD locus. Plasmids pIBAl and pIBA7 were obtained from Prof. Kechun Zhang (University of

Minnesota) (Zhang et ai , 2011). These plasmids were transformed into each AyqhD strain by adding the plasmids 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 pg/mL kanamycin and 100 pg/mL ampicillin to select for double plasmid transformants.

Isobutyrate production runs

Individual colonies of isobutyrate production strains were picked as biological replicates and inoculated into 300 pL of M9 medium containing 1% glucose, 50 pg/mL kanamycin, and 100 pg/mL ampicillin. In the first production screen, cells were grown overnight to saturation, and were inoculated the next morning into 2.5 mL of M9 medium containing 4% glucose, 0.5% yeast extract (from an autoclaved 10% stock), 0.5 g calcium carbonate (autoclaved powder), 100 pg/mL ampicillin, 50 pg/mL kanamycin, and 0.1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) in a 24 well deepwell plate. The plate was incubated at 30°C with 300 rpm shaking. Cell supernatants (1 mL) were harvested for isobutyric acid analysis after 24 and 48 hours.

In the second production screen, cells were grown overnight to saturation as described, and were inoculated the next morning into 2.5 mL M9 medium containing 4% glucose, 0.5 g calcium carbonate, 100 μg/mL ampicillin, 50 μg/mL kanamycin, 0.1 mM IPTG (no yeast extract), and either no added isobutyric acid, or sodium isobutyrate to a final concentration of 7.5 g/L, in a 24 well deepwell plate. The plate was incubated at 37°C and 1 mL samples of cell supernatants were harvested for isobutyric acid analysis after 24 and 50 hours.

Fed-batch fermentations were performed with strains K-12 MG1655, IBUA7-9, and IBUA8-3 with AyqhD and harboring plasmids pIBAl and pIBA7 in 1 L bioreactors. The batch phase medium (400 mL) contained 2 g/L ammonium sulfate, 5.6 g/L potassium phosphate dibasic, 0.12 g/L Antifoam 204, 2% glucose, 0.1 g/L zinc chloride, 0.15 g/L iron(II) sulfate heptahydrate, 1.5 g/L trisodium citrate, 2 mM magnesium sulfate, 0.125 mM calcium chloride, and 1 mL/L of a trace element solution containing 2.0 g/L aluminum sulfate octadecahydrate, 0.75 g/L cobalt(II) sulfate hexahydrate, 2.5 g/L copper(II) sulfate pentahydrate, 0.5 g/L boric acid, 24 g/L manganese(II) sulfate monohydrate, 3.0 g/L sodium molybdate dihydrate, 2.5 g/L nickel(II) sulfate trihydrate, and 2 mL/L of 30% sulfuric acid. The feed solution contained 600 g/L glucose, 18 g/L magnesium sulfate heptahydrate, 3 mL/L of trace element solution, 0.1 g/L zinc chloride, 0.15 g/L iron(II) sulfate heptahydrate, 1.5 g/L trisodium citrate, 0.2 mM IPTG, 100 μg/mL ampicillin, and 50 μg/mL kanamycin. The bioreactors were maintained at 30°C, pH 7.0 using 15% ammonium hydroxide, with air sparged at 1 vvm (1 L air per L liquid volume per minute), and the stirrer set to 800 rpm during the batch phase and 1000 rpm during the feed phase. In a first fermentation run, IPTG was added to a concentration of 0.2 mM after 22 hours and in the feeding phase at a concentration of 0.2 mM in the feeding solution, whereas in the second run IPTG was added 1 hour after the start of the feeding phase to a concentration of 0.1 mM, with a concentration of 0.1 mM in the feeding solution.

A screen of a larger selection of evolved isolates harboring deletions in yqhD and plasmids pIBAl and pIBA7 was performed using Feed-in-Time (FIT) medium (m2p-labs, Baesweiler, Germany) diluted 1 : 1 with 200 mM MOPS buffer, herein referred to as V2 FIT medium.

Individual colonies from transformation plates were first inoculated into 96-well deepwell plates containing 300 μί of M9 supplemented with 1% glucose, 100 mg/L ampicillin, and 50 mg/L kanamycin. The next morning, 20 μί was transferred from the preculture plate into corresponding wells of a 24-well deepwell plate containing 2.4 mL of V2 FIT plus 50 mg/L kanamycin and 100 mg/L ampicillin. Cultures were grown in quadruplicates at 30°C and with 300 rpm shaking. Culture supernatants were harvested after 48 hours for analysis. HPLC analysis of isobutyric acid

1 mL of cell culture was centrifuged at 16000 x g for 2 minutes and the supernatant was passed through a 0.2 μιη syringe filter. Supernatants were injected (30 μΙ_) onto an Aminex HPX-87H ion exclusion column held at 30°C on a Dionex UltiMate HPLC system equipped with a Shodex RI-101 refractive index detector held at 45°C. The mobile phase was 5 mM sulfuric acid and was kept at a constant flow rate of 0.6 mL/min. Isobutyric acid was found to elute at approximately 21.7 minutes with this method. Concentrations were calculated using a standard calibration curve (linear response with R ² = 0.9999).

Multiplex automated genome engineering (MAGE)

Genomic point mutants were generated using MAGE (Wang et ai , 2009), which involves multiple cycles of electroporation of cells expressing the β protein of λ Red recombinase with single stranded DNA oligonucleotides. The single-stranded oligonucleotides are believed to behave like Okazaki fragments during DNA replication, and their use enables a high enough efficiency of allelic replacement to preclude needing to select for cells that received the mutation.

In this study, K-12 MG1655 or knockout strains of K-12 MG1655 were transformed with pMA7SacB (Lennen et al. , NAR 2015), a plasmid that harbors the β subunit of λ Red recombinase and Dam under control of an arabinose-inducible promoter, and SacB to enable removing the plasmid by sucrose counterselection following the identification of a desired mutant. K-12 MG1655/pMA7SacB was grown in 15 mL of LB medium plus 100 μg/mL ampicillin to mid-exponential phase at 37°C, induced for 10 minutes with 0.2% L-arabinose, chilled in an ice water bath, and washed and concentrated 3 times with autoclaved chilled MilliQ water in a typical electrocompetent cell preparation. 50 pmol of oligonucleotide was added to a 50 μί aliquot of cells in a 1 mm gap electroporation cuvette, and cells were electroporated at 1.8 kV. Cells were immediately recovered in 1 mL LB and the entire volume of cells was used to inoculate the next 15 mL LB culture. Cells were grown to mid- exponential phase and the remainder of the procedure repeated, and recovered cells following electroporation were outgrown overnight to allow full genome segregation. The following morning, cells were plated on LB medium. Colonies appearing on LB medium were then screened for the presence of the desired introduced mutation. Colonies were resuspended in water for use as a template in a quantitative PCR (qPCR) with a HotStart Taq master mix containing SYBR Green. To achieve discrimination of a mutated base via the cycle threshold, both wild-type and mutant forward primers were designed and run as separate reactions with the same reverse primer binding approximately 80-100 bp downstream of the mutation. The mutant forward primer had the last base designed to be complementary to the mutated base and an additional mutation at the -3 position from the 3' end of the primer such that primer binding would be maximally destabilized with the wild-type base. The wild-type primer typically had the -3 position from the 3' end of the primer mutated to offer additional destabilization with the mutant base. This allowed discrimination of the desired mutant or wild-type base for each screened isolate by qualitatively observing a reversal in the fluorescence vs. cycle threshold curves by qPCR with the two primer sets. Individual isolates were verified to have the desired mutant sequence in the genome with no adjacent off-target mutations by Sanger sequencing.

Cross-compound tolerance screening

96 well deepwell plates containing 300 μΙ_ of M9 + 1% glucose were inoculated directly from cryogenic stocks made from precultures for the secondary screening of ALE isolates and were grown overnight at 37°C with 300 rpm shaking. The next day, cells were diluted 1 : 100 into 96 well half-deepwell plates containing the following final concentrations of each chemical in M9 + 1% glucose:

Plates were cultivated in a Growth Profiler for 48 hours as described for screening of ALE isolates. Green pixel integrated values from each well were converted to OD ₅₀ o values using a calibration curve and the resulting OD ₅₀ o Vs. elapsed time data was processed using custom scripts to determine the time required for each culture to reach an OD of 1.0 (t _0D i) - This value is a combined measure of growth rate and lag time in each culture. The median value was taken for biological triplicates of each isolate and was normalized to the median t _0D i for K-12 MG1655 controls (5 replicates). The ratio of t ₀ Di(evoived)/toDi(wiid-ty _P e) is presented.

Analysis of growth parameters (growth rate and lag time)

For data obtained with the Biolector microbioreactor system, self-baselined growth series were imported directly into a custom software platform that automatically detects growth phases and exports growth rates and lag times. In an earlier version of the software (values labeled in columns with "(1)", a line was fit to a detected linear region in semilog space to determine the growth rate. An updated version of the software (values labeled in columns with "(2)") implemented a direct exponential fit of a detected growth phase in linear space, resulting in higher weighting of the least squares fit to regions of the curve exhibiting higher growth. Additionally, the updated version of the software implemented an adaptive smoothing algorithm that split the data into variable sized windows that minimize the standard deviation of growth values within a time interval, and generated spline fits between points. Finally, the updated version of the software discarded regions where growth curves were fit but the signal-to-noise ratio was less than 1, to eliminate automatic detection of false growth phases. While automatic detection succeeded in detecting and fitting the dominant growth phase more than 95% of the time, all data was additionally manually curated to ensure that the main growth phase was always selected and that false growth phases were not detected when growth was essentially absent.

For data obtained with the Growth Profiler, improved image analysis was additionally implemented to obtain the updated growth parameters. For values labeled in columns with "(1)", integrated pixel values (which were later converted to OD ₅₀₀ using a calibration curve) were obtained directly from image analysis capabilities in the Growth Profiler software. For values labeled in columns with "(2)", a new algorithm was implemented that automatically detected the pixel integration region in each well in each image by locating the darkest pixels in each well. These values were converted to OD ₆₀₀ with a calibration run in the same manner. This eliminated a slowly oscillating frequency that was sometimes observed in the original data and made calculating growth rates extremely difficult or inaccurate. This oscillation was believed to be due to alterations in brightness due to very small differences in where the shaker stopped when scanning the bottom of the plates. Results

Wild-type tolerance to isobut ric acid

E. coli K-12 MG1655 exhibited a steadily decreasing growth rate as a function of isobutyric acid concentration, with very little growth observed above 10 g/L (Table 3). Lag times began increasing above 1 g/L and steadily increased with increasing isobutyric acid concentration. Table 3. Growth of K-12 MG1655 in varying concentrations of isobutyric acid (neutralized). mean (1) std. error (1) mean (2) std. error (2) isobutyric acid

(g/L) μ (h ¹ ) 1| _¾ (h) μ (h ¹ ) 1| _¾ (h) μ (h ¹ ) 1| _¾ (h) μ (h ¹ ) 1| _¾ (h)

0 0.732 1.0 0.019 0.1 0.653 0.5 0.010 0.0

0.5 0.738 0.9 0.027 0.1 0.635 0.3 0.004 0.0

1 0.684 1.0 0.027 0.0 0.620 0.6 0.012 0.1

2.5 0.540 1.5 0.039 0.2 0.512 1.2 0.023 0.3

5 0.361 3.3 0.030 0.9 0.361 3.3 0.011 1.5

7.5 0.279 5.0 0.015 0.4 0.295 5.3 0.016 0.7

10 0.266 7.3 0.015 0.4 0.258 6.7 0.038 0.7

15 0.196 12.1 0.009 0.2 0.180 10.7 0.019 1.0

Based on these results and aiming for an initial growth rate of approximately 0.5 h ^"1 , it was decided to begin evolutions at a concentration of 3 g/L isobutyric acid. Resequencinq of tolerant isolates

Variants detected in isobutyric evolved strains are presented in Table 4. Each strain name corresponds to the chemical the strain was isolated from, the population the strain was isolated from, and the original number of the strain assigned during primary screening (e.g. IBUA1-7 is an isobutyric acid-evolved strain isolated from population 1). In each table, strains are arranged such that all that were isolated from the same population are presented in the same rows. Strains with an asterisk (*) following their name are hypermutator strains, and only the mutation identified that can be associated with generating the hypermutator phenotype (here only in IBUA3-2, with an insertion in the intergenic region between secA and mutT and those mutations that are shared with other mutations in the same gene in other strains are shown.

Characterization of selected isolates

Each re-sequenced isolate was characterized using the Biolector system for growth at the screening concentration of chemical (12.5 g/L isobutyric acid) in biological triplicates. Tables showing the calculated average growth rates and lag times for each isolate are shown in Table 5. Standard errors are standard deviations about the mean of the growth rate and lag time for the three independent biological replicates.

Table 5. Growth rates and lag times of re-sequenced isobutyric acid evolved isolates in M9 + 12.5 g/L isobutyric acid (neutralized). mean (1) std. error (1) mean (2) std. error (2) strain μ (h ¹ ) tlag (h) μ (h ¹ ) tlag (h) μ (h ¹ ) tlag (h) μ (h ¹ ) tlag (h)

MG1655 0.171 18.3 0.031 0.7 0.179 18.8 0.001 2.1

IBUA1-7 0.450 9.0 0.037 0.7 0.421 8.5 0.008 0.3

IBUA1-9 0.240 3.4 0.033 1.9 0.174 -8.4 0.112 13.9

IBUA2-1 0.490 9.8 0.014 0.5 0.451 9.2 0.011 0.5

IBUA2-6 0.372 8.1 0.025 0.4 0.424 8.6 0.031 0.4

IBUA2-9 0.471 8.5 0.091 0.7 0.400 7.2 0.048 1.0

IBUA3-2 0.476 12.8 0.062 2.7 0.412 11.7 0.076 4.4

IBUA3-10 0.303 4.5 0.081 1.4 0.201 -3.6 0.075 8.9

IBUA4-1 0.331 5.7 0.069 1.2 0.235 -0.1 0.096 6.6

IBUA4-8 0.417 6.4 0.105 0.6 0.313 4.2 0.011 1.0

IBUA4-9 0.378 6.3 0.043 0.7 0.311 4.5 0.019 1.0

IBUA5-2 0.491 8.4 0.072 1.1 0.383 6.3 0.058 3.3

IBUA5-6 0.481 8.2 0.054 1.8 0.403 6.9 0.011 2.7

IBUA6-7 0.380 9.3 0.049 0.7 0.359 8.8 0.034 1.1

IBUA6-9 0.507 8.5 0.050 0.3 0.527 8.7 0.028 0.7

IBUA7-6 0.288 4.9 0.014 1.2 0.211 -0.5 0.040 1.8

IBUA7-7 0.447 7.1 0.016 0.4 0.363 5.2 0.055 0.9

IBUA7-9 0.357 6.3 0.051 0.7 0.191 -3.9 0.096 6.5

IBUA8-3 0.376 7.4 0.037 0.8 0.326 5.9 0.042 1.4

IBUA8-4 0.367 7.2 0.058 1.5 0.344 6.5 0.017 0.4

IBUA8-10 0.407 7.4 0.051 1.4 0.385 7.0 0.006 0.4

Large differences in growth behavior amongst evolved isolates can be noted. Better growing strains are defined by both the slope of the curve (higher growth rate) and at what time the cultures begin growing (reduced lag time). The majority of isolates exhibit at least a 200% increase in growth rate and a 50% reduction in lag time vs. the wild-type strain in 12.5 g/L isobutyric acid. Mutations that occur independently across multiple populations, or that appear fixed in a highly variable population, are likely causative and of highest interest. These include mutations in pykF, which are dominated by loss-of-function mutations (e.g. frameshift mutations and IS element insertions). The presence of clear loss-of-function mutations in these populations infers loss-of-function of pykF in the coding mutations present as well, e.g. in IBUA1-9. Many mutations in different RNA polymerase subunits (RpoB, RpoC) also appear causative, for example the IBUA4 population exhibits large heterogeneity in the 3 sequenced isolates, with the only mutation being conserved amongst isolates being RpoB

H526Y. This same mutation occurred independently in isolates from 3 additional populations. Knockout strain growth performance

Probable loss-of-function mutations were identified from re-sequencing results as described in methods. More obvious loss-of-function mutations were found (frameshift mutations and IS element insertions) in pykF in several populations, with the remainder of mutations in pykF being coding mutations or in the intergenic region upstream of pykF. It can therefore be inferred that these latter mutations likely also partially or fully inactivate PykF activity or abolish or reduce its expression via disruption of the promoter. Additional probable loss-of- function mutations were identified in two other genes, rpoS and yobF. For rpoS, a premature stop codon was found in IBUA5-2, a large 32 amino acid deletion was found in IBUA5-6, and a frameshifting 1 bp deletion was found IBUA6-7. For yobF, IS element insertions were observed in all sequenced isolates from population IBUA2. These individual gene knockouts were cleanly reconstructed alone and in combinations. Initially, single and double knockouts were screened with the Growth Profiler at two concentrations: 6.3 g/L and 12.5 g/L isobutyric acid. Average growth rates and lag times for three biological replicates are shown in Table 6. Table 6. Growth rates and lag times of single and double gene knockouts in M9 + 6.3 g/L isobutyric acid and M9 + 12.5 g/L isobutyric acid (neutralized) as measured in the Growth Profiler testing format.

6.3 g/L isobutyric acid

mea n (l) std. £ ?rror (1) mean (2) std. error (2)

Strain μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h)

MG1655 0.569 8.2 0.024 0.1 0.455 10.2 0.007 0.3

MG1655 pykF::kan 0.633 5.0 0.033 0.2 0.483 7.0 0.010 0.0

MG1655 rpoS::kan 0.678 24.2 0.092 5.1 0.449 26.8 0.038 4.9

MG1655 yobF::kan 0.531 7.8 0.031 0.2 0.413 9.9 0.003 0.2

MG1655 ApykF rpoS::kan 0.664 4.9 0.057 0.1 0.463 6.7 0.007 0.2

MG1655 ApykF yobF::kan 0.623 5.3 0.021 0.0 0.492 7.3 0.011 0.1

12.5 g/L iso outyric acid

mea n (l) std. £ ?rror (1) mean (2) std. error (2)

Strain μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h)

MG1655 0.318 21.8 0.012 1.9 0.224 23.9 0.012 1.3

MG1655 pykF::kan 0.347 10.5 0.029 0.1 0.292 12.7 0.002 0.1

MG1655 rpoS::kan 0.000 - 0.000 - 0.000 - 0.000 -

MG1655 yobF::kan 0.290 21.7 0.074 1.2 0.225 23.8 0.009 1.8

MG1655 ApykF rpoS::kan 0.386 9.0 0.039 0.1 0.318 10.8 0.006 0.1

MG1655 ApykF yobF::kan 0.348 11.1 0.030 0.1 0.294 12.9 0.002 0.1 In this testing format, it was found that deletion of pykF primarily decreased lag time compared to the wild-type when grown with 6.3 g/L or 12.5 g/L isobutyric acid. Deletion of rpoS by itself dramatically increased the lag time in 6.3 g/L isobutyric acid, and resulted in no growth in 12.5 g/L isobutyric acid. Deletion of yobF did not result in a growth phenotype vs. K-12 MG1655 at either concentration. K-12 MG1655 ApykF rpoS : : kan had a slightly higher growth rate in 12.5 g/L isobutyric acid than K-12 MG1655 pykFr.kan, with a similarly reduced lag time.

These strains were re-tested in the Biolector testing format together with a selection of evolved strains in M9 + 12.5 g/L isobutyric acid. The average growth rates and lag times for three biological replicates are shown in Table 7. The growth rate of the pykF single deletion strain was more than double that of K-12 MG1655 with a significantly reduced lag time, and recuperated 57 to 73% of the growth rate observed in the four tested evolved strains. The pykF rpoS double deletion strain exhibited the best overall features, with a growth rate that was 67 to 87% of the evolved strains and with the lowest lag time. The pykF yobF double deletion strain also performed better than the pykF deletion alone, but with a lag time similar to the pykF single deletion strain.

Based on these results, it was decided to construct the triple deletion strain K-12 MG1655 ApykF ArpoS yobFr.kan and to test it in the Biolector together with the same double deletion strains already tested (Table 8). No additional increase in growth rate or reduction in lag time was observed versus the double knockouts already constructed.

Table 7. Growth rates and lag times of single and double gene knockouts in M9 + 12.5 g/L isobutyric acid (neutralized) as measured in the Biolector testing format. mean (1) std. error (1) mean (2) std. error (2)

Strain μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ In ^-1 ) t (h)

MG1655 0.102 28.9 0.024 0.8 0.148 23.0 0.016 0.8

IBUA2-9 0.396 7.1 0.025 0.6 0.472 8.3 0.025 0.5

IBUA5-6 0.389 6.1 0.017 0.8 0.474 7.4 0.029 0.3

IBUA7-9 0.312 4.3 0.009 0.6 0.232 1.1 0.062 2.4

IBUA8-3 0.306 4.8 0.015 0.4 0.361 6.2 0.020 0.3

MG1655 pykF::kan 0.224 9.4 0.023 1.3 0.262 11.5 0.008 0.4

MG1655 rpoS::kan - - - - 0.026 - 0.012 -

MG1655 yobF::kan 0.226 17.7 0.052 2.9 0.199 17.5 0.027 0.8

MG1655 ApykF rpoS::kan 0.265 6.7 0.019 1.3 0.299 7.9 0.010 0.5

MG1655 ApykF yobF::kan 0.271 10.2 0.031 1.3 0.284 10.9 0.012 0.3 Table 8. Growth rates and lag times of double and triple gene knockouts in M9 + 12.5 g/L isobutyric acid (neutralized) as measured in the Biolector testing format. mean (1) std. error (1) mean (2) std. error (2)

Strain μ In ^-1 ) t (h) μ In ^-1 ) t (h) μ (h- ¹ ) t (h) μ In ^-1 ) t _laB (h)

MG1655 ApykF rpoS::kan 0.270 11.1 0.006 1.6 0.287 11.7 0.007 1.6

MG1655 ApykF yobF::kan 0.238 13.5 0.020 0.7 0.265 14.9 0.022 0.9

MG1655 ApykF ArpoS yobF::kan 0.259 12.1 0.011 2.5 0.225 10.3 0.022 3.5

The Keio collection of gene knockouts is a commercial collection of knockouts in nearly all non-essential genes and ORFs in E. coll strain BW25113. This strain is a K-12 derivative and possesses known mutations relative to the K-12 MG1655 background. All Keio collection strains with knockouts in genes that were found to be mutated in Table 1 were screened for growth against the BW25113 control in M9 + 1% glucose + 6.3 g/L or 12.5 g/L isobutyric acid (neutralized) in the Growth Profiler screening format. Initial (preliminary) qualitative observations based on averaged growth curves for 3 biological replicate cultures are shown in Table 9, with calculated growth rates shown in Table 10. Gene deletion strains of rph and yjcF exhibited increased growth rates in both 6.3 g/L and 12.5 g/L isobutyrate. Gene deletion strains of phoU exhibited an increased growth rate only at 6.3 g/L isobutyrate, while deletion of cheR increased the growth rate only in 12.5 g/L isobutyrate. Knockout mutations from Keio strains have already been PI phage transduced into K-12 MG1655 and are being validated and screened for growth in the Biolector format. Knockouts will additionally be constructed in the best performing knockout strain(s) obtained from earlier efforts.

Table 9: Initial (preliminary) qualitative evaluation of growth rates of Keio collection knockout strains compared to the background strain BW25113 in 6.3 g/L and 12.5 g/L isobutyrate as measured in the Growth Profiler testing format.

Growth rate effect vs. BW25113

Strain genotype 6.3 g/L isobutyrate 12.5 g/L isobutyrate

BW25113 cheR::kan Decreased increased

BW25113 phoUr.kan Increased none

BW25113 bglGr.kan Decreased none

BW25113 yedVr.kan Decreased decreased

BW25113 ybbWr.kan None none

BW25113 yiiOr.kan None decreased

BW25113 ydhZr.kan Abolished abolished

BW25113 yaiPr.kan Decreased none

BW25113 dtpCr.kan None none

BW25113 ydbA_l ::kan highly variable highly variable

BW25113 rphr.kan Increased increased

BW25113 sapFr.kan Decreased decreased

BW25113 yjcFr.kan Increased increased Table 10. Growth rates of Keio collection knockouts in M9 + 12.5 g/L isobutyric acid (neutralized) as measured in the Growth Profiler testing format.

Mechanisms of isobutyrate tolerance in knockout strains

Descriptions of genes disrupted in mutants with improved growth in high concentrations of isobutyrate are provided in Table 11. Loss-of-function of pykF results in a reduced cellular flux through lower glycolysis, reduced pyruvate accumulation and acetate formation, and increased flux through the pentose phosphate pathway (Al Zaid Siddiquee et al. , 2004). Without being limited to theory, reduced pyruvate formation may be beneficial and linked to the feedback resistance mutations found in the IBUA8 population in the small subunits of acetohydroxybutanoate synthase/acetolactate synthase (AHAS) (see nvestigation of mutations in IBUA8' below).

Table 11: Descriptions of genes disrupted in mutants with improved growth in high concentrations of isobutyrate

Gene name Description

pykF Pyruvate kinase I

rpoS RNA polymerase, sigma S (sigma 38) factor yobF small protein involved in stress response phoU negative regulator of the Pho regulon Production of isobutyric acid in evolved isolates

Isobutyric acid production has been demonstrated in E. coli from pyruvate via 5 steps (Zhang et a/. , 2011), the first 4 of which are common to isobutanol production as well (Atsumi et a/., 2007). First, pyruvate can be converted to (2S)-hydroxy-2-methyl-3-oxobutanoic acid with AlsS (acetolactate synthase) heterologously expressed from Bacillus subtilis. Second, this product can be reduced and isomerized to (2R)-2,3-dihydroxy-3-methylbutanoic acid by native E. coli IlvC (acetohydroxy acid isomeroreductase). Third, this product can be dehydrated to 2-ketovaline by native E. coli IlvD (dihydroxy acid dehydratase). Fourth, this product can be decarboxylated to isobutyraldehyde by Lactococcus lactis KIVD (2-ketoacid decarboxylase). Finally, this product can be oxidized to isobutyric acid promiscuously by a number of aldehyde dehydrogenases. The most active enzymes for this activity out of 7 tested were determined to be 3-hydroxypropionaldehyde dehydrogenase AldH from E. coli, and phenylacetaldehyde dehydrogenase PadA from E. coli (Zhang et a/. , 2011). Deletion of a native alcohol dehydrogenase which actively converts isobutyraldehyde to isobutanol (and is typically overexpressed when the desired product is isobutanol), YqhD, increases conversion to isobutyric acid and reduces production of isobutanol as a side product (Zhang et a/. , 2011).

To test isobutyric acid production in evolved isolates, production strains were first generated in a selection of evolved strains backgrounds and in the K-12 MG1655 and K-12 BW25113 controls. To achieve this, knockouts of yqhD were constructed in control strains BW25113 and K-12 MG1655, and in evolved isolates IBUA2-9, IBUA7-9, and IBUA8-3. Plasmids pIBAl and pIBA7 (Zhang et a/. , 2011) were obtained from Prof. Kechun Zhang at the University of Minnesota. pIBAl contains B. subtilis alsS and E. coli ilvD in an artificial operon under control of a Piac promoter (lactose or isopropyl β-D-l-thiogalactopyranoside (IPTG) inducible) and a kanamycin resistance gene. pIBA7 contains the KIVD gene from L. lactis and E. coli pad A, also in an artificial operon under control of a P| _ac promoter and an ampicillin resistance gene. These plasmids were transformed into the yqhD knockout strains.

To produce isobutyric acid, these strains were grown in M9 glucose with added yeast extract at 30°C and M9 glucose without added yeast extract at 37°C, with solid calcium carbonate added to the cultures to keep the pH constant, and 0.1 mM IPTG added from inoculation to induce the pathway genes being expressed from the plasmids. Non-induced controls were included for the K-12 MG1655 and IBUA2-9 background strains. Isobutyric acid was measured after 24 hours and 48 hours growth (Table 12). No significantly increased titers of isobutyric acid could be observed under these conditions in any of the evolved strain backgrounds compared to the control strains (BW25113 and MG1655). IBUA7-9 and IBUA8- 3 generated statistically equivalent titers of isobutyric acid, while IBUA2-9 produced a significantly lower titer than the control strains. Yeast extract was added to these cultures, which is likely not commercially feasible due to its high expense relative to the value of isobutyric acid.

Table 12: Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured after 24 h and 48 h in M9 glucose medium containing yeast extract at 30°C.

To test for production under conditions more representative of the anticipated conditions reguired for economical production, as well as to investigate the effect of toxic concentrations of isobutyric acid on additional production potential, strains were grown in M9 glucose without added yeast extract at 37°C, with or without the addition of 7.5 g/L isobutyric acid from inoculation. Calcium carbonate and IPTG were added as previously described. Blank media wells were included to allow determination of the isobutyric acid concentration when accounting for evaporation, and the values measured in blank wells were subtracted from measured concentrations in order to determine the amount that was produced on top of what was added to the cultures, for cultures with added isobutyric acid. Isobutyric acid was measured after 24 hours and 50 hours growth (Table 13).

Table 13: Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured after 24 h and 48 h in M9 glucose medium with or without added isobutyric acid at 37°C.

- isobutyric acid + 7.5 g/L isobutyric acid

24 h 50 h 24 h 50 h background strain titer (g/L) titer (g/L) titer (g/L) titer (g/L)

K-12 BW25113 0.00 ± 0.00 1.73 ± 0.24 0.02 ± 0.02 0.00 ± 0.10

K-12 MG1655 0.00 ± 0.00 1.43 ± 0.42 0.01 ± 0.07 -0.12 ± 0.07

IBUA2-9 0.00 ± 0.00 0.00 ± 0.00 -0.05 ± 0.05 -0.28 ± 0.04 IBUA7-9 0.00 ± 0.00 2.11 ± 0.17 0.00 ± 0.03 -0.24 ± 0.05

IBUA8-3 1.30 ± 0.04 1.62 ± 0.09 1.45 ± 0.12 2.12 ± 0.15

While lower titers were realized, likely due to the lack of yeast extract (which also supports evidence of probable starvation for an amino acid, likely isoleucine), differences in performance can be noted between control strains and evolved strain backgrounds. In M9 glucose without added isobutyric acid, only the IBUA8-3 strain background produces a detectable titer after 24 hours, indicative of an increased productivity. While its final titer after 50 hours was not significantly higher than control strains, IBUA7-9 exhibited an increased titer after 50 hours compared to both control strains. In the presence of 7.5 g/L isobutyric acid from the time of inoculation, only strain IBUA8-3 could further produce additional isobutyric acid, and at titers similar to or even higher than in the absence of added isobutyric acid.

Based on this promising result, fed-batch fermentations were conducted for the control strain background K-12 MG1655, IBUA7-9, and IBUA8-3 in a minimal salts medium supplemented with glucose at 30°C (Table 14). Some variability was seen between biological duplicate fermentations, however one replicate fermentation for each of IBUA8-3 and IBUA7-9 achieved over 2-fold improvements in isobutyric acid titers compared to either K-12 MG1655 control.

Table 14: Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from fed-batch fermentation runs after 45.5 hours in a minimal medium supplemented with glucose at 30°C. final titer

strain / replicate (q/L)

K-12 MG1655 / 1 6.47

K-12 MG1655 / 2 8.18

IBUA8-3 / 1 18.83

IBUA8-3 / 2 10.41

IBUA7-9 / 1 5.07

IBUA7-9 / 2 18.67

Due to the variability in the first run, fed-batch fermentations were repeated with a change i IPTG induction conditions and concentration, notably automatic addition of an IPTG pulse 1 hour after the start of the feeding phase to bring the concentration to 0.1 mM, plus addition of 0.1 mM IPTG to the feed solution. Again K-12 MG1655, IBUA7-9, and IBUA8-3 backgrounds possessing the yqhD deletion and plasmids pIBAl and pIBA7 were tested, however only one replicate of IBUA7-9 was successfully run (the other strains had two successful replicates). In this run, the K-12 MG1655 background produced much less isobutyric acid than previously, possibly due to earlier induction which has a negative effect on K-12 MG1655 physiology. However IBUA7-9 and IBUA8-3 backgrounds exhibited 12.6- fold and 6.1-fold higher production titers, respectively, in addition to significantly enhanced yields with respect to biomass formed (Table 15).

Table 15: Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from fed-batch fermentation runs after 47.5 hours in a minimal medium supplemented with glucose at 30°C, with altered induction conditions as described in the text.

Finally, it was sought to screen a wider range of evolved strain backgrounds harboring the yqhD deletion and plasmids pIBAl and pIBA7 for isobutyric acid production. This allows the identification of mutations that both confer tolerance and still enable high endogenous production. Isolates from some populations proved more recalcitrant to various genetic engineering methods (namely PI transduction and transformation required to generate the yqhD knockout and to transform the production plasmids), therefore these were left out of the screen. Strains were screened in a minimal slow-release glucose medium (described in Methods) and supernatants were analyzed for isobutyric acid after culturing for 24 hours and 48 hours (Table 16). Strains derived from the IBUA7 population (particularly IBUA7-9) and the IBUA8 population, which had previously been selected for further testing, were among the best performing strains with their final titers after 48 hours. Additionally, the IBUA3-10, IBUA6-7, and IBUA4-8 backgrounds produced over 2 g/L. Notably, IBUA3-10, IBUA4-8, and all IBUA7 strains share the RpoB-H526Y mutation. Thus it can be surmised that this mutation is causative for both higher tolerance while also allowing a high level of isobutyrate production. The IBUA6-7 population possessed an alternative mutation in RpoB, RpoB- A1183V. Two strains, IBUA3-2 and IBUA6-9 harbored different mutations on residue D622 of RpoC. IBUA3-2 was however a mutator strain possessing numerous additional mutations that are likely responsible for its higher production levels than IBUA6-9. The RpoC-D622A mutation has previously been studied and found to render RpoC insensitive to the strigent response alarmone ppGpp. Thus while this was effective as a mechanism of isobutyrate tolerance, this effect is counteractive toward endongenous production of isobutyrate. All IBUA2 derived strains, which also exhibited abolished or diminished isobutyrate production harbored an alternative RpoC mutation, RpoC-T757K.

Table 16: Isobutyric acid titers in background strains harboring the AyqhD mutation and plasmids pIBAl and pIBA7, measured from screening in a minimal slow-release glucose medium after 48 at 30°C.

Investigation of mutations in IBUA8

The performance of the IBUA8-3 strain background relative to the other tested strain backgrounds resulted in a closer investigation of mutations that could enable higher production levels. Examining isolates from the IBUA8 population, IBUA8-3 and IBUA8-4 appear genetically identical and both have IlvH-L9F mutations, while IBUA8-10 harbors an IlvN-N17H mutation. All isolates also harbor a GlyQ-E48D mutation. IlvH and IlvN are both small regulatory subunits of different active isoforms of acetohydroxybutanoate

synthase/acetolactate synthase (AHAS) in E. coli. There are a total of three isoforms, with AHAS III being composed of Ilvl and IlvH, AHAS I being composed of IlvB and IlvN, and AHAS II being composed of IlvG and IlvM. In E. coli K-12 lineages, ilvG is a pseudogene due to the presence of an internal frameshift mutation and AHAS II activity is abolished. The three AHAS isoforms catalyzes the first common steps in the biosynthesis of the branched chain amino acids, L-valine, L-isoleucine, and L-leucine, through complex allosteric inhibition mediated by the small regulatory subunits of each isoform (IlvH, IlvN, and IlvO). Both AHAS I and AHAS III are inhibited by valine, while AHAS III is nearly completely inhibited by leucine. Because both functional isoforms are inhibited by valine, E. coli K-12 is sensitive to the addition or accumulation of valine due to isoleucine starvation (De Felice et ai. , 1979), and has also been shown to undergo oscillations in isoleucine starvation under normal growth conditions (Andersen et ai , 2001).

Notably, the mutations found in evolved isolates in both IlvH and IlvN are in the N-terminal regions of these proteins. It has previously been shown that only the first 14-25 amino acids of IlvH are required for activation of AHAS III, and that N- and C-terminal truncated mutants do not exhibit valine inhibition (Zhao et a/. , 2013). L9A and L9V mutants of E. coli IlvH have been shown to reduce valine inhibition of AHAS III (Kaplun et ai , 2006), and specific feedback-resistant N-terminal mutations of E. coli IlvH (notably in residues 14 and 17) have been described for the purpose of producing L-valine (US Patent No. 6,737,255 B2).

Feedback-resistant mutations of E. coli IlvN, including the N17K mutation, have been described for the purpose of producing branched-chain amino acids (EP 1 942 183 Al). To investigate whether the IlvH-L9F and IlvN-N17H mutations are directly causative for tolerance to isobutyrate, these mutations were generated in K-12 MG1655 and K-12 MG1655 ApykF by MAGE. The results of growth testing in 12.5 g/L isobutyrate are shown in Table 17. Neither mutants in ilvH and ilvN exhibited improved growth rates in isobutyrate relative to the wild-type strain, and the same mutations in the ApykF background strain also did not exhibit an improved growth rate.

To investigate if the evolved isolates and the IlvH-L9F and IlvN-N17H mutations exhibited feedback resistance toward exogenous valine and leucine, mutants were tested for growth in M9 medium + 1% glucose + 1 g/L of L-valine or L-leucine (Table 18). Notably, IBUA8-3 has a significant growth rate increase and a dramatically reduced lag time in the presence of exogenous valine. IBUA8-10 also had better growth behavior but to a much lesser extent. These phenotypes could be seen to be largely due to the mutations in IlvH and IlvN, as similar trends were observed for cleanly constructed mutant strains with only those mutations. Inhibition by leucine does not produce as strong a phenotype, however IBUA8-10 exhibited the greatest reduction in lag time and largest increase in growth rate. Preliminary data indicated that this was not reconstituted by the IlvN mutation, however the IlvH mutant also exhibited a significantly reduced lag time. Therefore it can be concluded that the IlvH- L9F and IlvH-N17H are feedback resistant to inhibition by L-valine and to L-leucine.

Because it was highly unlikely to be coincidental that two different N-terminal mutations result in feedback resistance of inhibition of acetolactate synthase by L-valine and/or L- leucine, additional combinations of mutations were tested (Table 19). A wild-type reversion mutant of IlvH was constructed in evolved isolate IBUA8-3 ("IBUA8-3 /V -/- revert"), and the GlyQ-E48D mutation was also constructed in strains harboring IlvH-L9F and IlvN-N17H, either with or without ApykF, to determine if the IlvH mutation is important in the context of other mutations in the strain (see Table 4). It was found that the GlyQ-E48D mutation improved growth in the presence of 12.5 g/L isobutyrate only when it was together with the pykF deletion. The combination of the GlyQ-E48D, IlvH-L9F, and ApykF mutations did not improve growth over the strain that only harbored GlyQ-E48D and ApykF. The IlvH reversion mutant of IBUA8-3 exhibited unusual behavior, with an initial growth phase to a low cell density at a growth rate similar to IBUA8-3, then with an approximately 14 hour lag phase before growth resumed at a nearly equivalent rate.

When the reversion mutant was grown in 12.5 g/L isobutyrate with the addition of 1 g/L L- isoleucine, dual phase growth was eliminated and the IBUA8-3 reversion mutant grew identically to IBUA8-3 (Table 20). Taken together, this data indicates that the IlvH-L9F mutation alleviates a growth inhibition due to L-isoleucine starvation, likely as a result of L- valine and/or L-leucine accumulation, and that this L-isoleucine starvation and L-valine and/or L-leucine accumulation occurs as a result of a combination of the ApykF, GlyQ-E48D, and at least one other mutation in the strain background. Because IBUA8-10 also evolved a an AHAS feedback resistance mutation in parallel in the population, it can be concluded that the condition of isoleucine starvation or valine accumulation is particularly induced by the combination of the ApykF, GlyQ-E48D, and the increased expression of pyrE resulting from the 82 bp deletion within the 5' terminal region of rph (the increased growth rate imparted by these mutations likely necessitated a higher biosynthetic flux toward isoleucine than was otherwise possible without an AHAS feedback resistance mutation), as this was the only remaining common mutation between IBUA8-3 and IBUA8-10. It is further evident by comparing data in Tables 17 and 18, that isobutyrate induces L-isoleucine starvation, given that supplementation of 1 g/L L-isoleucine in medium containing 12.5 g/L isobutyrate results in an approximately 2.5-fold higher growth rate and an approximately 56% reduction in lag time in strain K-12 MG1655.

Table 17: Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyric acid (neutralized), as measured in the Biolector testing format.

Mean std. error

Strain μ (h ¹ ) t, _a „ (h) μ (h ¹ ) t, _a „ (h)

K-12 MG1655 0.163 18.0 0.041 3.8

IBUA8-3 0.314 3.2 0.018 0.5

MG1655 ApykF 0.237 5.5 0.028 1.2

MG1655 ilvH-L9F 0.247 22.2 0.063 1.1

MG1655 ilvN-N17H 0.173 22.4 0.031 2.3 MG1655 ApykF ilvH-L9F 0.235 8.4 0.020

MG1655 ApykF ilvN-N17H 0.238 6.7 0.013

Table 18: Growth rates and lag times of selected strains in M9 + 1 g/L L-valine or L-leucine, as measured in the Biolector testing format.

1 g/L va line

Mean std . error

Strain μ (h ¹ ) ¾ _a „ (h) μ (h ¹ ) ¾ _a „ (h)

K-12 MG1655 0.691 23.5 0.022 1.4

IBUA8-3 0.984 2.0 0.075 0.4

IBUA8-10 0.715 16.7 0.058 1.0

MG1655 ilvH-L9F 0.646 1.9 0.022 0.3

MG1655 ilvN-N17H 0.601 19.8 0.020 0.7

1 g/L leucine

Mean std. error

Strain μ (h ¹ ) ¾ _a „ (h) μ (h ¹ ) t, _a „ (h)

K-12 MG1655 0.557 7.6 0.016 0.6

IBUA8-3 0.761 6.1 0.011 0.4

IBUA8-10 0.727 4.5 0.003 0.2

MG1655 ilvH-L9F 0.530 5.3 0.007 0.5

MG1655 ilvN-N 17H 0.600 7.4 0.006 0.3

Table 19: Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyrate, measured in the Biolector testin format.

Table 20: Growth rates and lag times of selected strains in M9 + 12.5 g/L isobutyrate + 1 g/L L-isoleucine, as measured in the Biolector testing format.

mean std. error

Strain μ (h ¹ ) tlag (h) μ (h ¹ ) tlag (h)

K-12 MG1655 0.213 14.7 0.083 4.2

IBUA8-3 0.432 8.2 0.018 0.6

MG1655 ApykF 0.240 11.2 0.052 6.7

MG1655 glyQ-E48D 0.171 16.1 0.023 0.6

MG1655 ApykF glyQ-E48D 0.273 6.0 0.010 0.4

IBUA8-3 ilvH-revert 0.398 8.0 0.018 0.1

In the context of production of isobutyric acid, where IBUA8-3 was found to be the best producing evolved isolate out of three tested in the absence of added amino acids (e.g. yeast extract), AHAS feedback resistance mutations may improve native production of balanced levels of valine, leucine, and isoleucine during production. The production pathway employed uses a heterologous AHAS (AlsS) with a higher activity for utilizing pyruvate as a substrate, as well as overexpressed E. coli IlvD, which is also used for biosynthesis of L-valine and L- isoleucine. This may lead to accumulation of L-valine which then feedback-inhibits flux through native E. coli AHAS I and III, which is also required for biosynthesis of isoleucine. Feedback resistance mutations would be expected to allow maximum conversion of pyruvate to acetolactate through all possible enzymes. This effect can be responsible for the improved production of isobutyrate in IBUA8-3 in the absence of yeast extract, which contains exogenous isoleucine.

Cross-compound tolerance testing

Every secondary screened evolved isolate from the isobutyric acid evolution was grown in the presence of every other compound in the study as indicated in the Methods. The normalized toDi(evoived strain)/toDi(wiid-type) are presented as a heat map in Table 21. Lower values are are indicative a larger improvement in growth of the evolved isolate (left column) in that chemical condition (top row), whereas higher values are indicative of a lower improvement or decrease in growth compared to the wild-type. Averaged ratios across conditions and strains shown at the right and bottom of the plot allow for overall by-chemical and by-strain trends to be observed. Strain names that are followed by an asterisk (*) were not re-sequenced, and strain names in italics were found to be hypermutator strains.

The majority of evolved isolates exhibited improved growth in the presence of other monocarboxylic acids. This included 23/24 strains for coumarate (the only exception was hypermutator strain IBUA1-5), 21/24 strains for octanoate (all except population IBUA1 isolates), and 19/20 strains for hexanoate (nearly all except IBUA3 isolates) . The majority of strains (17 to 18 out of 24) exhibited improved tolerance to the diacids (glutarate and adipate), as well. It is believed that many of the mutations in rpoB and rpoC confer broad acid tolerance. The isolates which performed the best across all chemical classes were IBUA8- 3, IBUA8-4, and IBUA8-10. Table 21: Normalized t ₀ Di(evoived toDi(wiid-ty _P e) values for isobutyrate-evolved isolates grown in the presence of inhibitory concentrations of 12 different chemicals.

Additionally, each evolved isolate was tested for cross-tolerance toward other straight short- chain and branched short-chain carboxylic acids and alcohols of potential biotechnological interest. First, K-12 MG1655 was tested in the Growth Profiler screening format for growth in the presence of a range of concentrations of each compound : butyric acid, 2-methylbutyric acid, valeric acid, isovaleric acid (3-methylbutyric acid), 4-methylvaleric acid, 2- methylhexanoic acid, 1-propanol, 2-propanol (isopropanol), isobutanol, 3-methyl-l-butanol, 2-methyl-l-butanol, and 1-pentanol. Variable concentrations of these compounds elicited growth inhibition in E. coll K-12 MG1655 (Table 22). Based on these results, a screening concentration was selected for the evolved isolates for which wild-type cells could achieve at a growth rate of 0.2-0.3 h ^"1 (versus uninhibited growth at 0.7-0.9 h ^"1 in M9 glucose minimal medium). These concentrations were: 15 g/L butyrate, 13 g/L 2-methylbutyrate, 8 g/L valerate, 10 g/L isovalerate, 5 g/L 4-methylbutyrate, 5.5 g/L 2-methylhexanoate, 18 g/L 1- propanol, 25 g/L 2-propanol, 8 g/L isobutanol, 5.5 g/L 3-methyl-l-butanol, 5.5 g/L 2-methyl- l-butanol, and 5 g/L 1-pentanol. The isobutyrate-evolved isolates grown in the straight- and branched-chain carboxylic acids are shown in Tables 23 and 24. A large number of evolved isolates exhibit greatly improved growth rates and often-reduced lag times in all of these compounds compared with K-12 MG1655. Large numbers of the isolates exhibited cross- tolerance toward isovalerate, 4-methylvalerate, and 2-methylbutyrate. More selected numbers of isolates exhibited cross-tolerance toward valerate, butyrate, and 2- methylhexanoate. The only isolates that exhibited cross-tolerance toward the majority of alcohols were those derived from the IBUA8 population (Table 25). Some additional isolates from the IBUA5 population, IBUA4-9, and IBUA6-7 also exhibited cross-tolerance toward 3- methyl-l-butanol (Table 26).

Table 22. Growth rates and lag times of K-12 MG1655 in varying concentrations of short branched- and straight-chain carboxylic acids and alcohols, as measured in the Growth Profiler testing format. mean (2) std. error (2) mean (2) std. error (2)

2-methylhexanoic acid

butyric acid (g/L) μ (π ^-1 ) t _laK (h) μ ( ^η ') t _lM (h) (g/L) μ ( ^η ') t _lM (h) μ ( ^η ') t _lM (h)

0 0.753 5.1 0.029 0.0 0 0.758 4.9 0.070 0.2

5 0.471 10.5 0.021 0.4 2 0.535 8.8 0.017 0.5

7.5 0.395 13.5 0.020 0.8 4 0.390 12.5 0.030 0.8

10 0.285 16.3 0.022 1.2 6 0.128 22.1 0.004 1.7

12.5 0.230 19.7 0.034 1.1 8 0.000 - 0.000 -

15 0.225 25.4 0.016 0.8 1-propanol (g/L) μ ( ^η ') (h) μ ( ^η ') (h)

20 0.000 - 0.000 - 0 0.819 4.9 0.061 0.1

2-methylbutyric acid

(g/L) μ ( ^η ') (h) μ ( ^η ') (h) 10 0.480 6.8 0.012 0.2

0 0.738 5.1 0.027 0.4 25 0.000 - 0.000 - 5 0.450 9.6 0.017 0.4 2-propanol (g/L) μθι ¹ ) (h) μθι ¹ ) (h)

7.5 0.369 12.9 0.015 0.7 0 0.723 4.8 0.175 0.2

10 0.230 17.9 0.016 1.3 10 0.551 5.9 0.035 0.3

15 0.146 31.7 0.022 2.4 25 0.209 17.2 0.062 4.3

20 0.000 - 0.000 - 40 0.000 - 0.000 - valeric acid (g/L) θι ¹ ) (h) θι ¹ ) (h) isobutanol (g/L) μθι ¹ ) (h) μθι ¹ ) (h)

0 0.786 5.2 0.043 0.2 0 0.677 4.5 0.026 0.1

5 0.265 14.8 0.020 1.3 5 0.470 7.5 0.014 0.0

7.5 0.152 21.8 0.004 1.7 7.5 0.262 13.9 0.017 0.6

10 0.065 41.3 0.027 12.2 10 0.000 - 0.000 -

15 0.000 0.000 3-methyl-l-butanol (g/L) μθι ¹ ) (h) μθι ¹ ) (h) isovaleric acid (g/L) θι ¹ ) (h) μθι ¹ ) (h) 0 0.706 4.6 0.006 0.2

0 0.678 4.8 0.033 0.1 2.5 0.578 7.1 0.037 0.3

5 0.398 11.9 0.014 0.5 5 0.307 20.2 0.023 0.3

7.5 0.251 16.7 0.017 1.0 7.5 0.000 #DIV/0! 0.000 #DIV/0!

10 0.195 21.1 0.008 1.7 2-methyl-l-butanol (g/L) μθι ¹ ) (h) μθι ¹ ) (h)

15 0.144 34.6 0.026 0.3 0 0.663 4.7 0.024 0.1

20 0.000 - 0.000 - 2.5 0.533 7.6 0.038 0.3

4-methylvaleric acid

(g/L) θι ¹ ) (h) μθι ¹ ) (h) 5 0.387 21.9 0.025 0.3

0 0.731 5.0 0.070 0.2 7.5 0.000 - 0.000 -

2 0.490 9.6 0.048 0.7 1-pentanol (g/L) μθι ¹ ) (h) μθι ¹ ) (h)

4 0.341 15.0 0.058 1.3 0 0.675 4.6 0.027 0.4

6 0.140 25.1 0.009 2.1 2 0.577 6.4 0.013 0.6

8 0.000 - 0.000 - 4 0.426 15.0 0.044 1.7

6 0.000 0.000

Table 23. Growth rates and lag times of K-12 MG1655 in specified inhibitory concentrations of butyrate, 2-methylbutyrate, and valerate, as measured in the Growth Profiler testing format.

15 g/L butyrate 13 g/L 2-methylbutyrate 8 g/L valerate | mean (2) std. error (2) mean (2) std. error (2) mean (2) std. error (2) strain μ(π ^-1 ) t (h) μ( ^η ') t (h) μ( ^η ') (h) μ( ^η ') t (h) μ( ^η ') t (h) μ( ^η ') (h)

MG1655 0.175 29.7 0.070 7.1 0.248 20.4 0.016 0.3 0.197 22.4 0.010 0.7

IBUA1-5 0.085 7.9 0.147 13.7 0.302 13.4 0.093 4.3 0.000 0.0 0.000 0.0

IBUA1-7 0.230 19.3 0.039 4.5 0.273 12.6 0.007 0.8 0.206 14.8 0.011 0.2

IBUA1-9 0.100 14.8 0.145 13.0 0.240 19.3 0.082 4.5 0.140 19.6 0.006 2.1

IBUA2-1 0.000 0.0 0.000 0.0 0.354 14.3 0.104 3.6 0.144 11.2 0.127 9.9

IBUA2-6 0.000 0.0 0.000 0.0 0.137 19.7 0.119 20.2 0.000 0.0 0.000 0.0

IBUA2-9 0.084 20.0 0.078 17.8 0.442 10.9 0.047 0.7 0.219 15.0 0.041 0.9 IBUA3-2 0.244 19.4 0.183 16.7 0.065 2.5

IBUA3-4 0.343 13.9 0.248 12.9 0.029 0.7

IBUA3-10 0.265 19.2 0.319 15.5 0.032 0.5

IBUA4-1 0.261 20.2 0.261 15.9 0.021 0.5

IBUA4-8 0.044 8.2 0.253 12.5 0.031 1.1

IBUA4-9 0.275 18.2 0.280 15.3 0.022 0.5

IBUA5-2 0.319 14.3 0.367 10.0 0.014 0.3

IBUA5-5 0.311 14.4 0.337 10.8 0.011 0.2

IBUA5-6 0.295 15.3 0.325 10.9 0.008 0.2

IBUA6-7 0.335 16.3 0.348 9.8 0.014 0.5

IBUA6-9 0.000 0.0 0.000 0.0 0.000 0.0

IBUA6-10 0.000 0.0 0.000 0.0 0.000 0.0

IBUA7-6 0.255 17.9 0.000 0.0 0.000 0.0

IBUA7-7 0.304 15.3 0.000 0.0 0.000 0.0

IBUA7-9 0.293 19.3 0.000 0.0 0.000 0.0

IBUA8-3 0.337 14.7 0.363 14.3 0.019 0.4

IBUA8-4 0.345 15.3 0.291 16.7 0.029 1.5

IBUA8-10 0.314 19.5 0.131 32.8 0.031 5.4

Table 24. Growth rates and lag times of K-12 MG1655 in specified inhibitory concentrations of isovalerate, 4-methylvalerate, and 2-methylhexanoate, as measured in the Growth Profiler testing format.

10 g/L isovalerate 5 g/L 4-methylvalerate 5.5 g/L 2-methylhexanoate mea n (2) std. error (2) mean (2) std. error (2) mea n (2) std. error (2) strain μ (h ¹ ) (h) (h) μ (h ¹ ) (h) (h) μ (h ¹ ) (h) μ θι ¹ ) (h)

MG1655 0 247 17.1 0.008 0.4 0 290 17.1 0.013 0.4 0 241 14.4 0.007 0.4

IBUA1-5 0 384 10.5 0.011 1.8 0 383 13.6 0.019 2.9 0 255 15.1 0.047 5.2

IBUA1-7 0 315 10.2 0.021 0.1 0 328 12.3 0.012 0.2 0 245 12.2 0.006 0.3

IBUA1-9 0 326 10.7 0.007 1.1 0 317 12.4 0.014 2.0 0 209 11.3 0.004 1.0

IBUA2-1 0 352 9.8 0.028 0.2 0 311 12.4 0.027 0.5 0 308 11.1 0.031 0.3

IBUA2-6 0 315 14.0 0.014 1.3 0 381 13.1 0.026 0.5 0 206 17.3 0.092 3.7

IBUA2-9 0 361 9.5 0.005 0.2 0 312 12.5 0.022 0.1 0 286 11.1 0.038 0.6

IBUA3-2 0 379 8.6 0.010 0.2 0 413 11.5 0.013 0.2 0 230 10.9 0.011 0.4

IBUA3-4 0 413 8.1 0.012 0.1 0 488 9.3 0.045 0.0 0 359 9.2 0.028 0.0

IBUA3-10 0 279 10.1 0.015 0.7 0 302 11.1 0.030 0.8 0 262 9.4 0.029 0.9

IBUA4-1 0 315 10.2 0.004 0.3 0 392 10.0 0.024 0.0 0 324 10.2 0.022 0.1

IBUA4-8 0 373 8.4 0.024 0.7 0 431 9.2 0.019 0.4 0 457 8.7 0.034 0.4

IBUA4-9 0 334 10.2 0.022 0.1 0 065 14.4 0.038 4.7 0 271 11.9 0.013 0.3

IBUA5-2 0 380 8.1 0.004 0.1 0 424 8.8 0.014 0.0 0 352 8.8 0.004 0.1

IBUA5-5 0 387 8.0 0.001 0.1 0 388 8.6 0.015 0.2 0 351 8.5 0.031 0.2

IBUA5-6 0 392 8.2 0.014 0.0 0 415 9.0 0.006 0.0 0 186 9.0 0.032 0.5

IBUA6-7 0 390 8.2 0.012 0.3 0 418 7.4 0.035 0.3 0 418 8.6 0.014 0.2 IBUA6-9 0.351 9.1 0.009 0.2 0.384 8.9 0.012 0.2 0.230 9.5 0.027 0.5

IBUA6-10 0.390 12.0 0.014 3.1 0.455 11.8 0.039 2.3 0.153 18.4 0.038 6.1

IBUA7-6 0.310 10.0 0.017 0.4 0.332 11.6 0.049 0.3 0.152 11.3 0.021 0.5

IBUA7-7 0.322 9.4 0.005 0.3 0.307 11.9 0.023 0.3 0.202 12.4 0.024 0.3

IBUA7-9 0.316 10.8 0.033 0.5 0.391 12.2 0.036 0.7 0.189 13.7 0.053 0.7

IBUA8-3 0.412 9.6 0.010 0.1 0.435 9.6 0.004 0.1 0.301 12.7 0.002 0.2

IBUA8-4 0.411 9.6 0.015 0.4 0.423 9.8 0.018 0.2 0.296 12.9 0.010 0.5

IBUA8-10 0.424 9.9 0.010 0.1 0.429 9.5 0.003 0.1 0.269 13.5 0.002 0.1

Table 25. Growth rates and lag times of K-12 MG1655 and evolved isolates derived from population IBUA8 in specified inhibitory concentrations of 1-propanol, 2-propanol, isobutanol, 2-methyl-l-butanol, and 1-pentanol, as measured in the Growth Profiler testing format.

18 g/L 1-propanol 25 g/L 2-propanol 8 g/L isobutanol mean (2) std. error (2) mean (2) std. error (2) mean (2) std. error (2) strain (h) μ (π ^-1 ) (h) μ (π ^-1 ) (h) μ (π ^-1 ) (h) μ (π ^-1 ) (h) μ ί ^{η 1} ) (h)

MG1655 0.202 22.4 0.005 0.5 0.283 13.9 0.036 1.1 0.407 14.8 0.035 0.5

IBUA8-3 0.227 18.5 0.022 1.0 0.323 12.8 0.033 0.7 0.608 19.0 0.027 0.9

IBUA8-4 0.265 18.5 0.023 2.5 0.373 11.6 0.012 0.8 0.633 17.3 0.027 1.9

IBUA8-10 0.256 24.0 0.043 0.8 0.283 17.1 0.018 1.3 0.493 22.8 0.028 2.8

5.5 g/L 2-methyl-l-butanol 5 g/L 1-pentanol

mean (2) std. error (2) mean (2) std. error (2)

strain μ (π ^-1 ) (h) μ ί ^{η 1} ) (h) μ (π ^-1 ) (h) μ ί ^{η 1} ) (h)

MG1655 0.396 24.1 0.023 2.0 0.444 17.2 0.014 2.4

IBUA8-3 0.865 28.2 0.019 1.1 0.801 24.0 0.018 0.8

IBUA8-4 0.814 25.8 0.088 3.0 0.816 21.9 0.028 3.1

IBUA8-10 0.289 9.8 0.500 17.0 0.477 19.7 0.413 17.1

Table 26. Growth rates and lag times of K-12 MG1655 and selected evolved isolates in 5.5 g/L 3-methyl-l-butanol, as measured in the Growth Profiler testing format.

5.5 g/L 3-methyl-l-butanol |

mean (2) std. error (2)

strain μ (π ^-1 ) (h) μ (π ^-1 ) t (h)

MG1655 0.404 23.1 0.026 3.4

IBUA4-9 0.638 27.6 0.072 1.7

IBUA5-2 0.657 25.3 0.127 3.6

IBUA5-5 0.660 27.0 0.044 0.7 IBUA5-6 0.675 28.4 0.054 1.5

IBUA6-7 0.627 14.5 0.019 0.8

IBUA8-3 0.702 19.9 0.013 0.5

IBUA8-4 0.796 19.7 0.016 2.6

IBUA8-10 0.743 23.0 0.039 0.8

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