WO2015002916A1 - Régulation post-transcriptionnelle des voies biosynthétiques - Google Patents

Régulation post-transcriptionnelle des voies biosynthétiques Download PDF

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WO2015002916A1
WO2015002916A1 PCT/US2014/044989 US2014044989W WO2015002916A1 WO 2015002916 A1 WO2015002916 A1 WO 2015002916A1 US 2014044989 W US2014044989 W US 2014044989W WO 2015002916 A1 WO2015002916 A1 WO 2015002916A1
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seq
polypeptide
nucleic acid
conditions
acid sequence
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PCT/US2014/044989
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Arthur Leo Kruckeberg
Robert A. Larossa
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Butamax Advanced Biofuels Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the fields of industrial microbiology and alcohol production.
  • Embodiments of the invention relate to the the use of suitable post-transcriptional strategies for differential expression of genes during propagation and production phases to achieve regulated production via an engineered pathway in microorganisms.
  • Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
  • the methods comprise: A) contacting a recombinant host cell comprising a first heterologous polynucleotide and a second heterologous nucleotide, the first heterologous polynucleotide nucleic acid sequence encoding a biocatalyst polypeptide with a nonsense or frameshift allele and the second heterologous polynucleotide comprising i) a promoter nucleic acid sequence; and ii) a nucleic acid sequence encoding a suppressor tRNA specific to a nonsense or frameshift allele of the first heterologous polynucleotide, with a carbon substrate under a first set of conditions; and B) contacting the recombinant host cell with a carbon substrate under a second set of conditions; wherein the first set of conditions and second set of conditions differ and wherein the nucleic acid sequence encoding the suppressor tRNA is differentially expressed under the first set of conditions than under the second set of conditions.
  • the methods comprise A) contacting a recombinant host cell comprising a first heterologous polynucleotide and a second heterologous polynucleotide, the first heterologous polynucleotide comprising a nucleic acid sequence encoding a biocatalyst polypeptide, and the second heterologous polynucleotide comprising i) a promoter nucleic acid sequence; and ii) a nucleic acid sequence encoding an anti-sense RNA specific to the mRNA encoded by the first heterologous polynucleotide,with a carbon substrate under a first set of conditions; and B) contacting the recombinant host cell with a carbon substrate under a second set of conditions; wherein the first set of conditions and second set of conditions differ and wherein the nucleic acid sequence encoding the anti-sense RNA is differentially expressed under the first set of conditions than under the second set of conditions; and wherein the host cell produces but
  • the first and/or second set of conditions is an environmental or a process cue.
  • the environmental cue or process cue activates a transcriptional activator for a promoter described herein.
  • the environmental or process cue activates a transcriptional repressor for a promoter described herein.
  • the transcriptional repressor is a sulfonylurea- responsive repressor.
  • the transcriptional repressor is a Tet (tetracycline) repressor.
  • the host cell produces a fermentation product such as butanol or 2- butanone under at least one of the first set or the second set of conditions.
  • the biocatalyst polypeptide is expressed through the use of a constutive promoter.
  • the first set of conditions and the second set of conditions differ in at least one of source of carbon substrate, dissolved oxygen concentration, temperature, pH, glucose concentration, or fermentation product concentration such as butanol or 2-butanone concentration.
  • the dissolved oxygen concentration is greater during the first set of conditions than during the second set of conditions.
  • one set of conditions is anaerobic.
  • the second set of conditions is anaerobic.
  • the average glucose concentration is lower in the first set of conditions than during the second set of conditions.
  • the average glucose concentration is at least about 5 times lower in the first set of conditions than during the second set of conditions, at least about 50 times lower in the first set of conditions than during the second set of conditions, at least about 100 times lower in the first set of conditions than in the second set of conditions, or at least about 1000 times lower in the first set of conditions than in the second set of conditions.
  • the rate of butanol production is lower under the first set of conditions than under the second set of conditions.
  • the source of carbon substrate for the first set of conditions differs from that of the second set of conditions.
  • the source of the carbon substrate for the first set of conditions is molasses.
  • the source of the carbon substrate for the second set of conditions is corn mash.
  • heterologous polynucleotides comprising i) a promoter nucleic acid sequences; and ii) a nucleic acid sequence encoding suppressor tR A molecules are provided herein and may be employed in the disclosed methods.
  • heterologous polynucleotides comprising i) a promoter nucleic acid sequences; and ii) a nucleic acid sequence encoding anti-sense R A molecules are provided herein and may be employed in the disclosed methods.
  • isolated polynucleotides provided herein comprise: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding a suppressor tRNA; wherein the nucleic acid sequence of (b) is operably associated to the nucleic acid sequence of (a) such that the suppressor tRNA is differentially expressed during the production phase and the propagation phase of a fermentation process.
  • isolated polynucleotides provided herein comprise: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding an anti-sense RNA; wherein the nucleic acid sequence of (b) is operably associated to the nucleic acid sequence of (a) such that the anti-sense RNA is differentially expressed during the production phase and the propagation phase of a fermentation process.
  • differential expression of an anti-sense RNA or a suppressor tRNA results in differential expression of a biocatalyst polypeptide.
  • the expression of the biocatalyst polypeptide is higher in the production phase than in the propagation phase of fermentation.
  • the biocatalyst polypeptide catalyzes a substrate to product conversion in a butanol or 2-butanone biosynthetic pathway.
  • the biocatalyst polypeptide is selected from a group of enzymes having the following Enzyme Commission Numbers: EC 2.2.1.6, EC 1.1.1.86, EC 4.2.1.9, EC 4.1.1.72, EC 1.1.1.1, EC 1.1.1.265, EC 1.1.1.2, EC 1.2.4.4, EC 1.3.99.2, EC 1.2.1.57, EC 1.2.1.10, EC 2.6.1.66, EC 2.6.1.42, EC 1.4.1.9, EC 1.4.1.8, EC
  • the biocatalyst polypeptide catalyzes a substrate to product conversion in an isobutanol biosynthetic pathway.
  • the biocatalyst polypeptide is acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain alpha-keto acid decarboxylase, branched-chain alcohol dehydrogenase, branched-chain keto acid
  • dehydrogenase butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, transaminase, valine dehydrogenase, valine decarboxylase, omega transaminase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, isobutyryl-CoA mutase, acetolactate decarboxylase, acetonin aminase, butanol dehydrogenase, butyraldehyde dehydrogenase, acetoin kinase, acetoin phosphate aminase, aminobutanol phosphate
  • the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance.
  • the biocatalyst polypeptide is Sedl protein or Spil protein or a homolog thereof. The isolated polynucleotide of claim 28, 29, or 31 wherein the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance.
  • the biocatalyst polypeptide comprises at least about 80%, at least about 85%>, at least about 90%>, at least about 95%, at least about 99%, or at least about 100% identity to SEQ ID NO: 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, or 435.
  • a biocatalyst polypeptide may be a biosynthetic pathway polypeptide, a cell integrity polypeptide, a propagation polypeptide, a glycerol biosynthesis pathway polypeptide, by-product producing polypeptide, or an NADPH-generating polypeptide.
  • the biocatalyst polypeptide catalyzes the substrate to product conversion of pyruvate to acetolactate.
  • the biocatalyst polypeptide catalyzes the substrate to product conversion of pyruvate to acetolactate and comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% identity to SEQ ID NO: 1 or a variant or active fragment thereof.
  • the promoter nucleic acid sequence comprises at least about
  • the promoter nucleic acid sequence comprises at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 35
  • the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 775, 776, 777, or 778 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 90%>, at least about 95%, at least about 99% or 100% identity identity to SEQ ID NO: 686.
  • the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 384, 360, 386, or 331 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 772 or 773 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof.
  • the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 388, or 173 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 768 or 769. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof.
  • the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 711 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 151 or a fragment thereof.
  • the promoter nucleic acid sequence comprises at least about
  • the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177 or a fragment thereof.
  • the promoter nucleic acid sequence comprises at least about
  • the acetolactate synthase has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 1 or a variant or active fragment thereof.
  • the acetolactate synthase is B. subtilis AlsS or a variant or active fragment thereof.
  • the expression of the biocatalyst polypeptide is higher in the propagation phase than in the production phase of fermentation.
  • the biocatalyst polypeptide is a biosynthetic pathway polypeptide or a cell integrity polypeptide.
  • the biocatalyst polypeptide is a propagation polypeptide, an isobutanol pathway by-product polypeptide, a glycerol biosynthesis pathway, or a polypeptide of an NADPH generating pathway.
  • the biocatalyst polypeptide is a phosphoketolase.
  • the phosphoketolase is derived from Lactobacillus plantarum.
  • the biocatalyst polypeptide is a phosphotransacetylase. In some embodiments, the phosphotransacetylase is derived from Lactobacillus plantarum. In some embodiments, biocatalyst polypeptide is an acetolactate reductase. In some embodiments, the acetolactate reductase is YMR226C. In some embodiments, the biocatalyst polypeptide is an aldehyde dehydrogenase. In some embodiments, the biocatalyst polypeptide is ALD6. In some embodiments, the biocatalyst polypeptide is an enzyme of the oxidative pentose phosphate pathway. In some embodiments, the biocatalyst polypeptide is glucose-6-phosphate
  • biocatalyst polypeptide is glycerol 3-phosphate dehydrogenase.
  • the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95% at least about 99% or 100% identity to SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163, or a fragment thereof.
  • the nucleic acid sequence encoding a biocatalyst polypeptide is codon-optimized for expression in a specific host cell.
  • the recombinant host cells comprise biocatalyst polypeptides.
  • the host cell is a bacteria, cyanobacteria, filamentous fungi, or yeast cell.
  • the host cell is a bacterial or cyanobacterial cell.
  • the genus of the host cell is Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus,
  • the host cell is a filamentous fungi or yeast cell.
  • the genus of the host cell is Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces,
  • the host cell is Saccharomyces cerevisiae.
  • the host cell comprises reduced or eliminated expression of endogenous pyruvate decarboxylase.
  • the reduced or eliminated expression of decarboxylase is caused by gene deletion, disruption, or mutation.
  • the gene disrupted or deleted is PDCl, PDC5, PDC6, or a combination thereof.
  • the host cell comprises reduced or eliminated expression of an endogenous enzyme having aldehyde dehydrogenase activity, glycerol-3 -phosphate dehydrogenase activity, acetolactate reductase activity, or a polypeptide affecting Fe-S cluster biosynthesis.
  • the reduced or eliminated expression of an endogenous enzyme having aldehyde dehydrogenase activity, glycerol-3 -phosphate dehydrogenase activity, acetolactate reductase activity, or a polypeptide affecting Fe-S cluster biosynthesis is caused by gene deletion, disruption, or mutation.
  • recombinant yeast host cells comprising an isobutanol biosynthetic pathway and at least one modified expression construct that differentially expresses a polypeptide under conditions in which propagation of biomass is favored over production of isobutanol whereby isobutanol production under the conditions is substantially reduced as compared to a host cell without the modified expression construct under the same conditions.
  • the modified expression construct comprises SEQ ID NO: 711.
  • the modified expression construct comprises a polynucleotide encoding a polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate.
  • the polynucleotide encoding a polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 2.
  • the polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100%) identity to SEQ ID NO: 1.
  • the modified expression construct comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%) identity to SEQ ID NO: 790.
  • the yeast host cell is Saccharomyces cerevisiae.
  • Also disclosed herein are methods for the production of a fermentation product comprising: (a) providing a disclosed recombinant host cell; (b) contacting the host cell with fermentable carbon substrate in a fermentation medium under conditions whereby the
  • fermentation product is produced; and (c) optionally, recovering the fermentation product.
  • the methods further comprise propagating the host cell under conditions whereby the host cell propagates prior to the contacting of (b).
  • the conditions whereby the fermentation product are produced are anaerobic.
  • the conditions whereby the fermentation product are produced are microaerobic.
  • the fermentation product is selected from the group consisting of: butanol, 2-butanone, propanol, isopropanol, and ethanol.
  • the fermentation product is butanol or 2-butanone.
  • the fermentation product is isobutanol.
  • the fermentation product is isobutanol and the isolated polynucleotide comprises the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177 or a fragment thereof and the biocatalyst polypeptide is acetolactate synthase or ketol-acid reductoisomerase .
  • uncoupling growth and production occurs not necessarily from differential expression, but from changing conditions or activity of a cell pathway, protein, or other component by the addition or deletion of another component.
  • the addition, deletion or action of the other component may be combined with differential expression or may result in control over growth and/or production independent of differential expression.
  • Figure 1 depicts different isobutanol biosynthetic pathways. The steps labeled
  • a may be catalyzed, for example, by acetolactate synthase
  • b may be catalyzed, for example, by acetohydroxyacid reductoisomerase.
  • c may be catalyzed, for example, by acetohydroxy acid dehydratase
  • d may be catalyzed, for example, by branched- chain keto acid decarboxylase
  • e may be catalyzed, for example, by branched chain alcohol dehydrogenase
  • f ' may be catalyzed, for example, by branched chain keto acid dehydrogenase
  • g may be catalyzed, for example, by acylating aldehyde dehydrogenase
  • h may be catalyzed, for example, by transaminase or valine dehydrogenase
  • i may be catalyzed, for example, by valine decarboxylase
  • j may be catalyzed, for example, by omega transaminase.
  • Figure 2 depicts a genetic switch in which a suppressor tRNA is produced in response to a process or environmental cue, and wherein the suppressor tRNA rescues the translation of a pathway polypeptide containing a nonsense or a frameshift allele.
  • Figure 3 depicts a genetic switch in which an anti-sense RNA is produced in response to a process or environmental cue, and wherein the anti-sense RNA blocks the translation of a pathway polypeptide.
  • Figure 4 depicts a genetic switch in which a suppressor tRNA is produced in response to a process or environmental cue, and wherein the suppressor tRNA rescues the translation of a propagation polypeptide containing by a nonsense or a frameshift allele.
  • Figure 5 depicts a genetic switch in which an anti-sense RNA is produced in response to a process or environmental cue, and wherein the anti-sense RNA blocks the translation of a propagation polypeptide.
  • Figure 6 depicts a genetic switch in which the production of a suppressor tR A is blocked in response to a process or environmental cue, and wherein the translation of a pathway polypeptide containing by a nonsense or a frameshift allele is blocked due to the absence of the suppressor tRNA.
  • Figure 7 depicts a genetic switch in which the production of an anti-sense RNA is blocked in response to a process or environmental cue, and wherein the lack of production of the anti-sense RNA results in the translation of a pathway polypeptide.
  • Figure 8 depicts a genetic switch in which the production of a suppressor tRNA is blocked in response to a process or environmental cue, and wherein the translation of a propagation polypeptide containing by a nonsense or a frameshift allele is blocked due to the absence of the suppressor tRNA.
  • Figure 9 depicts a genetic switch in which the production of an anti-sense RNA is blocked in response to a process or environmental cue, and wherein the lack of production of the anti-sense RNA results in the translation of a propagation polypeptide.
  • Figure 10 depicts that isobutanol is synthesized at 26 and 37 hrs and that by 50 hrs of cell culture, isobutanol accumulation ceases.
  • Figure 11 depicts rate of isobutanol production, in g/L/hr, over fermentation time
  • Figure 12 depicts carbon dioxide evolution rate over fermentation time.
  • Figure 13 depicts oxygen consumption over fermentation time.
  • Industrial fermentation processes with yeast may employ a stage of biomass production in order to provide sufficient biocatalyst for the fermentation stage to have desired yield and production rate.
  • Ethanologen S. cerevisiae for example is typically propagated using fed-batch technology, in which low sugar concentrations and non-limiting aeration favor respiratory metabolism with high biomass yields, e.g., Y xs ⁇ 0.5 g biomass/g glucose.
  • the maintenance of low sugar concentrations in a fed-batch regime may be particularly important for a Crabtree-positive yeast like S. cerevisiae, in which the fraction of respiratory metabolism on overall metabolism is negatively correlated with increasing extracellular glucose concentrations.
  • the butanol produced may inhibit growth during the propagation phase of a production process and may add cost and inefficiency to either or both the
  • Applicants have addressed the stated problems by identifying various promoter nucleic acid sequences which provide differential expression of a suppressor tR A or an anti- sense R A specific for a gene of interest, and, as a result, differential expression of genes of interest under different conditions, thus providing a strategy for differential expression during biocatalyst propagation and fermentation product production phases.
  • recombinant host cells that produce fermentation products and comprise promoter sequences that provide differential expression in the propagation vs. production phases of a process, as well as methods for using the same. Promoters that can be used to provide such differential expression, and means of identifying such promoters, are described in Int'l Appl. No. PCT/US2012/072186, which is incorporated herein by reference in its entirety.
  • nucleic acid sequences to govern the differential expression of genes of interest during biocatalyst propagation and fermentation product production phases. Furthermore, hybrid sequences comprising nucleic acid sequences derived from more than one promoter region are provided.
  • nucleic acid sequences described herein may be employed as promoters for the differential expression of various polypeptides relevant to propagation and/or production and are hereinafter referred to from time to time as "genetic switches.”
  • the sequences and methods disclosed herein thus allow (i) biocatalyst polypeptides such as polypeptides which catalyze the substrate to product conversions of a biosynthetic pathway such as a butanol or 2-butanone biosynthetic pathway to be preferentially expressed during the production phase of fermentation through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, (ii) biocatalyst polypeptides beneficial for cell integrity to be preferentially expressed during the production phase of fermentation through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, (iii) biocatalys
  • recombinant host cells described herein produce butanol or 2- butanone from plant derived carbon sources. Accordingly, provided herein are methods for the production of butanol or 2-butanone using recombinant host cells comprising isolated
  • polynucleotides comprising promoter nucleic acid sequences controlling suppressor tRNAs and/or anti-sense RNAs that differentially regulate the expression of associated genes during the propagation and production phases of a fermentation process.
  • a polypeptide catalyzing the first step in a butanol biosynthetic pathway can be preferentially expressed during the production phase.
  • a polypeptide catalyzing a substrate to product conversion in an isobutanol biosynthetic pathway can be preferentially expressed during the production phase.
  • acetolactate synthase can be preferentially expressed during the production phase.
  • ketol-acid reductoisomerase can be
  • composition, mixture, process, method, article, or apparatus refers to an inclusive or and not to exclusive or.
  • "or” refers to an inclusive or and not to exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • invention or "present invention” is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.
  • the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like.
  • the term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
  • the term “about” means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.
  • suppressor tRNA refers to a tRNA molecule with a mutation in a gene encoding a tRNA that suppresses mutations in polynucleotides encoding missense, nonsense, or frameshift alleles. See, e.g., Tucker, S.D., et ah, Biochimie 71(6): 729-39 (1989).
  • the suppressor tRNA suppresses the effect of a mutation on the coding sequence, allowing transcription to proceed.
  • a tRNA that recognizes the codon "UAC” in the mRNA and inserts tyrosine into the growing polypeptide chain is encoded by the wild-type tyrT gene.
  • a mutation in the tyrT gene can alter the specificity of the tRNA from recognizing "UAC” to recognizing the stop codon "UAG", and therefore, instead of terminating the transcript when the stop codon is reached, the suppressor tRNA inserts a tryrosine in the polypeptide chain at the position of the stop codon.
  • suppressor tRNA molecules include those that suppress the UAG stop codon (Amber suppressors), the UGA stop codon (Opal suppressors), the UAA stop codon (Ochre suppressors), and frameshift suppressors (that recognize a codon of a different number or base pairs than the usual three base pairs, e.g., a codon of 4 base pairs).
  • anti-sense RNA refers to a single-stranded RNA molecule that is complementary to a messenger RNA strand that is transcribed within a cell. Anti-sense RNA can be introduced into a cell, where it can inhibit translation of its complementary mRNA strand by binding to the complementary mRNA and blocking the translation machinery.
  • nonsense mutation refers to a point mutation in a DNA sequence that creates a premature stop codon (a nonsense codon in the transcribed mRNA) in an open reading frame, and results in a truncation of the transcribed amino acid sequence.
  • Non-limiting examples of nonsense mutations include the UAG stop codon, the UGA stop codon, and the UAA stop codon.
  • missense mutation refers to a point mutation in a DNA sequence that changes the amino acid specified for the corresponding position in the polypeptide encoded by a gene and its mRNA.
  • the term "frameshift mutation” is a mutation that is caused by insertions or deletions of one or more nucleotides in a DNA sequence, wherein the insertion or deletion is not evenly divisible by three. The insertion or deletion therefore changes how the codons are grouped, and results in a completely different translation from the original sequence.
  • the term "environmental cue” or “process cue” refers to any change in cellular environmental conditions, including, but not limited to, changes in fermentable carbon substrates, oxygen levels, glucose levels, pH, temperature, and butanol levels.
  • the environmental cue or process cue activates a transcriptional activator for a promoter described herein.
  • the environmental or process cue activates a transcriptional repressor for a promoter described herein.
  • transcriptional activator refers to a protein that increases the transcription of one or more genes.
  • transcriptional repressor refers to a protein that prevents the binding of
  • the transcriptional repressor is a sulfonylurea-responsive repressor as described in U.S. Pat. No. 8,257,956, which is incorprated herein by reference in its entirety.
  • the transcriptional repressor is a Tet (tetracycline) repressor, as described in U.S. Pat. No. 8,257,956, which is incorprated herein by reference in its entirety.
  • growth phase or “propagation phase” refers to the process steps during which yeast biomass is produced and inoculum build-up occurs.
  • production phase refers to the fermentation process steps during which a desired fermentation product, including, but not limited to butanol, isobutanol, 1-butanol, 2- butanol and/or 2-butanone production, occurs.
  • biomass refers to the cell biomass of the fermentation product-producing microorganism, typically provided in units g/L dry cell weight (dew).
  • the term "fermentation product” includes any desired product of interest, including lower alkyl alcohols including, but not limited to butanol, lactic acid, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, 1,3-propane-diol, ethylene, glycerol, isobutyrate, etc.
  • lower alkyl alcohol refers to any straight-chain or branched, saturated or unsaturated, alcohol molecule with 1-10 carbon atoms.
  • butanol refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.
  • Isobutanol is also known as 2-methyl-l-propanol.
  • butanol biosynthetic pathway refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.
  • isobutanol biosynthetic pathways are disclosed in U.S. Patent No. 7,851,188, which is incorporated by reference herein.
  • Components of the pathways consist of all substrates, cofactors, byproducts, intermediates, end- products, and enzymes in the pathways.
  • 2-butanone biosynthetic pathway refers to an enzyme pathway to produce 2-butanone.
  • propagation polypeptide includes polypeptides associated with the production of biomass, and polypeptides associated with the performance of an enzyme that is associated with the production of biomass.
  • biocatalyst polypeptide includes polypeptides associated with the substrate to product conversions of an indicated biosynthetic pathway, for example a butanol or 2-butanone biosynthetic pathway, and polypeptides associated with the propagation or performance of a biocatalyst that is associated with the indicated biosynthetic pathway, including, but not limited to, cell integrity polypeptides and propagation polypeptides.
  • a polypeptide that is a part of an NADPH generating pathway or a polypeptide that is part of a non-butanol NADH consuming product pathway may be biocatalyst polypeptides.
  • biosynthetic pathway polypeptide includes polypeptides that catalyze substrate to product conversions of a recited biosynthetic pathway.
  • cell integrity polypeptide includes polypeptides involved in cell integrity, including polypeptides required for constituting the cellular architecture.
  • a "recombinant microbial host cell” is defined as a host cell that has been genetically manipulated.
  • recombinant microbial host cells have been genetically manipulated to express a biosynthetic production pathway, wherein the host cell either produces a biosynthetic product in greater quantities relative to an unmodified host cell or produces a biosynthetic product that is not ordinarily produced by an unmodified host cell.
  • fermentable carbon substrate refers to a carbon source capable of being metabolized by the microorganisms such as those disclosed herein.
  • suitable fermentable carbon substrates include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch, cellulose, or lignocellulose, hemicellulose; one-carbon substrates, fatty acids; and a combination of these.
  • “Fermentation medium” means the mixture of water, sugars (fermentable carbon substrates), dissolved solids, microorganisms producing fermentation products, fermentation product and all other constituents of the material held in the fermentation vessel in which the fermentation product is being made by the reaction of fermentable carbon substrates to fermentation products, water and carbon dioxide (C0 2 ) by the microorganisms present. From time to time, as used herein the term “fermentation broth” and “fermentation mixture” can be used synonymously with “fermentation medium.”
  • microaerobic conditions means conditions with low levels of dissolved oxygen.
  • the oxygen level may be less than about 1% of air-saturation.
  • anaerobic conditions means conditions in the absence of oxygen. It will be understood that in many fermentation processes, an initial amount of oxygen is present at the onset of the process, but such oxygen is depleted over the course of the fermentation such that the majority of the process takes place in the absence of detectable oxygen.
  • yield refers to the amount of product per amount of carbon source in g/g.
  • the yield may be exemplified for glucose as the carbon source. It is understood unless otherwise noted that yield is expressed as a percentage of the theoretical yield.
  • theoretical yield is defined as the maximum amount of product that can be generated per total amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isopropanol is 0.33 g/g. As such, a yield of isopropanol from glucose of 0.297 g/g would be expressed as 90% of theoretical or 90% theoretical yield. It is understood that while in the present disclosure the yield is exemplified for glucose as a carbon source, the invention can be applied to other carbon sources and the yield may vary depending on the carbon source used. One skilled in the art can calculate yields on various carbon sources.
  • acetohydroxyacid synthase acetolactate synthase
  • acetolactate synthetase acetolactate synthetase
  • Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB07802.1 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), CAB15618 (SEQ ID NO: 789), Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:3), M73842 (SEQ ID NO:4)), and
  • Lactococcus lactis (GenBank Nos: AAA25161 (SEQ ID NO:5), L16975 (SEQ ID NO:6)).
  • KARI ketol-acid reductoisomerase
  • acetohydroxy acid isomeroreductase will be used interchangeably and refer to enzymes capable of catalyzing the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate.
  • Example KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP 418222 (SEQ ID NO: 7), NC 000913 (SEQ ID NO: 8)),
  • KARIs include Anaerostipes caccae KARI variants "K9G9", “K9D3", and "K9JB4P" (SEQ ID NOs: 167, 166, and 791 respectively). In some embodiments, KARI utilizes NADH. In some embodiments, KARI utilizes NADPH.
  • DHAD acetohydroxy acid dehydratases
  • E. coli GenBank Nos: YP 026248 (SEQ ID NO: 15), NC 000913 (SEQ ID NO: 16)), S. cerevisiae (GenBank Nos: NP_012550 (SEQ ID NO: 17), NC 001142 (SEQ ID NO: 18)), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO: 19), BX957219 (SEQ ID NO: 20)), B. subtilis
  • DHADs include S. mutans variant "12 V5" (SEQ ID NO: 792)
  • KIVD a-ketoiso valerate decarboxylase or “2-ketoisovalerate decarboxylase”
  • Example branched-chain a-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166 (SEQ ID NO: 23), AY548760 (SEQ ID NO: 24); CAG34226 (SEQ ID NO: 25), AJ746364 (SEQ ID NO: 26), Salmonella typhimurium (GenBank Nos: NP_461346 (SEQ ID NO: 27), NC_003197 (SEQ ID NO: 28)), Clostridium acetobutylicum (GenBank Nos: NPJ49189 (SEQ ID NO: 29), NC_001988 (SEQ ID NO: 30)), M.
  • Lactococcus lactis GenBank Nos: AAS49166 (SEQ ID NO: 23), AY548760 (SEQ ID NO: 24
  • CAG34226 SEQ ID NO: 25
  • ADH alcohol dehydrogenase
  • Alcohol dehydrogenases may be "branched chain alcohol dehydrogenases” or may be referred to as “butanol dehydrogenases.”
  • Example alcohol dehydrogenases suitable for embodiments disclosed herein may be known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases, for example, according to published preference for NADH (typically 1.1.1.1) or NADPH (typically 1.1.1.2) as cofactors.
  • NADH typically 1.1.1.1
  • NADPH typically 1.1.1.2
  • Such enzymes are available from a number of sources, including, but not limited to, S.
  • NC 000913 SEQ ID NO: 36
  • C. acetobutylicum GenBank Nos: NP 349892 (SEQ ID NO: 37), NC 003030 (SEQ ID NO: 38); NP 349891 (SEQ ID NO: 39), NC 003030 (SEQ ID NO: 40)
  • Pyrococcus furiosus GenBank Nos: AAC25556, AF013169
  • Acinetobacter sp. GenBank Nos: AAG10026, AF282240
  • Rhodococcus ruber GenBank Nos: CAD36475, AJ491307)
  • A. xylosoxidans SEQ ID NO: 798
  • horse liver ADH SEQ ID NO: 799
  • Butanol dehydrogenase enzymes are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307). Additional NADP dependent enzymes are known, and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169). Additionally, a butanol dehydrogenase is available from Escherichia coli (GenBank Nos: NP 417484, NC_000913) and a cyclohexanol dehydrogenase is available from Acinetobacter sp. (GenBank Nos: AAG10026, AF282240).
  • Butanol dehydrogenases are additionally available from, for example, C. acetobutylicum (GenBank NOs: NP_149325, NC_001988; note: this enzyme possesses both aldehyde and alcohol dehydrogenase activity); NP 349891, NC 003030; and NP_349892, NC_003030) and E. coli (GenBank NOs: NP_417484, NC_000913).
  • branched-chain keto acid dehydrogenase refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), typically using NAD + (nicotinamide adenine dinucleotide) as an electron acceptor.
  • Example branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. Such branched- chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B.
  • subtilis GenBank Nos: CAB14336 (SEQ ID NO: 41), Z99116 (SEQ ID NO: 42); CAB14335 (SEQ ID NO: 43), Z99116 (SEQ ID NO: 44); CAB14334 (SEQ ID NO: 45), Z99116 (SEQ ID NO: 46); and CAB14337 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48)) and Pseudomonas putida
  • acylating aldehyde dehydrogenase refers to an enzyme that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using either NADH or NADPH as an electron donor.
  • Example acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO: 57), AF157306 (SEQ ID NO: 58)), C. acetobutylicum (GenBank Nos: NPJ49325 (SEQ ID NO: 59),
  • NC 001988 (SEQ ID NO: 60); NP 149199 (SEQ ID NO: 61), NC 001988 (SEQ ID NO: 62)), P. putida (GenBank Nos: AAA89106 (SEQ ID NO: 63), U13232 (SEQ ID NO: 64)), and Thermus thermophilus (GenBank Nos: YP 145486 (SEQ ID NO: 65), NC 006461 (SEQ ID NO: 66)).
  • transaminase refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, using either alanine or glutamate as an amine donor.
  • Example transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, E.
  • GenBank Nos: YP 026231 (SEQ ID NO: 67), NC 000913 (SEQ ID NO: 68)) and Bacillus licheniformis (GenBank Nos: YP_093743 (SEQ ID NO: 69), NC_006322 (SEQ ID NO: 70)).
  • sources for glutamate-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026247 (SEQ ID NO: 71), NC 000913 (SEQ ID NO: 72)), S. cerevisiae (GenBank Nos: NP_012682 (SEQ ID NO: 73), NC_001142 (SEQ ID NO:
  • NC 000916 SEQ ID NO: 76
  • valine dehydrogenase refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor.
  • Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP_628270 (SEQ ID NO: 77), NC_003888 (SEQ ID NO: 78)) and B.
  • valine decarboxylase refers to an enzyme that catalyzes the conversion of L-valine to isobutylamine and C0 2 .
  • Example valine decarboxylases are known by the EC number 4.1.1.14. Such enzymes are found in Streptomyces, such as for example, Streptomyces viridifaciens (GenBank Nos: AAN10242 (SEQ ID NO: 81), AY116644 (SEQ ID NO: 82)).
  • omega transaminases refers to an enzyme that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor.
  • Example omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 83), AY330220 (SEQ ID NO: 84)), Ralstonia eutropha (GenBank Nos: YP_294474 (SEQ ID NO: 85), NC_007347 (SEQ ID NO: 86)), Shewanella oneidensis (GenBank Nos: NP_719046 (SEQ ID NO: 87), NC_004347 (SEQ ID NO: 88)), and P. putida (GenBank Nos: AAN66223 (SEQ ID NO: 89), AE016776
  • acetyl-CoA acetyltransferase refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (Co A).
  • Example acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well.
  • Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli
  • Saccharomyces cerevisiae GenBank Nos: NP_015297 (SEQ ID NO: 99), NC_001148 (SEQ ID NO: 100)).
  • 3-hydroxybutyryl-CoA dehydrogenase refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA.
  • hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide (NADH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3- hydroxybutyryl-CoA. Examples may be classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with a substrate preference for (S)-3- hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C.
  • 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP 349314 (SEQ ID NO: 101), NC 003030 (SEQ ID NO: 102)), B.
  • subtilis GenBank NOs: AAB09614 (SEQ ID NO: 103), U29084 (SEQ ID NO: 104)), Ralstonia eutropha (GenBank NOs: YP 294481 (SEQ ID NO: 105), NC_007347 (SEQ ID NO: 106)), and Alcaligenes eutrophus (GenBank NOs: AAA21973 (SEQ ID NO: 107), J04987 (SEQ ID NO: 108)).
  • crotonase refers to an enzyme that catalyzes the conversion of 3- hydroxybutyryl-CoA to crotonyl-CoA and H 2 0.
  • Example crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and may be classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively.
  • Crotonases are available from a number of sources, for example, E. coli (GenBank NOs: NP 415911 (SEQ ID NO: 109), NC 000913 (SEQ ID NO: 110)), C.
  • acetobutylicum GenBank NOs: NP 349318 (SEQ ID NO: 111), NC 003030 (SEQ ID NO: 112)
  • B. subtilis GenBank NOs: CAB13705 (SEQ ID NO: 113), Z99113 (SEQ ID NO: 114)
  • Aeromonas caviae GenBank NOs: BAA21816 (SEQ ID NO: 115), D88825 (SEQ ID NO: 116)
  • butyryl-CoA dehydrogenase refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA.
  • Example butyryl-CoA dehydrogenases may be NADH-dependent, NADPH-dependent, or flavin-dependent and may be classified as E.C.
  • Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP 347102 (SEQ ID NO: 117), NC_ 003030 (SEQ ID NO: 118))), Euglena gracilis (GenBank NOs:
  • isobutyryl-CoA mutase refers to an enzyme that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme Bi 2 as cofactor.
  • Example isobutyryl-CoA mutases are known by the EC number 5.4.99.13.
  • Streptomyces cinnamonensis GenBank Nos: AAC08713 (SEQ ID NO: 125), U67612 (SEQ ID NO: 126); CAB59633 (SEQ ID NO: 127), AJ246005 (SEQ ID NO: 128)), S coelicolor (GenBank Nos: CAB70645 (SEQ ID NO: 129), AL939123 (SEQ ID NO: 130); CAB92663 (SEQ ID NO: 131), AL939121 (SEQ ID NO: 132)), and Streptomyces avermitilis (GenBank Nos: NP_824008 (SEQ ID NO: 133), NC 003155 (SEQ ID NO: 134); NP 824637 (SEQ ID NO: 135), NC 003155 (SEQ ID NO: 136)).
  • butyraldehyde dehydrogenase refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as co factor.
  • Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841 , AF157306) and C. acetobutylicum (GenBank NOs: NPJ49325, NC_001988).
  • acetolactate decarboxylase refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin.
  • Example acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774, AY722056).
  • acetoin aminase or "acetoin transaminase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 3-amino-2- butanol.
  • Acetoin aminase may utilize the cofactor pyridoxal 5'-phosphate or NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate).
  • NADH reduced nicotinamide adenine dinucleotide
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • the resulting product may have (R) or (S) stereochemistry at the 3 -position.
  • the pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor.
  • the NADH- and NADPH-dependent enzymes may use ammonia as a second substrate.
  • a suitable example of an NADH dependent acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688).
  • An example of a pyridoxal-dependent acetoin aminase is the amine :pyruvate aminotransferase (also called amine :pyruvate transaminase) described by Shin and Kim, J. Org. Chem. 67:2848-2853 (2002)).
  • acetoin kinase refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin.
  • Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymes that catalyze the analogous reaction on the similar substrate
  • acetoin phosphate aminase refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of phosphoacetoin to 3-amino-2- butanol O-phosphate.
  • Acetoin phosphate aminase may use the cofactor pyridoxal 5 '-phosphate, NADH or NADPH.
  • the resulting product may have (R) or (S) stereochemistry at the 3-position.
  • the pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate.
  • the NADH and NADPH-dependent enzymes may use ammonia as a second substrate.
  • a pyridoxal phosphate-dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta et ah, Appl. Environ. Microbial. (57:4999-5009 (2001)).
  • aminobutanol phosphate phospho lyase also called “amino alcohol O- phosphate lyase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3 -amino-2 -butanol O-phosphate to 2-butanone.
  • Amino butanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5'-phosphate.
  • enzymes that catalyze the analogous reaction on the similar substrate l-amino-2-propanol phosphate (Jones et al., Biochem J. 754: 167-182 (1973)).
  • U.S. Appl. Pub. No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.
  • aminobutanol kinase refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3-amino-2 -butanol to 3-amino-2butanol O- phosphate.
  • Amino butanol kinase may utilize ATP as the phosphate donor.
  • enzymes catalyzing this reaction on 3-amino-2-butanol there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and l-amino-2- propanol (Jones et al, supra).
  • U.S. Appl. Pub. No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.
  • butanediol dehydrogenase and “acetoin reductase” refer to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol.
  • Butanedial dehydrogenases are a subset of the broad family of alcohol dehydrogenases.
  • Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product.
  • (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085, D86412).
  • (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP 830481, NC_004722;
  • butanediol dehydratase dial dehydratase or "propanediol dehydratase” refer to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone.
  • Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B12; although vitamin B12 may refer also to other forms of cobalamin that are not coenzyme B12).
  • Adenosyl cobalamin- dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (Note all three subunits are required for activity)], and Klebsiella pneumonia (GenBank Nos: AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064).
  • dial dehydratases include, but are not limited to, B12-dependent dial dehydratases available from Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza et al, J.
  • pyruvate decarboxylase refers to an enzyme that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenases are known by the EC number 4.1.1.1. These enzymes are found in a number of yeast, including Saccharomyces cerevisiae (GenBank Nos: CAA97575 (SEQ ID NO: 137), CAA97705 (SEQ ID NO: 138), CAA97091 (SEQ ID NO: 139)).
  • phosphoketolase refers to an enzyme that catalyzes the conversion of xyulose 5 -phosphate to glyceraldehyde 3 -phosphate and acetyl phosphate.
  • phosphoketolases are known by the EC number 4.1.2.9. In some embodiments, the
  • phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181).
  • phosphotransacetylase refers to an enzyme that catalyzes the conversion of acetyl-CoA and phosphate to CoA and acetyl phosphate.
  • the phosphotransacetylases are known by the EC number 2.3.1.8.
  • the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 178; amino acid SEQ ID NO: 179).
  • polypeptide is intended to encompass a singular "polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” may be used instead of, or
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known
  • polypeptides provided herein including, but not limited to biosynthetic pathway polypeptides, cell integrity polypeptides, propagation polypeptides, and other enzymes comprise full-length polypeptides and active fragments thereof.
  • a polypeptide of the invention may be of a size of about 10 or more, 20 or more,
  • Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three- dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • polypeptides of the present invention are derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
  • active variant refers to polypeptides of the present invention and include any polypeptides that are capable of catalyzing the indicated substrate to product conversion.
  • variants of polypeptides of the present invention include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions and/or additions.
  • Derivatives of polypeptides of the present invention are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.
  • variant polypeptides may also be referred to herein as "polypeptide analogs.”
  • a “derivative" of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • a "fragment” is a unique portion of polypeptide used in the invention which is identical in sequence to but shorter in length than the parent full-length sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous amino acid residues.
  • a fragment may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 100 or 200 amino acids of a polypeptide as shown in a certain defined sequence.
  • active fragment when used in reference to a polypeptide, is a portion of a polypeptide which retains the functionality of the subject polypeptide, but comprises less than the entire sequence of the polypeptide.
  • recombinant variants encoding these same or similar polypeptides can be synthesized or selected by making use of the "redundancy" in the genetic code.
  • Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a host cell system.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they can be result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements.
  • Constant amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • "non-conservative" amino acid substitutions can be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids.
  • “Insertions” or “deletions” are preferably in the range of about 1 to about 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
  • Polypeptides suitable for use in the present invention and fragments thereof are encoded by polynucleotides.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA virally-derived RNA
  • pDNA plasmid DNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond ⁇ e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • Polynucleotides according to the present invention further include such molecules produced synthetically.
  • Polynucleotides of the invention may be native to the host cell or heterologous.
  • a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • a polynucleotide or polypeptide sequence can be referred to as "isolated,” in which it has been placed in an environment other than its native environment or is produced synthetically or is a non-naturally occurring, or engineered, sequence.
  • isolated in which it has been placed in an environment other than its native environment or is produced synthetically or is a non-naturally occurring, or engineered, sequence.
  • heterologous polynucleotide encoding a polypeptide or polypeptide fragment having enzymatic activity (e.g., the ability to convert a substrate to xylulose) contained in a vector is considered isolated for the purposes of the present invention.
  • Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • An isolated polynucleotide fragment in the form of a polymer of DNA can be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
  • gene refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5' non- coding sequences) and following (3' non-coding sequences) the coding sequence.
  • a "coding region” or “ORF” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' non-translated regions, and the like, are not part of a coding region.
  • Suitable regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem- loop structures.
  • promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.
  • Constitutive promoters Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.”
  • Example constitutive promoters are provided herein and/or are known to those of skill in the art.
  • Constitutive promoters include, but are not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1.
  • Other constitutive promoters are known in the art It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • fragments of different lengths of the sequences provided may have identical promoter activity, thus, reference to, for example "HEM13 promoter”, will be understood to encompass a sequence provided herein or any fragment of the promoter region of the HEM 13 gene which has identical promoter activity or a substantially similar effect on expression of a target polypeptide or production of an indicated product.
  • HEM13 promoter will be understood to encompass a sequence provided herein or any fragment of the promoter region of the HEM 13 gene which has identical promoter activity or a substantially similar effect on expression of a target polypeptide or production of an indicated product.
  • the disclosed nucleic acid sequences should not be construed as limited solely to the provided sequence.
  • the polynucleotide or nucleic acid is DNA.
  • DNA In the case of
  • DNA a polynucleotide comprising a nucleic acid, which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments are "operably associated” or “operably linked” or “coupled” if induction of promoter function results in the transcription of mRNA and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the
  • An "expression construct”, as used herein, comprises a promoter nucleic acid sequence operably linked to a coding region for a polypeptide and, optionally, a terminator nucleic acid sequence.
  • RNA for example, in the form of messenger RNA (mRNA).
  • R A of the present invention may be single stranded or double stranded.
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance.
  • Host organisms containing the transformed nucleic acid fragments are referred to as "recombinant" or
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Differentially expressed” refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene depending on the environment of the host cell.
  • differentially expressed gene may be overexpressed or underexpressed as compared to the expression level under other conditions. In one aspect, it refers to a differential that is 1, 2, 3, 4, 5, 10 , or 20 times higher or lower than the expression level detected in a reference environment.
  • the term "differentially expressed” also refers to nucleotide sequences in a cell which are expressed where silent or not expressed in a control environment or not expressed where expressed in a control cell.
  • Plasmid and vector refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.
  • Expression cassette refers to a specific construct containing a gene and having elements in addition to the gene that allow for expression of that gene.
  • “Native” refers to the form of a polynucleotide, gene, or polypeptide as found in nature with its own regulatory sequences, if present.
  • Endogenous refers to the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism.
  • polynucleotide includes a native polynucleotide in its natural location in the genome of an organism.
  • Endogenous gene includes a native gene in its natural location in the genome of an organism.
  • Endogenous polypeptide includes a native polypeptide in its natural location in the organism.
  • Heterologous refers to a polynucleotide, gene, or polypeptide not normally found in the host organism but that is introduced into the host organism.
  • Heterologous polynucleotide may include a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native polynucleotide.
  • Heterologous gene includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene.
  • heterologous gene can include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host.
  • Heterologous polypeptide includes a native polypeptide that is reintroduced into the source organism in a form that is different from the corresponding native polypeptide.
  • nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence of the present invention can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et ah, Comp. Appl. Biosci. (5:237-245 (1990). In a sequence alignment the query and subject sequences are both DNA sequences.
  • R A sequence can be compared by converting U's to T's.
  • the result of the global sequence alignment is in percent identity.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10%> is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5 ' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • Polypeptides used in the invention are encoded by nucleic acid sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences described elsewhere in the specification, including active variants, fragments or derivatives thereof.
  • active variant refers to polynucleotides of the present invention and include any polynucleotides that encode biocatalyst polypeptides used in the invention that retain their respective enzymatic activities or structure.
  • Variants of polynucleotides of the present invention include polynucleotides with altered nucleotide sequences due to base pair substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • Derivatives of polynucleotides of the present invention are polynucleotides which have been altered so that the polypeptides they encode exhibit additional features not found on the native polypeptide. Examples include
  • polynucleotides that encode fusion proteins.
  • Variant polynucleotides may also be referred to herein as "polynucleotide analogs.”
  • a "derivative" of a polynucleotide refers to a subject polynucleotide having one or more nucleotides chemically derivatized by reaction of a functional side group. Also included as “derivatives" are those polynucleotides which contain one or more naturally occurring nucleotide derivatives. For example, 3-methylcytidine may be substituted for cytosine; ribothymidine may be substituted for thymidine; and N4-acetylcytidine may be substituted for cytosine.
  • a "fragment" when used in reference to a promoter sequence is a unique portion of the promoter nucleic acid sequence or the nucleic acid sequence encoding the biocatalyst polypeptide used in the invention which is identical in sequence to but shorter in length than the parent nucleic acid sequence.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides.
  • a fragment used as a probe, primer, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide or amino acid.
  • Fragments may be preferentially selected from certain regions of a molecule.
  • a polynucleotide fragment may comprise a certain length of contiguous nucleotides selected from the first 100 or 200 nucleotides of a polynucleotide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • codon degeneracy refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide.
  • the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • codon optimized coding region means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
  • Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code” which shows which codons encode which amino acids is reproduced herein as Table A. As a result, many amino acids are designated by more than one codon.
  • amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet.
  • This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.
  • Codon preference or codon bias differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at http://www.kazusa.or.jp/codon/ (visited March 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 February 2002], are reproduced below as Table B.
  • Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly.
  • various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from
  • Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as "synthetic gene designer"
  • a gene encoding a biocatalyst polypeptide contains a frameshift mutation, such that the polypeptide is not properly translated in the absence of a suppressor tRNA molecule.
  • a gene encoding a biocatalyst polypeptide contains a nonsense mutation, such that the polypeptide is not fully translated in the absence of a suppressor tRNA molecule.
  • the nonsense mutation in the gene coding the biocatalyst polypeptide is the the UAG stop codon, the UGA stop codon, and/or the UAA stop codon.
  • the promoter activity is sensitive to one or more physiochemical differences between propagation and production stages of fermentation. In embodiments, the promoter activity is sensitive to the dissolved oxygen concentration. In embodiments, the promoter activity is sensitive to the glucose concentration. In some embodiments, the promoter activity is sensitive to the dissolved oxygen concentration. In embodiments, the promoter activity is sensitive to the glucose concentration.
  • the promoter activity is sensitive to the source of the fermentable carbon substrate. In some embodiments, the promoter activity is sensitive to the concentration of butanol in fermentation medium. In some embodiments, the promoter activity is sensitive to the pH in the fermentation medium. In some embodiments, the promoter activity is sensitive to the
  • the promoter activity provides for differential expression in propagation and production stages of fermentation. Production and Propagation
  • Promoter nucleic acid sequences useful in the invention include those identified using "promoter prospecting" described and exemplified herein including those that comprise nucleic acid sequences which are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequences of SEQ ID NOs: 141-163, including variants, fragments or derivatives thereof that confer or increase sensitivity to fermentation conditions, such as, the concentration of oxygen, butanol, isobutyraldehyde, isobutyric acid, acetic acid, or a fermentable carbon substrate in the fermentation medium.
  • a subset of these suitable promoter nucleic acid sequences is given in Tables 1 and 2 below.
  • the promoter nucleic acid sequence is sensitive to the concentration of fermentable carbon substrates in fermentation medium.
  • the promoter nucleic acid sequence is sensitive to the concentration of a fermentable carbon substrate selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides, fatty acids, and mixtures thereof.
  • promoter nucleic acid sequences suitable for use in the invention comprise nucleotide sequences that are at least about 80%, 85%, 90%, 95%, 96%o, 97%o, 98%), 99%o or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 140 [IMA1/YGR287], [HXK1], SEQ ID NO: 141 [HXK2], SEQ ID NO: 142
  • promoter nucleic acid sequences suitable for use in the invention are selected from the group consisting of: SEQ ID NO: 140 [IMA1/YGR287], [HXK1], SEQ ID NO: 141 [HXK2], SEQ ID NO: 142 [SLT2], SEQ ID NO: 143 [YHR210c], SEQ ID NO: 144 [YJL171c], SEQ ID NO: 145 [PUNl], SEQ ID NO: 146 [PRE8], SEQ ID NO: 147 [COS3], SEQ ID NO: 148 [DIAl], SEQ ID NO: 149 [YNR062C], SEQ ID NO: 150 [PRE10], SEQ ID NO: 151 [AIM45], SEQ ID NO: 152 [ZRT1], SEQ ID NO: 153 [ZRT2], SEQ ID NO: 154 [PH084], SEQ ID NO: 155 [PCLl], SEQ ID NO: 156 [ARGl], SEQ ID NO: 157
  • the promoter nucleic acid sequence drives transcription of a suppressor tRNA.
  • the expression of the suppressor tRNA allows for the translation of the biocatalyst polypeptide that contains a nonsense, missense, or frameshift allele. See Figures 2 and 6.
  • the promoter nucleic acid sequence drives transcription of a suppressor tRNA.
  • the expression of the suppressor tRNA allows for the translation of the propagation polypeptide propagation polypeptide that a nonsense, missense, or frameshift allele. See Figures 4 and 8.
  • the promoter nucleic acid sequence drives transcription of anti-sense RNA.
  • the transcription of the anti-sense RNA blocks the translation of a biocatalyst polypeptide. See Figures 3 and 7.
  • the promoter nucleic acid sequence drives transcription of anti-sense RNA.
  • the transcription of the anti-sense RNA blocks the translation of a propagation polypeptide. See Figures 5 and 9.
  • the expression of the suppressor tRNA or anti-sense RNA molecule is increased by a transcriptional activator in response to an environmental cue. In embodiments, the expression of the suppressor tRNA or antisense RNA molecule is blocked by a transcriptional repressor in response to an environmental cue.
  • a sulfonylurea-responsive tetracycline promoter and/or a sulfonylurea-responsive tetracycline activator is used in an ALS sulfonylurea-resistant background as described in, for example, Falco, S.C., et ah, Genetics. 109(1): 21-35 (1985).
  • an inactive sulfonylurea or an ALS inactive sulfonylurea that binds to a sulfonylurea responsive activator or repressor derived from TET genes is used.
  • the promoter nucleic acid sequence is sensitive to the
  • a distinguishing characteristic between the propagation and production stages is the presence of high (for example, greater than about 5% of air-saturation) dissolved oxygen concentrations during most of the propagation phase, and low (for example, less than about 5% or less than about 3%) dissolved oxygen concentrations, frequently even anaerobic conditions, in most of the production phase. Consequently, in embodiments, "high” vs. "low” dissolved oxygen concentrations results in the increase or decrease of expression of suppressor tRNAs and/or anti-sense RNAs that result in an increase or decrease of expression of biocatalyst polypeptides of interest in the propagation vs. the production stage of the process.
  • biocatalyst polypeptides include, but are not limited to, acetohydroxyacid synthase (AHAS), glucose-6-phosphate dehydrogenase (ZWFl), phosphoketolase (XPK), and glycerol-3- phosphate dehydrogenase (GPD), described elsewhere herein.
  • AHAS acetohydroxyacid synthase
  • ZWFl glucose-6-phosphate dehydrogenase
  • XPK phosphoketolase
  • GPD glycerol-3- phosphate dehydrogenase
  • differential control of promoters and the suppressor tRNAs and/or anti-sense RNAs that they control result in lower expression of desired biocatalyst polypeptides and consequently the gene products (biocatalyst polypeptides) under aerobic than under anaerobic conditions.
  • biocatalyst polypeptides comprise, but are not limited to the acetohydroxyacid synthase (AHAS) of the butanol biosynthesis pathway.
  • Saccharomyces cerevisiae promoter nucleic acid sequences affected by aerobic or anaerobic conditions are shown in Table 3.
  • the promoter is a HEM 13 (SEQ ID NO: 176) promoter or active fragment thereof.
  • the promoter is a HES1 (SEQ ID NO: 177) promoter or active fragment thereof.
  • the promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNA they control result in lower expression of polynucleotides encoding biosynthetic pathway polypeptides during the propagation phase and increased expression during production phase, for example acetohydroxyacid synthase.
  • Suitable promoter nucleic acid sequences are provided herein and/or are available in the art (Boles, E., W. Lehnert, et al, Eur. J. Biochem. 217(1): 469-77 (1993); Heux, S., A. Cadiere, et al, FEMS Yeast Res. 8(2): 217-24 (2008); Kresnowati, M. T., W. A. van Winden, et al., Mol Syst Biol 2: 49 (2006); Kundaje, A., X. Xin, et al, PLoS Comput. Biol 4(11): el000224 (2008); Lai, L. C, A. L.
  • Promoter nucleic acid sequences useful in the invention include those comprising sequences provided herein and those that comprise sequences which are at least about 80%, 85%>, 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences given in Table 3, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen.
  • the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 775
  • biocatalyst polypeptide comprises at least about 90%>, at least about 95%>, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.
  • Table 3 Example Candidate Promoter Sequences for Higher Expression in Low Oxygen
  • promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNAs that they control result in a lower expression of desired polynucleotides and consequently the encoded products under anaerobic than under aerobic conditions.
  • polynucleotides comprise, but are not limited to, polypeptides which may produce by-products of the butanol biosynthesis pathway such as isobutyric acid or DHMB. Saccharomyces cerevisiae promoter nucleic acid sequences affected by such conditions are shown in Table 4.
  • the promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNAs that they control result in lower expression of genes encoding by-product producing polypeptides during the propagation phase and lower expression during production phase, for example YMR226C or aldehyde dehydrogenases, including, but not limited to, ALD6.
  • Promoter nucleic acid sequences useful in the invention comprise those provided herein and those which comprise sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of Table 4, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen.
  • a distinguishing condition between the propagation and production phases is the presence of low glucose concentrations during the propagation phase and the presence of excess glucose during the production phase. Consequently "high” vs. "low” glucose concentrations could be used to express/repress suppressor tNRA and/or anti-sense RNA expression, and therefore biocatalyst polypeptide expression, in the propagation vs. production phase.
  • biocatalyst polypeptides of interest to differentially control their expression include, but are not limited to, acetohydroxyacid synthase (AHAS), glucoses- phosphate dehydrogenase (ZWF1), phosphoketolase (XPK), glycerol-3 -phosphate
  • the hexose transporter gene family in S. cerevisiae contains the sugar transporter genes HXT1 to HXT17, GAL2 and the glucose sensor genes SNF3 and RGT2.
  • the proteins encoded by HXT1 to HXT4 and HXT6 to HXT7 are considered to be the major hexose transporters in S. cerevisiae.
  • the expression of most of the HXT glucose transporter genes is known to depend on the glucose concentration (Ozcan, S. and M. Johnston, Microbiol. Mol. Biol. Rev. 63(3): 554-69 (1999)). Consequently their promoters are provided herein for differential expression of genes under "high” or "low” glucose concentrations.
  • promoter nucleic acid sequences comprising sequences from the promoter region of HXT2 (SEQ ID NO: 384), HXT5 (SEQ ID NO: 360), HXT6 (SEQ ID NO: 386), or HXT7 (SEQ ID NO: 331) are employed for higher expression under glucose-limiting conditions, and lower expression under glucose-excess conditions.
  • HXT5, HXT6 and HXT7 show also strong expression with growth on ethanol, in contrast to HXT2 (Diderich, J. A., Schepper, M., et al, J. Biol. Chem. 274(22): 15350-9 (1999).
  • HXT5 and HXT6 expression showed variability (Rintala, E., M. G. Wiebe, et al, BMC Microbiol. 8: 53 (2008)), however, equipped with this disclosure, one of skill in the art is readily able to make and test such promoter constructs under conditions relevant for a desired production process.
  • Promoter nucleic acid sequences useful in the invention comprise those provided herein and those that comprise nucleic acid sequences which are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of HXT2 (SEQ ID NO: 384), HXT5 (SEQ ID NO: 360), HXT6 (SEQ ID NO: 386), or HXT7 (SEQ ID NO: 331), including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen.
  • the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 772 [HXT5] or 773 [HXT7] or a fragment or derivative thereof.
  • the biocatalyst polypeptide comprises at least about 90%>, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.
  • HXT1 is the promoter for glucose-based control of gene expression, providing high expression under conditions of high glucose.
  • HXT3 may have promise in promoter strength, but may also show some low expression under very low glucose concentrations (Brauer, M. J., C. Huttenhower, et al. Mol Biol Cell 19(1): 352-67 (2008)).
  • promoter nucleic acid sequences useful in the invention comprise those provided herein and those that comprise nucleic acid sequences which are at least about 80%>, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of HXT1 (SEQ ID NO: 168), HXT3 (SEQ ID NO: 169), HXT4 (SEQ ID NO: 388), or EN02 (SEQ ID NO: 173), including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen.
  • the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 768 [HXT3], 769 [HXT1], or 711 [hybrid promoter comprising HXT1 promoter nucleic acid sequence] or a fragment or derivative thereof.
  • the biocatalyst polypeptide comprises at least about 90%, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.
  • a distinguishing condition between the propagation and production phases is the pH.
  • Saccharomyces cerevisiae YKL096W-A (CWP2) promoter (SEQ ID NO: 389) or YER150W (SPI1) promoter (SEQ ID NO: 190) are employed to govern differential expression in processes where the pH is different in different phases. Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process.
  • promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 389 or SEQ ID NO: 190), including variants, fragments or derivatives thereof that confer or increase sensitivity to pH.
  • a distinguishing condition between the propagation and production phases is the temperature.
  • YLL026W (HSP104) promoter (SEQ ID NO: 392) are employed to govern differential expression in processes where the temperature is different in different phases. Temperature sensitive promoters and candidate temperature sensitive promoters are also available in the art (Becerra, M., et ah, Comp Func Genomics 4(4): 366-75 (2003) and Al-Fageeh, MB, et ⁇ , Biochem J J 7(2):247-59 (2006), both incorporated by reference). Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process.
  • promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%>, 85%, 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 391 or SEQ ID NO: 392, including variants, fragments or derivatives thereof that confer or increase sensitivity to temperature.
  • Butanol
  • a distinguishing condition between the propagation and production phases is the concentration of fermentation product such as butanol or 2-butanone.
  • promoter nucleic acid sequences from the Saccharomyces cerevisiae YOL151W (GRE2) promoter (SEQ ID NO: 393) or from the YOR153W (PDR5) promoter (SEQ ID NO: 394) are employed to govern differential expression in processes where the butanol level is different in different phases. Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process.
  • promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 393 or SEQ ID NO: 394, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of butanol.
  • hybrid promoters which comprise nucleic acid sequences from more than one promoter region can be constructed and employed in
  • a Roxl binding site eg. "ATTGT” or a sequence comprising a Rox 1 binding site (e.g., SEQ ID NO: 395)
  • ATTGT a Roxl binding site
  • SEQ ID NO: 395 a sequence comprising a Rox 1 binding site
  • a Roxl binding site may already be present for the HXT1 promoter based on bioinformatic analysis (Maclsaac, K. D., T. Wang, et al, (2006).
  • Hybrid promoter nucleic acid sequences may comprise FBAl promoter sequences
  • a hybrid promoter nucleic acid sequence comprises nucleic acid sequences that are at least about 80%>, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FBAl (SEQ ID NO: 779 or SEQ ID NO: 770) or a variant, fragment or derivative thereof.
  • a hybrid promoter nucleic acid sequence comprises nucleic acid sequences that are at least about 80%, 85%, 90%>, 95%, 96%, 97%, 98%, 99% or 100% identical to HXT1 (SEQ ID NO: 168) or DAN1 (SEQ ID NO: 186) promoters or a variant, fragment or derivative thereof.
  • the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to P[FBA1 ::DAN1_AR314) (SEQ ID NO: 686) or a variant, fragment or derivative thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to P[FBA1 ::HXT1_331] (SEQ ID NO: 711) or a variant, fragment or derivative thereof.
  • the biocatalyst polypeptide comprises at least about 90%>, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.
  • an expression construct comprises SEQ ID NO: 711 or a polynucleotide encoding a polypeptide having at least about 90%>, at least about 95%, at least about 99% or at 100% identity to SEQ ID NO: 1 or both.
  • an expression construct comprises at least about 90%>, at least about 95%, at least about 99% or at 100%) identity to SEQ ID NO: 790.
  • a recombinant host cell comprises such an expression construct, and may further comprise an isobutanol biosynthetic pathway. In embodiments, such a recombinant host cell may be employed in methods wherein it is contacted with a carbon substrate under conditions whereby isobutanol is produced and optionally recovered.
  • RNA transcript comparison between microbial cells grown under selected propagation conditions and cells grown under selected production conditions. Promoters associated with RNA transcripts that are upregulated or downregulated during the propagation phase as compared to during the production phase are suitable for use in the invention.
  • Promoters associated with RNA transcripts that are upregulated or downregulated during the production phase as compared to the propagation phase are also suitable for use in the invention.
  • Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially expressed during the production phase of fermentation, comprising:
  • Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially transcribed less during the production phase of fermentation, comprising:
  • Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially inhibited during the biomass propagation phase, comprising:
  • the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected are introduced into a reporter construct.
  • the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected are introduced into a fluorescent reporter construct.
  • the fluorescent reporter construct expresses green fluorescent protein (GFP).
  • the propagation conditions comprise growing the microorganism comprising a reporter construct in fermentation medium comprising low concentrations of a fermentable carbon substrate and the production conditions comprise growing the microorganism in fermentation medium comprising high concentrations of the same fermentable carbon substrate.
  • the fermentable carbon substrate is selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides, fatty acids, and mixtures thereof.
  • the propagation conditions comprise growing the microorganism comprising a reporter construct in fermentation medium comprising a high concentration of dissolved oxygen and the production conditions comprise growing the microorganism in fermentation medium comprising a low concentration of dissolved oxygen.
  • the methods for identifying promoter nucleic acid sequences for use in the invention further comprise performing a literature search for candidate nucleic acid sequences.
  • literature references such as Li B.-Z., et al., J. Ind.
  • Microbiol. Biotechnol. 57:43-55 (2010) provide information as to which genes are expressed during fed-batch fermentation.
  • Li et al. examined the transcriptional profile of yeast taken from industrial ethanol fermentations (both continuous and fed-batch using 80% corn mash and 20% grain mash as feedstocks). They sampled in the seed stage, during early "main” fermentation, and late main fermentation. They found strong up-regulation of genes involved in reserve carbohydrate metabolism and protein folding (the unfolded protein response).
  • HXK1 and GLK1 encoding the other hexokinase isozymes; gluconeogenic genes; high-affinity glucose transporters
  • HXK2 response observed by Li et al. differs than what was observed in the promoter prospecting experiments in the Examples discussed below.
  • promoter nucleic acid sequences suitable for use in the invention comprise nucleotide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) or 100%) identical to a sequence selected from the group consisting of: [MSN2], [MSN4], [HAP1], [ROX1], [HAP2], [HAP3], [HAP4], [HAP5], [UPC2], [PYC1], [GPP1], [TKLl],
  • promoter nucleic acid sequences suitable for use in the invention can be selected from the group consisting of: [MSN2], [MSN4], [HAP1], [ROX1], [HAP2], [HAP3], [HAP4], [HAP5], [UPC2], [PYC1], [GPPl], [TKLl], [TALI], [YGR032C], [COX5b], [MDH2], [YHR210], and [PCKl] or a variant, fragment or derivative thereof.
  • microorganism is a yeast microorganism.
  • an isobutanologen yeast strain known in the art or disclosed herein may be used for an isobutanol production run using the fermentation process developed for that strain and for the scale of fermentors used.
  • the process may include (i) a biomass production phase, during which biomass is formed with a high yield on a carbon source feedstock (eg. beet molasses), and (ii) a fermentation phase, in which a carbon source feedstock (eg. corn mash) is fermented to isobutanol.
  • a carbon source feedstock eg. corn mash
  • RNA can then be extracted from the cell pellet by methods known in the art (eg. using Trizol reagent, Life Technologies, Grand Island NY), followed by RNA analysis using standard methods (for example with a BioAnalyzer 2100; Agilent Technologies, Inc., Santa Clara CA) to determine a rRNA peak ratio of 1.8 or higher.
  • RNA-Seq also known as Whole Transcriptome Shotgun Sequencing
  • the RNA from each timepoint could be enriched for mRNA using an oligo(dT) technology.
  • the enriched pool would then be reverse-transcribed to cDNA, which is then fragmented to the appropriate length for the sequence method to be used.
  • the fragmented cDNA is then prepared for sequencing (e.g., amplified to create a library) and then the DNA sequences of the fragments would be determined.
  • the resulting raw dataset may be composed of many (typically in the millions) short sequence reads for each timepoint.
  • Bio-informatic analysis would then be erformed to align these sequences, for example by use of a reference genome sequence available in the art. With adequate sequence coverage, then, the read depth for all genes in the genome would be determined for each sample point across the fermentation process. Using bio-informatic methods known in the art, these are converted into numerical descriptions of gene expression levels. Genes with particular expression patterns are identified by methods such as cluster analysis, which reports the fold change in abundance of groups of transcripts at the various timepoints, relative to a reference point (for example, the reference could be the last timepoint in the production phase). Genes with properties of potential utility are identified, for example those with low abundance throughout the biomass production phase and high abundance during the fermentation phase (or at least during certain periods within the fermentation phase).
  • Nucleic acid sequences corresponding to the promoters of those genes could then be tested as candidates for regulated and predictable expression of heterologous genes during the fermentation process. They could also be engineered further; for example, regulatory elements within them could be transferred to other promoters (e.g., strong glycolytic promoters) to confer on them the regulatory properties of the identified genes.
  • One desirable feature of the polynucleotides, recombinant host cells, and methods disclosed herein is that accumulation of inhibitory by-products due to flux via enzymes of the butanol production pathway can be avoided during the growth phase.
  • a non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is isobutyric acid.
  • Another non-limiting example with regard to an inhibitory byproduct produced via enzymes of the isobutanol biosynthetic pathway is isobutyraldehyde.
  • Yet another non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is acetic acid.
  • Some acetolactate synthase enzymes demonstrate a significant oxygenase side reaction in which molecular oxygen electrophilically attacks a highly reactive carbanion/enamine to form a peroxy-adduct that decomposes to ThDP and peracetic acid. See Tse, M.T. and Schloss, J.V., Biochemistry 52: 10398-10403 (1993).
  • the peracetic acid can further react with pyruvate to form two moles of acetate.
  • carbon lost to the by-products adds to a lower yield of biocatalyst on the employed carbon substrate.
  • Another desirable feature of some embodiments is that with oxygen supply, reducing equivalents produced in metabolic pathways leading to pyruvate can be oxidized to a greater extent by the respiratory chain rather than by a biosynthetic pathway such as a butanol biosynthetic pathway. A higher fraction of pyruvate can transit the mitochondrial membrane and be further metabolized by pyruvate dehydrogenase and the tricarboxylic acid cycle.
  • Another desirable feature of some embodiments is that without oxygen supply, more reducing equivalents produced in metabolic pathways leading to pyruvate may be oxidized by the butanol production pathway and fewer by the glycerol production pathway. This way a lower yield of glycerol and a higher yield of butanol may be achieved.
  • Yet another desirable feature of some embodiments is that in case of excess pyruvic acid production, pyruvic acid can be excreted into the medium. See van Maris, A.J., et ah, Appl. Environ. Micriobiol. 70: 159-166 (2004). However, pyruvic acid even at very high concentrations is not growth inhibiting.
  • compositions and methods provided herein provide preferential expression of the acetolactate synthase of an isobutanol production pathway during the production phase.
  • the biocatalyst polypeptide encoded by the isolated polynucleotide of the invention confers host cell tolerance to the fermentation product. In another embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide of the invention confers host cell tolerance to fermentation by-products. In one embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to butanol. In another embodiment, the biocatalyst polypeptide encoded by the isolated
  • polynucleotide confers host cell tolerance to isobutyraldehyde.
  • biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to isobutyric acid.
  • biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to acetic acid.
  • a gene encoding a biocatalyst polypeptide contains a frameshift mutation, such that the polypeptide is not properly translated in the absence of a suppressor tRNA molecule.
  • a gene encoding a biocatalyst polypeptide contains a nonsense or missense mutation, such that the polypeptide is not fully translated in the absence of a suppressor tRNA molecule.
  • the nonsense mutation in the gene coding the biocatalyst polypeptide is the the UAG stop codon, the UGA stop codon, and/or the UAA stop codon.
  • Biosynthetic pathways for the production of isobutanol include those described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference.
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions: - a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;
  • a-ketoisovalerate to isobutyraldehyde which may be catalyzed, for example, by a branched-chain keto acid decarboxylase;
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • valine ⁇ -ketoisovalerate to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase;
  • valine to isobutylamine which may be catalyzed, for example, by valine decarboxylase;
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • the isobutanol biosynthetic pathway comprises the substrate to product conversions shown as steps k, g, and e in Figure 1.
  • Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Appl. Pub. No. 2008/0182308, which is incorporated herein by reference.
  • the 1-butanol biosynthetic pathway comprises the following substrate to product conversions:
  • butyryl-CoA crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase;
  • butyryl-CoA to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase;
  • Biosynthetic pathways for the production of 2-butanol include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference.
  • the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:
  • alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;
  • the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:
  • alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;
  • Biosynthetic pathways for the production of 2-butanone include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference.
  • the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:
  • alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;
  • the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:
  • alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;
  • Another embodiment of the invention is directed to an isolated polynucleotide comprising:
  • nucleic acid sequence of (b) a nucleic acid sequence encoding a biocatalyst polypeptide; wherein the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).
  • the biocatalyst polypeptide comprises or is selected from the group of enzymes having the following Enzyme Commission Numbers: EC 2.2.1.6, EC 1.1.1.86, EC 4.2.1.9, EC 4.1.1.72, EC 1.1.1.1, EC 1.1.1.265, EC 1.1.1.2, EC 1.2.4.4, EC
  • the biocatalyst polypeptide which catalyzes the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol utilize NADH as a cofactor.
  • enzymes from the biosynthetic pathway are localized to the cytosol.
  • enzymes from the biosynthetic pathway that are usually localized to the mitochondria are localized to the cytosol.
  • an enzyme from the biosynthetic pathway is localized to the cytosol by removing the mitochondrial targeting sequence.
  • mitochondrial targeting is eliminated by generating new start codons as described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference in its entirety.
  • the biocatalyst polypeptide is KARL In some embodiments, the biocatalyst polypeptide is KARL In some
  • KARI preferentially utilizes NADH as a cofactor.
  • the biocatalyst polypeptide is ADH.
  • ADH preferentially utilizes NADH as a cofactor.
  • the biocatalyst polypeptide is KIVD, is some embodiments.
  • the biocatalyst polypeptide is DHAD. In some embodiments, the biocatalyst polypeptide is ALS.
  • dehydratases including those from Lactococcus lactis (SEQ ID NO: 794) and Streptococcus mutans (SEQ ID NO: 793) and variants thereof, eg. S. mutans I2V5 (SEQ ID NO: 792).
  • Ketoisovalerate decarboxylases include those derived from Lactococcus lactis (SEQ ID NO: 795), Macrococcus caseofyticus (SEQ ID NO: 797) and Listeria grayi (SEQ ID NO: 796).
  • SEQ ID NO: 795 Lactococcus lactis
  • SEQ ID NO: 797 Macrococcus caseofyticus
  • Listeria grayi SEQ ID NO: 796.
  • Alcohol dehydrogenases include SadB from Achromobacter xylosoxidans (SEQ ID NO: 798) disclosed in U.S. Patent 8,188,250, incorporated herein by reference.
  • Additional alcohol dehydrogenases include horse liver ADH (SEQ ID NO: 799) and Beijerinkia indica ADH (SEQ ID NO: 800), and those that utilize NADH as a cofactor.
  • KARI enzymes are described for example, in U.S. Patent Nos. 7,910, 342 and 8,129,162; U.S. Publication No. 2010/0197519; International Publication No. WO 2012/129555, all of which are incorporated by reference.
  • KARIs include Pseudomonas fluorescens KARI (SEQ ID NO: 801) and variants thereof and Anaerostipes caccae KARI (SEQ ID NO: 802) and variants thereof (eg.
  • a butanol biosynthetic pathway comprises a) a ketol-acid reductoisomerase that has a K m for NADH less than about 300 ⁇ , less than about 100 ⁇ , less than about 50 ⁇ , less than about 20 ⁇ or less than about 10 ⁇ ; b) an alcohol dehydrogenase that utilizes NADH as a cofactor; or c) both a) and b).
  • a ketol-acid reductoisomerase that has a K m for NADH less than about 300 ⁇ , less than about 100 ⁇ , less than about 50 ⁇ , less than about 20 ⁇ or less than about 10 ⁇
  • an alcohol dehydrogenase that utilizes NADH as a cofactor
  • Another embodiment of the invention is directed to an isolated polynucleotide comprising: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding a biocatalyst polypeptide for cell integrity.
  • the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).
  • the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance.
  • the biocatalyst polypeptide is YER150W (SPIl) (nucleic acid SEQ ID NO: 190; amino acid SEQ ID NO: 397), or a homolog thereof.
  • the biocatalyst polypeptide is encoded by a cell wall integrity gene activated by Rlml such as a polypeptide listed in Table 5 or a homolog thereof.
  • cell integrity polypeptides are preferentially expressed during the production phase. While not wishing to be bound by theory, it is believed that expression of such polypeptides may contribute to improved tolerance of a host cell to a fermentation product (e.g., butanol), thus improving production.
  • a fermentation product e.g., butanol
  • Another embodiment of the invention is directed to an isolated polynucleotide comprising:
  • nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).
  • the propagation polypeptide is phosphoketolase. In some embodiments, the propagation polypeptide is phosphotransacetylase. Example phosphoketolases and phosphotransacetylases are described in PCT Publication No. WO/2011/159853 and U.S. App. Pub. No. 20120156735 Al, incorporated by reference herein. In some embodiments, the phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181). In some embodiments, the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181). In some embodiments, the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181). In some embodiments, the phosphotransacetylase is eu
  • Lactobacillus plantarum (nucleic acid SEQ ID NO: 178; amino acid SEQ ID NO: 179).
  • host cells comprising such nucleic acid sequences encoding biocatalyst polypeptides for cell propagation have reduced or eliminated pyruvate decarboxylase activity.
  • DHMB can be produced as a result of a side-reaction that occurs when host cells are genetically manipulated to include a biosynthetic pathway, e.g., a biosynthetic pathway that involves the production of acetolactate.
  • a biosynthetic pathway e.g., a biosynthetic pathway that involves the production of acetolactate.
  • the presence of DHMB indicates that not all of the pathway substrates are being converted to the desired product. Thus, yield may be lowered.
  • DHMB present in the fermentation media may have inhibitory effects on product production.
  • DHMB can decrease the activity of enzymes in the biosynthetic pathway or have other inhibitory effects on cell growth and/or productivity during fermentation.
  • described herein are isolated polynucleotides resulting in lower expression of DHMB during the production phase of fermentation than in the propagation phase.
  • the ability of a host cell to convert acetolactate to DHMB can be reduced or eliminated, for example, by reducing the expression of a polypeptide having acetolactate reductase activity.
  • the polypeptide having acetolactate reductase activity is YMR226C (nucleic acid SEQ ID NO: 182, amino acid SEQ ID NO: 183) or a homo log thereof.
  • the last step in the biosynthesis of isobutanol via a pyruvate-utilizing biosynthetic pathway is the conversion of isobutyraldehyde to isobutanol ( Figure 1).
  • a side reaction in this pathway is the conversion of isobutyraldehyde to isobutyric acid which results in reduced amounts of isobutyraldehyde available to convert into isobutanol and reduced isobutanol yield. Reducing or eliminating the conversion of isobutyraldehyde to isobutyric acid may result in increased amounts of isobutyraldehyde available for conversion to isobutanol.
  • a recombinant host cell of the invention can be S.
  • ALD2 nucleic acid SEQ ID NO: 436; amino acid SEQ ID NO: 437
  • ALD3 nucleic acid SEQ ID NO: 438; amino acid SEQ ID NO: 439
  • ALD4 nucleic acid SEQ ID NO: 440; amino acid SEQ ID NO: 441)
  • ALD5 nucleic acid SEQ ID NO: 442; amino acid SEQ ID NO: 443
  • ALD6 nucleic acid SEQ ID NO: 184; amino acid SEQ ID NO: 185
  • a recombinant host cell can be Kluyveromyces lactis, and a polypeptide having aldehyde dehydrogenase activity can be KLLA0F00440, KLLA0E23057, KLLA0D 10021, KLLA0D09999G, or combinations thereof.
  • a recombinant host cell can be Pichia stipitis, and a polypeptide having aldehyde dehydrogenase activity can be ALD2, ALD3, ALD4, ALD5, ALD7, or combinations thereof.
  • a recombinant host cell can be Lactobacillus plantarum, and a polypeptide having aldehyde dehydrogenase activity can be AldH.
  • a recombinant host cell can be E. coli, and a polypeptide having aldehyde dehydrogenase activity can be aldA, aldB, or combinations thereof.
  • Endogenous NAD-dependent glycerol-3 -phosphate dehydrogenase is a key enzyme in glycerol synthesis, converting dihydroxyacetone phosphate (DHAP) to glycerol-3 - phosphate and playing a role in cellular oxidation of NADH.
  • Yeast strains may have one or more genes encoding NAD-dependent glycerol-3 -phosphate dehydrogenase (GPD).
  • GPD1 and GPD2 are functional homologs for NAD-dependent glycerol-3 -phosphate dehydrogenase.
  • the biocatalyst polypeptide is GPD1 (nucleic acid SEQ ID NO: 444; amino acid SEQ ID NO: 445) or GPD2 (nucleic acid SEQ ID NO: 446; amino acid SEQ ID NO: 447), or a homolog thereof.
  • the biocatalyst polypeptide is an enzyme of the oxidative pentose phosphate pathway.
  • the biocatalyst polypeptide is glucoses- phosphate dehydrogenase (ZWF1, nucleic acid SEQ ID NO: 448; amino acid SEQ ID NO: 449), 6-phosphoglucononolactonase (SOL3: nucleic acid SEQ ID NO: 450; amino acid SEQ ID NO: 451; or SOL4: SEQ ID NO: 452; amino acid SEQ ID NO: 453), or 6-phosphogluconate dehydrogenase nucleic acid (GND1 : nucleic acid SEQ ID NO: 454; amino acid SEQ ID NO: 455; GND2: nucleic acid SEQ ID NO: 456; amino acid SEQ ID NO: 457).
  • ZWF1 glucoses- phosphate dehydrogenase
  • SOL3 nucleic acid SEQ ID NO: 450; amino acid SEQ ID NO: 4
  • ZWF1 is preferentially expressed in propagation.
  • the reducing equivalents (such as NADH) produced in glycolysis are utilized by the biosynthetic production pathway during the production phase.
  • NADH reducing equivalents
  • 2 molecules of NADH produced during glycolysis are consumed per molecule of isobutanol produced. While not wishing to be bound by theory, it is believed that reduced expression of ZWF1 during such production processes may result in decreased excess NADPH production and consequently decreased by-product production.
  • the genetic manipulations of a recombinant host cell disclosed herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202).
  • Non-limiting examples of host cells for use in the invention include bacteria, cyanobacteria, filamentous fungi and yeasts.
  • the recombinant host cell of the invention is a bacterial or a cyanobacterial cell.
  • the recombinant host cell comprises or is selected from the group consisting of: Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus, Streptomyces, Zymomonas, Escherichia, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Serratia, Shigella, Alcaligenes, Erwinia, Paenibacillus, and Xanthomonas .
  • the recombinant host cell is E. coli, S. cerevisiae, or L. plantarum.
  • the recombinant host cell of the invention is a filamentous fungi or yeast cell.
  • the recombinant host cell comprises or is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zy go saccharomyces, Galactomyces,
  • the host cell does not express an enzyme or has reduced expression of an enzyme having the following Enzyme Commission Number: EC 4.1.1.1.
  • the yeast is crabtree-positive.
  • Crabtree-positive yeast cells demonstrate the crabtree effect, which is a phenomenon whereby cellular respiration is inhibited when a high concentration of glucose is present in aerobic culture medium.
  • Suitable crabtree- positive yeast are viable in culture and include, but are not limited to, Saccharomyces,
  • Suitable species include, but are not limited to,
  • Saccharomyces cerevisiae Schizosaccharomyces pombe, Kluyveromyces thermotolerans, Candida glabrata, Issatchenkia orientalis.
  • Crabtree-positive yeast cells may be grown with high aeration and in low glucose concentration to maximize respiration and cell mass production, as known in the art, rather than butanol production. Typically the glucose concentration is kept to less than about 0.2 g/L.
  • the aerated culture can grow to a high cell density and then be used as the present production culture.
  • yeast cells that are capable of producing butanol may be grown and concentrated to produce a high cell density culture.
  • the yeast is Crabtree-negative.
  • Crabtree-negative yeast cells do not demonstrate the crabtree effect when a high concentration of glucose is added to aerobic culture medium, and therefore, in crabtree-negative yeast cells, alcoholic fermentation is absent after an excess of glucose is added.
  • Suitable Crabtree-negative yeast genera are viable in culture and include, but are not limited to, Hansenula, Debaryomyces, Yarrowia, Rhodotorula, and Pichia.
  • Suitable species include, but are not limited to, Candida utilis, Hansenula nonfermentans, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia stipitis, and Pichia pastoris.
  • Suitable microbial hosts include, but are not limited to, members of the genera
  • Suitable hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida,
  • the host cell is Saccharomyces cerevisiae.
  • S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S.
  • cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm ProTM yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMaxTM Green yeast, FerMaxTM Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.
  • Recombinant microorganisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to a desired product can be constructed using techniques well known in the art.
  • genes encoding the enzymes of one of the isobutanol biosynthetic pathways of the invention for example, acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain a-keto acid decarboxylase, and branched-chain alcohol
  • dehydrogenase can be obtained from various sources, as described above.
  • Methods of obtaining desired genes from a genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries can be created by restriction endonuclease digestion and can be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. 4,683,202) to obtain amounts of DNA suitable for transformation using
  • vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE ® (Madison, WI), Invitrogen Corp. (Carlsbad, CA), Stratagene (La Jolla, CA), and New England Biolabs, Inc. (Beverly, MA).
  • EPICENTRE ® Madison, WI
  • Invitrogen Corp. Carlsbad, CA
  • Stratagene La Jolla, CA
  • New England Biolabs, Inc. Bact al.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.
  • Initiation control regions or promoters which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements, including those used in the Examples, is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL 10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli,
  • yeast recombinant host cells a number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1; and the following inducible promoters suitable for use in yeast: GAL1, GAL 10, OLE1, and CUP1.
  • yeast promoters include hybrid promoters UAS(PGKl)-FBAlp, UAS(PGKl)-EN02p, UAS(FBAl)- PDClp, UAS(PGKl)-PDClp, and UAS(PGK)-OLElp.
  • Promoters, transcriptional terminators, and coding regions can be cloned into a yeast 2 micron plasmid and transformed into yeast cells (Ludwig, et al. Gene, 132: 33-40, 1993; US Appl. Pub. No. 20080261861A1).
  • Adjusting the amount of gene expression in a given host may be achieved by varying the level of transcription, such as through selection of native or artificial promoters.
  • techniques such as the use of promoter libraries to achieve desired levels of gene transcription are well known in the art.
  • Such libraries can be generated using techniques known in the art, for example, by cloning of random cDNA fragments in front of gene cassettes (Goh et al. (2002) AEM 99, 17025), by modulating regulatory sequences present within promoters (Ligr et al. (2006) Genetics 172, 2113),or by mutagenesis of known promoter sequences (Alper et al. (2005) PNAS, 12678; Nevoigt et al. (2006) AEM 72, 5266).
  • Termination control regions can also be derived from various genes native to the hosts. Optionally, a termination site can be unnecessary or can be included.
  • Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation.
  • the complete and annotated sequence of pRK404 and three related vectors-pR 437, pR 442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid, 50: 74-79, 2003).
  • Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.
  • thermosensitive variant of the broad-host-range replicon pWVlOl has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin, et al., J. Bacteriol, 174: 5633-5638 (1992)).
  • pVE6002 plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria
  • transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®.
  • 2007/0259410A1 and 2007/0292927A1 both incorporated by reference, disclose the engineering of recombinant microorganisms for production of 2-butanol. Multiple pathways are described for biosynthesis of isobutanol and 2- butanol. The methods disclosed in these publications can be used to engineer the recombinant host cells of the present invention. The information presented in these publications is hereby incorporated by reference in its entirety.
  • the host cells comprising a biosynthetic pathway as provided herein may further comprise one or more additional modifications.
  • U.S. Appl. Pub. No. 2009/0305363, incorporated herein by reference discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity.
  • modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate -utilizing biosynthetic pathway. Other modifications are described in PCT. Pub. No. WO2012/129555, incorporated herein by reference. Modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In embodiments, the polypeptide having acetolactate reductase activity is YMR226C of Saccharomyces cerevisae or a homolog thereof.
  • Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity.
  • the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homolog thereof.
  • a genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Appl. Pub. No. 2011/0124060, incorporated herein by reference.
  • the pyruvate decarboxylase that is deleted or downregulated is selected from the group consisting of: PDC1, PDC5, PDC6, or combinations thereof.
  • host cells contain a deletion or downregulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3 -phosphate to glycerate 1,3, bisphosphate.
  • the enzyme that catalyzes this reaction is glyceraldehyde-3 -phosphate dehydrogenase.
  • Recombinant host cells may further comprise (a) at least one heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity; and (b)(i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe-S cluster biosynthesis; and/or (ii) at least one heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster biosynthesis, described in PCT Publication No.
  • the polypeptide affecting Fe-S cluster biosynthesis is encoded by AFTl, AFT2, FRA2, GRX3, or CCCl .
  • the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFTl L99A, AFTl L102A, AFTl C291F, or AFTl C293F.
  • a recombinant host cell comprising promoter nucleic acid sequences may be subjected to different conditions, such as conditions
  • differential expression of a suppressor tRNA and/or anti-sense RNA will result in differential expression of a target polynucleotide or its encoded polypeptide, which may be confirmed using methods known in the art and/or provided herein.
  • Differential expression of a polynucleotide encoding a biocatalyst polypeptide can be confirmed by comparing transcript levels under different conditions using reverse transcriptase polymerase chain reaction (RT-PCR) or real-time PCR using methods known in the art and/or exemplified herein.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a reporter such as green fluorescent protein (GFP) can be used in combination with flow cytometry to confirm the capability of a promoter nucleic acid sequence to affect expression under different conditions.
  • the activity of a biocatalyst polypeptide may be determined under different conditions to confirm the differential expression of the polypeptide.
  • ALS is the biocatalyst polypeptide
  • the activity of ALS present in host cells subjected to different conditions may be determined (using, for example, methods described in Westerfeld, W.W., J. Biol. Chem. 161:495-502 (1945), modified as described in the Examples herein).
  • a difference in ALS activity can be used to confirm differential expression of the ALS.
  • differential expression of a biocatalyst polypeptide can be confirmed indirectly by measurement of downstream products or byproducts. For example, a decrease in production of isobutyraldehyde may be indicative of differential ALS expression.
  • spectrophotometric measurement of optical density can be used as an indicator of biomass.
  • Measurement of pathway products or by-products including, but not limited to butanol concentration, DHMB concentration, or isobutyric acid can be carried out using methods known in the art and/or provided herein such as high pressure liquid chromatography (HPLC; for example, see PCT. Pub. No. WO2012/129555, incorporated herein by reference)
  • HPLC high pressure liquid chromatography
  • the rate of biomass increase, the rate of glucose consumption, or the rate of butanol production can be determined, for example by using the indicated methods.
  • Biomass yield and product (eg. butanol) yield can likewise be determined using methods disclosed in the art and/or herein.
  • Another embodiment of the present invention is directed to methods for producing various fermentation products including, but not limited to, lower alkyl alcohols. These methods employ the recombinant host cells of the invention.
  • the method of the present invention comprises providing a recombinant host cell as discussed above, contacting the recombinant host cell with a fermentable carbon substrate in a fermentation medium under conditions whereby the fermentation product is produced and, optionally, recovering the fermentation product.
  • a process for producing fermentation products may comprise multiple phases.
  • process may comprise a first biomass production phase, a second biomass production phase, a fermentation production phase, and an optional recovery phase.
  • processes provided herein comprise more than one, more than two, or more than three phases.
  • process conditions may vary from phase to phase.
  • one phase of a process may be substantially aerobic, while the next phase may be substantially anaerobic.
  • Other differences between phases may include, but are not limited to, source of carbon substrate (e.g., feedstock from which the fermentable carbon is derived), carbon substrate (e.g.,. glucose) concentration, dissolved oxygen, pH, temperature, or concentration of fermentation product (e.g., butanol).
  • Promoter nucleic acid sequences and nucleic acid sequences encoding biocatalyst polypeptides and recombinant host cells comprising such promoter nucleic acid sequences may be employed in such processes.
  • a biocatalyst polypeptide is preferentially expressed in at least one phase.
  • the propagation phase generally comprises at least one process by which biomass is increased.
  • the temperature of the propagation phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C.
  • the pH in the propagation phase may be at least about 4, at least about 5, at least about 5.5, at least about 6, or at least about 6.5.
  • the propagation phase continues until the biomass concentration reaches at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 50, at least about 70, or at least about 100 g/L.
  • the average glucose or sugar concentration is about or less than about 2 g/L, about or less than about 1 g/L, about or less than about 0.5 g/L or about or less than about 0.1 g/L.
  • the dissolved oxygen concetration may average as undetectable, or as at least about 10%, at least about 20%, at least about 30%, or at least about 40%.
  • a stage of the propagation phase comprises contacting a recombinant yeast host cell with at least one carbon substrate at a temperature of about 30 to about 35°C and a pH of about 4 to about 5.5, until the biomass concentration is in the range of about 20 to about 100 g/L.
  • the dissolved oxygen level over the course of the contact may average from about 20 to 40% (0.8 - 3.2 ppm).
  • the source of the carbon substrate may be molasses or corn mash, or pure glucose or other sugar, such that the glucose or sugar concentration is from about 0 to about 1 g/L over the course of the contacting or from about 0 to about 0.1 g/L.
  • a recombinant yeast host cell may be subjected to a further process whereby recombinant yeast at a concentration of about 0.1 g/L to about 1 g/L is contacted with at least one carbon substrate at a temperature of about 25 to about 35°C and a pH of about about 4 to about 5.5 until the biomass concentration is in the range of about 5 to about 15 g/L.
  • the dissolved oxygen level over the course of the contact may average from undetectable to about 30% (0-2.4 ppm).
  • the source of the carbon substrate may be corn mash such that the glucose concentration averages about 2 to about 30 g/L over the course of contacting.
  • propagation phase may comprise one, two, three, four, or more stages, and that the above non-limiting example stages may be practiced in any order or combination.
  • the production phase typically comprises at least one process by which a product is produced.
  • the average glucose concentration during the production phase is at least about 0.1, at least about 1, at least about 5, at least about 10 g/L, at least about 30 g/L, at least about 50 g/L, or at least about 100 g/L.
  • the temperature of the production phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C.
  • the pH in the production phase may be at least about 4, at least about 5, or at least about 5.5.
  • the production phase continues until the product titer reaches at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 30 g/L, at least about 35 g/L or at least about 40 g/L.
  • the dissolved oxygen concetration may average as less than about 5%, less than about 1%, or as negligible such that the conditions are substantially anaerobic.
  • recombinant yeast cells at a concentration of about 0.1 to about 6 g/L are contacted with at least one carbon substrate at a concentration of about 5 to about 100 g/L, temperature of about 25 to about 30°C, pH of about 4 to about 5.5.
  • the dissolved oxygen level over the course of the contact may be negligible on average, such that the contact occurs under substantially anaerobic conditions.
  • the source of the carbon substrate may mash such as corn mash, such that the glucose concentration averages about 10 to about 100 g/L over the course of the contacting, until it is substantially completely consumed.
  • the glucose concentration is about 100-fold to about 1000-fold higher in the production phase than in the propagation phase.
  • the glucose concentration in production is at least about 5X, at least about 10X, at least about 5 OX, at least about 100X, or at least about 500X higher than that in propagation.
  • the temperature in the propagation phase is about 5 to about 10 degrees lower in the production phase than in the propagation phase.
  • concentration is anaerobic in the production phase and microaerobic to aerobic in the propagation phase.
  • the conditions for propagating a host cell and/or producing a fermentation product utilizing a host cell may vary according to the host cell being used.
  • the method for producing a fermentation product is performed under anaerobic conditions.
  • the method for producing a fermentation product is performed under microaerobic conditions.
  • the process may be further refined to take advantage of the differential expression afforded thereby.
  • the genetic switch provides preferential expression in high glucose conditions
  • one of skill in the art will be able to readily determine the glucose levels necessary to maintain minimal expression.
  • the glucose concentration in the phase of the process under which minimal expression is desired can be controlled so as to maintain minimal expression.
  • polymer-based slow-release feed beads available, for example, from Kuhner Shaker, Basel, Switzerland
  • a similar strategy can be employed to refine the propagation or production phase conditions relevant to the differential expression using the compositions and methods provided herein.
  • Carbon substrates may include, but are not limited to, monosaccharides (such as fructose, glucose, mannose, rhamnose, xylose or galactose), oligosaccharides (such as lactose, maltose, or sucrose), polysaccharides such as starch, maltodextrin, or cellulose, fatty acids, or mixtures thereof and unpurified mixtures from renewable feedstocks such as corn mash, cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt.
  • Other carbon substrates may include ethanol, lactate, succinate, or glycerol.
  • the carbon substrate may also be a one carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.
  • methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity.
  • methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion, et al, Microb. Growth CI Compd., [Int. Symp.], 7th (1993), 415 32, Editor(s): Murrell, J.
  • Candida will metabolize alanine or oleic acid (Suiter, et al., Arch. Microbiol. 755:485-489 (1990)).
  • the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
  • preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars.
  • Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof.
  • Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof.
  • fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Appl. Pub. No. 2007/0031918 Al, which is herein incorporated by reference.
  • Biomass in reference to a carbon source refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid.
  • Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste.
  • biomass examples include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
  • crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
  • the carbon substrates may be provided in any media that is suitable for host cell growth and reproduction.
  • media that can be used include M122C, MOPS, SOB, TSY, YMG, YPD, 2XYT, LB, Ml 7, or M9 minimal media.
  • Other examples of media that can be used include solutions containing potassium phosphate and/or sodium phosphate. Suitable media can be supplemented with NADH or NADPH.
  • the method for producing a fermentation product results in a titer of at least about 20 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 30 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L or 40 g/L of fermentation product.
  • the rate of production of a fermentation product is increased.
  • the rate of biomass production is increased.
  • the yield of fermentation product is increased.
  • the yield of biomass is increased.
  • Non- limiting examples of lower alkyl alcohols which may be produced by the methods of the invention include butanol (for example, isobutanol), propanol, isopropanol, and ethanol. In one embodiment, isobutanol is produced.
  • the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of theoretical. In one embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 25% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 40% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 50% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 75% of theoretical.
  • Non- limiting examples of lower alkyl alcohols produced by the recombinant host cells of the invention include butanol, isobutanol, propanol, isopropanol, and ethanol. In one embodiment, the recombinant host cells of the invention produce isobutanol. In another embodiment, the recombinant host cells of the invention do not produce ethanol.
  • Bioproduced butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 4P:639-648 (1998), Groot, et al, Process. Biochem. 27:61-75 (1992), and references therein).
  • solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like.
  • the butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.
  • distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
  • the butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol.
  • the butanol containing fermentation broth is distilled to near the azeotropic composition.
  • the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation.
  • the decanted aqueous phase may be returned to the first distillation column as reflux.
  • the butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
  • the butanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation.
  • the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent.
  • the butanol-containing organic phase is then distilled to separate the butanol from the solvent.
  • Distillation in combination with adsorption can also be used to isolate butanol from the fermentation medium.
  • the fermentation broth containing the butanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic
  • distillation in combination with pervaporation may be used to isolate and purify the butanol from the fermentation medium.
  • the fermentation broth containing the butanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo, et al., J. Membr. Sci. 245: 199-210 (2004)).
  • ISPR In situ product removal
  • extractive fermentation can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields.
  • One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction.
  • the fermentation medium which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the
  • the fermentation medium form a biphasic mixture.
  • the butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.
  • Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 2009/0305370, the disclosure of which is hereby incorporated in its entirety.
  • U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase.
  • the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C 12 to C 22 fatty alcohols, C 12 to C 22 fatty acids, esters of C 12 to C 22 fatty acids, C 12 to C 22 fatty aldehydes, and mixtures thereof.
  • the extractant(s) for ISPR can be non-alcohol extractants.
  • the ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.
  • an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.
  • the alcohol can be formed by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst capable of esterfiying the alcohol with the organic acid.
  • the organic acid can serve as an ISPR extractant into which the alcohol esters partition.
  • the organic acid can be supplied to the fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol.
  • the catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock.
  • alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel.
  • Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant.
  • the extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel.
  • the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration.
  • unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.
  • In situ product removal can be carried out in a batch mode or a continuous mode.
  • a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process.
  • the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium.
  • the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level.
  • the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel.
  • the ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved.
  • the ester-containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.
  • PCR means polymerase chain reaction
  • OD optical density
  • OD600 optical density measured at a wavelength of 600 nm
  • kDa kilodaltons
  • g can also mean the gravitation constant
  • bp means base pair(s)
  • kbp means kilobase pair(s)
  • kb means kilobase
  • % means percent
  • % w/v means weight/volume percent
  • % v/v means volume/volume percent
  • HPLC means high performance liquid chromatography
  • g/L means gram per liter
  • ' ⁇ g/L means microgram per liter
  • ng ⁇ L means nanogram per
  • Microarray analysis identified a number of genes that were up-regulated (up to 200-fold) and highly expressed in one or more timepoints during production, but not in the propagation sample. Twelve of these were selected for further study; their promoters were fused to the green fluorescent protein (GFP) as a reporter for expression, and their transcriptional activity was monitored during fermentation (including scaled-down models of simultaneous saccharification and fermentation). The twelve genes are tabulated in Table 1.
  • GFP green fluorescent protein
  • IMAl encoding isomaltase (involved in fermentation of residual sugars produced by a-amylase-catalyzed starch hydrolysis), genes induced by cell wall damage, genes involved in thermotolerance and halotolerance, in pseudohyphal growth (known to be induced by isobutanol), and genes encoding proteasomal subunits (the proteasome degrades misfolded proteins, which increase in abundance under certain kinds of stress).
  • promoters that were identified through promoter prospecting would not necessarily have been selected based on a rational, a priori, approach. It may be necessary to periodically repeat promoter prospecting experiments, as isobutanologen strains and processes evolve. Also, promoters of genes that displayed dynamic expression profiles during this promoter prospecting experiment may also be screened for utility in a second round of testing.
  • the induction of the IMAl gene may be in response to maltrins present in the corn mash.
  • Yeast has an active transcriptional response to corn oil fatty acid fractions, particularly oleic acid, resulting in the activation of genes involved in peroxisomal biogenesis and function. This response is overridden by glucose repression.
  • GRE2 encoding 3-methylbutanal reductase
  • PDR5 encoding a drug-efflux pump
  • heat shock genes Upon fusing the GRE2 promoter to a gene encoding GFP, it was determined that GFP expression is indeed activated by isobutanol challenge.
  • a CEN.PK gpd2- pdc- yeast strain (PNY1504, described in US Appn. Pub. No. 20120237988, incorporated herein by reference) was grown in 3 g/L glucose + 3 g/L ethanol salt medium for ⁇ 24 hrs. 13-15 mL of each 250 mL culture was transferred to -270 mL medium in a 2 L, baffled, vented flask at an OD 2.0-2.5. Four 2L flasks were started for each propagation tank. 24 hrs after inoculating the 2 L flasks.
  • YEP stock solution 200 g/L peptone, 100 g/L yeast extract
  • 300 mL of sterile, virgin, 90-95% Cognis oleyl alcohol was added and the flasks were returned to the shaker for ⁇ 20 hrs.
  • the aqueous phase and oleyl alcohol phase were allowed to settle for ⁇ 5 minutes.
  • the aqueous phase was pooled together from all 12 flasks and ⁇ 1.2 L was distributed to three pressure cans that were used to inoculate each propagation tank.
  • SSF Simultaneous Saccharification and Fermentation
  • RNA species were quantified using Agilent arrays by standard methods. Data was averaged and statistical analysis was performed. The abundance of RNA transcripts were interpreted with regard to physiology. Hence, relevant physiological data is summarized for the propagation tank first. Physiology
  • RNAs that are regulated by the anoxic conditions of the fermentation tank.
  • the promoters of such RNAs could be useful switches (e.g., OFF -> ON) or amplifier modules (e.g., LOW -> HIGH) that elevate expression from a significant basal level. Both "switch” and “amplifier” promoter elements are desirable.
  • ORFs Two ORFs (YJL 171 C and YGR287C) were transcribed at 26 hrs and retained elevated expression levels while DIA1 was transcribed at 37 hrs and was still highly expressed at 50 hrs. There are also choices to throw a switch at 26 hrs (IMD2), 37 hrs (CHA1 and YJL195 and 50 hrs (PRM6).
  • the fraction is 40/45, 38/45, and 35/45 at 26, 37 and 50 hr EFT, respectively.
  • translation is critical during exponential growth (26 hrs/production) but not to the other three conditions (15 hr, 37 hr, and 50 hr) which are more akin to stationary phase.
  • the biocatalyst pathway polypeptide alsS is chosen to measure differential expression using a genetic switch.
  • An alsS polynucleotide is designed that contains a nonsense mutation that results in no translation of the full-length, functional biocatalyst protein and is under the control of a constitutive promoter.
  • a second polynucleotide is designed containing a promoter identified in this application as differentially activated in response to a specific environmental cue and a suppressor tRNA specific for the nonsense mutation of the biocatalyst polypeptide. In the absence of the cue, the suppressor tRNA is not expressed, and the biocatalyst polypeptide is not translated. In the presense of the cue, the suppressor tRNA is expressed, and the biocatalyst polypeptide is translated.
  • the biocatalyst pathway polypeptide alsS is chosen to measure differential expression using a genetic switch.
  • An alsS polynucleotide is designed that is under the control of a constitutive promoter.
  • a second polynucleotide is designed containing a promoter identified in this application as differentially activated in response to a specific environmental cue and an anti- sense RNA specific for the alsS mRNA.
  • the anti-sense RNA is not expressed, and the biocatalyst polypeptide is translated.
  • the anti-sense RNA is expressed, and the biocatalyst polypeptide is not translated.

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Abstract

L'invention concerne l'utilisation de diverses stratégies post-transcriptionnelles pour l'expression différentielle de gènes pendant les phases de propagation et de production d'un processus de fermentation. L'invention concerne également des cellules hôtes recombinantes comprenant des séquences d'acides nucléiques promoteurs identifiées et des procédés de production de produits de fermentation les utilisant.
PCT/US2014/044989 2013-07-03 2014-07-01 Régulation post-transcriptionnelle des voies biosynthétiques WO2015002916A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021229429A3 (fr) * 2020-05-11 2022-02-24 Lallemand Hungary Liquidity Management Llc Cellule hôte de levure recombinante exprimant une hydrolase
CN116496936A (zh) * 2023-03-02 2023-07-28 湖南酵本生物医药有限公司 一种醋酸杆菌和含有该菌的菌剂、以及它们的应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006024892A1 (fr) * 2004-09-02 2006-03-09 Suntory Limited Methode d'analyse de genes de levures industrielles
WO2008098227A2 (fr) * 2007-02-09 2008-08-14 The Regents Of The University Of California Production de biocarburant par des micro-organismes recombinants
US20090203139A1 (en) * 2007-12-21 2009-08-13 E.I. Du Pont De Nemours And Company Production of four carbon alcohols using improved strain
US20120064561A1 (en) * 2010-02-17 2012-03-15 Butamax(Tm) Advanced Biofuels Llc Activity of fe-s cluster requiring proteins
WO2012122465A2 (fr) * 2011-03-09 2012-09-13 Gevo, Inc. Microorganismes de levure à accumulation réduite de 2,3-butanediol pour production améliorée de carburants, produits chimiques et acides aminés
WO2013016717A2 (fr) * 2011-07-28 2013-01-31 Butamax(Tm) Advanced Biofuels Llc Enzymes céto-isovalérate décarboxylase et leurs procédés d'utilisation
WO2013086222A2 (fr) * 2011-12-09 2013-06-13 Butamax(Tm) Advanced Biofuels Llc Procédé pour éliminer des alcools en produit d'un bouillon de fermentation
WO2013090837A2 (fr) * 2011-12-16 2013-06-20 Invista North America S.A.R.L. Procédés de fabrication de produits chimiques à 6-carbones par allongement de chaîne carbonée dépendant de coa associée à un stockage de carbone

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006024892A1 (fr) * 2004-09-02 2006-03-09 Suntory Limited Methode d'analyse de genes de levures industrielles
WO2008098227A2 (fr) * 2007-02-09 2008-08-14 The Regents Of The University Of California Production de biocarburant par des micro-organismes recombinants
US20090203139A1 (en) * 2007-12-21 2009-08-13 E.I. Du Pont De Nemours And Company Production of four carbon alcohols using improved strain
US20120064561A1 (en) * 2010-02-17 2012-03-15 Butamax(Tm) Advanced Biofuels Llc Activity of fe-s cluster requiring proteins
WO2012122465A2 (fr) * 2011-03-09 2012-09-13 Gevo, Inc. Microorganismes de levure à accumulation réduite de 2,3-butanediol pour production améliorée de carburants, produits chimiques et acides aminés
WO2013016717A2 (fr) * 2011-07-28 2013-01-31 Butamax(Tm) Advanced Biofuels Llc Enzymes céto-isovalérate décarboxylase et leurs procédés d'utilisation
WO2013086222A2 (fr) * 2011-12-09 2013-06-13 Butamax(Tm) Advanced Biofuels Llc Procédé pour éliminer des alcools en produit d'un bouillon de fermentation
WO2013090837A2 (fr) * 2011-12-16 2013-06-20 Invista North America S.A.R.L. Procédés de fabrication de produits chimiques à 6-carbones par allongement de chaîne carbonée dépendant de coa associée à un stockage de carbone

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021229429A3 (fr) * 2020-05-11 2022-02-24 Lallemand Hungary Liquidity Management Llc Cellule hôte de levure recombinante exprimant une hydrolase
CN116496936A (zh) * 2023-03-02 2023-07-28 湖南酵本生物医药有限公司 一种醋酸杆菌和含有该菌的菌剂、以及它们的应用

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