WO2014151645A1 - Procédé de maximisation de croissance de biomasse et de rendement en butanol par rétrocontrôle - Google Patents

Procédé de maximisation de croissance de biomasse et de rendement en butanol par rétrocontrôle Download PDF

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WO2014151645A1
WO2014151645A1 PCT/US2014/026162 US2014026162W WO2014151645A1 WO 2014151645 A1 WO2014151645 A1 WO 2014151645A1 US 2014026162 W US2014026162 W US 2014026162W WO 2014151645 A1 WO2014151645 A1 WO 2014151645A1
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butanol
set point
catalyzed
controlled variable
coa
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PCT/US2014/026162
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English (en)
Inventor
Ritu Bhalla
Steven D. DOIG
Kakasaheb Suresh KONDE
Vaibhav Sambhaji Nikam PATIL
Ranjan Patnaik
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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
    • 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 field of industrial microbiology and the fermentative production of butanol and isomers thereof. More specifically, the invention relates to methods to implement feedback control strategies in order to maximize biomass production and butanol yield by genetically modified butanologens.
  • 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. Accordingly, there is a high demand for butanol, as well as for efficient production methods.
  • One production method which has the potential to reduce environmental impact includes the production of butanol utilizing fermentation by recombinant microorganisms.
  • oxygen uptake rate OUR, mmoles/l/hr
  • carbon dioxide production rate CER, mmoles/l/hr
  • respiratory quotient RQ is calculated as the ratio of CER to OUR.
  • Biomass generation by maintaining RQ near 1 was demonstrated in fed-batch ethanol fermentation (Aiba et al., Biotechnol. Bioeng. 18: 1001- 1016 (1976), Cooney et al, Biotechnology and Bioengineering 19: 55-67 (1977)).
  • Another way of tracking the critical growth rate is to allow a small but measurable production of ethanol (Chen et al, Automatica 31 : 55-65 (1995); Rani and Rao, Bioprocess Engineering 21 : 77-88 (1999);
  • the carbon flux of sugar substrate has to be less than the threshold value.
  • the threshold value of carbon flux in organisms under aerobic conditions depends on competition between PDC and pyruvate dehydrogenase complex (PDH) enzymes. Unmodified
  • microorganisms prefer to maximize biomass generation under normal culture condition, thus, PDH and PDC activities are optimally expressed.
  • isobutanologen (PDC-) strain
  • PDC is deleted
  • the PDH pathway remains intact
  • isobutanol production pathway enzymes are introduced.
  • the first enzyme to act in the isobutanol production pathway is acetolactate synthase (ALS).
  • ALS acetolactate synthase
  • the carbon flux distribution for biomass growth and for the isobutanol pathway under aerobic conditions depends on the relative activity of ALS instead of the PDH enzyme.
  • the physiological behavior of a recombinant isobutanologen is different from an unmodified S. cerevisiae due to the effect of the deletion of PDC genes and introduction of heterologous isobutanol pathway enzymes.
  • the carbon flux has to channel through the PDH pathway efficiently to improve biomass yield and minimize carbon flux to isobutanol pathway leakages.
  • Pathway leakage products can include isobutanol and isobutyric acid, which can adversely affect biomass growth rate and the final biomass achieved.
  • the isobutanol yield and productivity can be adversely affected by accumulation of pathway intermediates ⁇ e.g., glycerol and isobutyric acid).
  • the optimal operating regime (growth and production) for an ethanologen may not be the optimal operating regime for an isobutanologen.
  • the processes comprise (a) providing a fermentation medium and a recombinant microorganism comprising an engineered butanol biosynthetic pathway in a fermentor whereby fermentation occurs under variable conditions wherein one or more of said conditions has a set point which the condition may be controlled to whereby a controlled variable is provided and wherein one or more variable fermentation conditions may be manipulated to adjust the controlled variable; (b) monitoring one or more controlled variables; and (c) adjusting one or more variable fermentation conditions to maintain the set point of the controlled variable, wherein the biomasss of the recombinant microorganism is increased during fermentation.
  • the set point of the controlled variable is dynamically adjusted based on byproduct level.
  • the processes comprise (a) providing a fermentation medium in a first fermentor comprising a recombinant microorganism comprising an engineered butanol biosynthetic pathway; (b) monitoring a controlled variable of the first fermentor, wherein the controlled variable has a set point; (c) adjusting a manipulated variable of the first fermentor based on the set point of the monitored controlled variable; (d) providing a fermentation medium in a second fermentor comprising a recombinant microorganism comprising an engineered butanol biosynthethic pathway; (e) monitoring the controlled variable of the second fermentor, wherein the controlled variable has a set point; and (f) adjusting the manipulated variable of the second fermentor based on the adjustment made to the first fermentor; wherein the adjustment of the manipulated variable in the second fermentor increases the biomass of the recombinant microorganism in the second fermentor.
  • the first fermentor is smaller than the second fermentor.
  • the first fermentor is smaller than the second ferment
  • microorganism comprises two stages.
  • the first stage can, for example, be a biomass growth phase
  • the second stage can, for example, be a transition phase from the growth phase to a butanol production phase for the recombinant microorganism.
  • the first stage is glucose limited (e.g., less than about 5 grams per liter (g/L)).
  • the first stage is aerobic.
  • the second stage is glucose excess (e.g., 5 g/L to about 50 g/L).
  • the second stage is microaerobic.
  • the controlled variable for increasing biomass can be selected from the group consisting of respiratory quotient (RQ), specific oxygen uptake rate (Sp. OUR), specific carbon dioxide evolution rate (Sp. CER), pH, biomass level, specific growth rate, oxygen partial pressure (p0 2 ), rate of heat generation, specific butanol production rate, specific byproduct production rate, and combinations thereof.
  • the manipulated variable for increasing biomass is selected from the group consisting of feed rate, feed composition, air flow rate, air composition, stirring rate, pressure, and combinations thereof.
  • processes for increasing production of butanol from a recombinant microorganism during fermentation comprise (a) providing a fermentation medium and a recombinant microorganism comprising an engineered butanol biosynthetic pathway in a fermentor whereby fermentation occurs under variable conditions wherein one or more of said conditions has a set point which the condition may be controlled to whereby a controlled variable is provided and wherein one or more variable fermentation conditions may be manipulated to adjust the controlled variable; (b) monitoring one or more controlled variables; and (c) adjusting one or more variable fermentation conditions to maintain the set point of the controlled variable, wherein the butanol production of the recombinant microorganism is increased during fermentation.
  • the set point of the controlled variable is dynamically adjusted based on byproduct level.
  • the processes comprise (a) providing a fermentation medium in a first fermentor comprising a recombinant microorganism comprising an engineered butanol biosynthetic pathway; (b) monitoring a controlled variable of the first fermentor, wherein the controlled variable has a set point; (c) adjusting a manipulated variable of the first fermentor based on the set point of the monitored controlled variable; (d) providing a fermentation medium in a second fermentor comprising a recombinant microorganism comprising an engineered butanol biosynthethic pathway; (e) monitoring the controlled variable of the second fermentor, wherein the controlled variable has a set point; and (f) adjusting the manipulated variable of the second fermentor based on the adjustment made to the first fermentor; wherein the adjustment of the manipulated variable increases butanol production of the recombinant microorganism in the second fermentor.
  • the first fermentor is smaller than the second fermentor.
  • the set point of the controlled variable of the first fermentor is equal to the set point of the controlled variable of the second fermentor.
  • the controlled variable for increasing butanol production is selected from the group consisting of respiratory quotient (RQ), specific oxygen uptake rate (Sp. OUR), specific carbon dioxide evolution rate (Sp. CER), pH, carbon dioxide to butanol production ratio, specific butanol production rate, oxygen partial pressure (p0 2 ), rate of heat generation, temperature, byproduct concentration, and combinations thereof.
  • the manipulated variable for increasing butanol production is selected from the group consisting of feed rate, feed composition, air flow rate, air composition, stirring rate, pressure, and combinations thereof.
  • the recombinant microorganism can comprise a butanol biosynthetic pathway selected from the group consisting of (a) a 1 -butanol biosynthetic pathway; (b) a 2-butanol biosynthetic pathway; and (c) an isobutanol biosynthetic pathway.
  • the 1 -butanol biosynthetic pathway comprises at least one polypeptide that performs one of the following substrate to product conversions: (a) acetyl-CoA to acetoacetyl-CoA, as catalyzed by acetyl-CoA acetyltransferase; (b) acetoacetyl-CoA to 3- hydroxybutyryl-CoA, as catalyzed by 3-hydroxybutyryl-CoA dehydrogenase; (c) 3- hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed by crotonase; (d) crotonyl-CoA to butyryl- CoA, as catalyzed by butyryl-CoA dehydrogenase; (e) butyryl-CoA to butyraldehyde, as catalyzed by butyraldehyde dehydrogenase; and (
  • the 2-butanol biosynthetic pathway comprises at least one polypeptide that performs one of the following substrate to product conversions: (a) pyruvate to alpha- acetolactate, as catalyzed by acetolactate synthase; (b) alpha-acetolactate to acetoin, as catalyzed by acetolactate decarboxylase; (c) acetoin to 2,3-butanediol, as catalyzed by butanediol dehydrogenase; (d) 2,3-butanediol to 2-butanone, as catalyzed by butanediol dehydratase; and (e)
  • the isobutanol biosynthetic pathway comprises at least one
  • polypeptide that performs one of the following substrate to product conversions: (a) pyruvate to acetolactate, as catalyzed by acetolactate synthase; (b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by ketol-acid reductoisomerase; (c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, as catalyzed by dihydroxyacid dehydratase; (d) a-ketoisovalerate to isobutyraldehyde, as catalyzed by a branched chain keto acid decarboxylase; and (e) isobutyraldehyde to isobutanol, as catalyzed by branched-chain alcohol dehydrogenase.
  • the recombinant microorganism is from a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,
  • compositions, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive or and not to an 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” as used herein 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 claims as presented or as later amended and supplemented, or in the specification.
  • 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. In one embodiment, the term “about” means within 10% of the reported numerical value, or within 5% of the reported numerical value.
  • butanol refers to the butanol isomers 1-butanol (1-
  • butanol can include, but are not limited to, fuels (e.g., bio fuels), a fuel additive, an alcohol used for the production of esters that can be used as diesel or biodiesel fuel, as a chemical in the plastics industry, an ingredient in formulated products such as cosmetics, and a chemical intermediate.
  • fuels e.g., bio fuels
  • a fuel additive e.g., an alcohol used for the production of esters that can be used as diesel or biodiesel fuel
  • an ingredient in formulated products such as cosmetics
  • butanol may also be used as a solvent for paints, coatings, varnishes, resins, gums, dyes, fats, waxes, resins, shellac, rubbers, and alkaloids.
  • bio-produced means that the molecule (e.g., butanol) is produced from a renewable source (e.g., the molecule can be produced during a fermentation process from a renewable feedstock).
  • a renewable source e.g., the molecule can be produced during a fermentation process from a renewable feedstock.
  • bio-produced isobutanol can be isobutanol produced by a fermentation process from a renewable feedstock.
  • Molecules produced from a renewable source can further be defined by the 14 C/ 12 C isotope ratio.
  • a 14 C/ 12 C isotope ratio in range of from 1 :0 to greater than 0: 1 indicates a bio-produced molecule, whereas a ratio of 0: 1 indicates that the molecule is fossil derived.
  • a recombinant host cell comprising an "engineered alcohol production pathway"
  • a host cell refers to a host cell containing a modified pathway that produces alcohol in a manner different than that normally present in the host cell. Such differences include production of an alcohol not typically produced by the host cell, or increased or more efficient production.
  • heterologous biosynthetic pathway refers to an enzyme pathway to produce a product in which at least one of the enzymes is not endogenous to the host cell containing the biosynthetic pathway.
  • butanol biosynthetic pathway refers to the enzymatic pathway to produce 1 -butanol, 2-butanol, or isobutanol.
  • 1 -butanol biosynthetic pathway refers to an enzymatic pathway to produce 1 -butanol.
  • a "1 -butanol biosynthetic pathway” can refer to an enzyme pathway to produce 1 -butanol from acetyl-coenzyme A (acetyl-CoA).
  • acetyl-CoA acetyl-CoA
  • 1 -butanol biosynthetic pathways are disclosed in U.S. Patent Application Publication No. 2008/0182308 and
  • 2-butanol biosynthetic pathway refers to an enzymatic pathway to produce 2- butanol.
  • a "2-butnaol biosynthetic pathway” can refer to an enzyme pathway to produce 2-butanol from pyruvate.
  • 2-butanol biosynthetic pathways are disclosed in U.S. Patent No. 8,206,970, U.S. Patent Application Publication No. 2007/0292927, International Publication Nos. WO 2007/130518 and WO 2007/130521 , which are herein incorporated by reference in their entireties.
  • isobutanol biosynthetic pathway refers to an enzymatic pathway to produce isobutanol.
  • An “isobutanol biosynthetic pathway” can refer to an enzyme pathway to produce isobutanol from pyruvate.
  • isobutanol biosynthetic pathways are disclosed in U.S. Patent No. 7,851 , 188, U.S. Application Publication No. 2007/0092957, and International Publication No. WO 2007/050671 , which are herein incorporated by reference in their entireties. From time to time "isobutanol biosynthetic pathway” is used synonymously with “isobutanol production pathway.”
  • extract refers to one or more organic solvents which can be used to extract butanol from a fermentation broth.
  • the term "effective titer" as used herein, refers to the total amount of a particular alcohol (e.g. , butanol) produced by fermentation per liter of fermentation medium.
  • the total amount of butanol includes: (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; and (iii) the amount of butanol recovered from the gas phase, if gas stripping is used.
  • the term "effective rate" as used herein, refers to the total amount of butanol produced by fermentation per liter of fermentation medium per hour of fermentation.
  • the term "specific productivity" refers to the grams of butanol isomer produced per gram of dry cell weight of cells per unit time.
  • growth rate refers to the rate at which the microorganisms grow in the culture medium.
  • the growth rate of the recombinant microorganisms can be monitored, for example, by measuring the optical density at 600 nanometers.
  • the doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate.
  • separation is synonymous with “recovery” and refers to removing a chemical compound from an initial mixture to obtain the compound in greater purity or at a higher concentration than the purity or concentration of the compound in the initial mixture.
  • aqueous phase refers to the aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.
  • fermentation broth then specifically refers to the aqueous phase in biphasic fermentative extraction.
  • organic phase refers to the non-aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.
  • carbon substrate or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
  • Non-limiting examples of carbon substrates include, but are not limited to, monosaccharides, disaccharides, oligosaccharides, polysaccharides, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, glucose, fructose, lactose, sucrose, xylose, arabinose, dextrose, cellulose, methane, amino acids, or mixtures thereof.
  • Frermentation broth as used herein means the mixture of water, sugars
  • reactionable carbon sources dissolved solids (if present), microorganisms producing alcohol, product alcohol and all other constituents of the material in which product alcohol is being made by the reaction of sugars to alcohol, water and carbon dioxide (C0 2 ) by the microorganisms present.
  • reaction medium and “fermented mixture” can be used synonymously with “fermentation broth.”
  • a fermentor refers to any container, containers, or apparatus that are used to ferment a substrate.
  • a fermentor can contain a fermentation medium and
  • microorganism capable of fermentation.
  • the term "fermentation vessel” refers to the vessel in which the fermentation reaction is carried out whereby alcohol such as butanol is made from sugars. "Fermentor” can be used herein interchangeable with “fermentation vessel.”
  • fixation product includes any desired product of interest, including, but not limited to 1 -butanol, isobutanol, etc.
  • Biomass refers to a natural product containing a hydrolysable starch that provides a fermentable sugar, including any cellulosic or lignocellulosic material and materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides, disaccharides, and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipids. Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source. For example, biomass can 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 include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, 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.
  • feedstock as used herein means a product containing a fermentable carbon source. Suitable feedstock include, but are not limited to, rye, wheat, corn, corn mash, cane, cane mash, sugar cane, barley, cellulosic material, lignocellulosic material, and mixtures thereof.
  • biomass refers to the mass of the culture, e.g., the amount of recombinant microorganisms, typically provided in units of grams per liter (g/1) dry cell weight (dew).
  • biomass yield refers to the ratio of microorganism biomass produced (i.e., cell biomass production) to carbon substrate consumed.
  • aerobic conditions means growth conditions in the presence of oxygen.
  • microaerobic conditions means growth conditions with low levels of oxygen (i.e., below normal atmospheric oxygen levels).
  • anaerobic conditions means growth conditions in the absence of oxygen.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • a polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5 ' and 3 ' sequences and the coding sequences.
  • the polynucleotide can be composed of any
  • polyribonucleotide or polydeoxyribonucleotide which can be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • Polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
  • a polynucleotide sequence can be referred to as "isolated,” in which it has been removed from its native environment.
  • a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having ALS activity 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. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • 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.”
  • reduced activity refers to any measurable decrease in a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the reduced activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein.
  • a reduced activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.
  • Reduced activity of an enzyme refers to down-regulation, whether partial or total, of the activity of the enzyme as compared to the activity of the wildtype enzyme.
  • Down-regulation may occur when a native gene has a "disruption" or "modification,” referring to an insertion, deletion, or targeted mutation within a portion of that gene, that results in e.g. , a complete gene knockout such that the gene is deleted from the genome and no protein is translated or a translated subunit protein having an insertion, deletion, amino acid substitution or other targeted mutation.
  • the location of the modification in the protein may be, for example, within the N-terminal portion of the protein or within the C-terminal portion of the protein.
  • the modified protein will have impaired activity with respect to the protein that was not disrupted, and can be non-functional.
  • Reduced activity in an enzyme could also result via manipulating the upstream regulatory domains or by use of sense, antisense or RNAi technology, etc.
  • Another mechanism of reducing activity of an enzyme is introduction of a mutation that alters kinetic properties of the enzyme (e.g., reducing the affinity for a substrate, lowering the k cat , etc.).
  • an eliminated activity refers to the complete abolishment of a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity.
  • a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein.
  • An eliminated activity includes a biological activity of a polypeptide that is not measurable when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity.
  • An eliminated activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.
  • PDC- refers to a cell that has a genetic modification to inactivate or reduce expression of a gene encoding pyruvate decarboxylase (PDC) so that the cell substantially or completely lacks pyruvate decarboxylase enzyme activity. If the cell has more than one expressed (active) PDC gene, then each of the active PDC genes can be inactivated or have minimal expression thereby producing a PDC- cell.
  • PDC pyruvate decarboxylase
  • the term "specific activity" as used herein is defined as the units of activity in a given amount of protein. Thus, the specific activity is not directly measured but is calculated by dividing 1) the activity in units/ml of the enzyme sample by 2) the concentration of protein in that sample, so the specific activity is expressed as units/mg, where an enzyme unit is defined as moles of product formed/minute.
  • the specific activity of a sample of pure, fully active enzyme is a characteristic of that enzyme.
  • the specific activity of a sample of a mixture of proteins is a measure of the relative fraction of protein in that sample that is composed of the active enzyme of interest.
  • isolated nucleic acid molecule isolated nucleic acid fragment
  • genetic construct are used interchangeably and mean a polymer of RNA or DNA that is single or double-stranded, optionally containing synthetic, non natural or altered nucleotide bases.
  • An isolated nucleic acid fragment in the form of a polymer of DNA can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • amino acid refers to the basic chemical structural unit of a protein or polypeptide.
  • the abbreviations in Table 1 are used herein to identify specific amino acids.
  • the term "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.
  • “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of a
  • a "foreign” gene refers to a gene not normally found in the host microorganism, but that is introduced into the host microorganism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native microorganism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • “native” refers to the form of a polynucleotide, gene, or polypeptide as found in nature with its own regulatory sequences, if present.
  • coding sequence or “coding region” refers to a DNA sequence that encodes for a specific amino acid sequence.
  • 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.
  • Endogenous 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 transcribed and translated from a native polynucleotide or gene in its natural location in the genome of an organism.
  • heterologous when used in reference to a polynucleotide, a gene, or a polypeptide refers to a polynucleotide, gene, or polypeptide not normally found in the host organism. "Heterologous” also 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, e.g., not in its natural location in the organism's genome. The heterologous polynucleotide or gene can be introduced into the host organism by, e.g., gene transfer.
  • a heterologous gene can include a native coding region with non-native regulatory regions that is reintroduced into the native host.
  • a 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.
  • a "heterologous" polypeptide or polynucleotide can also include an engineered polypeptide or polynucleotide that comprises a difference from the "native" polypeptide or polynucleotide, e.g., a point mutation within the endogenous polynucleotide can result in the production of a
  • heterologous polypeptide As used herein a “chimeric gene,” a “foreign gene,” and a
  • transgene can all be examples of “heterologous” genes.
  • a "transgene” is a gene that has been introduced into the genome by a
  • recombinant genetic expression element refers to a nucleic acid fragment that expresses one or more specific proteins, including regulatory sequences preceding (5 ' non-coding sequences) and following (3 ' termination sequences) coding sequences for the proteins.
  • a chimeric gene is a recombinant genetic expression element.
  • the coding regions of an operon can form a recombinant genetic expression element, along with an operably linked promoter and termination region.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression can also refer to translation of mRNA into a polypeptide.
  • heterologous gene refers to expression that is higher than endogenous expression of the same or related gene.
  • a heterologous gene is overexpressed if its expression is higher than that of a comparable endogenous gene.
  • overexpression refers to an increase in the level of nucleic acid or protein in a host cell.
  • overexpression can result from increasing the level of transcription or translation of an endogenous sequence in a host cell or can result from the introduction of a heterologous sequence into a host cell.
  • Overexpression can also result from increasing the stability of a nucleic acid or protein sequence.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host microorganism, resulting in genetically stable inheritance.
  • Host microorganisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” microorganisms.
  • Plasmid refers 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 can 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 vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
  • 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” can be used instead of, or interchangeably with any of these terms.
  • a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • variant and mutant are synonymous and refer to a polypeptide differing from a specifically recited polypeptide by one or more amino acid insertions, deletions, mutations, and substitutions, created using, e.g., recombinant DNA techniques, such as mutagenesis.
  • Guidance in determining which amino acid residues can be replaced, added, or deleted without abolishing activities of interest can be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, e.g., yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.
  • Engineerered polypeptide refers to a polypeptide that is synthetic, i.e., differing in some manner from a polypeptide found in nature.
  • polynucleotide 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 silent changes which produce various restriction sites, can be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence can be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.
  • mutations can be used to reduce or eliminate expression of a target protein and include, but are not limited to, deletion of the entire gene or a portion of the gene, inserting a DNA fragment into the gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less enzymatically active protein is expressed.
  • Amino acid substitutions can be 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 the 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 can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, 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” can be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed can 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.
  • biomass growth phase biomass growth is optimized.
  • the biomass growth phase can be run, for example, under aerobic glucose limited conditions (e.g., less than 5 g/L of glucose in at least 5% 0 2 ) in order to minimize the production of byproducts.
  • the biomass growth phase can comprise a transition phase, which can occur prior to the second phase of the fermentation.
  • butanol production phase In the second phase, referred to herein as a "butanol production phase,” butanol (e.g., isobutanol) production is optimized.
  • the butanol production phase can be run, for example, under microaerobic to anaerobic conditions of excess glucose (e.g., about 5 to about 30 g/L of glucose in about 0.01% 0 2 to 5% 0 2 for microaerobic conditions and 0% 0 2 for anaerobic conditions) in order to maximize butanol production.
  • the biomass growth phase can comprise a transition phase, which can occur prior to the butanol production phase of the fermentation.
  • the transition phase can change from aerobic conditions to microaerobic conditions while transitioning from glucose limited conditions to glucose excess conditions.
  • the transition phase can allow for the induction of the butanol pathway enzymes before transitioning the biocatalyst for butanol production. Controlled and manipulated variables for use in each of these phases are provided herein
  • the concentration of glucose in the butanol production phase can be, for example, about 3 to about 50 g/L, about 3 to about 45 g/L, about 3 to about 40 g/L, about 3 to about 35 g/L, about 3 to about 30 g/L about 3 to about 25 g/L, about 3 to about 20 g/L, about 3 to about 15 g/L, about 3 to about 10 g/L, about 5 to about 50 g/L, about 5 to about 45 g/L, about 5 to about 40 g/L, about 5 to about 35 g/L, about 5 to about 30 g/L, about 5 to about 25 g/L, about 5 to about 20 g/L, about 5 to about 15 g/L, about 5 to about 10 g/L, about 10 to about 50 g/L, about 10 to about 45 g/L, about 10 to about 40 g/L, about 10 to about 35 g/L, about 10 to about 30 g/L, about 10 to about 30 g
  • concentration of glucose in the butanol production phase can also be, for example, about 15 to about 50 g/L, about 15 to about 45 g/L, about 15 to about 40 g/L, about 15 to about 30 g/L, about 15 to about 25 g/L or about 15 to about 20 g/L.
  • the controlled variable in the biomass growth phase of fermentation is selected from the group consisting of respiratory quotient (RQ), specific oxygen uptake rate (Sp. OUR), specific carbon dioxide evolution rate (Sp. CER), pH, specific growth rate, oxygen partial pressure (p0 2 ), rate of heat generation, specific isobutanol production rate, specific byproduct (e.g., isobutyric acid) production rate, and combinations thereof.
  • RQ respiratory quotient
  • Sp. OUR specific oxygen uptake rate
  • Sp. CER specific carbon dioxide evolution rate
  • pH specific growth rate
  • oxygen partial pressure p0 2
  • rate of heat generation specific isobutanol production rate
  • specific byproduct e.g., isobutyric acid
  • the controlled variable in the butanol production phase of fermentation is selected from the group consisting of respiratory quotient (RQ), specific oxygen uptake rate (Sp. OUR), specific carbon dioxide evolution rate (Sp. CER), pH, carbon dioxide to butanol production ratio, specific butanol production rate, oxygen partial pressure (p0 2 ), rate of heat generation, specific byproduct production rate, and combinations thereof.
  • RQ respiratory quotient
  • Sp. OUR specific oxygen uptake rate
  • Sp. CER specific carbon dioxide evolution rate
  • pH carbon dioxide to butanol production ratio
  • specific butanol production rate specific butanol production rate
  • oxygen partial pressure (p0 2 ) oxygen partial pressure
  • rate of heat generation specific byproduct production rate, and combinations thereof.
  • the controlled variable in the butanol production phase of fermentation is RQ.
  • the controlled variable can have a "set point,” which refers to a value or range of values in which it is beneficial to maintain the controlled variable.
  • the set point of the controlled variable is constant throughout a biomass growth phase.
  • the set point of the controlled variable is constant throughout a butanol production phase.
  • the set point is one constant value or range of values throughout a biomass growth phase and a second constant value or range of values throughout a butanol production phase.
  • the set point of the controlled variable is dynamically adjusted during a biomass growth phase and/or a butanol production phase, e.g., based on byproduct level.
  • the controlled variable in the biomass growth phase can be dynamically adjusted based on the specific isobutyric acid production rate and/or the specific isobutanol production rate during the growth phase.
  • the controlled variable is RQ (e.g., RQ set point is 1.2)
  • the set point for RQ can decrease (e.g., from 1.2 to 1.1) to ensure increased biomass growth.
  • the controlled variable in the butanol production phase can be dynamically adjusted based on the carbon dioxide (C0 2 ) to isobutanol ratio and/or the isobutanol to glucose ratio.
  • RQ e.g., RQ set point is 10
  • the set point for RQ can be decreased (e.g., from 10 to 8) to ensure increased butanol production.
  • Specific carbon dioxide evolution rate (Sp. CER, millimoles/g/hr) and specific oxygen uptake rate (Sp. OUR, millimoles/g/hr) can be calculated by measuring flow rate, inlet and exhaust gas composition of air (C0 2 , 0 2 etc.), using, for example, mass spectrometry and/or cell density measurements.
  • Specific carbon dioxide evolution rate is the ratio of carbon dioxide produced (air flow rate multiplied by difference between outlet and inlet carbon dioxide concentration) to cell density per unit time.
  • Specific oxygen uptake rate is the ratio of oxygen consumed (air flow rate multiplied by difference between inlet and outlet oxygen concentration) to cell density.
  • Sp. OUR is measured directly, e.g., using exhaust gas analysis.
  • Sp. CER is measured directly, e.g., using exhaust gas analysis.
  • the Sp. OUR set point during a biomass growth phase is about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, or about 3 to about 5 millimoles per grams of cell per hour.
  • the Sp. OUR set point during a butanol production phase is
  • 0 to about 1.5 0 to about 1.3, 0 to about 1.1, 0 to about 1.0, 0 to about 0.9, 0 to about 0.8, 0 to about 0.7, 0 to about 0.6, 0 to about 0.5, about 0.05 to about 1.5, about 0.05 to about 1.3, about 0.05 to about 1.1, about 0.05 to about 1.0, about 0.05 to about 0.9, about 0.05 to about 0.8, about 0.05 to about 0.6, or about 0.05 to about 0.5 millimoles per grams of cell per hour.
  • the Sp. CER set point during a biomass growth phase is about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5.5, about 1 to about 4, about 1 to about 3, about 2.5 to about 10, about 2.5 to about 9, about 2.5 to about 8, about 2.5 to about 7, about 2.5 to about 6, about 2.5 to 5, about 2.5 to about 4, about 2.5 to about 3, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to 5, about 2 to about 4, or about 2 to about 3 millimoles per grams of cells per hour (mmol/g cell/hr).
  • the Sp. CER set point during a butanol production phase is about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3.5, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 2 to about 3.5, about 2.5 to about 10, about 2.5 to about 9, about 2.5 to about 8, about 2.5 to about 7, about 2.5 to about 6, about 2.5 to about 5, about 2.5 to about 4, about 2.5 to about 3, or about 2.5 to about 3.5 millimoles per grams of cell per hour (mmol/g cell/hr).
  • Respiratory quotient is ratio of CER and OUR. Only the inlet and outlet gas composition from mass spectrometry are required to calculate RQ for a given constant air flow rate.
  • RQ is used as a control variable that couples the oxygen uptake rate with the carbon flux through the bioreactor system.
  • RQ is intrinsically independent of scale. RQ can be measured, for example, using exhaust gas analysis.
  • the RQ set point during a biomass growth phase is about
  • the RQ set point during the transition phase of the biomass growth phase can be about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 1.5 to about 2, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 2.5 to about 5, about 2.5 to about 4, about 2.5 to about 3, about 3 to about 5, about 3 to about 4, about 3.5 to about 5, about 3.5 to about 4.
  • the RQ set point during a butanol production phase is about 2 to about 100, about 2 to about 75, about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 4 to about 75, about 4 to about 100, about 4 to about 50, about 4 to about 40, about 4 to about 30, about 4 to about 20, about 4 to about 10, about 5 to about 100, about 5 to about 75, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 10, about 6 to about 100, about 6 to about 75, about 6 to about 50, about 6 to about 40, about 6 to about 30, about 6 to about 20, or about 6 to about 10.
  • the pH set point during a biomass growth phase is about
  • the pH set point during a butanol production phase is about
  • Oxygen partial pressure (p0 2 ) can be measured directly using p0 2 probe.
  • the p0 2 set point during a biomass growth phase is about 5 to about 40%, about 5 to about 35%, about 5 to about 30%>, about 5 to about 25%, about 5 to about 20%, about 5 to about 15%), or about 5 to about 10%>, about 10 to about 40%>, about 10 to about 35%, about 10 to about 30%), about 10 to about 25%, about 10 to about 20%>, or about 10 to about 15%.
  • the p0 2 set point during a butanol production phase is 0 to about 10%, 0 to about 9 %, 0 to about 8%, 0 to about 7%, 0 to about 6%, 0 to about 5 %, 0 to about 4 %), 0 to about 3 %, 0 to about 2 %, or 0 to about 1%.
  • Specific growth rate can be estimated from cell density or OD 6 oo data or indirectly from OUR data from empirical model.
  • Rate of heat generation can be estimated from rate of heat removal from chiller data such as chiller flow rate, and/or the inlet and outlet temperature from chiller.
  • the specific butanol, e.g., isobutanol, and isobutyric production rates can be estimated from HPLC/GC data and/or exhaust gas analysis by mass spectrometry and cell density data.
  • the specific butanol production rate during a biomass growth phase is about 0 to about 0.01 grams per grams of cells per hour (g/g cell/hr), about 0 to about 0.005 g/g cell/hr, about 0 to about 0.0025 g/g cell/hr, or about 0.00025 to about 0.001 g/g cell/hr.
  • the specific butanol production rate during a butanol production phase is about 0.1 to about 0.3 g/g cell/hr, about 0.1 to about 0.4 g/g cell/hr , about 0.1 to about 0.5 g/g cell/hr , about 0.1 to about 0.6 g/g cell/hr , about 0.1 to about 0.7 g/g cell/hr , about 0.1 to about 0.8 g/g cell/hr, about 0.1 to about 0.9 g/g cell/hr, about 0.1 to about 1 g/g cell/hr, about 0.2 to about 0.3 g/g cell/hr, about 0.2 to about 0.4 g/g cell/hr, about 0.2 to about 0.5 g/g cell/hr, about 0.2 to about 0.6 g/g cell/hr, about 0.2 to about 0.7 g/g cell/hr, about 0.2 to about 0.8 g/g cell/hr, about
  • the controlled variable is a byproduct
  • the byproduct is selected from the group consisting of isobutyrate, isobutyric acid, dihydroxyisovalerate, ketoisovalerate, isobutyraldehyde, lactate, acetolactate, acetate, formate, glycerol, and combinations thereof.
  • the byproduct is isobutyric acid.
  • the specific isobutyric acid production rate during a biomass growth phase is about 0.001 to about 0.2 g/g cell/hr, about 0.001 to about 0.01 g/g cell/hr, about 0.001 to about 0.005 g/g cell/hr, about 0.0025 to about 0.02 g/g cell/hr, about 0.0025 to about 0.01 g/g cell/hr, or about 0.0025 to about 0.005 g/g cell/hr.
  • the carbon dioxide to butanol production molar ratio set point during a butanol production phase is about 2 to about 20, about 2 to about 10, about 2 to about 5, about 2 to about 3, about 2 to about 2.5, about 2 to about 2.25, about 1.5 to about 2, about 1.5 to about 10, about 1.5 to about 5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2.25, or about 1.5 to about 2.
  • the temperature set point during a butanol production phase is about 30 degrees Celsius. In some embodiments, the temperature set point during butanol production phase is about 20 to about 38 degrees Celsius, about 20 to about 35 degrees Celsius, about 20 to about 32 degrees Celsius, about 20 to about 30 degrees Celsius, about 24 to about 38 degrees Celsius, about 24 to about 35 degrees Celsius, about 24 to about 32 degrees Celsius, about 24 to about 30 degrees Celsius, about 25 to about 38 degrees Celsius, about 25 to about 35 degrees Celsius, about 25 to about 32 degrees Celsius, about 25 to about 30 degrees Celsius, about 28 to about 38 degrees Celsius, about 28 to about 35 degrees Celsius, about 28 to about 32 degrees Celsius, about 28 to about 30 degrees Celsius, about 30 to about 38 degrees Celsius, about 30 to about 35 degrees Celsius, or about 30 to about 32 degrees Celsius.
  • the manipulated variable in the biomass growth phase of fermentation is selected from the group consisting of feed rate, feed composition, air flow rate, air composition, stirring rate, pressure, and combinations thereof.
  • the manipulated variable in the butanol production phase of fermentation is selected from the group consisting of feed rate, feed composition, air flow rate, air composition, stirring rate, pressure, and combinations thereof.
  • Table 2 (below) indicates how particular manipulated variables can be adjusted in response to particular controlled variables.
  • Table 2 Interaction matrix of manipulation and controlled variables
  • '+' sign indicates manipulation variable and controlled variables act in same direction, while the '-' sign indicates manipulation and controlled variables act in inverse direction.
  • the '+/-' sign indicates manipulation and controlled variable can act in the same direction or in inverse directions, depending upon operation condition.
  • RQ can be measured during the biomass growth phase and/or the butanol production phase of fermentation.
  • RQ can be controlled, e.g., maintained at a set-point, in either or both of these phases by, for example, changing carbon feed rate, feed composition, air flow rate, air composition, stirring rate, etc.
  • RQ can be increased by increasing the carbon feed rate, increasing the feed composition (e.g., % glucose in feed, glucose to ethanol ratio in feed), decreasing the air flow rate, decreasing inlet gas composition (e.g., air to nitrogen ratio, air to oxygen ratio, etc.), decreasing the stirring rate, and/or a combination of these manipulations and/or those shown in Table 2.
  • feed composition e.g., % glucose in feed, glucose to ethanol ratio in feed
  • inlet gas composition e.g., air to nitrogen ratio, air to oxygen ratio, etc.
  • the carbon feed rate can be increased (e.g., from 2 to 2.5 mL/hr), the feed composition can be increased (e.g., 40 to 50% glucose in feed, 10 to 12 glucose to ethanol ratio in feed), the air flow rate can be decreased (e.g, from 1 to 0.8 liter per minute (LPM)), the inlet gas composition can be decreased (e.g., air to nitrogen ratio decreased from 10 to 8, air to oxygen ratio increased from 10 to 12), the stirring rate can be decreased (e.g., from 500 to 400 rotations per minute (rpm)), and/or any combination of these manipulated variables can be changed.
  • LPM 0.8 liter per minute
  • RQ can be decreased by decreasing the carbon feed rate, decreasing the feed composition (e.g., % glucose in feed, glucose to ethanol ratio in feed), increasing air flow rate, increasing inlet gas composition (e.g., air to nitrogen ratio, air to oxygen ratio, etc.), increasing stirring rate, and/or a combination of these manipulations and/or those shown in Table 2.
  • decreasing the feed composition e.g., % glucose in feed, glucose to ethanol ratio in feed
  • increasing air flow rate e.g., increasing inlet gas composition (e.g., air to nitrogen ratio, air to oxygen ratio, etc.), increasing stirring rate, and/or a combination of these manipulations and/or those shown in Table 2.
  • the carbon feed rate can be decreased (e.g., from 2 to 1.5 mL/hr)
  • the feed composition can be decreased (e.g., 40 to 30% glucose in feed, 10 to 8 glucose to ethanol ratio in feed)
  • the air flow rate can be increased (e.g, from 1 to 1.2 LPM)
  • the inlet gas composition can be increased (e.g., air to nitrogen ratio increased from 10 to 12, air to oxygen ratio decreased from 10 to 8)
  • the stirring rate can be increased (e.g., from 500 to 600 rpm), and/or any combination of these manipulated variables can be changed.
  • a biomass generation protocol can be established to maximize biomass yield and productivity in the aerobic growth phase and optimize butanol (e.g., isobutanol) yield and productivity in the microaerobic to anaerobic production phase using RQ as a controlled variable.
  • RQ e.g., isobutanol
  • the range of optimal RQ can depend on several parameters such as cell density, cell yield, cell productivity, and yield of byproducts such as isobutanol and isobutyric acid, which can affect cell growth rate and reduce cell yield.
  • the carbon flux can be optimally funneled for biomass generation by performing carbon limited fed-batch condition with RQ as a control variable.
  • the carbon flux can be optimally diverted through the butanol (e.g., isobutanol) pathway by maintaining RQs that maximize butanol yield in a butanologen under microaerobic to anaerobic conditions.
  • butanol e.g., isobutanol
  • the recombinant microorganism comprising an engineered butanol biosynthetic pathway can be grown aerobically under carbon (e.g., glucose) limited conditions, and butanol production is under microaerobic condition with carbon limited/excess conditions. The transition between aerobic growth and microaerobic production can be done under optimal aerobic carbon excess conditions.
  • cells can be grown aerobically under glucose limited condition.
  • the feed rates are manipulated such that RQ is maintained at about 0.67 to about 1.5, such that maximum sugar carbon flux is diverted to biomass generation.
  • RQ can be about 0.9 to about 1.2 or about 0.95 to about 1.05.
  • RQ can drop below set point due to at least increased OUR of cells at a particular feed flow rate. Therefore, feeding can be increased to match the RQ set point.
  • the feed rate can increase exponentially to match the RQ set point, which, in turn, can match the exponential growth of cells.
  • the cells can be primed with glucose excess and or optimal nutrition conditions to minimize lag time in the production stage, i.e., to activate butanol (e.g., isobutanol) pathway enzymes and recondition cellular metabolism suitable for optimal isobutanol production.
  • butanol e.g., isobutanol
  • the glucose excess condition during the transition phase can be achieved in a fermenter by increasing the RQ set point to about 1.5 to about 3.0 under aerobic to microaerobic conditions, and the manipulated variable to achieve RQ can be carbon feed to prime the butanol pathway enzymes.
  • a suitable period of time is about 5 hours to about 20 hours or about 8 hours to about 15 hours.
  • microaerobic to anaerobic conditions can be maintained by increasing RQ to about 2 to infinity as per production condition requirements.
  • microaerobic conditions can overcome potential redox and/or energy imbalances of the recombinant microorganism, which can reduce pathway byproducts and improve butanol (e.g., isobutanol) yield.
  • aerobic conditions after a certain threshold value can result in a decrease in butanol yield due to a carbon flux diversion to growth.
  • the degree of microaerobicity can depend on the recombinant microorganism and the optimal pathway redox balance.
  • the RQ is about 2 to about 50, about 4 to about 20, or about 5 to about 10.
  • the culture can be switched to production phase through a change in aeration conditions by changing from aerobic conditions to microaerobic/anaerobic conditions.
  • the metabolic pathways of microorganisms may be genetically modified to produce butanol. These pathways may also be modified to reduce or eliminate undesired metabolites, and thereby improve yield of the product alcohol.
  • the production of butanol by a microorganism is disclosed, for example, in U.S. Patent Nos. 7,851,188; 7,993,889; 8,178,328, 8,206,970; U.S. Patent Application Publication Nos.
  • the microorganism is genetically modified to comprise a butanol biosynthetic pathway or a biosynthetic pathway for a butanol isomer, such as 1 -butanol, 2-butanol, or isobutanol.
  • At least one, at least two, at least three, at least four, or at least five polypeptides catalyzing substrate to product conversions in the butanol biosynthetic pathway are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, all the polypeptides catalyzing substrate to product conversions of the butanol biosynthetic pathway are encoded by heterologous polynucleotides in the
  • microorganisms comprising a butanol biosynthetic pathway may further comprise one or more additional genetic modifications as disclosed in U.S. Patent Application Publication No. 2013/0071898, which is herein incorporated by reference in its entirety.
  • the microorganism may be bacteria, cyanobacteria, filamentous fungi, or yeasts.
  • Suitable microorganisms capable of producing product alcohol (e.g., butanol) via a biosynthetic pathway include a member of the genera Clostridium,
  • Zymomonas Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces, Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia, Trichosporon, Yamadazyma, or Saccharomyces.
  • recombinant microorganisms may be selected from the group consisting of Escherichia coli, Alcaligenes eutrophus, Bacillus lichenifonnis, Paenibacillus macerans, Rhodocuccus erythropolis,
  • the genetically modified microorganism is yeast.
  • the genetically modified microorganism is a crabtree-positive yeast selected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species of Candida.
  • Species of crabtree-positive yeast include, but are not limited to,
  • Saccharomyces cerevisiae Saccharomyces kluyveri, Schizosaccharomyces pombe,
  • Saccharomyces bayanus Saccharomyces mikitae, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces castelli, Zygosaccharomyces rouxii, Zygosaccharomyces bailli, and Candida glabrata.
  • the host cell is Saccharomyces cerevisiae. Saccharomyces cerevisiae 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.
  • the microorganism may be immobilized or encapsulated.
  • the microorganism may be immobilized or encapsulated using alginate, calcium alginate, or polyacrylamide gels, or through the induction of bio film formation onto a variety of high surface area support matrices such as diatomite, celite, diatomaceous earth, silica gels, plastics, or resins.
  • ISPR may be used in combination with immobilized or encapsulated microorganisms. This combination may improve productivity such as specific volumetric productivity, metabolic rate, product alcohol yields, and tolerance to product alcohol.
  • immobilization and encapsulation may minimize the effects of the process conditions such as shearing on the microorganisms.
  • Biosynthetic pathways for the production of isobutanol include those as described by Donaldson et al. in U.S. Patent No. 7,851,188; U.S. Patent No. 7,993,388; and International Publication No. WO 2007/050671, which are incorporated herein by reference.
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • step b) the acetolactate from step a) to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase;
  • step b) the 2,3-dihydroxyisovalerate from step b) to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;
  • step d) the a-ketoisovalerate from step c) to isobutyraldehyde, which may be catalyzed, for example, by a branched-chain a-keto acid decarboxylase;
  • step d) the isobutyraldehyde from step d) to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • step b) the acetolactate from step a) to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase;
  • step b) the 2,3-dihydroxyisovalerate from step b) to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;
  • step c) the ⁇ -ketoisovalerate from step c) to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase;
  • step d) the valine from step d) to isobutylamine, which may be catalyzed, for example, by valine decarboxylase;
  • step f) the isobutylamine from step e) to isobutyraldehyde, which may be catalyzed by, for example, omega transaminase;
  • step f) the isobutyraldehyde from step f) to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.
  • the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetolactate which may be catalyzed, for example, by acetolactate synthase
  • acetolactate from step a) to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase
  • step b) the 2,3-dihydroxyisovalerate from step b) to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;
  • step d) the a-ketoisovalerate from step c) to isobutyryl-CoA, which may be catalyzed, for example, by branched-chain keto acid dehydrogenase;
  • step d) the isobutyryl-CoA from step d) to isobutyraldehyde, which may be catalyzed, for example, by acylating aldehyde dehydrogenase; and,
  • step f) the isobutyraldehyde from step e) to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.
  • Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Patent Application Publication No. 2008/0182308 and WO2007/041269, which are incorporated herein by reference.
  • the 1-butanol biosynthetic pathway comprises the following substrate to product conversions:
  • acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl-CoA acetyltransferase;
  • step b) the acetoacetyl-CoA from step a) to 3-hydroxybutyryl-CoA, which may be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;
  • step b) the 3-hydroxybutyryl-CoA from step b) to crotonyl-CoA, which may be catalyzed, for example, by crotonase;
  • step d) the crotonyl-CoA from step c) to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase;
  • butyryl-CoA from step d) to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase;
  • step f) the butyraldehyde from step e) to 1-butanol, which may be catalyzed, for example, by butanol dehydrogenase.
  • Biosynthetic pathways for the production of 2-butanol include those described by Donaldson et al. in U.S. Patent No. 8,206,970; U.S. Patent Application Publication Nos. 2007/0292927 and 2009/0155870; International Publication Nos. WO
  • the 2-butanol biosynthetic pathway comprises the following substrate to product conversions: a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;
  • step b) the alpha-acetolactate from step a) to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;
  • step b) the acetoin from step b) to 3-amino-2-butanol, which may be catalyzed, for example, acetoin aminase;
  • step d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase;
  • step d) the 3-amino-2-butanol phosphate from step d) to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase;
  • step f) the 2-butanone from step e) to 2-butanol, which may be catalyzed, for example, by butanol dehydrogenase.
  • the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:
  • a) pyruvate to alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • step b) the alpha-acetolactate from step a) to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;
  • step b) the acetoin to 2,3-butanediol from step b), which may be catalyzed, for example, by butanediol dehydrogenase;
  • step c) the 2,3-butanediol from step c) to 2-butanone, which may be catalyzed, for example, by diol dehydratase; and,
  • Biosynthetic pathways for the production of 2-butanone include those described in U.S. Patent No. 8,206,970 and U.S. Patent Application Publication Nos. 2007/0292927 and 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
  • acetoin which may be catalyzed, for example, by acetolactate decarboxylase
  • step b) the acetoin from step b) to 3-amino-2-butanol, which may be catalyzed, for example, acetoin aminase;
  • step d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase;
  • step d) the 3-amino-2-butanol phosphate from step d) to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase.
  • the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:
  • a) pyruvate to alpha-acetolactate which may be catalyzed, for example, by acetolactate synthase;
  • step b) the alpha-acetolactate from step a) to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;
  • step b) the acetoin from step b) to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase;
  • step d) the 2,3-butanediol from step c) to 2-butanone, which may be catalyzed, for example, by diol dehydratase.
  • acetolactate synthetase (abbreviated “ALS”) are used interchangeably herein to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and C0 2 .
  • 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, Z99122, NCBI (National Center for
  • a suitable acetolactate synthase can comprise SEQ ID NO: 123 from Bacillus subtilis.
  • ketol-acid reductoisomerase (“KARI")
  • acetohydroxy acid isomeroreductase "acetohydroxy acid reductoisomerase”
  • acetohydroxy acid reductoisomerase will be used interchangeably and refer to enzymes capable of catalyzing the reaction of (S)-acetolactate to 2,3- dihydroxyiso valerate.
  • 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, NC_000913), Saccharomyces cerevisiae (GenBank Nos: NP_013459, NC_001144),
  • KARIs include Anaerostipes caccae KARI variants "K9G9” (SEQ ID NO: 128), "K9D3" (SEQ ID NO: 129), and "K9JB4P" (SEQ ID NO: 127).
  • Ketol-acid reductoisomerase (KARI) enzymes are described in U.S. Patent Nos. 7,910,342 and 8,129,162; U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376,
  • KARIs disclosed therein are those from Lactococcus lactis (SEQ ID NO: 126), Anaerostipes caccae (SEQ ID NO: 125) and variants thereof; Pseudomonas fluorescens (SEQ ID NO: 124) and PF5 mutants thereof; Vibrio cholera, and Pseudomonas aeruginosa PAOl .
  • the KARI utilizes NADH.
  • the KARI utilizes NADPH.
  • the KARI utilizes NADH or NADPH.
  • DHAD DHAD
  • E. coli GenBank Nos: YP_026248, NC000913
  • Saccharomyces cerevisiae GenBank Nos:
  • NP_012550, NC 001142 M. maripaludis (GenBank Nos: CAF29874, BX957219), B. subtilis (GenBank Nos: CAB14105, Z99115), L. lactis (SEQ ID NO:131), and N. crassa.
  • DHADs dihydroxyacid dehydratases
  • KIVD 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, AY548760; CAG34226, AJ746364), Salmonella typhimurium (GenBank Nos: NP_461346, NC_003197), Clostridium acetobutylicum (GenBank Nos: NPJ49189, NC_001988), M. caseolyticus (SEQ ID NO: 134), and L. grayi (SEQ ID NO: 133).
  • a suitable branched-chain a-keto acid decarboxylases can comprise SEQ ID NO: 132 from Lactococcus lactis.
  • ADH branched-chain alcohol dehydrogenase
  • S. cerevisiae GenBank Nos: NP 010656, NC OOl 136,
  • Achromobacter xylosoxidans SEQ ID NO: 135).
  • Alcohol dehydrogenases also include horse liver ADH (SEQ ID NO: 136) and Beijerinkia indica ADH (SEQ ID NO: 137) (as described by U.S. Patent Application Publication No. 2011/0269199, which is incorporated herein by reference).
  • butanol dehydrogenase refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of isobutyraldehyde to isobutanol or the conversion of 2-butanone and 2-butanol.
  • Butanol dehydrogenases are a subset of a broad family of alcohol dehydrogenases.
  • Butanol dehydrogenase may be NAD- or NADP-dependent.
  • the NAD-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from
  • Rhodococcus ruber GenBank Nos: CAD36475, AJ491307).
  • the NADP dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169).
  • 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).
  • dehydrogenase also refers to an enzyme that catalyzes the conversion of butyraldehyde to 1- butanol, using either NADH or NADPH as cofactor.
  • Butanol dehydrogenases are 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_417-484, 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, Z99116; CAB14335, Z99116; CAB14334, Z99116; and CAB14337, Z99116
  • Pseudomonas putida GenBank Nos: AAA65614, M57613; AAA65615, M57613; AAA65617, M57613; and AAA65618, M57613
  • 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, AF157306), C.
  • acetobutylicum GenBank Nos: NPJ49325, NC_001988; NP 149199, NC_001988), P. putida (GenBank Nos: AAA89106, U13232), and Thermus thermophilus (GenBank Nos: YP_145486, NC 006461).
  • 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. coli (GenBank Nos: YP 026231, NC 000913) and Bacillus licheniformis (GenBank Nos: YP 093743, NC 006322). Examples of sources for glutamate-dependent enzymes include, but are not limited to, E.
  • valine dehydrogenase refers to an enzyme that catalyzes the conversion of ⁇ -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, NC_003888) and B. subtilis (GenBank Nos: CAB14339, Z99116).
  • 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, AY116644).
  • 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, AY330220), Ralstonia eutropha (GenBank Nos: YP_294474, NC_007347), Shewanella oneidensis (GenBank Nos: NP_719046, NC_004347), and P. putida (GenBank Nos: AAN66223, 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
  • 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, NC_003030), B. subtilis (GenBank NOs: AAB09614, U29084), Ralstonia eutropha (GenBank NOs: YP_294481, NC_007347), and Alcaligenes eutrophus (GenBank NOs: AAA21973, J04987).
  • 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, NC 000913), C. acetobutylicum (GenBank NOs: NP_349318, NC_003030), B. subtilis (GenBank NOs: CAB13705, Z99113), and
  • Aeromonas caviae GenBank NOs: BAA21816, D88825.
  • 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:
  • Streptomyces collinus GenBank NOs: AAA92890, U371357
  • Streptomyces coelicolor GenBank NOs: CAA22721, AL939127
  • 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).
  • 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, U67612; CAB59633, AJ246005
  • S coelicolor GenBank Nos: CAB70645, AL939123; CAB92663, AL939121
  • Streptomyces avermitilis GenBank Nos: NP_824008, NC_003155; NP_824637, NC_003155.
  • 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.
  • 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
  • dihydroxy acetone for example, include enzymes known as EC 2.7.1.29 (Garcia- Alles, et al., Biochemistry 45: 13037-13046, 2004).
  • 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.
  • aminobutanol phosphate phospholyase also called “amino alcohol diphosphate 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 ah, Biochem J. 754: 167-182, 1973).
  • U.S. Patent Application Publication 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-2-butanol 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 1- amino-2-propanol (Jones, et ah, supra).
  • U.S. Patent Application Publication No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.
  • butanediol dehydrogenase also known as “acetoin reductase” refers 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 also known as “diol dehydratase” or
  • propanediol dehydratase refers 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), AF 102064).
  • Other suitable 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
  • GenBank Nos: CAC82542 medium subunit
  • AJ297723 GenBank Nos: CAD01091 (small subunit), AJ297723
  • enzymes from Lactobacillus brevis particularly strains CNRZ 734 and CNRZ 735, Speranza, et ah, J. Agric. Food Chem. 45:3476-3480, 1997), and nucleotide sequences that encode the corresponding enzymes.
  • Methods of dial dehydratase gene isolation are well known in the art (e.g., U.S. Patent No. 5,686,276).
  • 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, CAA97705, CAA97091).
  • host cells comprising an isobutanol biosynthetic pathway as provided herein may further comprise one or more additional modifications.
  • U.S. Patent Application Publication No. 2009/0305363 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.
  • the host cells comprise modifications to reduce glycerol-3 -phosphate
  • modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway.
  • Other modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity.
  • acetolactate reductase activity refers to the activity of any polypeptide having the ability to catalyze the conversion of acetolactate to DHMB.
  • Such polypeptides can be determined by methods well known in the art and disclosed herein.
  • DHMB refers to 2,3-dihydroxy-2-methyl butyrate.
  • DHMB includes "fast DHMB,” which has the 2S, 3S configuration, and “slow DHMB,” which has the 2S, 3R configurate.
  • Kaneko et ah Phytochemistry 39: 115-120 (1995), which is herein incorporated by reference in its entirety and refers to fast DHMB as anglyceric acid and slow DHMB as tiglyceric acid.
  • the polypeptide having acetolactate reductase activity is YMR226C of Saccharomyces cerevisiae 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.
  • aldehyde dehydrogenase activity refers to any polypeptide having a biological function of an aldehyde dehydrogenase.
  • polypeptides include a polypeptide that catalyzes the oxidation (dehydrogenation) of aldehydes.
  • Such polypeptides include a polypeptide that catalyzes the conversion of isobutyraldehyde to isobutyric acid.
  • polypeptides also include a polypeptide that corresponds to Enzyme Commission Numbers EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5. Such polypeptides can be determined by methods well known in the art and disclosed herein.
  • aldehyde oxidase activity refers to any polypeptide having a biological function of an aldehyde oxidase.
  • Such polypeptides include a polypeptide that catalyzes carboxylic acids from aldehydes.
  • polypeptides include a polypeptide that catalyzes the conversion of isobutyraldehyde to isobutyric acid.
  • polypeptides also include a polypeptide that corresponds to Enzyme Commission Number EC 1.2.3.1. Such polypeptides can be determined by methods well known in the art and disclosed herein.
  • the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homolog thereof.
  • the pyruvate decarboxylase that is deleted or down-regulated is selected from the group consisting of: PDC1, PDC 5, PDC6, and combinations thereof.
  • the pyruvate decarboxylase is selected from PDC1 pyruvate decarboxylase from Saccharomyces cerevisiae, PDC5 pyruvate decarboxylase from Saccharomyces cerevisiae, PDC6 pyruvate decarboxylase from Saccharomyces cerevisiae, pyruvate decarboxylase from Candida glabrata, PDC1 pyruvate decarboxylase from Pichia stipites, PDC2 pyruvate decarboxylase from Pichia stipites, pyruvate decarboxylase from Kluveromyces lactis, pyruvate decarboxylase from Yarrowia lipo
  • host cells contain a deletion or down- regulation 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.
  • WIPO publication number WO 2011/103300 discloses recombinant host cells comprising (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.
  • the polypeptide affecting Fe-S cluster biosynthesis is encoded by AFT I, AFT2, FRA2, GRX3, or CCC1.
  • the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT 1 C293F.
  • host cells may comprise heterologous polynucleotides encoding a polypeptide with phosphoketolase activity and/or a heterologous polynucleotide encoding a polypeptide with phosphotransacetylase activity.
  • any particular nucleic acid molecule or polypeptide may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence described herein.
  • the term "percent identity” as known in the art is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and
  • yeast e.g., Saccharomyces cerevisiae
  • Methods for gene expression in yeast are known in the art (e.g., Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology, Part A, 2004, Christine Guthrie and Gerald R. Fink, eds., Elsevier Academic Press, San Diego, CA).
  • Expression of genes in yeast typically requires a promoter, followed by the gene of interest, and a transcriptional terminator.
  • yeast promoters can be used in constructing expression cassettes for genes encoding an isobutanol biosynthetic pathway, including, but not limited to constitutive promoters FBA, GPD, ADHl, and GPM, and the inducible promoters GALl, GAL 10, and CUPl .
  • Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERGlOt, GAL It, CYC1, and ADHl .
  • suitable promoters, transcriptional terminators, and the genes of an isobutanol biosynthetic pathway can be cloned into E.
  • co/z-yeast shuttle vectors and transformed into yeast cells as described in U.S. App. Pub. No. 2010/0129886. These vectors allow strain propagation in both E. coli and yeast strains. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host.
  • plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, MD), which contain an E. coli replication origin (e.g., pMBl), a yeast 2 ⁇ origin of replication, and a marker for nutritional selection.
  • the selection markers for these four vectors are His3 (vector pRS423), Tr l (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426).
  • Construction of expression vectors with genes encoding polypeptides of interest can be performed by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast.
  • the gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast.
  • a yeast vector DNA is digested ⁇ e.g., in its multiple cloning site) to create a "gap" in its sequence.
  • a number of insert DNAs of interest are generated that contain a > 21 bp sequence at both the 5' and the 3' ends that sequentially overlap with each other, and with the 5' and 3' terminus of the vector DNA.
  • a yeast promoter and a yeast terminator are selected for the expression cassette.
  • the promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5' end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3' end of the linearized vector.
  • the "gapped" vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids.
  • the presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells.
  • the plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g., TOP 10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can
  • integration into the yeast genome also takes advantage of the homologous recombination system in yeast.
  • a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR- amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5 ' and 3 ' of the genomic area where insertion is desired.
  • the PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker.
  • the promoter-coding region X-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning.
  • the full cassette, containing the promoter-coding regionX- terminator-iy 43 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5' and 3' of location "Y" on the yeast chromosome.
  • the PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.
  • Additional carbon substrates may include, but are not limited to, monosaccharides such as fructose, oligosaccharides such as lactose, maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt.
  • Other carbon substrates can include ethanol, lactate, succinate, or glycerol.
  • the carbon substrate may also be one-carbon substrates 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.], 7 th (1993), 415-32, Editors: Murrell, J. Collin, Kelly, Don P.; Publisher: Intercept, Andover, UK).
  • 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.
  • the 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 can be derived from renewable grain sources through
  • fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. 2007/0031918 Al, which is herein incorporated by reference.
  • Biomass when used in reference to carbon substrate, refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.
  • Biomass can also comprise additional components, such as protein and/or lipid.
  • Biomass can 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.
  • fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of an enzymatic pathway described herein.
  • the butanologen produces butanol at least 90% of effective yield, at least 91%> of effective yield, at least 92% of effective yield, at least 93% of effective yield, at least 94% of effective yield, at least 95% of effective yield, at least 96% of effective yield, at least 97% of effective yield, at least 98% of effective yield, or at least 99% of effective yield.
  • the butanologen produces butanol at about 55% to at about 75% of effective yield, about 50% to about 80% of effective yield, about 45% to about 85% of effective yield, about 40% to about 90% of effective yield, about 35% to about 95% of effective yield, about 30%) to about 99% of effective yield, about 25% to about 99% of effective yield, about 10%) to about 99% of effective yield, or about 10% to about 100% of effective yield.
  • the cells are grown at a temperature of 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C or 40°C. In some embodiments, the cells are grown at a temperature of about 25°C to about 40°C in an appropriate medium.
  • Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth or broth that includes yeast nitrogen base, ammonium sulfate, and dextrose (as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains.
  • Other defined or synthetic growth media can also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.
  • agents known to modulate catabolite repression directly or indirectly e.g., cyclic adenosine 2', 3 '-monophosphate (cAMP), can also be incorporated into the fermentation medium.
  • cAMP cyclic adenosine 2', 3 '-monophosphate
  • Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred for the initial condition.
  • Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0.
  • about pH 5.0 to about pH 8.0 is used for the initial condition.
  • Suitable pH ranges for the fermentation of other microorganisms are between about pH 3.0 to about pH 7.5.
  • about pH 4.5 to about pH 6.5 is used for the initial condition.
  • Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentation.
  • the culture conditions are such that the fermentation occurs without respiration.
  • cells can be cultured in a fermenter under micro-aerobic or anaerobic conditions.
  • Butanol, or other products can be produced using a batch method of fermentation.
  • a classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation.
  • a variation on the standard batch system is the fed-batch system.
  • Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments at the fermentation progresses.
  • Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media.
  • Batch and fed-batch fermentations are common and well known in the art and examples can be found in Thomas D.
  • Butanol, or other products may also be produced using continuous fermentation methods.
  • Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing.
  • Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
  • Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration.
  • butanol or other products
  • production of butanol, or other products can be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable.
  • cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for butanol production.
  • 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 the processes described herein 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 isobutanol.
  • 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 or to a separate stripping column.
  • 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
  • ISPR In situ product removal
  • 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. Nos. 2009/0305370 and 2011/0097773, the disclosures of which are hereby incorporated in their entirety.
  • 2009/0305370 and 2011/0097773 describe 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, polyunsaturated (and mixtures thereof) C 12 to C22 fatty alcohols, C 12 to C22 fatty acids, esters of C 12 to C22 fatty acids, C12 to C22 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.
  • an alcohol ester can be formed by contacting the alcohol in a fermentation medium with an organic acid ⁇ e.g., fatty acids) and a catalyst capable of esterifying 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.
  • Carboxylic acids that are produced during the fermentation can additionally be esterified with the alcohol produced by the same or a different catalyst.
  • 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.
  • the presence and/or concentration of isobutanol in the culture medium can be determined by a number of methods known in the art (see, for example, U.S. Patent 7,851,188, incorporated by reference).
  • HPLC high performance liquid chromatography
  • a specific high performance liquid chromatography (HPLC) method utilizes a Shodex SH-1011 column with a Shodex SHG guard column, both may be purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H 2 SO 4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50 °C.
  • Isobutanol has a retention time of 46.6 min under the conditions used.
  • GC gas chromatography
  • HP-F NOWax column (30 m X 0.53 mm id, 1 ⁇ film thickness, Agilent Technologies, Wilmington, DE), with a flame ionization detector (FID).
  • the carrier gas is helium at a flow rate of 4.5 mL/min, measured at 150 °C with constant head pressure; injector split is 1 :25 at 200 °C; oven temperature is 45 °C for 1 min, 45 to 220 °C at 10 °C/min, and 220 °C for 5 min; and FID detection is employed at 240 °C with 26 mL/min helium makeup gas.
  • the retention time of isobutanol is 4.5 min.
  • 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
  • Saccharomyces cerevisiae strain PNY0827 is used as the host cell for further genetic manipulation for PNY2115.
  • PNY0827 refers to a strain derived from Saccharomyces cerevisiae which has been deposited at the ATCC under the Budapest Treaty on September 22, 2011 at the American Type Culture Collection, Patent Depository 10801 University Boulevard, Manassas, VA 20110-2209 and has the patent deposit designation PTA-12105.
  • a deletion cassette was PCR-amplified from pLA54 (SEQ ID NO: 1) which contains a P TE Fi-kanMX4-TEFlt cassette flanked by loxP sites to allow homologous recombination in vivo and subsequent removal of the KANMX4 marker.
  • PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and primers BK505 (SEQ ID NO: 2) and BK506 (SEQ ID NO: 3).
  • the URA3 portion of each primer was derived from the 5' region 180bp upstream of the URA3 ATG and 3' region 78bp downstream of the coding region such that integration of the kanMX4 cassette results in replacement of the URA3 coding region.
  • the PCR product was transformed into PNY0827 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were selected on YEP medium supplemented 2% glucose and 100 ⁇ g/ml Geneticin at 30°C. Transformants were screened by colony PCR with primers LA468 (SEQ ID NO: 4) and LA492 (SEQ ID NO: 5) to verify presence of the integration cassette.
  • NYLA98 which has the genotype MATa/ ⁇ ORA3/ura3 AoxP-kanMX4-loxP.
  • haploids NYLA98 was sporulated using standard methods (Codon AC, Gasent-Ramirez JM, Benitez T. Factors which affect the frequency of sporulation and tetrad formation in
  • Tetrads were dissected using a micromanipulator and grown on rich YPE medium supplemented with 2% glucose. Tetrads containing four viable spores were patched onto synthetic complete medium lacking uracil supplemented with 2% glucose, and the mating type was verified by multiplex colony PCR using primers AK109-1 (SEQ ID NO: 6), AK109-2 (SEQ ID NO: 7), and AK109-3 (SEQ ID NO: 8). The resulting identified haploid strain called NYLA103, which has the genotype: MATa ura3A::loxV-kanMX4-loxV, and NYLA106, which has the genotype: MATa ura 3 A: :loxP-kanMX4-loxP.
  • HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 9) and primer oBP453 (SEQ ID NO: 10), containing a 5' tail with homology to the 5' end of HIS3 Fragment B.
  • HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO: 11), containing a 5' tail with homology to the 3' end ofHIS3 Fragment A, and primer oBP455 (SEQ ID NO: 12) containing a 5 ' tail with homology to the 5 ' end of HIS3 Fragment U.
  • HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 13), containing a 5' tail with homology to the 3' end ofHIS3 Fragment B, and primer oBP457 (SEQ ID NO: 14), containing a 5' tail with homology to the 5' end of HIS3 Fragment C.
  • HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 15), containing a 5' tail with homology to the 3' end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 16). PCR products were purified with a PCR Purification kit (Qiagen).
  • HIS 3 Fragment AB was created by overlapping PCR by mixing HIS 3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 9) and oBP455 (SEQ ID NO: 12).
  • HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO: 13) and oBP459 (SEQ ID NO: 16). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen).
  • the HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 9) and oBP459 (SEQ ID NO: 16). The PCR product was purified with a PCR Purification kit (Qiagen).
  • Competent cells of NYLA106 were transformed with the HIS3 ABUC PCR cassette and were plated on synthetic complete medium lacking uracil supplemented with 2% glucose at 30 °C. Transformants were screened to verify correct integration by replica plating onto synthetic complete medium lacking histidine and supplemented with 2% glucose at 30°C.
  • Genomic DNA preps were made to verify the integration by PCR using primers oBP460 (SEQ ID NO: 17) and LA135 (SEQ ID NO: 18) for the 5' end and primers oBP461 (SEQ ID NO: 19) and LA92 (SEQ ID NO: 20) for the 3' end.
  • the URA3 marker was recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30°C following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD -URA medium to verify the absence of growth.
  • the resulting identified strain, called PNY2003 has the genotype: MATa ura3A::loxP-kanMX4-loxP his3A.
  • a deletion cassette was PCR- amplified from pLA59 (SEQ ID NO: 21), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using Phusion High Fidelity PCR Master Mix (New England
  • each primer was derived from the 5' region 50bp downstream of the PDCl start codon and 3' region 50bp upstream of the stop codon such that integration of the URA3 cassette results in replacement of the PDCl coding region but leaves the first 50bp and the last 50bp of the coding region.
  • the PCR product was transformed into PNY2003 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 2% glucose at 30°C.
  • Transformants were screened to verify correct integration by colony PCR using primers LA337 (SEQ ID NO: 24), external to the 5' coding region and LA135 (SEQ ID NO: 18), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA692 (SEQ ID NO: 25) and LA693 (SEQ ID NO: 26), internal to the PDCl coding region.
  • the URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 27) containing the CRE recombinase under the GALl promoter and plated on synthetic complete medium lacking histidine and supplemented with 2% glucose at 30°C.
  • Transformants were plated on rich medium supplemented with 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 2% glucose to verify absence of growth.
  • the resulting identified strain, called PNY2008 has the genotype: MATa ura3A::loxV-kanMX4-loxV his 3 A pdclA lox?71/66.
  • a deletion cassette was PCR- amplified from pLA59 (SEQ ID NO: 21), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using Phusion High Fidelity PCR Master Mix (New England
  • the PDC5 portion of each primer was derived from the 5' region 50bp upstream of the PDC5 start codon and 3' region 50bp downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire PDC5 coding region.
  • the PCR product was transformed into PNY2008 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C.
  • Transformants were screened to verify correct integration by colony PCR using primers LA453 (SEQ ID NO: 30), external to the 5' coding region and LA135 (SEQ ID NO: 18), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA694 (SEQ ID NO: 31) and LA695 (SEQ ID NO: 32), internal to the PDC5 coding region.
  • the URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 27) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30°C.
  • Transformants were plated on rich YEP medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth.
  • the resulting identified strain, called PNY2009 has the genotype: MATa ura3A::loxP-kanMX4-loxP his3A /3 ⁇ 4 ciA::loxP71/66
  • the FRA2 deletion was designed to delete 250 nucleotides from the 3 ' end of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding sequence intact. An in- frame stop codon was present 7 nucleotides downstream of the deletion.
  • the four fragments for the PCR cassette for the scarless FRA2 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, CA).
  • FRA2 Fragment A was amplified with primer oBP594 (SEQ ID NO: 33) and primer oBP595 (SEQ ID NO: 34), containing a 5' tail with homology to the 5' end of FRA2 Fragment B.
  • FRA2 Fragment B was amplified with primer oBP596 (SEQ ID NO: 35), containing a 5' tail with homology to the 3' end of FRA2 Fragment A, and primer oBP597 (SEQ ID NO: 36), containing a 5' tail with homology to the 5' end of FRA2 Fragment U.
  • FRA2 Fragment U was amplified with primer oBP598 (SEQ ID NO: 37), containing a 5' tail with homology to the 3' end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO: 38), containing a 5' tail with homology to the 5' end of FRA2 Fragment C.
  • FRA2 Fragment C was amplified with primer 0BP6OO (SEQ ID NO: 39), containing a 5' tail with homology to the 3' end of FRA2 Fragment U, and primer 0BP6OI (SEQ ID NO: 40).
  • PCR products were purified with a PCR Purification kit (Qiagen).
  • FRA2 Fragment AB was created by overlapping PCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying with primers oBP594 (SEQ ID NO: 33) and oBP597 (SEQ ID NO: 36).
  • FRA2 Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U and FRA2
  • the resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen).
  • the FRA2 ABUC cassette was created by overlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO: 33) and 0BP6OI (SEQ ID NO: 40).
  • the PCR product was purified with a PCR Purification kit (Qiagen).
  • the scarless deletion cassette obtained above was transformed into PNY2009 using standard techniques and plated on synthetic complete medium lacking uracil and supplemented with 1% ethanol.
  • Genomic DNA preps were made to verify the integration by PCR using primers oBP602 (SEQ ID NO: 41) and LAI 35 (SEQ ID NO: 18) for the 5' end, and primers oBP602 (SEQ ID NO: 41) and oBP603 (SEQ ID NO: 42) to amplify the whole locus.
  • the URA3 marker was recycled by plating on synthetic complete medium supplemented with 1% ethanol and 5-FOA (5-Fluoroorotic Acid) at 30°C following standard protocols.
  • the loxP71 -URA3-loxP66 marker was PCR-amplified using Phusion DNA polymerase (New England BioLabs; Ipswich, MA) from pLA59 (SEQ ID NO: 21), and transformed along with the LA811x817 (SEQ ID NOs: 43, 44) and LA812x818 (SEQ ID NOs: 45, 46) 2-micron plasmid fragments (amplified from the native 2-micron plasmid from CEN.PK 113-7D; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre) into strain PNY2037 on SE -URA plates at 30°C.
  • Phusion DNA polymerase New England BioLabs; Ipswich, MA
  • LA811x817 SEQ ID NOs: 43, 44
  • LA812x818 SEQ ID NOs: 45, 46
  • 2-micron plasmid fragments amplified from the native 2-micron plasmid from C
  • the resulting strain PNY2037 2 ⁇ ::1 ⁇ 71- ⁇ 3-1 ⁇ 66 was transformed with pLA34 (pRS423 were) (also called, pLA34) (SEQ ID NO: 27) and selected on SE -HIS -URA plates at 30°C. Transformants were patched onto YP-1% galactose plates and allowed to grow for 48 hrs at 30°C to induce Cre recombinase expression. Individual colonies were then patched onto SE -URA, SE -HIS, and YPE plates to confirm URA3 marker removal.
  • the resulting identified strain, PNY2050 has the genotype: MATa wra3A::loxP-kanMX4-loxP, his3 /3 ⁇ 4 ciA::loxP71/66 /3 ⁇ 4 c5A::loxP71/66 fra2A 2-micron.
  • pdc6A :(UAS)PGKl-P[FBAl]-KIVD
  • PNY2050 was as follows.
  • the PDC 1 portion of each primer was derived from 60bp of the upstream of the coding sequence and 50bp that are 53bp upstream of the stop codon.
  • the PCR product was transformed into PNY2050 using standard genetic techniques and trans formants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers 681 (SEQ ID NO: 57), external to the 3' coding region and 92 (SEQ ID NO: 58), internal to the URA3 gene. Positive transformants were then prepped for genomic DNA and screened by PCR using primers N245 (SEQ ID NO: 59) and N246 (SEQ ID NO: 60).
  • the URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 27) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30°C. Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth.
  • the resulting identified strain called PNY2090 has the genotype MATa ura3A::loxP, his3A, pdclA::loxP71/66, pdc5A::loxP71/66 fra2A 2-micron pdclA::P[PDCl]-ALS
  • Pdc6A (UAS PG l-PrFBAll-KIVD
  • an integration cassette was PCR- amplified from pLA78 (SEQ ID NO: 53), which contains the kivD gene from the species Listeria grayi with a hybrid FBA1 promoter and a TDH3 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using KAPA HiFi and primers 896 (SEQ ID NO: 61) and 897 (SEQ ID NO: 62).
  • the PDC6 portion of each primer was derived from 60bp upstream of the coding sequence and 59bp downstream of the coding region.
  • the PCR product was transformed into PNY2090 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers 365 (SEQ ID NO: 63) and 366 (SEQ ID NO: 64), internal primers to the PDC6 gene. Transformants with an absence of product were then screened by colony PCR N638 (SEQ ID NO: 65), external to the 5' end of the gene, and 740 (SEQ ID NO: 66), internal to the FBA1 promoter. Positive transformants were than the prepped for genomic DNA and screened by PCR with two external primers to the PDC6 coding sequence.
  • pdc6A :(UAS)PGKl-P[FBAl]-KIVD
  • an integration cassette was PCR-amp lifted from pLA65 (SEQ ID NO: 54), which contains the alcohol dehydrogenase from the species Beijerinckii with an ILV5 promoter and a ADHl terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using KAPA HiFi and primers 856 (SEQ ID NO: 67) and 857 (SEQ ID NO: 68).
  • the ADHl portion of each primer was derived from the 5' region 50 bp upstream of the ADHl start codon and the last 50 bp of the coding region.
  • the PCR product was transformed into PNY2093 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers BK415 (SEQ ID NO: 69), external to the 5' coding region and N1092 (SEQ ID NO: 70), internal to the BiADH gene.
  • the resulting identified strain called PNY2101 has the genotype MATa ura3A::loxP his3A pdc5A::loxP71/66 fra2A 2-micron pdclA::P[PDCl]-ALS
  • Fra2A :PriLV51-ADH
  • PCPv-amp lifted from pLA65 (SEQ ID NO: 54), which contains the alcohol dehydrogenase from the species Beijerinckii indica with an ILV5 promoter and an ADH1 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using KAPA HiFi and primers 906 (SEQ ID NO: 71) and 907 (SEQ ID NO: 72). The FRA2 portion of each primer was derived from the first 60bp of the coding sequence starting at the ATG and 56bp downstream of the stop codon.
  • the PCR product was transformed into PNY2101 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers 667 (SEQ ID NO: 73), external to the 5' coding region and 749 (SEQ ID NO: 74), internal to the ILV5 promoter.
  • the URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 27) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30°C.
  • Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth.
  • the resulting identified strain, called PNY2110 has the genotype MATa ura3A::loxP his3A pdc5A::loxP66/71 2-micron pdclA::P[PDCl]-ALS
  • pdc6A (UAS)PGKl-P[FBAl]-KIVD
  • a deletion cassette was PCR amplified from pLA59 (SEQ ID NO: 21), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using KAPA HiFi and primers LA512 (SEQ ID NO: 47) and LA513 (SEQ ID NO: 48).
  • the GPD2 portion of each primer was derived from the 5 'region 50bp upstream of the GPD2 start codon and 3' region 50bp downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire GPD2 coding region.
  • the PCR product was transformed into PNY2110 using standard genetic techniques and
  • transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers LA516 (SEQ ID NO: 49) external to the 5' coding region and LAI 35 (SEQ ID NO: 18), internal to URA3. Positive transformants were then screened by colony PCR using primers LA514 (SEQ ID NO: 50) and LA515 (SEQ ID NO: 51), internal to the GPD2 coding region.
  • the URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 27) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30°C. Transformants were plated on rich medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth.
  • the resulting identified strain has the genotype MATa ura3A::loxP his3A pdc5A::loxP66/71 fra2A 2- micron pdclA::P[PDCl]-ALS
  • PNY2145 was constructed from PNY2115 by the additional integration of a phosphoketolase gene cassette at the pdc5A locus and by replacing the native AMNl gene with a codon optimized verison of the ortholog from CEN.PK. Integration constructs are further described below.
  • pdc5A :FB A(L8 -xpk 1 -CYC 1 t-loxP71/66
  • SEQ ID NO: 76 was PCR amplified using primers N1341 and N1338 (SEQ ID NOs: 77 and 78), generating a 3.1 kb product.
  • the loxP-flanked URA3 gene cassette from pLA59 (SEQ ID NO: 21) was amplified with primers N1033c and N1342 (SEQ ID NOs: 79 and 80), generating a 1.6 kb product.
  • the xpkl and URA3 PCR products were fused by combining them without primers for an additional 10 cycles of PCR using Phusion DNA polymerase.
  • the resulting reaction mix was then used as a template for a PCR reaction with KAPA Hi Fi and primers N1342 and N1364 (SEQ ID NOs: 80 and 81).
  • a 4.2 kb PCR product was recovered by purification from an electrophoresis agarose gel (Zymo kit).
  • FBA promoter variant L8 (SEQ ID NO: 82) was amplified using primers N1366 and N1368 (SEQ ID NOs: 83 and 84).
  • the xpkl ::URA3 PCR product was combined with the FBA promoter by additional rounds of PCR.
  • the resulting product was phosphorylated with polynucleotide kinase and ligated into pBR322 that had been digested with EcoRV and treated with calf intestinal phosphatase.
  • the ligation reaction was transformed into E. coli cells (Stbl3 competent cells from Invitrogen).
  • the integration cassette was confirmed by sequencing.
  • the plasmid was used as a template in a PCR reaction with Kapa HiFi and primers N1371 and N1372 (SEQ ID NOs: 85 and 86).
  • the PCR product was isolated by phenol-chloroform extraction and ethanol precipitation (using standard methods; eg. Maniatas, et al).
  • Transformants were grown in rich medium supplemented with 1% ethanol to derepress the recombinase. Marker removal was confirmed for single colony isolates by patching to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. Loss of the recombinase plasmid, pJT254, was confirmed by patching the colonies to synthetic complete medium lacking histidine and supplemented with 1% ethanol. Proper marker removal was confirmed by PCR (primers N160SeqF5 (SEQ ID NO: 90) and BK380. One resulting clone was designated PNY2293. amnlA::AMNl(vVloxP71/66
  • AMN1 gene from CEN.PK2 an integration cassette containing the CEN.PK ⁇ Vi promoter, AMNl(y) gene (nucleic acid SEQ ID NO: 91; amino acid SEQ ID NO: 92), and CEN.PK.4MVi terminator was assembled by SOE PCR and subcloned into the shuttle vector pLA59.
  • the AMNl(y) gene was ordered from DNA 2.0 with codon-optimization for S. cerevisiae.
  • the completed pLA67 plasmid contained: 1) pUC19 vector backbone sequence containing an E. coli replication origin and ampicillin resistance gene; 2) URA3 selection marker flanked by loxP71 and loxP66 sites; and 3) PAMNi(CEN.PK)- ⁇ M ⁇ _term AMNi(CEN.PK) expression cassette
  • PCR amplification of the AMNl(y)- ⁇ oxPl ⁇ -URA3- ⁇ oxP66 cassette was done by using KAPA HiFi from Kapa Biosystems, Woburn, MA and primers LA712 (SEQ ID NO: 94) and LA746 (SEQ ID NO: 95).
  • the PCR product was transformed into PNY2293 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30 °C. Transformants were observed under magnification for the absence of a clumping phenotype with respect to the control (PNY2293).
  • the URA3 marker was recycled using the pJT254 Cre recombinase plasmid as described above.
  • a resulting identified strain, PNY2145 has the genotype: MATa ura3A::loxP his3A pdc5A::P[FBA(L8)]-XPK
  • Strain PNY 1621 was constructed from strain PNY2145.
  • the chimeric gene on chromosome XII in PNY2145 consisting of the PDC1 promoter, alsS coding region, CYC I terminator, and loxP71/66 site was deleted from 750 bp upstream of the alsS coding region to the first base of native PDC1 3 ' UTR region.
  • the region was deleted using CRE-lox mediated marker removal.
  • the region was replaced with a chimeric gene comprised of the
  • the native PDC1 terminator was used to complete the chimeric gene.
  • Plasmids were introduced into the strain for expression of KARI and DHAD (pLH804::L2V4, plasmid SEQ ID NO:97) and KivD and ADH (pRS413 : :BiADH-kivD_Lg(y), plasmid SEQ ID NO:98).
  • pLH804::L2V4 was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans containing the L2V4 mutation (nt position 5356-3641) expressed from the yeast TEF1 mutant 7 promoter (nt 5766-5366; Nevoigt et al. 2006.
  • pRS413 : :BiADH-kivD_Lg(y) was constructed to contain a chimeric gene having the coding region of the kivD gene from Listeria grayi codon optimized for expression in Saccharomyces cerevisiae (nt position 2902-4548) expressed from the UAS(PGKl) FBA1 promoter (nt 2169-2893) and followed by the TDH3 terminator (nt 4560-5139) for expression of KivD, and a chimeric gene having the coding region of the adh gene from Beijerinckia indica codon optimized for expression in Saccharomyces cerevisiae (nt 6853-7896) expressed from the yeast PDC1 promoter (nt 5983-6852) and followed by the ADH1 terminator (nt 7905-8220) for expression of ADH.
  • the resulting strain was designated PNY 1621.
  • PNY2310 was generated by transforming strain PNY2145 with plasmids pLH804-
  • Plasmid pLH804-L2V4 (SEQ ID NO:97) is a yeast-E. coli shuttle vector based on pHR81 (ATCC#87541). It contains genes for the expression of KARI variant K9JB4P (SEQ ID NO: 138) and DHAD variant L2V4.
  • Plasmid pRS413::BiADH-kivD (SEQ ID NO:98) is a yeast-E. coli shuttle vector based on pRS413 (ATCC#87518). It contains genes for the expression of BiADH and kivD.
  • Plasmid trans formants were selected by plating on synthetic complete medium lacking uracil and histidine with 1% (v/v) ethanol as the carbon source. Colonies were transferred to fresh plates by patching. After two days, cells from the patches were transferred to plates containing synthetic complete medium (minus uracil and histidine) with 2% (w/v) glucose as the carbon source. The resulting strain was designated PNY2310.
  • a deletion cassette was PCR- amplified from pLA59 (SEQ ID NO: 21), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker.
  • PCR was done by using Phusion High Fidelity PCR Master Mix (New England
  • the GPD2 portion of each primer was derived from the 5' region 50bp upstream of the GPD2 start codon and 3' region 50bp downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire GPD2 coding region.
  • the PCR product was transformed into PNY2050 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C.
  • the resulting identified strain, PNY2056 has the genotype: MATa ura3A: :loxP-kanMX4-loxP hi 3 /3 ⁇ 4 ciA::loxP71/66 /3 ⁇ 4 c5A::loxP71/66 fra2A 2-micron gpd2A.
  • PNY1558 was derived from PNY2056 by integrating a phosphoketolase and phosphotransacetylase expression cassette at the pdc5A::loxP locus in PNY2056 to create a C2- independent strain.
  • the phosphoketolase and phosphotransacetylase expression cassette, P[TEF(M4)]-xpkl+P[EN01]-eutD was from pRS423::TEF(M4)- xpkl+ENOl-eutD (SEQ ID NO: 76; US20120237988, incorporated herein by reference), which has the xpkl gene from Lactobacillus plantarum expressed from the yeast TEF1 mutant 4 promoter (Nevoigt et al. 2006.
  • phosphotransacetylase expression cassette was amplified from pRS423::TEF(M4)-xpkl+EN01- eutD using primers oBP962 (SEQ ID NO:99) and oBP963 (SEQ ID NO: 100), each containing an EcoRI restriction site.
  • the resulting PCR product and pLA59 (SEQ ID NO:21) were ligated together after digestion with EcoRI.
  • pLA59 contains a URA3 marker flanked by degenerate loxP sites.
  • the URA3-xpk-eutD integration cassette from the resulting plasmid was amplified with oBP988 (SEQ ID NO: 101), containing a 5' tail with homology to the sequence upstream of PDC5, and oBP989 (SEQ ID NO: 102), containing a 5' tail with homology to the sequence downstream of PDC5.
  • PNY2056 was transformed with the resulting PCR product and transformants were selected for growth on synthetic complete media lacking uracil supplemented with 1% ethanol at 30C.
  • a glucose-regulated hybrid promoter was constructed by cloning a 168 base-pair glucose repressor sequence from the S. cerevisiae HXT1 promoter into the S. cerevisiae FBA1 promoter. Two constructs were built, one for initial hybrid promoter testing using the green fluorescent protein ZsGreen (Clontech; Mountain View, CA; Matz et al, Nature Biotechnology (1999) 17:969; Lukyanov et al, ]BC (2000) 275 (34):25879) gene as the reporter and the second construct for integration of the URA3::P[FBAl ::HXTl-331]-alsS cassette into the
  • the gene for ZsGreen was PCR amplified from plasmid pZsGreen (Clontech,
  • the glucose-repressor sequence from the HXT1 promoter was PCR amplified from a genomic DNA template prepared from S. cerevisiae strain BY4743 using primers N1424 (SEQ ID NO: 106) and N1425 (SEQ ID NO: 107) (based on the S288c genomic sequence; ATCC #204508) each containing a 5' engineered Blpl restriction site resulting in a 190 base pair PCR product. Amplification was carried out using a hot start DNA polymerase (Phusion - New England BioLabs). The resulting PCR product was restriction digested with Blpl and
  • the ligated DNA was transformed into E. coli TOP 10 chemically competent cells and plated on a selective medium. Plasmid DNA from several transformants was isolated and the presence and direction of the HXT1 glucose repressor insert screened by sequencing using primers N1314 (SEQ ID NO: 108) and N1323 (SEQ ID NO: 109). The new construct was designated pJT331 (SEQ ID NO: 110).
  • the hybrid FBA1 promoter was designated P[FBA1 : :HXT1_331] (SEQ ID NO: 111).
  • P[FBA1 ::HXT1_331] was PCR amplified from plasmid pJT331 using primers N1453 (EQ ID NO : 112) and N 1454 (SEQ ID NO : 113) containing engineered Notl and Pad restriction sites, respectively. Amplification was carried out using a hot start DNA polymerase (Phusion - New England BioLabs). The 798 base pair PCR product was restriction digested with Notl and Pad and directionally cloned into the integration vector pBP2662 (SEQ ID NO: l 14) previously restriction digested with Notl and Pad. The ligated DNA was transformed into E. coli TOP 10 chemically competent cells and plated on a selective medium.
  • Clones containing the new construct were isolated by PCR colony screening several transformants using forward and reverse check primers N1434 (SEQ ID NO: 115) and N 1446 (SEQ ID NO: 116), respectively. Plasmids prepared from several clones were sequenced to verify the promoter sequence insert using primers N1434, N1445 (SEQ ID NO: 117), N1446, and N1459 (SEQ ID NO: 118). The new construct was designated pJT337 (SEQ ID NO: l 19).
  • Plasmid pJT337 was restriction digested with Pmel and Sail to release a 4,595 base pair linear integration cassette containing URA3 and P[FBAl ::HXTl_331]-a/&S' expression cassettes flanked by upstream and downstream sequences homologous to the Apdcl ::loxP71/66 locus.
  • the linearized integration cassette was transformed into S. cerevisiae strain PNY1558 for integration by homologous recombination using a standard yeast transformation protocol.
  • the transformed cells were plated onto SE 1.0% medium minus uracil for selection of
  • Apdcl ::loxP71-URA3-loxP66::P[FBAl ::HXTl_331]alsS integrants were verified by PCR colony screening for the 5 ' and 3 ' ends of the integration site using primers N1463 (SEQ ID NO: 120), N1464 (SEQ ID NO: 121), N1434 and N1446.
  • the URA marker was removed from the integration site by cleavage at the loxP sites by expression of cre-recombinase from plasmid pJT254.
  • URA3 marker removal was verified in several clones by plating on selective and non-selective media where a negative growth phenotype on media lacking uracil was evidence for marker removal.
  • Plasmid pHR81 : :ILV5p-K9JB4P (SEQ ID NO: 140) is a yeast-E. coli shuttle vector based on pHR81 (ATCC#87541). It contains the gene for the expression of KARI variant K9JB4P (SEQ ID NO: 138).
  • Plasmid pLA84 (SEQ ID NO:141) is a yeast-E. coli shuttle vector based on pRS423 (ATCC#77104).
  • Plasmid transformants were selected by plating on synthetic complete medium lacking uracil and histidine with 1% (v/v) ethanol as the carbon source. Colonies were transferred to fresh plates by patching. After two days, cells from the patches were transferred to plates containing synthetic complete medium (minus uracil and histidine) with 2% (w/v) glucose as the carbon source. The resulting strain was designated PNY2289.
  • This culture was used to inoculate 90 ml of fresh synthetic complete medium (with 2 g/1 ethanol and 2 g/1 glucose) in 250 ml flask and grown for 24 hrs at 30°C and 200 rpm. After 24 hrs, cells were harvested by centrifugation at 4000 rpm for 5 minutes and re-suspended at an initial OD 6 oonm of 20 in synthetic complete medium containing 2 g/1 ethanol and 20% v/v glycerol (Sigma). These cells were distributed in aliquots of 1 ml in screw cap tubes and frozen using slow freezers and stored at -80 °C (glycerol stocks) until further use.
  • Seed culture was prepared in three stages: pre-seed, seed 1, and seed 2.
  • Pre-seed culture was started by inoculating 3 vials of 20 OD glycerol stock (described above) in 100 ml of filter sterilized pre-seed medium (containing 0.65 g/1 amino acid dropout without Histidine, Uracil and Leucine (Clonetech), 60 mg/1 Leucine (Sigma), 6.5 g/1 yeast nitrogen base without amino acids (Difco), 5 g/1 glucose, 5 g/1 ethanol, 19.5 g/1 4-Morpholineethane-sulphonic acid (Sigma), 50 mg/1 Ampicillin (Himedia; Mumbai, India) with pH adjusted to 5.5 using 1M H 2 S0 4 ) in 500 ml flask and grown at 30°C and 250 rpm for 24 hrs.
  • Seed 1 stage was initiated by adding 40 ml of pre-seed culture in 200 ml of filter sterilized seed flask medium (containing 6.7 g/1 yeast nitrogen base without amino acids, 2.8 g/1 amino acid drop out mix without histidine, uracil and leucine (Clonetech), 200 mg/1 of leucine, 40 mg/1 of tryptophan, 2 g/1 yeast extract (Becton Dickinson), 4 g/1 peptone (Becton Dickinson), 19.5 g/1 4-Morpholineethane-sulphonic acid (Sigma), 5 g/1 glucose, 5 g/1 ethanol and 50 mg/1 Ampicillin) with pH adjusted to 5.5 using 1M H2SO4) in 1L flask and incubated at 30°C and 250 rpm for 24 hrs.
  • 80 ml of seed 1 culture was inoculated in 400 ml of fresh seed flask medium (as described above) in 2 L flask and incubated at 30°C and 250
  • the growth phase was initiated by inoculating 2 L Biostat B Plus vessel fermenter containing 1 L growth medium (containing 2 g/1 Yeast Extract, 1 g/1 Ammonium sulphate, 5 g/1 Potassium sulphate monobasic, 2 g/1 Magnesium sulphate heptahydrate, 1 ml of 1000X Delft Trace element solution, 0.5 ml of Antifoam agent (Sigma), 50 mg/1 Ampicillin, 2 ⁇ g/l Biotin with pH adjusted to 5.5 using 1 M H 2 SO 4 /2 M NaOH) at target cell density of 1 g/L by centrifuging cells at 4000 rpm from seed 2 stage.
  • 1000X Delft trace Mineral solution contains 15 g/1 EDTA, 4.5 g/L Zinc sulphate heptahydrate, 0.84 g/1 Manganese chloride dehydrate, 0.3 g/1
  • the air flow rate was fixed constant at 1 LPM.
  • Dissolved oxygen was maintained at 30% by varying stirrer speed from 500 to 1200 rpm.
  • the temperature was maintained at 30°C, and pH was maintained at 5.5 using 2 M NaOH.
  • Substrate (glucose) feeding was done using different fed-batch feeding strategies.
  • the fermenter was started with initial sugar of 5 g/L glucose and 5 g/L ethanol.
  • the exponential feeding was done at rate of 0.07 hr "1 growth rate after exhaustion of glucose in batch mode.
  • the feed composition used was 400 g/L glucose and 75 g/L ethanol.
  • the harvest time of fermenter was decided by saturation in feeding rate and cell density. 10 mL samples were taken periodically for analysis each time. A portion of sample was used for dry cell weight measurement, and the remaining sample was centrifuged and stored in -80°C for further analysis using HPLC.
  • Table 7 Growth phase-effect of feeding strategy on cell density and byproduct profile of isobutanologen PNY2289
  • cells were harvested by centrifugation at 4000 rpm for 5 minutes and re-suspended at an initial OD 6 oonm of 20 in synthetic complete medium containing 2 g/1 ethanol and 20% v/v glycerol (Sigma). These cells were distributed in aliquots of 1 ml in screw cap tubes and frozen using slow freezers and stored at -80°C (glycerol stocks) until use.
  • Seed culture was prepared in three stages: pre-seed, seed 1, and seed 2.
  • Pre-seed culture was started by inoculating 3 vials of 20 OD glycerol stock (described above) in 100 ml of filter sterilized pre-seed medium (containing 0.65 g/1 Amino acid dropout without Histidine, Uracil and Leucine (Clonetech), 60 mg/1 Leucine (Sigma), 6.5 g/1 yeast nitrogen base without amino acids (Difco), 5 g/1 glucose, 5 g/1 ethanol, 19.5 g/1 4-Morpholineethane-sulphonic acid (Sigma), 50 mg/1 Ampicillin (Himedia) with pH adjusted to 5.5 using 1M H2SO4) in 500 ml flask and grown at 30°C and 250 rpm for 24 hrs.
  • pre-seed culture was started by inoculating 3 vials of 20 OD glycerol stock (described above) in 100 ml of filter sterilized pre-seed medium (
  • Seed 1 stage was initiated by adding 40 ml of pre- seed culture in 200 ml of filter sterilized seed flask medium (containing 6.7 g/1 Yeast nitrogen base without amino acids (Difco), 2.8 g/1 amino acid drop out mix without histidine, uracil and leucine (Clonetech), 200 mg/1 of leucine, 40 mg/1 of tryptophan, 2 g/1 yeast extract (Becton Dickinson), 4 g/1 peptone (Becton Dickinson), 19.5 g/1 4-Morpholineethane-sulphonic acid (Sigma), 5 g/1 glucose, 5 g/1 ethanol, and 50 mg/1 Ampicillin) with pH adjusted to 5.5 using 1M H2SO4) in 1L flask and incubated at 30°C and 250 rpm for 24 hrs.
  • filter sterilized seed flask medium containing 6.7 g/1 Yeast nitrogen base without amino acids (Difco), 2.8 g/1 amino acid drop out mix without histidine,
  • seed 1 culture was inoculated in 400 ml of fresh seed flask medium (as described above) in 2L flask and incubated at 30°C and 250 rpm for 24 hrs.
  • the growth phase was initiated by inoculating 2 L Biostat B Plus vessel fermenter containing 1 L growth medium (containing 8 g/1 Potassium phosphate monobasic, 8 g/1
  • 1000X Delft vitamin solution contains 0.05 g/1 Biotin (D-), 1 g/1 Calcium D(+) panthotenate, 1 g/1 Nicotinic acid, 25 g/1 myo-Inositol (for microbiology), 1 g/1 Thiamine Hydrochloride, 1 g/1 Pyridoxal
  • Two exponential feeding base runs were performed. In both the exponential feeding runs, fermenters were started with initial sugar of 5 g/L glucose and 5 g/L ethanol. The feeding was started after exhaustion of glucose in batch mode. The exponential feeding was done at a growth rate of 0.1 hr -1 in the first run and 0.15 hr -1 in second run.
  • the feed composition used was 400 g/L glucose, 75 g/L ethanol and 8 g/L of yeast extract for RQ based feeding.
  • the feed composition used was 400 g/L glucose for exponential feeding.
  • the harvest time of the fermenter was decided by saturation in feeding rate and cell density. The samples were taken periodically for analysis with 10 mL of sample each time.
  • cells were harvested by centrifugation at 4000 rpm for 5 minutes and re-suspended at an initial OD 6 oonm of 20 in synthetic complete medium containing 2 g/1 ethanol and 20% v/v glycerol (Sigma). These cells were distributed in aliquots of 1 ml in screw cap tubes and frozen using slow freezers and stored at -80°C (glycerol stocks) until use.
  • Seed culture was prepared in three stages: pre-seed, seed 1, and seed 2.
  • Pre-seed culture was started by inoculating 3 vials of 20 OD glycerol stock (described above) in 100 ml of filter sterilized pre-seed medium (containing 0.65 g/1 Amino acid dropout without Histidine,
  • Seed 1 stage was initiated by adding 40 ml of pre- seed culture in 200 ml of filter sterilized seed flask medium (containing 6.7 g/1 Yeast nitrogen base without amino acids (Difco), 2.8 g/1 amino acid drop out mix without histidine, uracil and leucine (Clonetech), 200 mg/1 of leucine, 40 mg/1 of tryptophan, 2 g/1 yeast extract (Becton Dickinson), 4 g/1 peptone (Becton Dickinson), 19.5 g/1 4-Morpholineethane-sulphonic acid (Sigma), 20 g/1 glucose, 5 g/1 ethanol, and 50 mg/1 Ampicillin) with pH adjusted to 5.5 using 1M H 2 SO 4 ) in 1L flask and incubated at 30°C and 250 rpm for 24 hrs.
  • 80 ml of seed 1 culture was inoculated in 400 ml of fresh seed flask medium (as described above) in 2L flask and in
  • the growth phase was initiated by inoculating 2 L Biostat B Plus vessel fermenter containing 1 L growth medium (containing 8 g/1 Potassium phosphate monobasic, 8 g/1
  • 1000X Delft vitamin solution contains 0.05 g/1 Biotin (D-), 1 g/1 Calcium D(+) panthotenate, 1 g/1 Nicotinic acid, 25 g/1 myo-Inositol (for microbiology), 1 g/1 Thiamine Hydrochloride, 1 g/1 Pyridoxal
  • the harvest time of the fermenter was decided by saturation in feeding rate and cell density.
  • the samples were taken periodically for analysis with 10 mL of sample each time. A portion of sample was used for dry cell weight measurement, and the remaining sample was centrifuged and stored in -80 °C for further analysis using HPLC.
  • the cell yield on glucose was decreased by increasing RQ in growth phase.
  • the isobutanol was increased and isobutyric acid was decreased as moving from low RQ (1.25) to high RQ (2.0) in the growth phase.
  • 1000X Delft trace Mineral solution contains 15 g/1 EDTA, 4.5 g/L Zinc sulphate heptahydrate, 0.84 g/1 Manganese chloride dehydrate, 0.3 g/1 Cobalt(II)chloride hexahydrate, 0.3 g/1 Copper (II) sulphate pentahydrate, 0.4 g/1 Di-sodium molybdenum dehydrate, 4.5 g/1 Calcium chloride dehydrate, 3 g/1 Iron sulphate heptahydrate, 1 g/1 Boric acid, 1 ml/1 Potassium iodide per liter of solution made in water. 800 ml of oleyl alcohol was added to the production phase fermenter as an extractant.
  • the temperature was maintained at 30°C, and pH was maintained at 5.5 using 10 M NaOH.
  • the dissolved oxygen was not controlled during the production phase.
  • the feed composition used was 50% glucose (% w/v). Sampling was done periodically, and samples were stored in -80°C freezer for further analysis using HPLC and GC.
  • Table 10 Production phase- effect of RQ on isobutanol titer, yield, productivity, and glycerol profile for isobutanologen PNY1621 at the end of a 21 hour production.

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Abstract

L'invention concerne des méthodes de maximisation de la production de biomasse et du rendement en butanol (par exemple l'isobutanol) pendant la fermentation de microorganismes recombinants. La production de biomasse pendant une phase de croissance de biomasse et/ou la production de butanol pendant une phase de production de butanol peuvent être maximisées par réglage d'une variable manipulée, telle que la vitesse d'alimentation, en réponse à une variable contrôlée, telle que le quotient respiratoire (RQ).
PCT/US2014/026162 2013-03-15 2014-03-13 Procédé de maximisation de croissance de biomasse et de rendement en butanol par rétrocontrôle WO2014151645A1 (fr)

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Citations (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028476A1 (fr) 1994-04-15 1995-10-26 Midwest Research Institute Zymomonas de recombinaison pour la fermentation du pentose
US5514583A (en) 1994-04-15 1996-05-07 Midwest Research Institute Recombinant zymomonas for pentose fermentation
US5686276A (en) 1995-05-12 1997-11-11 E. I. Du Pont De Nemours And Company Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism
US5712133A (en) 1994-04-15 1998-01-27 Midwest Research Institute Pentose fermentation by recombinant zymomonas
US6284453B1 (en) * 1999-09-29 2001-09-04 Steven Anthony Siano Method for controlling fermentation growth and metabolism
US6432688B1 (en) 1999-01-18 2002-08-13 Daicel Chemical Industries, Ltd. Amino alcohol dehydrogenase converts keto alcohol to amino alcohol and amino alcohol to keto alcohol
US20070031918A1 (en) 2005-04-12 2007-02-08 Dunson James B Jr Treatment of biomass to obtain fermentable sugars
WO2007041269A2 (fr) 2005-09-29 2007-04-12 E. I. Du Pont De Nemours And Company Production par fermentation de quatre alcools carbonés
US20070092957A1 (en) 2005-10-26 2007-04-26 Donaldson Gail K Fermentive production of four carbon alcohols
US7223575B2 (en) 2000-05-01 2007-05-29 Midwest Research Institute Zymomonas pentose-sugar fermenting strains and uses thereof
US20070259410A1 (en) 2006-05-02 2007-11-08 Donaldson Gail K Fermentive production of four carbon alcohols
US20080261230A1 (en) 2007-04-18 2008-10-23 Der-Ing Liao Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
US20080274525A1 (en) 2007-05-02 2008-11-06 Bramucci Michael G Method for the production of 2-butanol
WO2008137406A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production d'isobutanol
WO2008137402A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production de 1-butanol
WO2009013066A1 (fr) * 2007-06-15 2009-01-29 Hitec Zang Gmbh Procédé de fermentation de cultures cellulaires
US20090155870A1 (en) 2006-05-02 2009-06-18 Donaldson Gail K Fermentive production of four carbon alcohols
US20090163376A1 (en) 2007-12-20 2009-06-25 E.I. Du Pont De Nemours And Company Ketol-acid reductoisomerase using nadh
US20090203099A1 (en) 2007-10-30 2009-08-13 E. I. Du Pont De Nemours And Company Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate
US20090246846A1 (en) 2008-03-27 2009-10-01 E. I. Du Pont De Nemours And Company Zymomonas with improved xylose utilization
US20090269823A1 (en) 2008-04-28 2009-10-29 E.I. Du Pont De Nemours And Company Butanol dehydrogenase enzyme from the bacterium achromobacter xylosoxidans
US20090305370A1 (en) 2008-06-04 2009-12-10 E.I. Du Pont De Nemours And Company Method for producing butanol using two-phase extractive fermentation
US20090305363A1 (en) 2008-06-05 2009-12-10 E. I. Du Pont De Nemours And Company Enhanced pyruvate to acetolactate conversion in yeast
US20100081154A1 (en) 2008-09-29 2010-04-01 Butamax(Tm) Advanced Biofuels Llc IDENTIFICATION AND USE OF BACTERIAL [2Fe-2S] DIHYDROXY-ACID DEHYDRATASES
US20100120105A1 (en) 2008-10-27 2010-05-13 Butamax (Tm) Advanced Biofuels Llc Carbon pathway optimized production hosts for the production of isobutanol
US7741119B2 (en) 2006-09-28 2010-06-22 E. I. Du Pont De Nemours And Company Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production
WO2010075241A1 (fr) 2008-12-22 2010-07-01 E. I. Du Pont De Nemours And Company Zymomonas avec utilisation de xylose améliorée dans des conditions de stress pour la production de l'éthanol
US20100197519A1 (en) 2007-12-20 2010-08-05 E. I. Du Pont De Nemours And Company Ketol-acid reductoisomerase using nadh
WO2011041415A1 (fr) 2009-09-29 2011-04-07 Butamax(Tm) Advanced Biofuels Llc Production fermentative d'isobutanol à l'aide de céto-acide réducto-isomérases à efficacité élevée
US20110097773A1 (en) 2009-04-13 2011-04-28 Butamax(Tm) Advanced Biofuels Llc Method for producing butanol using extractive fermentation
US20110124060A1 (en) 2009-09-29 2011-05-26 Butamax(Tm) Advanced Biofuels Llc Yeast production host cells
US7993388B2 (en) 2001-09-17 2011-08-09 Ev3 Peripheral, Inc. Stent with offset cell geometry
WO2011103300A2 (fr) 2010-02-17 2011-08-25 Butamax(Tm) Advanced Biofuels Llc Amélioration de l'activité de protéines nécessitant l'agrégat fe-s
US20110269199A1 (en) 2009-12-29 2011-11-03 Butamax(Tm) Advanced Biofuels Llc Alcohol dehydrogenases (adh) useful for fermentive production of lower alkyl alcohols
US20110313206A1 (en) 2005-10-26 2011-12-22 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
US8241878B2 (en) 2008-09-29 2012-08-14 Butamax(Tm) Advanced Biofuels Llc Recombinant yeast host cell with Fe-S cluster proteins and methods of using thereof
US20120237988A1 (en) 2010-09-07 2012-09-20 Butamax(Tm) Advanced Biofuels Llc Butanol strain improvement with integration of a polynucleotide encoding a polypeptide that catalyzes pyruvate to acetolactate conversion
WO2012129555A2 (fr) 2011-03-24 2012-09-27 Butamax (Tm) Advanced Biofuels Llc Cellules hôtes et procédés de production d'isobutanol
US20120258873A1 (en) 2011-04-06 2012-10-11 Butamax(Tm) Advanced Biofuels Llc Reduction of 2,3-dihydroxy-2-methyl butyrate (dhmb) in butanol production

Patent Citations (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028476A1 (fr) 1994-04-15 1995-10-26 Midwest Research Institute Zymomonas de recombinaison pour la fermentation du pentose
US5514583A (en) 1994-04-15 1996-05-07 Midwest Research Institute Recombinant zymomonas for pentose fermentation
US5712133A (en) 1994-04-15 1998-01-27 Midwest Research Institute Pentose fermentation by recombinant zymomonas
US5686276A (en) 1995-05-12 1997-11-11 E. I. Du Pont De Nemours And Company Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism
US6432688B1 (en) 1999-01-18 2002-08-13 Daicel Chemical Industries, Ltd. Amino alcohol dehydrogenase converts keto alcohol to amino alcohol and amino alcohol to keto alcohol
US6284453B1 (en) * 1999-09-29 2001-09-04 Steven Anthony Siano Method for controlling fermentation growth and metabolism
US7223575B2 (en) 2000-05-01 2007-05-29 Midwest Research Institute Zymomonas pentose-sugar fermenting strains and uses thereof
US7993388B2 (en) 2001-09-17 2011-08-09 Ev3 Peripheral, Inc. Stent with offset cell geometry
US20070031918A1 (en) 2005-04-12 2007-02-08 Dunson James B Jr Treatment of biomass to obtain fermentable sugars
WO2007041269A2 (fr) 2005-09-29 2007-04-12 E. I. Du Pont De Nemours And Company Production par fermentation de quatre alcools carbonés
US20080182308A1 (en) 2005-09-29 2008-07-31 Donaldson Gail K Fermentive production of four carbon alcohols
US20070092957A1 (en) 2005-10-26 2007-04-26 Donaldson Gail K Fermentive production of four carbon alcohols
US20110313206A1 (en) 2005-10-26 2011-12-22 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
US8178328B2 (en) 2005-10-26 2012-05-15 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
US7993889B1 (en) 2005-10-26 2011-08-09 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
WO2007050671A2 (fr) 2005-10-26 2007-05-03 E. I. Du Pont De Nemours And Company Production fermentaire d'alcools a quatre atomes de carbone
US7851188B2 (en) 2005-10-26 2010-12-14 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
US20110111472A1 (en) 2005-10-26 2011-05-12 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
WO2007130521A2 (fr) 2006-05-02 2007-11-15 E. I. Du Pont De Nemours And Company Production par fermentation d'alcools à quatre atomes de carbone
US8206970B2 (en) 2006-05-02 2012-06-26 Butamax(Tm) Advanced Biofuels Llc Production of 2-butanol and 2-butanone employing aminobutanol phosphate phospholyase
US20070259410A1 (en) 2006-05-02 2007-11-08 Donaldson Gail K Fermentive production of four carbon alcohols
US20090155870A1 (en) 2006-05-02 2009-06-18 Donaldson Gail K Fermentive production of four carbon alcohols
WO2007130518A2 (fr) 2006-05-02 2007-11-15 E. I. Du Pont De Nemours And Company Production par fermentation d'alcools à quatre atomes de carbone
US20070292927A1 (en) 2006-05-02 2007-12-20 Donaldson Gail K Fermentive production of four carbon alcohols
US7741119B2 (en) 2006-09-28 2010-06-22 E. I. Du Pont De Nemours And Company Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production
US20110250610A1 (en) 2007-04-18 2011-10-13 Butamax(Tm) Advanced Biofuels Llc Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
US20080261230A1 (en) 2007-04-18 2008-10-23 Der-Ing Liao Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
US7910342B2 (en) 2007-04-18 2011-03-22 Butamax(Tm) Advanced Biofuels Llc Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
WO2008137403A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production de 2-butanol
WO2008137402A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production de 1-butanol
WO2008137406A1 (fr) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Procédé de production d'isobutanol
US20080274525A1 (en) 2007-05-02 2008-11-06 Bramucci Michael G Method for the production of 2-butanol
WO2009013066A1 (fr) * 2007-06-15 2009-01-29 Hitec Zang Gmbh Procédé de fermentation de cultures cellulaires
US20090203099A1 (en) 2007-10-30 2009-08-13 E. I. Du Pont De Nemours And Company Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate
US8129162B2 (en) 2007-12-20 2012-03-06 Butamax(Tm) Advanced Biofuels Llc Ketol-acid reductoisomerase using NADH
US20090163376A1 (en) 2007-12-20 2009-06-25 E.I. Du Pont De Nemours And Company Ketol-acid reductoisomerase using nadh
US20100197519A1 (en) 2007-12-20 2010-08-05 E. I. Du Pont De Nemours And Company Ketol-acid reductoisomerase using nadh
US20090246846A1 (en) 2008-03-27 2009-10-01 E. I. Du Pont De Nemours And Company Zymomonas with improved xylose utilization
US20090269823A1 (en) 2008-04-28 2009-10-29 E.I. Du Pont De Nemours And Company Butanol dehydrogenase enzyme from the bacterium achromobacter xylosoxidans
US20090305370A1 (en) 2008-06-04 2009-12-10 E.I. Du Pont De Nemours And Company Method for producing butanol using two-phase extractive fermentation
US20090305363A1 (en) 2008-06-05 2009-12-10 E. I. Du Pont De Nemours And Company Enhanced pyruvate to acetolactate conversion in yeast
US8241878B2 (en) 2008-09-29 2012-08-14 Butamax(Tm) Advanced Biofuels Llc Recombinant yeast host cell with Fe-S cluster proteins and methods of using thereof
US20100081154A1 (en) 2008-09-29 2010-04-01 Butamax(Tm) Advanced Biofuels Llc IDENTIFICATION AND USE OF BACTERIAL [2Fe-2S] DIHYDROXY-ACID DEHYDRATASES
US20100120105A1 (en) 2008-10-27 2010-05-13 Butamax (Tm) Advanced Biofuels Llc Carbon pathway optimized production hosts for the production of isobutanol
WO2010075241A1 (fr) 2008-12-22 2010-07-01 E. I. Du Pont De Nemours And Company Zymomonas avec utilisation de xylose améliorée dans des conditions de stress pour la production de l'éthanol
US20110097773A1 (en) 2009-04-13 2011-04-28 Butamax(Tm) Advanced Biofuels Llc Method for producing butanol using extractive fermentation
US20110124060A1 (en) 2009-09-29 2011-05-26 Butamax(Tm) Advanced Biofuels Llc Yeast production host cells
WO2011041415A1 (fr) 2009-09-29 2011-04-07 Butamax(Tm) Advanced Biofuels Llc Production fermentative d'isobutanol à l'aide de céto-acide réducto-isomérases à efficacité élevée
US20110269199A1 (en) 2009-12-29 2011-11-03 Butamax(Tm) Advanced Biofuels Llc Alcohol dehydrogenases (adh) useful for fermentive production of lower alkyl alcohols
WO2011103300A2 (fr) 2010-02-17 2011-08-25 Butamax(Tm) Advanced Biofuels Llc Amélioration de l'activité de protéines nécessitant l'agrégat fe-s
US20120237988A1 (en) 2010-09-07 2012-09-20 Butamax(Tm) Advanced Biofuels Llc Butanol strain improvement with integration of a polynucleotide encoding a polypeptide that catalyzes pyruvate to acetolactate conversion
WO2012129555A2 (fr) 2011-03-24 2012-09-27 Butamax (Tm) Advanced Biofuels Llc Cellules hôtes et procédés de production d'isobutanol
US20130071898A1 (en) 2011-03-24 2013-03-21 Butamax(Tm) Advanced Biofuels Llc Host cells and methods for production of isobutanol
US20120258873A1 (en) 2011-04-06 2012-10-11 Butamax(Tm) Advanced Biofuels Llc Reduction of 2,3-dihydroxy-2-methyl butyrate (dhmb) in butanol production

Non-Patent Citations (46)

* Cited by examiner, † Cited by third party
Title
"Enzyme Nomenclature", 1992, ACADEMIC PRESS
"Methods in Yeast Genetics", 2005, COLD SPRING HARBOR LABORATORY PRESS, pages: 201 - 202
ADEN ET AL.: "Report NREL/TP-510-32438", June 2002, NATIONAL RENEWABLE ENERGY LABORATORY, article "Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover"
AIBA ET AL., BIOTECHNOL. BIOENG., vol. 18, 1976, pages 1001 - 1016
AIBA S ET AL: "FED BATCH CULTURE OF SACCHAROMYCES-CEREVISIAE A PERSPECTIVE OF COMPUTER CONTROL TO ENHANCE THE PRODUCTIVITY IN BAKERS YEAST CULTIVATION", BIOTECHNOLOGY AND BIOENGINEERING, WILEY & SONS, HOBOKEN, NJ, US, vol. 18, no. 7, 1 January 1976 (1976-01-01), pages 1001 - 1016, XP002507590, ISSN: 0006-3592, DOI: 10.1002/BIT.260180712 *
AMBERG ET AL.: "Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual", 2005, COLD SPRING HARBOR PRESS
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1987, GREENE PUBLISHING ASSOC. AND WILEY-INTERSCIENCE
BELLION ET AL.: "Microb. Growth C1 Compd., [Int. Symp.", 1993, INTERCEPT, pages: 415 - 32
CHEN ET AL., AUTOMATICA, vol. 31, 1995, pages 55 - 65
CHRISTINE GUTHRIE AND GERALD R. FINK,: "Methods in Enzymology", vol. 194, 2004, ELSEVIER ACADEMIC PRESS
COONEY ET AL., BIOTECHNOLOGY AND BIOENGINEERING, vol. 19, 1977, pages 55 - 67
DOHERTY; MALONE: "Conceptual Design of Distillation Systems", 2001, MCGRAW HILL
DURRE, APPL. MICROBIOL. BIOTECHNOL., vol. 49, 1998, pages 639 - 648
FELDMANN ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 38, 1992, pages 354 - 61
GARCIA-ALLES ET AL., BIOCHEMISTRY, vol. 43, 2004, pages 13037 - 13046
GRIBSKOV, M. AND DEVEREUX, J.,: "Sequence Analysis Primer", 1991, STOCKTON
GRIFFIN, A. M., AND GRIFFIN, H. G.,: "Computer Analysis of Sequence Data", 1994, HUMANIA
GROOT ET AL., PROCESS. BIOCHEM., vol. 27, 1992, pages 61 - 75
GUO ET AL., J. MEMBR. SCI., vol. 245, 2004, pages 199 - 210
HAHNAI ET AL., APPL. ENVIRON., vol. 73, 2007, pages 7814 - 8
JONES ET AL., BIOCHEM J., vol. 134, 1973, pages 167 - 182
KANEKO ET AL., PHYTOCHEMISTRY, vol. 39, 1995, pages 115 - 120
KURIYAMA; KOBAYASHI, J. FERMENT. BIOENG., vol. 75, 1993, pages 364 - 367
LESK, A. M.,: "Computational Molecular Biology", 1988, OXFORD UNIVERSITY
LUKYANOV ET AL., BC, vol. 275, no. 34, 2000, pages 25879
MATZ ET AL., NATURE BIOTECHNOLOGY, vol. 17, 1999, pages 969
OHTA ET AL., APPL. ENVIRON. MICROBIOL., vol. 57, 1991, pages 893 - 900
PHILLIPP ET AL.,: "Manual of Methods for General Bacteriology", AMERICAN SOCIETY FOR MICROBIOLOGY
PREUSS ET AL., CHEMICAL ENGINEERING, vol. 78, 2000, pages 53 - 59
RANI; RAO, BIOPROCESS ENGINEERING, vol. 21, 1999, pages 77 - 88
RIEGER ET AL., J. GEN. MICROBIOL., vol. 129, 1983, pages 653 - 61
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SHEN; LIAO, METAB. ENG., vol. 10, 2008, pages 312 - 20
SHIN; KIM, J. ORG. CHEM., vol. 67, 2002, pages 2848 - 2853
SMITH, D. W.,: "Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC
SONNLEITNERT; KAPPELI, BIOTECH & BIOENG, vol. 28, 1986, pages 927 - 37
SPERANZA ET AL., J. AGRIC. FOOD CHEM., vol. 45, 1997, pages 3476 - 3480
SULTER ET AL., ARCH. MICROBIOL., vol. 153, 1990, pages 485 - 489
THOMAS D. BROCK: "Biotechnology: A Textbook of Industrial Microbiology Second Edition,", 1989, SINAUER ASSOCIATES, INC.
UNDERWOOD ET AL., APPL. ENVRION. MICROBIOL., vol. 68, 2002, pages 1071 - 81
VALENTINOTTI ET AL., CONTROL ENGINEERING PRACTICE, vol. 11, 2003, pages 665 - 674
VON HEINJE, G.,: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC
WEUSTHUIS ET AL., MICROBIOLOGY, vol. 140, 1994, pages 703 - 715
YASUTA ET AL., APPL. ENVIRON. MICROBIAL., vol. 67, 2001, pages 4999 - 5009
YEAST, vol. 20, 2003, pages 117 - 132
ZHANG ET AL., SCIENCE, vol. 267, 1995, pages 240 - 3

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3180440A4 (fr) * 2014-08-11 2018-01-10 Butamax Advanced Biofuels LLC Préparations de levures et leurs procédés de fabrication
US10280438B2 (en) 2014-08-11 2019-05-07 Butamax Advanced Biofuels Llc Method for the production of yeast
CN106987573A (zh) * 2017-04-01 2017-07-28 山东福洋生物制造工程研究院 一种生产麦芽寡糖基海藻糖合成酶、麦芽寡糖基海藻糖水解酶的方法以及生产海藻糖的方法
CN111386339A (zh) * 2017-11-30 2020-07-07 东丽株式会社 用于生产3-羟基己二酸、α-氢化己二烯二酸及/或己二酸的基因修饰微生物以及该化学产品的制造方法
CN111386339B (zh) * 2017-11-30 2024-05-10 东丽株式会社 用于生产3-羟基己二酸、α-氢化己二烯二酸及/或己二酸的基因修饰微生物以及该化学产品的制造方法
WO2019209241A1 (fr) 2018-04-23 2019-10-31 Dupont Nutrition Biosciences Aps Augmentation de l'exportation de 2'fucosyllactose à partir de cellules microbiennes par l'expression d'un acide nucléique hétérologue
WO2019209245A1 (fr) 2018-04-23 2019-10-31 Dupont Nutrition Biosciences Aps Augmentation de l'activité de transporteurs de 2' fucosyllactose endogènes à des cellules microbiennes
US12071614B2 (en) 2018-04-23 2024-08-27 Inbiose N.V. Increasing activity of 2′fucosyllactose transporters endogenous to microbial cells
WO2021086606A1 (fr) 2019-10-28 2021-05-06 Danisco Us Inc Cellules hôtes microbiennes pour la production d'hydrolases d'acide cyanurique hétérologues et d'hydrolases de biuret
CN113957101A (zh) * 2020-07-21 2022-01-21 山东福洋生物科技股份有限公司 一种重组大肠杆菌发酵生产肌醇的方法
CN113278537A (zh) * 2021-06-15 2021-08-20 天津科技大学 系列低产高级醇酿酒酵母及其构建方法与应用

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