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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01008—Glycerol-3-phosphate dehydrogenase (NAD+) (1.1.1.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01094—Glycerol-3-phosphate dehydrogenase (NAD(P)+)(1.1.1.94)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
  • C12Y101/05003—Glycerol-3-phosphate dehydrogenase (1.1.5.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
  • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
  • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
• • 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
• • 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
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

• polypeptide is intended to encompass a singular
• 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.
• adenine is complementary to thymine and cytosine is complementary to guanine
• cytosine is complementary to guanine
• GPD1 and GPD2 are functional homo logs. Any of the genes encoding GPD enzymes of yeast may be disrupted to reduce GPD activity in a yeast cell.
• GPD enzymes that can utilize NADH or NADPH and/or are feedback inhibited by glycerol-3 -phosphate can include both naturally occurring proteins and engineered proteins.
• NADH-utilizing or NADPH -utilizing GPD enzymes are described by EC 1.1.1.94 and have been found in Aspergillus oryzae, Candida versatilis, Escherichia coli, and Oryctolagus cuniculus.
• the GPD comprises a substitution of the residue corresponding to position 129 of SEQ ID NO: 195 (Phe in SEQ ID NO: 195) to an amino acid selected from the group consisting of G, A, R, and K.
• step b) the 3-hydroxybutyryl-CoA from step b) to crotonyl-CoA, which may be catalyzed, for example, by crotonase;
• butyryl-CoA from step d) to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase;
• 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;
• the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:
• KARIs disclosed therein are those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAOl, and Pseudomonas fluorescens PF5 mutants.
• the KARI utilizes NADH.
• the KARI utilizes NADPH.
• the KARI utilizes NADH or NADPH.
• valine dehydrogenase refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor.
• Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP_628270, NC_003888) and B. subtilis (GenBank Nos: CAB14339, Z99116).
• 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.
• dihydroxy acetone for example, include enzymes known as EC 2.7.1.29 (Garcia- Alles, et al, Biochemistry 45: 13037-13046, 2004).
• the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFTI L99A, AFTI L102A, AFTI C291F, or AFTI C293F.
• host cells may comprise heterologous polynucleotides encoding a polypeptide with phosphoketolase activity and/or a heterologous polynucleotide encoding a polypeptide with phosphotransacetylase activity as described in U.S. Patent Application No. 2012/0156735, incorporated herein by reference.
• Recombinant host cells disclosed herein are contacted with suitable carbon substrates, typically in fermentation media.
• 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.
• 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.
• 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.
• 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.
• a resulting identified strain, PNY2145 has the genotype: MATa ura3A::loxP his3A pdc5A::P[FBA(L8)]-XPK
• transformants were screened using two sets of primers oBP1342 (SEQ ID NO:6) and OBP1352 (SEQ ID NO:243) and oBP1357 (SEQ ID NO:248) and oBP1345 (SEQ ID NO:8) for confirmation of integration at both ends.
• the primers oBP1342 (SEQ ID NO:6) and oBP1345 (SEQ ID NO: 8) were designed from a region outside the cassette to confirm integration at the right site. Removal of URA3 Marker
• Example 1 these proteins were expressed in E. coli, and crude cell extracts were used to measure NADH K M values.
• Candida versatilis GPD did not yield significant measureable activity.
• the sequence of the Rickettsia enzyme was compared to the crystal structure of the human enzyme with NAD+ in the binding site.
• a notable feature of the human enzyme :NAD complex is the pi-stacking of phe41 and phe97 sandwiched around the adenine ring of NAD+ (pdb: 1X0X; Ou et al, 2006 J Mol Biol 357: 858-869).
• the pi-stacking is a very stable structure, and sequence alignment reveals that the homologous positions are conserved in yeast GPD1 sequence (phe73, phel29).

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