Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y401/00—Carbon-carbon lyases (4.1)
  • C12Y401/02—Aldehyde-lyases (4.1.2)
  • C12Y401/02009—Phosphoketolase (4.1.2.9)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • 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
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
  • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
• • 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
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/54—Acetic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
  • C12Y203/01008—Phosphate acetyltransferase (2.3.1.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03021—Glycerol-1-phosphatase (3.1.3.21)
• • 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

• the present disclosure relates to compositions and methods for producing acetyl-CoA derived compounds in engineered host cells.
• Acetyl coenzyme A is a key intermediate in the synthesis of essential biological compounds, including polyketides, fatty acids, isoprenoids, phenolics, alkaloids, vitamins, and amino acids.
• metabolites derived from acetyl-CoA are primary and secondary metabolites, including compounds of industrial utility.
• yeast acetyl-CoA is biosynthesized from pyruvate metabolism (FIG. 1). However, in this biosynthetic pathway, C0 2 is lost via the reactions catalyzed by pyruvate carboxylase and/or pyruvate dehydrogenase.
• one benefit of providing an alternative to pyruvate metabolism and lower glycolysis is that less C0 2 is produced in the decarboxylation of pyruvate, and thus more carbon can be captured in the end product, thereby increasing the maximum theoretical yield.
• a second benefit is that less NADH is produced, and therefore significantly less oxygen is needed to reoxidize it. This can be accomplished by expressing phosphoketolase (PK; EC 4.1.2.9) in conjunction with phosphoacetyltransferase (PTA; EC 2.3.1.8).
• PK and PTA catalyze the reactions to convert fructose-6-phosphate (F6P) or xylulose-5 -phosphate (X5P) to acetyl-CoA.
• F6P fructose-6-phosphate
• X5P xylulose-5 -phosphate
• PK draws from the pentose phosphate intermediate xyulose 5-phosphate, or from the upper glycolysis intermediate D- fructose 6-phosphate (F6P).
• PK splits X5P into glyceraldehyde 3-phosphate (G3P) and acetyl phosphate, or F6P into erythrose 4-phosphate (E4P) and acetyl phosphate.
• G3P glyceraldehyde 3-phosphate
• E4P erythrose 4-phosphate
• PTA then converts the acetyl phosphate into acetyl-CoA.
• G3P can re-enter lower glycolysis
• E4P can re-enter the pentose phosphate pathway or glycolysis by cycling through the non- oxidative pentose phosphate pathway network of transaldolases and transketolases.
• Sondregger et al. have also described the benefits of PK and PTA with respect to ethanol production in a xylose-utilizing yeast strain. See Sondregger et al, Applied and Environmental Microbiology 70(5):2892-2897 (2004), the contents of which are hereby incorporated by reference in their entirety.
• the heterologous phosphoketolase pathway (PK, PTA, and ADA) was introduced in S. cerevisiae to address low ethanol yields that result from overexpression of NAD(P)H-dependent xylose reductase and NAD + -dependent xylitol dehydrogenase from Pichia stipitis.
• oxidoreductase reactions caused an anaerobic redox balancing problem that manifested in the extensive accumulation of the reduced reaction intermediate xylitol, and thus, low ethanol yields.
• Redox metabolism was balanced by introducing the phosphoketolase pathway, which lead to the net reoxidation of one NADH per xylose converted to ethanol, and an
• compositions and methods provided herein address this need and provide related advantages as well.
• compositions and methods for the improved utilization of phosphoketolase (PK) and phosphotransacetylase (PTA) for the production of industrially useful compounds are based on the surprising discovery that phosphoketolase pathway-based acetate accumulation results from the enzyme-catalyzed hydrolysis of acetyl phosphate, the product of PK catalysis. Hydrolysis of acetyl phosphate is an undesirable side -reaction that can negatively impact production, via depletion of carbon, of any type of product derived from acetyl-CoA, including isoprenoids, polyketides, and fatty acids. By functionally disrupting native enzymes in the host cell that catalyze acetyl phosphate hydrolysis, acetate accumulation is reduced and carbon flux through the PK/PTA pathway towards acetyl-CoA production is increased.
• PK phosphoketolase
• PTA phosphotransacetylase
• compositions and methods provided herein are further based on the unexpected discovery of native enzymes in yeast that catalyze the hydrolysis of acetyl phosphate to acetate, namely GPP1/RHR2, and its closely related homolog GPP2/HOR2. Both of these enzymes have only been previously characterized as having glycerol- 1- phosphatase (EC 3.1.3.21; alternately referred to as "glycerol-3-phosphatase”) activity, and thus, the promiscuous acetyl-phosphatase activity of these enzymes is unexpected.
• deletion of one or both of the genes encoding RHR2 and HOR2 leads to a reduction in acetate accumulation, with deletion of the gene encoding RHR2 alone leading to a substantial reduction in acetate levels.
• deletion of the RHR2 gene in cells engineered to comprise PK, PTA and a mevalonate pathway resulted in a substantial increase in the production of farnesene, an acetyl-CoA derived isoprenoid.
• a genetically modified host cell comprising: a heterologous nucleic acid encoding a
• the genetically modified host cell further comprises a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8).
• a genetically modified host cell comprising: a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8); and a functional disruption of an endogenous enzyme that converts acetyl phosphate to acetate.
• the genetically modified host cell further comprises a heterologous nucleic acid encoding a phosphoketolase (PK; EC 4.1.2.9).
• the enzyme that converts acetyl phosphate to acetate is a glycerol- 1 -phosphatase (EC 3.1.3.21).
• the glycerol- 1 -phosphatase is selected from the group consisting of GPP1/RHR2, GPP2HOR2, and homologues and variants thereof.
• the genetically modified host cell comprises a functional disruption of GPP1/RHR2.
• the genetically modified host cell comprises a functional disruption of GPP2/HOR2.
• the genetically modified host cell comprises a functional disruption of both GPP1/RHR2 and GPP2/HOR2.
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an acylating acetylaldehyde dehydrogenase (ADA; EC 1.2.1.10).
• the genetically modified host cell further comprises a functional disruption of one or more enzymes of the native pyruvate dehydrogenase (PDH) - bypass.
• the one or more enzymes of the PDH-bypass are selected from acetyl-CoA synthetase 1 (ACS1), acetyl-CoA synthetase 2 (ACS2), and aldehyde dehydrogenase 6 (ALD6).
• the genetically modified host cell is capable of producing a heterologous acetyl-CoA derived compound.
• the heterologous acetyl-CoA derived compound is selected from the group consisting of an isoprenoid, a polyketide, and a fatty acid.
• the genetically modified host cell is capable of producing an isoprenoid.
• the genetically modified host cell comprises one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate.
• the one or more enzymes of the MEV pathway comprise an NADH-using HMG-CoA reductase.
• the one or more enzymes of the MEV pathway comprise an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA.
• the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetoacetyl- CoA with acetyl-CoA to form HMG-CoA.
• the one or more enzymes of the MEV pathway comprise an enzyme that converts HMG-CoA to mevalonate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5- phosphate to mevalonate 5 -pyrophosphate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5 -pyrophosphate to isopentenyl pyrophosphate.
• the one or more enzymes of the MEV pathway are selected from HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase.
• the host cell comprises a plurality of heterologous nucleic acids encoding all of the enzymes of the MEV pathway.
• the one or more heterologous nucleic acids encoding one or more enzymes of the MEV pathway are under control of a single transcriptional regulator.
• the one or more heterologous nucleic acids encoding one or more enzymes of the MEV pathway are under control of multiple heterologous transcriptional regulators.
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can condense IPP and/or DMAPP molecules to form a polyprenyl compound.
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.
• the enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound is selected from the group consisting of carene synthase, geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, a-pinene synthase, ⁇ -pinene synthase, ⁇ -terpinene synthase, terpinolene synthase,
• amorphadiene synthase amorphadiene synthase, a-farnesene synthase, ⁇ -farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nootkatone synthase, and abietadiene synthase.
• the isoprenoid is selected from the group consisting of a hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, sesquiterpene, and
• the isoprenoid is a sesquiterpene. In some embodiments, the isoprenoid is a C5-C20 isoprenoid. In some embodiments, the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, a-farnesene, ⁇ -farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, ⁇ -pinene, sabinene, ⁇ -terpinene, terpinolene, and valencene.
• a genetically modified host cell capable of producing an isoprenoid, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate; a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid encoding a phosphotransacetylase (PTA); and a functional disruption of a glycerol- 1 -phosphatase (EC 3.1.3.21).
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol-1- phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing an isoprenoid, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate; a heterologous nucleic acid encoding an acetylaldehyde dehydrogenase, acetylating (ADA); a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid encoding a phosphotransacetylase (PTA); and a functional disruption of a glycerol- 1 -phosphatase (EC 3.1.3.21).
• MMV mevalonate
• ADA acetylaldehyde dehydrogenase
• ADA acetylating
• PK phosphoketolase
• PTA phosphotransacetylase
• the glycerol-1- phosphatase is GPP1/RHR2, or a homologue or variant thereof. In some embodiments, the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing an isoprenoid, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate; a heterologous nucleic acid encoding an acetylaldehyde dehydrogenase, acetylating (ADA); a functional disruption of at least one enzyme of the native PDH-bypass selected from the group consisting of acetyl-CoA synthetase 1 (AC SI), acetyl-CoA synthetase 2 (ACS2), and aldehyde dehydrogenase 6 (ALD6); a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid encoding a
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing an isoprenoid, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate, wherein the one or more enzymes comprise a NADH-using HMG-CoA reductase; a heterologous nucleic acid encoding an acetylaldehyde dehydrogenase, acetylating (ADA); a functional disruption of at least one enzyme of the native PDH-bypass selected from the group consisting of acetyl-CoA synthetase 1 (AC SI), acetyl-CoA synthetase 2 (ACS2), and aldehyde dehydrogenase 6 (ALD6); a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid en
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing an isoprenoid, the cell comprising: one or more heterologous nucleic acids encoding a plurality of enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate, wherein the plurality of enzymes comprise an acetyl-CoA:malonyl-CoA acyltransferase; a heterologous nucleic acid encoding an acetylaldehyde dehydrogenase, acetylating (ADA); a functional disruption of at least one enzyme of the native PDH-bypass selected from the group consisting of acetyl-CoA synthetase 1 (AC SI), acetyl-CoA synthetase 2 (ACS2), and aldehyde dehydrogenase 6 (ALD6); a heterologous nucleic acid encoding a phosphoketolase (PK); a
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing an polyketide, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of polyketide biosynthetic pathway; a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid encoding a
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• a genetically modified host cell capable of producing a fatty acid, the cell comprising: one or more heterologous nucleic acids encoding one or more enzymes of fatty acid biosynthetic pathway; a heterologous nucleic acid encoding a phosphoketolase (PK); a heterologous nucleic acid encoding a phosphotransacetylase (PTA); and a functional disruption of a glycerol- 1 -phosphatase (EC 3.1.3.21).
• the glycerol- 1 -phosphatase is GPP1/RHR2, or a homologue or variant thereof.
• the glycerol- 1 -phosphatase is GPP2/HOR2, or a homologue or variant thereof.
• the genetically modified host cell provided herein is selected from the group consisting of a bacterial cell, a fungal cell, an algal cell, an insect cell, and a plant cell.
• the cell is a yeast cell.
• the yeast is Saccharomyces cerevisiae.
• the genetically modified host cell produces an increased amount of an acetyl-CoA derived compound ⁇ e.g., an isoprenoid, polyketide, or fatty acid) compared to a yeast cell not comprising a functional disruption of an endogenous enzyme that converts acetyl phosphate to acetate.
• an acetyl-CoA derived compound e.g., an isoprenoid, polyketide, or fatty acid
• heterologous acetyl-CoA derived compound in another aspect, provided herein are methods for producing a heterologous acetyl-CoA derived compound, the method comprising: culturing a population of genetically modified host cells, capable of producing a heterologous acetyl-CoA derived compound as described herein, in a medium with a carbon source under conditions suitable for making said heterologous acetyl-CoA derived compound; and recovering said heterologous acetyl-CoA derived compound from the medium.
• heterologous acetyl-CoA derived compound is selected from the group consisting of an isoprenoid, a polyketide, and a fatty acid.
• a method for increasing the production of acetyl-CoA or an acetyl-CoA derived compound in a host cell comprising:
• the method further comprises expressing in the host cell a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8).
• PTA phosphotransacetylase
• a method for increasing the production of acetyl-CoA in a host cell comprising: expressing in the host cell a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8); and functionally disrupting an endogenous enzyme that converts acetyl phosphate to acetate.
• the method further comprises expressing in the host cell a heterologous nucleic acid encoding a phosphoketolase (PK; EC 4.1.2.9).
• the enzyme that converts acetyl phosphate to acetate is a glycerol- 1 -phosphatase (EC 3.1.3.21).
• the glycerol- 1 -phosphatase is selected from GPP1/RHR2, GPP2/HOR2, and homologues and variants thereof.
• GPP1/RHR2, or a homologue or variant thereof is functionally disrupted.
• GPP2/HOR2, or a homologue or variant thereof is functionally disrupted.
• both GPP1/RHR2 and GPP2/HOR2, or both a homologue or variant of GPP1/RHR2 and a homologue or variant of GPP2/HOR2, are functionally disrupted.
• the host cell is selected from the group consisting of a bacterial cell, a fungal cell, an algal cell, an insect cell, and a plant cell.
• the host cell is a yeast cell.
• the yeast is Saccharomyces cerevisiae.
• the host cell produces an increased amount of acetyl-CoA or an acetyl-CoA derived compound compared to a yeast cell not comprising a functional disruption of an endogenous enzyme that converts acetyl phosphate to acetate.
• FIG. 1 provides a schematic representation of the pathways involved in the conversion of sugar (glucose and xylose) to acetyl-CoA, and acetyl-CoA derived compounds, in a yeast host cell.
• the bold arrows indicate the recombinant phosphoketolase pathway.
• Acetyl phosphate is an intermediate of the phosphoketolase (PK) / phosphotransacetyklase (PTA) pathway to acetyl-CoA, and is hydro lyzed to acetate by RHR2 and HOR2.
• PK phosphoketolase
• PTA phosphotransacetyklase
• G6P glucose-6-phosphate
• R5P ribulose-5-phosphate
• X5P xyulose-5- phosphate
• F6P fructose-6-phosphate
• E4P eryhtrose-4-phosphate
• FBP fructose- 1,6- biphosphate
• DHAP dihydroxyacetone phosphate
• G3P glyceraldehyde-3-phosphate
• PEP phosphoenolpyruvate
• ADA acetaldehyde dehydrogenase, acetylating
• ACP acetyl phosphate.
• FIG. 2 provides representative enzymes of the mevalonate pathway for isoprenoid production.
• AcCoA acetyl-CoA
• AcAcCoA acetoacetyl-CoA
• HMGCoA 3-hydroxy-3-methylglutaryl-CoA
• Mev5P mevalonate-5-phosphate
• Mev5DP mevalonate-5 -diphosphate
• IPP isopentenyl diphosphate
• DMAPP dimethylallyl
• FIGS. 3A-3B provides the sugar consumption (A) and acetate production (B) of wild-type (strain Y967, left) and recombinant yeast cells (middle, right) comprising: a heterologous acetaldehyde dehydrogenase acylating (Dz.eutE) and deletion of the native PDH-bypass (acslA acs2 A ald6A) (strain Y 12869; middle); and further comprising a heterologous phosphoketolase (Lm.PK) and phosphotransacetylase (Ck.PTA) (strain
• FIGS. 3C-3D provides the sugar consumption (C) and acetate production (D) of recombinant yeast cells comprising: a heterologous acetaldehyde dehydrogenase acylating (Dz.eutE) and deletion of the native PDH-bypass (acslA acs2 A ald6A) (strain Y12869; left); and further comprising a heterologous phosphoketolase (Lm.PK) (strain Y19390; middle) or phosphotransacetylase (Ck.PTA) (strain Y 19391; right).
• a heterologous acetaldehyde dehydrogenase acylating Dz.eutE
• acslA acs2 A ald6A a heterologous phosphoketolase
• Lm.PK heterologous phosphoketolase
• Ck.PTA phosphotransacetylase
• FIG. 4 provides a demonstration of acetyl phosphate hydrolysis in cell free extracts (CFE) of wild-type S. cerevisiae strain Y967 over a 120 minute timecourse. Shown are CFE only (left); CFE plus 30 mM sodium fluoride, a broad spectrum phosphatase inhibitor (middle); and CFE that has been heat inactivated (right).
• CFE cell free extracts
• FIG. 5 provides results of anion exchange chromatography on Y967 cell free extracts. Protein was eluted with a 0-100% gradient of buffer B (20 mM Tris-Cl pH 7, 1M NaCl, 10% glycerol) over 30 column volumes at a flow rate of 0.5 mL/minute, and 1 mL fractions were collected, analyzed by protein gel electrophoresis (FIG. 5B), and assayed for acetyl phosphatase activity (FIG. 5A). ACP, acetyl phosphate.
• FIG. 6 A provides results of anion exchange chromatography on fraction #10 of Y967.
• the most active fraction from this purification, # 14 was analyzed by mass spectrometry to determine the identity of the proteins in the fraction (FIG. 6B).
• RHR2 was identified as a phosphatase in the active fraction.
• FIG. 7 provides results of acetyl phosphatase activity assays on CFEs of a wild-type yeast strain (Y968) or recombinant yeast strains comprising a deletion of RHR2, HOR2 or both RHR2 and HOR2.
• FIGS. 8A-8C provides acetate levels (A), glycerol levels (B) and optical densities (C) of recombinant yeast strain populations.
• Strain Y12746.ms63909.ms64472 comprises a deletion of the PDH-bypass (acslA acs2 A ald6A), and heterologously expresses acetaldehyde dehydrogenase aceylating (Dz.eutE), phosphoketolase (Lm.PK),
• FIGS. 8D-8E provides acetate levels (D) and optical densities (E) of recombinant yeast strain populations.
• Strain Y12745 comprises a deletion of the PDH-bypass ⁇ acslA acs2 A ald6A), and heterologously expresses acetaldehyde dehydrogenase aceylating (Dz.eutE), phosphoketolase (Lm.PK), and phosphotransacetylase (Ck.PTA).
• Strain Y12746 rhr2 A is isogenic to strain Y 12746 but further comprises a deletion of RHR2 (rhr2 A ).
• FIG. 9 provides relative farnesene levels (top) and relative optical densities
• Y968 (right panel) is a wild-type yeast strain.
• Y12869.ms63907.ms64472 (“Y 12869"; 2 nd from right panel) comprises a deletion of the PDH-bypass (acslA acs2 A ald6A), and heterologously expresses acetaldehyde dehydrogenase aceylating (Dz.eutE) and genes in the farnesene production pathway, but does not express phosphoketolase or phosphotransacetylase.
• Y12746.ms63907.ms64472 (“Y12746"; 2 nd from left panel) comprises a deletion of the PDH-bypass (acslA acs2 A ald6A), and heterologously expresses acetaldehyde dehydrogenase aceylating (Dz.eutE) and genes in the farnesene production pathway, and uses phosphoketolase and phosphotransacetylase as a pathway to produce cytosolic acetyl-CoA, which is used for synthesis of farnesene.
• PDH-bypass acslA acs2 A ald6A
• Dz.eutE acetaldehyde dehydrogenase aceylating
• Y12745.ms63907.ms64472 (“Y12745”; left panel) comprises a deletion of the PDH-bypass (acslA acs2 A ald6A), and genes in the farnesene production pathway, and uses phosphoketolase and
• phosphotransacetylase as a pathway to produce cytosolic acetyl-CoA, which is used for synthesis of farnesene.
• heterologous refers to what is not normally found in nature.
• heterologous nucleotide sequence refers to a nucleotide sequence not normally found in a given cell in nature.
• a heterologous nucleotide sequence may be: (a) foreign to its host cell ⁇ i.e., is "exogenous” to the cell); (b) naturally found in the host cell ⁇ i.e., "endogenous") but present at an unnatural quantity in the cell ⁇ i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.
• heterologous enzyme refers to an enzyme that is not normally found in a given cell in nature.
• the term encompasses an enzyme that is: (a) exogenous to a given cell ⁇ i.e., encoded by a nucleotide sequence that is not naturally present in the host cell or not naturally present in a given context in the host cell); and (b) naturally found in the host cell (e.g., the enzyme is encoded by a nucleotide sequence that is endogenous to the cell) but that is produced in an unnatural amount (e.g., greater or lesser than that naturally found) in the host cell.
• the term “native” or “endogenous” as used herein with reference to molecules, and in particular enzymes and nucleic acids, indicates molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower, equal, or higher than the level of expression of the molecule in the native microorganism. It is understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.
• a “functional disruption” e.g., of a target gene, for example, one or more genes of the PDH-bypass
• the target gene is altered in such a way as to decrease in the host cell the activity of the protein encoded by the target gene.
• the functional disruption of a target gene results in a reduction by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the expression level of the target gene compared to its expression when not functionally disrupted.
• a target protein for example, a protein having acetyl phosphatase activity
• the functional disruption of a target protein results in a reduction by at least 5%, 10%>, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the activity or expression level of the target protein compared to its activity or expression when not functionally disrupted.
• the activity of the target protein encoded by the target gene is eliminated in the host cell.
• the activity of the target protein encoded by the target gene is decreased in the host cell.
• Functional disruption of the target gene may be achieved by deleting all or a part of the gene so that gene expression is eliminated or reduced, or so that the activity of the gene product is eliminated or reduced.
• Functional disruption of the target gene may also be achieved by mutating a regulatory element of the gene, e.g., the promoter of the gene so that expression is eliminated or reduced, or by mutating the coding sequence of the gene so that the activity of the gene product is eliminated or reduced.
• functional disruption of the target gene results in the removal of the complete open reading frame of the target gene.
• parent cell refers to a cell that has an identical genetic background as a genetically modified host cell disclosed herein except that it does not comprise one or more particular genetic modifications engineered into the modified host cell, for example, one or more modifications selected from the group consisting of: heterologous expression of an ADA, heterologous expression of an NADH-using HMG-CoA reductase, heterologous expression of an AACS, heterologous expression of a phosphoketolase, heterologous expression of a phosphotransacetylase, and heterologous expression of one or more enzymes of the mevalonate pathway.
• modifications selected from the group consisting of: heterologous expression of an ADA, heterologous expression of an NADH-using HMG-CoA reductase, heterologous expression of an AACS, heterologous expression of a phosphoketolase, heterologous expression of a phosphotransacetylase, and heterologous expression of one or more enzymes of the mevalonate pathway.
• production generally refers to an amount of an isoprenoid produced by a genetically modified host cell provided herein.
• production is expressed as a yield of isoprenoid by the host cell. In other embodiments, production is expressed as a productivity of the host cell in producing the isoprenoid.
• productivity refers to production of an isoprenoid by a host cell, expressed as the amount of isoprenoid produced (by weight) per amount of fermentation broth in which the host cell is cultured (by volume) over time (per hour).
• yield refers to production of an isoprenoid by a host cell, expressed as the amount of isoprenoid produced per amount of carbon source consumed by the host cell, by weight.
• acetyl-CoA derived compound refers to a compound which uses acetyl-CoA as a substrate in its biosynthesis.
• exemplary acetyl-CoA derived compounds include, but are not limited to, isoprenoids, polyketides, fatty acids, and alcohols.
• an acetyl-CoA derived compound is ethanol, for example, bioethanol produced from pentose substrates, as described in U.S. Patent No. 7,253,001, the contents of which are hereby incorporated by reference in their entirety.
• the term "variant” refers to a polypeptide differing from a specifically recited “reference” polypeptide (e.g., a wild-type sequence) by amino acid insertions, deletions, mutations, and substitutions, but retains an activity that is substantially similar to the reference polypeptide.
• the variant is created by recombinant DNA techniques, such as mutagenesis.
• a variant polypeptide differs from its reference polypeptide by the substitution of one basic residue for another (i.e. Arg for Lys), the substitution of one hydrophobic residue for another (i.e. Leu for He), or the substitution of one aromatic residue for another (i.e. Phe for Tyr), etc.
• variants include analogs wherein conservative substitutions resulting in a substantial structural analogy of the reference sequence are obtained.
• conservative substitutions include glutamic acid for aspartic acid and vice-versa; glutamine for asparagine and vice-versa; serine for threonine and vice-versa; lysine for arginine and vice-versa; or any of isoleucine, valine or leucine for each other.
• Host cells useful compositions and methods provided herein include archae, prokaryotic, or eukaryotic cells.
• Suitable prokaryotic hosts include, but are not limited, to any of a variety of gram-positive, gram-negative, or gram-variable bacteria. Examples include, but are not limited to, cells belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enter obacter, Erwinia, Escherichia, Lactobacillus, Lactococcus,
• Rhodobacter Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.
• prokaryotic strains include, but are not limited to: Bacillus subtilis, Bacillus
• the host cell is an Escherichia coli cell.
• Suitable archae hosts include, but are not limited to, cells belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma.
• Examples of archae strains include, but are not limited to: Archaeoglobus fulgidus, Halobacterium sp., Methanococcus jannaschii,
• thermoautotrophicum Methanobacterium thermoautotrophicum, Thermoplasma acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii, Pyrococcus abyssi, and Aeropyrum pernix.
• Suitable eukaryotic hosts include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells.
• yeasts useful in the present methods include yeasts that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc) and belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera,
• Pachysolen Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula,
• Saccharomyces Saccharomy codes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus,
• the host microbe is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis
• the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.
• the host microbe is Saccharomyces cerevisiae.
• the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1.
• the host microbe is a strain of Saccharomyces cerevisiae selected from the group consisting of PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1.
• Saccharomyces cerevisiae is PE-2.
• the strain of the Saccharomyces cerevisiae is PE-2.
• the strain of the Saccharomyces cerevisiae is PE-2.
• the strain of the Saccharomyces cerevisiae is PE-2.
• the strain of the Saccharomyces cerevisiae is PE-2.
• the strain of the Saccharomyces cerevisiae is PE-2.
• the strain of Saccharomyces cerevisiae is PE-2.
• Saccharomyces cerevisiae is CAT-1.
• Saccharomyces cerevisiae is BG-1.
• the host microbe is a microbe that is suitable for industrial fermentation.
• the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.
• the phosphoketolase pathway is activated in the genetically modified host cells provided herein by engineering the cells to express polynucleotides and/or polypeptides encoding phosphoketolase and, optionally,
• the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity.
• the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.
• the genetically modified host cells provided herein comprise both a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity and a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.
• Phosphoketolase (EC 4.1.2.9) catalyzes the conversion of xylulose 5- phosphate into glyceraldehyde 3-phosphate and acetyl phosphate; and/or the conversion of fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate.
• Phosphoketolase activity has been identified in several yeast strains growing with xylose as the sole carbon source but not in yeast strains grown with glucose (Evans and Ratledge, Arch. Microbiol. 139: 48-52; 1984).
• Inhibitors of phosphoketolase include, but are not limited to, erythrose 4- phosphate and glyceraldehyde 3-phosphate.
• polynucleotides, genes and polypeptides encoding phosphoketolase activity are known in the art and can be used in the genetically modified host cell provided herein.
• a polynucleotide, gene and/or polypeptide is the xylulose 5-phosphateketolase (XpkA) of Lactobacillus pentosus MD363 (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002).
• XpkA is the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and exhibits a specific activity of 4.455 ⁇ mol/min/mg (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002).
• PGP phosphoketolase pathway
• such a polynucleotide, gene and/or polypeptide is the phosphoketolase of Leuconostoc mesenteroides (Lee et al., Biotechnol Lett. 27(12);853-858 (2005)), which exhibits a specific activity of 9.9 ⁇ mol/min/mg and is stable at pH above 4.5 (Goldberg et al., Methods Enzymol.
• This phosphoketolase exhibits a Km of 4.7 mM for D- xylulose 5- phosphate and a Km of 29 mM for fructose 6-phosphate (Goldberg et al,
• Representative phosphoketolase nucleotide sequences of Leuconostoc mesenteroides includes accession number AY804190, and SEQ ID NO: 1 as provided herein.
• Representative phosphoketolase protein sequences of Leuconostoc mesenteroides include accession numbers YP 819405, AAV66077.1, and SEQ ID NO: 2 as provided herein.
• such a polynucleotide, gene and/or polypeptide is the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene xfp from B. lactis, as described, for example, in a pentose-metabolizing S. cerevisiae strain by Sonderegger et al. (Appl. Environ. Microbiol. 70: 2892-7; 2004).
• Bifidobacterium dentium ATCC 27678 (ABIX02000002.1 :2350400..2352877; EDT46356.1); Bifidobacterium animalis (NC_017834.1 : 1127580..1130057; YP_006280131.1); and
• Bifidobacterium pseudolongum (AY518216.1 :988..3465; AAR98788.1); Aspergillus nidulans FGSC A4 (CBF76492.1); Bifidobacterium longum (AAR98787.1);
• Bifidobacterium bifidum NCIMB 41171 (ZP 03646196.1); Bifidobacterium animalis subsp. lactis HN019 (ZP 02962870.1); Lactobacillus plantarum WCFS1 (NP_786060.1);
• Lactobacillus brevis subsp. gravesensis ATCC 27305 (ZP 03940142.1); Lactobacillus reuteri 100-23 (ZP_03073172.1); and Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (YP_818922.1).
• phosphoketolases include: (YP 001601863.1; Gluconacetobacter diazotrophicus Pal 5), (YP 001093221.1; Shewanella loihica PV-4), (YP_926792.1;
• Shewanella amazonensis SB2B Shewanella amazonensis SB2B
• Aspergillus niger Aspergillus niger
• XP_001263382.1 Neosartorya fischeri NRRL 181
• XP_001271080.1 Aspergillus clavatus NRRL 1
• XP_001213784.1 Aspergillus terreus NIH2624
• neoformans JEC21 (XP_759561.1; Ustilago maydis 521), (ZP_05027078.1; Microcoleus chthonoplastes PCC 7420), (YP 003101114.1; Actinosynnema mirum DSM 43827), (ZP_03568244.1; Atopobium rimae ATCC 49626), (YP 003180237.1; Atopobium parvulum DSM 20469), (ZP_03946928.1; Atopobium vaginae DSM 15829), (ZP_03296299.1; Collinsella stercoris DSM 13279), (AAR98787.1;
• Bifidobacterium angulatum DSM 20098) (ZP_03324204.1; Bifidobacterium catenulatum DSM 16992), (AAR98790.1; Bifidobacterium sp. CFAR 172), (AAR98789.1;
• Propionibacterium freudenreichii subsp. Shermanii Propionibacterium freudenreichii subsp. Shermanii), (NP_791495.1; Pseudomonas syringae pv. Tomato str. DC3000), (YP 003125992.1; Chitinophaga pinensis DSM 2588),
• Burkholderia phytofirmans PsJN Burkholderia phytofirmans PsJN
• (YP OO 1861620.1; Burkholderia phymatum STM815) (YP_002755285.1; Acidobacterium capsulatum ATCC 51196), (EDZ38884.1; Leptospin7/ «m sp. Group II '5-way CO')
• EES53204.1 Leptospirillum ferrodiazotrophum
• YP_172723.1 Synechococcus elongatus PCC 6301
• NP_681976.1 Thermosynechococcus elongatus BP -I
• YP l 14037.1
• Methylococcus capsulatus str. Bath (YP_002482577.1; Cyanothece sp. PCC 7425), (NP_442996.1; Synechocystis sp. PCC 6803), (YP_002482735.1; Cyanothece sp. PCC 7425), (ZP_04774866.1; Allochromatium vinosum DSM 180), (ZP_01453148.1;
• Mariprofundus ferrooxydans PV-l (ZP_04830548.1; Gallionella ferruginea ES-2),
• Oibberella zeae PH-1 Oibberella zeae PH-1
• EEU46265.1 Nectria haematococca mp VI 77-13-4
• AC024516.1 Metarhizium anisopliae
• XP_959985.1 Neurospora crassa OR74A
• XP OO 1904686.1 Podospora anserine
• YP_002220141.1 Acidithiobacillus ferrooxidans ATCC 53993
• YP OO 1220128.1; Acidiphilium cryptum JF -5 (YP OO 1471202.1; Thermotoga lettingae TMO), (YP_002352287.1; Dictyoglomus turgidum DSM 6724), (YP 571790.1; Nitrobacter hamburgensis XI 4), (ZP_01092401.1; Blastopirellula marina DSM 3645),
• Lactobacillus crispatus IV-VOl Lactobacillus crispatus IV-VOl
• ZP 04010922.1 Lactobacillus ultunensis DSM 16047
• ZP_05549961.1 Lactobacillus crispatus 125-2-CRN
• ZP_03951361.1 Lactobacillus gasseri IV-V03
• ZP_05744515.1 Lactobacillus iners DSM 13335
• YP_618635.1 Lactobacillus crispatus IV-VOl
• Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (ZP_03955917.1; Lactobacillus jensenii IV-VI6), (ZP 03942415.1; Lactobacillus buchneri ATCC 11577), (ZP O 1544800.1; Oenococcus oeni ATCC BAA- 1163), (NP_786060.1; Lactobacillus plantarum WCFSI), (Q937F6; XPKA LACPE), (YP 394903.1; Lactobacillus sakei subsp. sakei 23K),
• Streptococcus agalactiae NEM316 (ZP_04442854.1; Listeria grayi DSM 20601),
• Mycoplasma fermentans PG 18 (YP 002000006.1; Mycoplasma arthritidis 15 8L3-1), (YP OO 1256266.1; Mycoplasma agalactiae PG2), (YP OO 1988835.1; Lactobacillus casei BL23), (NP_786753.1; Lactobacillus plantarum WCFS 1), (ZP_04009976.1; Lactobacillus salivarius ATCC 11741), (YP_818922.1; Leuconostoc mesenteroides subsp.
• Bacteroides capillosus ATCC 29799) Bacteroides capillosus ATCC 29799), (XP 002180542.1; Phaeodactylum tricomutum CCAP 1055/1), (YP_568630.1; Rhodopseudomonas palustris BisB5), (YP_487462.1;
• Rhodopseudomonas palustris HaA2) (NP_947019.1; Rhodopseudomonas palustris
• Polaromonas naphthalenivorans CJ2 Polaromonas naphthalenivorans CJ2
• ZP_01464191.1 Stigmatella aurantiaca DW4/3-1
• YP OO 1267778.1 Pseudomonas putida FT
• YP_829644.1 Arthrobacter sp. FB24
• YP_002486392.1 Arthrobacter chlorophenolicus A6
• ZP_05816651.1 Sanguibacter keddieii DSM 10542
• YP_002883053.1 Beutenbergia cavemae DSM 12333
• paratuberculosis K-10 paratuberculosis K-10
• ZP_05224330.1 Mycobacterium intracellul are ATCC 13950
• YP OO 1703240.1 Mycobacterium abscessus
• ZP_00995133.1 Janibacter sp. HTCC2649
• ZP 291026.1 Thermobifida fusca YX
• ZP 04031845.1 Thermomonospora curvata DSM 43183
• ZP_04475514.1 Streptosporangium roseum DSM 43021
• Actinosynnema mirum DSM 43827 (NP_733508.1; Streptomyces coelicolor A3 (2)), (CAJ88379.1; Streptomyces ambofaciens ATCC 23877), (ZP_05536883.1; Streptomyces griseoflavus Tu4000), (ZP 05020421.1; Streptomyces sviceus ATCC 29083), (CBG67625.1; Streptomyces scabiei 87.22), (NP_822448.1; Streptomyces avermitilis MA-4680),
• Saccharopolyspora erythraea NRRL 2338 (YP_002282673.1; Rhizobium leguminosarum by. trifolii WSM2304), (YP_002977256.1; Rhizobium leguminosarum by. trifolii WSM1325), (YP OO 1979796.1; Rhizobium etli CI AT 652), (YP_470926.1; Rhizobium etli CFN 42), (YP_002540633.1; Agrobacterium radiobacter K84), (ZP 05182366.1; Brucella sp. 83/13), (ZP_04683384.1; Ochrobactrum intermedium LMG 3301), (YP_001373254.1;
• Ochrobactrum anthropi ATCC 49188 Ochrobactrum anthropi ATCC 49188
• YP_002961612.1 Ochrobactrum anthropi ATCC 49188
• Methylobacterium extorquens AM 1) (YP_674792.1; Mesorhizobium sp. BNCI),
• Methylobacterium extorquens DM4 (YP_002964777.1; Methylobacterium extorquens AM 1), (YP 002501292.1; Methylobacterium nodulans ORS 2060), (YP 002495265.1;
• Methylobacterium nodulans ORS 2060 (YP_001770387.1; Methylobacterium sp.4-46), (YP_002944712.1; Variovorax paradoxus SI 10), (ZP 01156757.1; Oceanicola granulosus HTCC2516), (ZP_01628787.1; Nodularia spumlgena CCY9414), (YP OO 1865546.1; Nostoc punctiforme PCC 73102), (YP_321015.1; Anabaena variabilis ATCC 29413),
• Prosthecochloris aestuarii DSM 271) (YP OO 1943369.1; Chlorobium limicola DSM 245), (NP_662409.1; Chlorobium tepidum TLS), (ZP_01386179.1; Chlorobium ferrooxidans DSM 13031), (YP_375422.1; Chlorobium luteolum DSM 273), (YP_285277.1; Dechloromonas aromatica RCB), (YP_314589.1; Thiobacillus denitrificans ATCC 25259), (YP_545002.1; Methyl obacillus flagellatus KT), (NP_842139.1; Nitrosomonas europaea ATCC 19718), (YP_748274.1; Nitrosomonas eutropha C91), (YP 411688.1; Nitrosospira multiformis ATCC 25196), (YP_344700.1; Nitrosoc
• Candidatus Protochlamydia amoebophila UWE25 (NP_435833.1; Sinorhizobium meliloti 1021), (ZP_04421874.1; Sulfurospirillum deleyianum DSM 6946), (NPJ07054.1;
• Phosphoketolases also useful in the compositions and methods provided herein include those molecules which are said to be “derivatives" of any of the
• phosphoketolases described herein Such a “derivative” has the following characteristics: (1) it shares substantial homology with any of the phosphoketolases described herein; and (2) is capable of catalyzing the conversion of X5P into glyceraldehyde 3-phosphate (G3P) and acetyl phosphate; or F6P into erythrose 4-phosphate (E4P) and acetyl phosphate.
• G3P glyceraldehyde 3-phosphate
• E4P erythrose 4-phosphate
• a derivative of a phosphoketolase is said to share "substantial homology" with the
• the amino acid sequences of the derivative is at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of the
• the genetically modified host cell provided herein comprises a heterologous nucleotide sequence encoding a phosphotransacetylase.
• Phosphotransacetylase (EC 2.3.1.8) converts acetyl phosphate into acetyl-CoA.
• Numerous examples of polynucleotides, genes and polypeptides encoding phosphotransacetylase activity are known in the art and can be used in the genetically modified host cell provided herein.
• such a polynucleotide, gene and/or polypeptide is the phosphotransacetylase from Clostridium kluyveri.
• Representative phosphotransacetylase nucleotide sequences of Clostridium kluyveri includes accession number NC 009706.1 : 1428554..1429555, and SEQ ID NO: 3 as provided herein.
• Representative phosphotransacetylase protein sequences of Clostridium kluyveri include accession number YP 001394780 and SEQ ID NO: 4 as provided herein.
• Other useful phosphotransacetylases include, but are not limited to, those from Lactobacillus reuteri (NC 010609.1 :460303..461277; YP 001841389.10); Bacillus subtilis
• Lactobacillus plantarum WCFS1 (NP_784550.1); Lactobacillus fermentum ATCC 14931 (ZP 03944466.1); Bacillus subtilis subsp. subtilis str. 168 (NP_391646.1); Methanosarcina thermophila (AAA72041.1); Clostridium thermocellum DSM 4150 (ZP 03152606.1);
• Clostridium acetobutylicum ATCC 824 (NP_348368.1); Clostridium kluyveri DSM 555 (YP 001394780.1); Veillonella parvula DSM 2008 (ZP 03855267.1); and Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 (YP_ 149725.1).
• phosphotransacetylases include: (ZP_05427766.1; Eubacterium saphenum ATCC 49989), (ZP 03627696.1; bacterium Ellin514), ), (ZP 03131770.1 ;
• Chthonio bacter flavus Ellin428) (YP 001878031.1; Akkermansia muciniphila TCCBAA- 835), (ZP_04562924.1; Citrobacter sp.30_2), (YP_001451936.1; Citrobacter koseri ATCC BAA-895), (YP_ 149725.1; Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150), (YP 001569496.1; Salmonella enterica subsp. anzonae serovar 62:z4,z23:—), (NP_416953.1; Escherichia coli str. K-12 substr. MG1655), (YP_002920654.1; Klebsiella pneumomae NTUH-K2044), (ZP_04637797.1; Yersinia intermedia ATCC 29909),
• Rhodococcus jostii RHA1 Rhodococcus jostii RHA1
• ZP_05479963.1 Streptomyces sp. AA4
• ZP 002761398.1 Gemmatimonas aurantiaca T-27
• ZP 04670189.1 Clostridiales bacterium 1_7_47FAA
• ZP_05493958.1 Clostridium papyrosolvens DSM 2782
• YP 003143506.1 Slackia heliotrinireducens DSM 20476
• ZP_05090822.1 Ruegeria sp.
• Arcobacter butzleri RM4018 (YP 003163236.1; Leptotrichia buccalis C-1013-b),
• NP_853403.1 Mycoplasma gallisepticum R
• NP_757889.1 Mycoplasma penetrans HF- 2
• YP l 16016.1 Mycoplasma hyopneumoniea 232
• YP 002960607.1 Mycoplasma conjunctivae
• YP_001256282.1 Mycoplasma agalactiae PG2
• BAH69503.1
• Mycoplasma fermentans PG18 (YP_278771.1; Mycoplasma synoviae 53), (NP_326068.1; Mycoplasma pulmonis UAB CTIP), (YP O 15865.1; Mycoplasma mobile 163K),
• Thermoanaero bacterium saccharolyticum (ZP_05336886.1; Thermoanaero bacterium thermosaccharolyticum SM 571), (NP_623097.1; Thermoanaero bacter tengcongensis MB4), (YP OO 1663354.1; Thermoanaero bacter sp. X514), (YP_002508771.1; Halothermoth rix orenii H 168), (YP 003190679.1; Desulfotomaculum acetoxidans DSM 771),
• Carboxydothermus hydrogenoformans Z-2901) (EY83551.1; Bacteroides sp.2 1 _33B), (ZP_02033408.1; Parabacteroides merdae ATCC 43184), (NP_905297.1; Porphyromonas gingivalis W83), (ZP 04056000.1; Porphyromonas uenonis 60-3), (ZP 04389884.1;
• Porphyromonas endodontalis ATCC 35406) (ZP_02068815.1; Bacteroides uniformis ATCC 8492), (ZP_03460749.1; Bacteroides eggerthii DSM 20697), (ZP 03676944.1; Bacteroides cellulosilyticus DSM 14838), (YP_097761.1; Bacteroides fragilis YCH46), (ZP_04545825.1; Bacteroides sp. Dl), (ZP_03643544.1; Bacteroides coprophilus DSM 18228),
• Epulopiscium sp. 'N.t. morphotype B (YP 003182082.1; Eggerthella lenta DSM 2243), (YP 003151027.1; Cryptobacterium curium DSM 15641), (YP 003143601.1; Slackia heliotrinireducens DSM 20476), (ZP_05498135.1; Clostridium papyrosolvens DSM 2782), (ZP 03152606.1; Clostridium thermocellum JW20), (YP 001180817.1; Caldicellulosiruptor saccharolyticus DSM 8903), (AAA72041.1; Methanosarcina thermophile), (NP_618482.1; Methanosarcina acetivorans C2A), (YP_305342.1; Methanosarcina barkeri str.
• Rhodobacteraceae bacterium KLH11 Rhodobacteraceae bacterium KLH11
• ZP_05786337.1 Silicibacter lacuscaerulensis ITI- 1157
• YP 001313586.1 Sinorhizobium medicae WSM419)
• NP_437512.1 Sinorhizobium meliloti 1021
• ZP_04682129.1 Ochrobactrum intermedium LMG 3301
• Roseobacter sp. AzwK-3b (ZP_01752570.1; Roseobacter sp. SK209-2-6), (ZP 02140073.1; Roseobacter litoralis Och 149), (YP 510789.1; Jannaschia sp. CCS1), (ZP 05073153.1; Rhodobacteral es bacterium HTCC2083), (YP_822367.1; Candidatus Solibacter usitatus Ellin6076), (ZP 01313101.1; Desulfuromon as acetoxidans DSM 684), (YP 357950.1;
• Campylobacter concisus 13826 (YP OO 1408221.1; Campylobacter curvus 525.92), (ZP_05363348.1; Campylobacter show ae RM3277), (ZP_03742933.1; Bifidobacterium pseudocatenulatum DSM 20438), (ZP_02918887.1; Bifidobacterium dentium ATCC 27678), (ZP_02028883.1; Bifidobacterium adolescentis L2-32), (ZP 04448100.1; Bifidobacterium angulatum DSM 20098), (ZP_03618886.1; Bifidobacterium breve DSM 20213),
• Corynebacterium glucuronolyticum ATCC 51867) (ZP_05708623.1; Corynebacterium genitalium ATCC 33030), (ZP 03977910.1; Corynebacterium lipophiloflavum DSM 44291), (ZP 03932064.1; Corynebacterium accolens ATCC 49725), (ZP 05366890.1;
• Corynebacterium tuberculostearicum SK141 (YP_002835817.1; Corynebacterium aunmucosum ATCC 700975), (YP_250020.1; Corynebacterium jeikeium K411),
• Corynebacterium kroppenstedtii DSM 44385) (ZP_03393297.1; Corynebacterium amycolatum SK46), (ZP_03718987.1; Neisseria flavescens NRL30031/H 210),
• Corynebacterium glutamicum (ZP 03994160.1; Mobiluncus mulieris ATCC 35243), (ZP_03922640.1; Mobiluncus curtisii ATCC 43063), (ZP 03716209.1; Eubacterium hallii DSM 3353), (ZP_03718143.1; Eubacterium hallii DSM 3353), (ZP 05614434.1;
• Roseburia intestinalis Ll-82 (YP_002937332.1; Eubacterium rectale ATCC 33656), (ZP_02074244.1; Clostridium sp. L2-50), (ZP_04455374.1; Shuttleworthia sacot DSM 14600), (ZP_03488480.1; Eubacterium biforme DSM 3989), (ZP_02078327.1; Eubacterium dolichum DSM 3991), (ZP_02077559.1; Eubacterium dolichum DSM 3991),
• Streptococcus mutans NN2025), (ZP_02920305.1; Streptococcus infantarius subsp.
• Lactobacillus vaginalis ATCC 49540 Lactobacillus vaginalis ATCC 49540
• (YP OO 1271004.1 Lactobacillus reuteri DSM 20016)
• (ZP_05745668.1 Lactobacillus antri DSM 16041)
• (YP_818931.1 Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293)
• ZP_04782044.1 Weissella paramesentero ides ATCC 33313
• (ZP O 1544468.1 Oenococcus oeniATCC BAA-1163
• (ZP_05737294.1 Granulicatella adiacens ATCC
• Camobacterium sp.ATT (ZP 05649755.1; Enterococcus gallinarum EG2),
• cremons MG1363) (YP_806234.1; Lactobacillus casei ATCC 334), (NP_784550.1; Lactobacillus plantarum WCFS1), (YP_794848.1; Lactobacillus brevis ATCC 367), (ZP_03954831.1; Lactobacillus hilgardii ATCC 8290), (BABI9267.1; Lactobacillus sanfranciscensis), (ZP_03958288.1; Lactobacillus ruminis ATCC 25644), (YP_536042.1; Lactobacillus salivarius UCC118), (ZP_05747635.1; Erysipelothrix rhusiopathiae ATCC 19414), (YP_803875.1; Pediococcus pentosaceus ATCC 25745), (ZP_02093784.1; Parvimonas micraATCC 33270),
• DC3000 (YP_258069.1; Pseudomonas fluorescens Pf-5), (YP_606637.1; Pseudomonas entomophila L48), (YP_002800579.1; Azotobacter vinelandii DJ), (YP 001171663.1; Pseudomonas stutzeri A1501), (NP_840385.1;
• Nitrosomonas europaea ATCC 19718) (YP_002801221.1; Azotobacter vinelandii DJ), (YP 002787111.1; Deinococcus deserti VCD115), (YP_603523.1; Deinococcus geothermalis DSM 11300), (NP_293799.1; Deinococcus radiodurans Rl), (YP_521550.1; Rhodoferax ferrireducens T118), (YP_530962.1; Rhodopseudo monas palustris BisB18), (YP_531882.1; Rhodopseudo monas palustris BisA53), (ZP_02367347.1; Burkholderia oklahomensis C6786), (YP_428079.1; Rhodospirillum rubrum ATCC 11170), (YP_530535.1;
• Rhodopseudo monas palustris BisB18 Rhodopseudo monas palustris BisB18
• NP 901200.1 Chromobacterium violaceum ATCC 12472
• ZP_03698345.1 Lutiella nitroferrum 2002
• YP OO 1279250.1 Psychrobacter sp. PRwf-1
• YP_579484.1 Psychrobacter cryohalolentis K5
• ZP 05618978.1 ;
• Enhydrobacter aerosaccus SK60 (ZP_05362319.1; Acinetobacter radioresistens SK82), (YP_045288.1; Acinetobacter sp. ADPl), (ZP_05823314.1; Acinetobacter sp. RUH2624), (ZP 03824416.1; Acinetobacter sp. ATCC 27244), (YP_001380280.1; Anaeromyxobacter sp. Fwl09-5 ⁇ (YP_466103.1; Anaeromyxobacter dehalogenans 2CP-C), (YP 088190.1;
• Haemophilus influenzae 3655) (YP_719012.1; Haemophilus somnus 129PT),
• NP_245642.1 Pasteur ella multocida subsp. multocida sir. Pm70
• ZP 05920444.1 ZP 05920444.1
• Pasteurella dagmatis ATCC 43325 (ZP OO 133992.2; Actinobacillus pleuropneumoniae serovar 1 str. 4074), (ZP_04753547.1; Actinobacillus minor NM305), (NP_873873.1;
• Haemophilus ducreyi 35000HP (ZP_04978908.1; Mannheimia haemolytica PHL213), (YP_002475022.1; Haemophilus parasuis SH0165), (ZP_05730581.1; Pantoea sp. At-9b), (YP OO 1907133.1; Erwinia tasmaniensis Etl/99), (YP_455287.1; Sodalis glossinidius str. 'morsitans'), (ZP_05723922.1; Dickeya dadantii Ech586), (YP 003258889.1;
• Pectobacterium wasabiae WPP163), (YP 002988159.1; Dickeya dadantii Ech703),
• NP_668938.1 Yersinia pestis KIM 10
• YP OO 1479543.1 Serratia proteamaculans 568
• YP 002934098.1 Edwardsiella ictaluri 93-146
• YP 002151502.1 Proteus mirabilis HI4320
• NP_930328.1 Photorhabdus luminescens subsp. laumondii TTOl
• Microcystis aerugmosa NIES-843 (YP_002485151.1; Cyanothece sp. PCC 7425),
• Desulfohalobium retbaense DSM 5692 (YP_003157577.1; Desulfomicrobium baculatum DSM 4028), (ZP 03737911.1; Desulfonatronospira thiodismutans AS03-1),
• PCC 8106 (ZP_03272899.1; Arthrospira maxima CS-328), (YP_845596.1; Syntrophobacter fumaroxidans MPOB), (ZP_04773932.1; Allochromatium vinosum DSM 180), (NP_869002.1; Rhodopirellula baltica SH 1), (YP_392571.1; Sulfurimonas denitrificans DSM 1251), (ZP 05071717.1; Campylobacter ales bacterium GD 1), (ZP_04421899.1; Sulfurospirillum deleyianum DSM 6946), (YP OO 1359295.1; Sulfurovum sp.
• Mycobacterium tuberculosis H37Rv Mycobacterium tuberculosis H37Rv
• NP_962819.1 Mycobacterium avium subsp.
• paratuberculosis K-10 paratuberculosis K-10
• ZP_05223872.1 Mycobacterium intracellular ATCC 13950
• YP_002764919.1 Rhodococcus erythropolis PR4
• ZP 702162.1 Rhodococcus jostii RHAI
• YP_121562.1 Nocardia farcinica IFM 10152
• ZP_04025361.1 Tsukamurella paurometabola DSM 20162
• YP_003275431.1 Gordonia bronchialis DSM 43247
• YP 003160610.1 Jonesia denitrificans DSM 20603
• ZP 05816650.1 Sanguibacter keddieii DSM 10542
• ZP_04368027.1 Cellulomonas flavigena DSM 20109
• Streptomyces albus 11074 (ZP_04997745.1; Streptomyces sp. Mgl), (ZP 05509147.1; Streptomyces sp. C), (ZP_05514718.1; Streptomyces hygroscopicus ATCC 53653),
• Mariprofundus ferrooxydans PV-l and (ZP_01307392.1; Bermanella marisrubri).
• Phosphotransacetylases also useful in the compositions and methods provided herein include those molecules which are said to be “derivatives" of any of the
• a derivative of a phosphotransacetylase is said to share "substantial homology" with the phosphotransacetylase if the amino acid sequences of the derivative is at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of the phosphotransacetylase.
• the genetically modified host cell provided herein comprises a functional disruption in an enzyme that converts acetyl phosphate to acetate.
• the enzyme is native to the host cell.
• the enzyme that converts acetyl phosphate to acetate is a glycerol- 1 -phosphatase (EC 3.1.3.21).
• the enzyme having glycerol- 1 -phosphatase activity is RHR2 (GPP1/RHR2; systematic name: YIL053W), or a homolog or variant thereof.
• GPP1/RHR2 is a constitutively expressed glycerol- 1 -phosphatase involved in glycerol biosynthesis, and is induced in response to both anaerobic and osmotic stress.
• GPP1/RHR2 gene of S. cerevisiae has been previously described. See, e.g., Norbeck et al., J Biol Chem 271(23): 13875-13881 (1996); and Pahlman et al., J Biol Chem 276(5): 3555- 3563 (2001). Gppl/Rhr2 has been previously described as catalyzing the following reaction:
• glycerol- 1 -phosphate + H20 glycerol + phosphate.
• Representative GPP1/RHR2 nucleotide sequences of Saccharomyces cerevisiae include accession number NM 001179403.1, and SEQ ID NO: 5 as provided herein.
• Representative Gppl/Rhr2 protein sequences of Saccharomyces cerevisiae include accession number NP 012211, and SEQ ID NO:6 as provided herein.
• HOR2 A closely related homolog of GPP1/RHR2 which also catalyzes the hydrolysis of acetyl phosphate to acetate is HOR2 (GPP2/HOR2; systematic name: YER062C).
• the sequence of the GPP2/HOR2 gene of S. cerevisiae has been previously described. See, e.g., Norbeck et ah, J. 13875- 13881 (1996); and Pahlman et al., J. of Biological Chemistry 276(5): 3555-3563 (2001).
• Representative GPP2/HOR2 nucleotide sequences of Saccharomyces cerevisiae include accession number NM 001178953.3, and SEQ ID NO:7 as provided herein.
• Representative Gppl/Rhr2 protein sequences of Saccharomyces cerevisiae include accession number NP 010984, and SEQ ID NO: 8 as provided herein.
• GPP1/RHR2 and/or GPP2/HOR2 in yeast other than S. cerevisiae can similarly be inactivated using the methods described herein.
• a polynucleotide, gene and/or polypeptide encoding acetyl-phosphatase activity e.g., RHR2 and/or HOR2
• RHR2 and/or HOR2 can be used to identify other polynucleotide, gene and/or polypeptide sequences or to identify homologs having acetyl-phosphatase activity in other host cells.
• Such sequences can be identified, for example, in the literature and/or in bioinformatics databases well known to the skilled person.
• the activity or expression of an endogenous enzyme that converts acetyl phosphate to acetate is reduced by at least about 50%.
• the activity or expression of an endogenous enzyme that converts acetyl phosphate to acetate is reduced by at least about 60%, by at least about 65%, by at least about 70%>, by at least about 75%, by at least about 80%>, by at least about 85%, by at least about 90%>, by at least about 95%>, or by at least about 99% as compared to a recombinant microorganism not comprising a reduction or deletion of the activity or expression of an endogenous enzyme that converts acetyl phosphate to acetate.
• the endogenous enzyme that converts acetyl phosphate to acetate is RHR2, or homologues thereof. In some embodiments, the endogenous enzyme that converts acetyl phosphate to acetate is HOR2, or homologues thereof.
• the genetically modified host cell comprises a mutation in at least one gene encoding acetyl-phosphatase activity (e.g., RHR2, HOR2 or a homolog or variant thereof), resulting in a reduction of activity of a polypeptide encoded by said gene.
• the genetically modified host cell comprises a partial deletion of a gene encoding acetyl-phosphatase activity (e.g., RHR2, HOR2 or a homolog or variant thereof), resulting in a reduction of activity of a polypeptide encoded by the gene.
• the genetically modified host cell comprises a complete deletion of a gene encoding acetyl-phosphatase activity (e.g., RHR2, HOR2 or a homolog or variant thereof), resulting in a reduction of activity of a polypeptide encoded by the gene.
• the genetically modified host cell comprises a modification of the regulatory region associated with the gene encoding acetyl-phosphatase activity (e.g., RHR2, HOR2 or a homolog or variant thereof), resulting in a reduction of expression of a polypeptide encoded by said gene.
• the genetically modified host cell comprises a modification of the transcriptional regulator resulting in a reduction of transcription of a gene encoding acetyl-phosphatase activity (e.g., RHR2, HOR2 or a homolog or variant thereof).
• disruption of one or more genes encoding a protein capable of catalyzing the conversion of acetyl phosphate to acetate is achieved by using a "disruption construct" that is capable of specifically disrupting such a gene (e.g., RHR2 or HOR2) upon introduction of the construct into the microbial cell, thereby rendering the disrupted gene non-functional.
• disruption of the target gene prevents the expression of a functional protein.
• disruption of the target gene results in expression of a non-functional protein from the disrupted gene.
• disruption of a gene encoding a protein capable of converting acetyl phosphate to acetate is achieved by integration of a "disrupting sequence" within the target gene locus by homologous recombination.
• the disruption construct comprises a disrupting sequence flanked by a pair of nucleotide sequences that are homologous to a pair of nucleotide sequences of the target gene locus (homologous sequences).
• the disrupting sequence prevents the expression of a functional protein, or causes expression of a non-functional protein, from the target gene.
• Disruption constructs capable of disrupting a gene may be constructed using standard molecular biology techniques well known in the art. See, e.g., Sambrook et al., 2001, Molecular Cloning— A Laboratory Manual, 3 rd edition, Cold Spring Harbor
• Parameters of disruption constructs that may be varied in the practice of the present methods include, but are not limited to, the lengths of the homologous sequences; the nucleotide sequence of the homologous sequences; the length of the disrupting sequence; the nucleotide sequence of the disrupting sequence; and the nucleotide sequence of the target gene.
• an effective range for the length of each homologous sequence is 50 to 5,000 base pairs. In particular embodiments, the length of each homologous sequence is about 500 base pairs.
• the homologous sequences comprise coding sequences of the target gene. In other embodiments, the homologous sequences comprise upstream or downstream sequences of the target gene. Is some embodiments, one homologous sequence comprises a nucleotide sequence that is
• homologous to a nucleotide sequence located 5' of the coding sequence of the target gene and the other homologous sequence comprises a nucleotide sequence that is homologous to a nucleotide sequence located 3' of the coding sequence of the target gene.
• the disrupting sequence comprises a nucleotide sequence encoding a selectable marker that enables selection of microbial cells comprising the disrupting sequence.
• the disruption construct has a dual function, i.e., to functionally disrupt the target gene and to provide a selectable marker for the identification of cells in which the target gene is functionally disrupted.
• a termination codon is positioned in-frame with and downstream of the nucleotide sequence encoding the selectable marker to prevent translational read-through that might yield a fusion protein having some degree of activity of the wild type protein encoded by the target gene.
• the length of the disrupting sequence is one base pair.
• Insertion of a single base pair can suffice to disrupt a target gene because insertion of the single base pair in a coding sequence could constitute a frame shift mutation that could prevent expression of a functional protein.
• the sequence of the disruption sequence differs from the nucleotide sequence of the target gene located between the homologous sequences by a single base pair.
• the single base pair substitution that is introduced could result in a single amino acid substitution at a critical site in the protein and the expression of a non-functional protein. It should be recognized, however, that disruptions effected using very short disrupting sequences are susceptible to reversion to the wild type sequence through spontaneous mutation, thus leading to restoration of acetyl-phosphatase function to the host strain.
• the disrupting sequences are longer than one to a few base pairs.
• a disrupting sequence of excessive length is unlikely to confer any advantage over a disrupting sequence of moderate length, and might diminish efficiency of transfection or targeting.
• Excessive length in this context is many times longer than the distance between the chosen homologous sequences in the target gene.
• the length for the disrupting sequence can be from 2 to 2,000 base pairs.
• the length for the disrupting sequence is a length approximately equivalent to the distance between the regions of the target gene locus that match the homologous sequences in the disruption construct.
• the disruption construct is a linear DNA molecule. In other embodiments, the disruption construct is a circular DNA molecule. In some embodiments, the disruption construct is a linear DNA molecule. In other embodiments, the disruption construct is a circular DNA molecule. In some
• the circular disruption construct comprises a pair of homologous sequences separated by a disrupting sequence, as described above.
• the circular disruption construct comprises a single homologous sequence.
• Such circular disruption constructs upon integration at the target gene locus, would become linearized, with a portion of the homologous sequence positioned at each end and the remaining segments of the disruption construct inserting into and disrupting the target gene without replacing any of the target gene nucleotide sequence.
• the single homologous sequence of a circular disruption construct is homologous to a sequence located within the coding sequence of the target gene.
• Disruption constructs can be introduced into a microbial cell by any method known to one of skill in the art without limitation. Such methods include, but are not limited to, direct uptake of the molecule by a cell from solution, or facilitated uptake through lipofection using, e.g., liposomes or immunoliposomes; particle-mediated transfection; etc. See, e.g., U.S. Patent No. 5,272,065; Goeddel et al, eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression— A
• the genetically modified host cells provided herein further comprise one or more heterologous nucleotide sequences encoding acylating acetaldehyde dehydrogenase (alternately referred to as “acetylaldehyde dehydrogenase, acetylating,” “acetylaldehyde dehydrogenase, acylating,” or ADA (EC 1.2.1.10)).
• heterologous nucleotide sequences encoding acylating acetaldehyde dehydrogenase (alternately referred to as "acetylaldehyde dehydrogenase, acetylating,” “acetylaldehyde dehydrogenase, acylating,” or ADA (EC 1.2.1.10)).
• compositions and methods provided herein include the following four types of proteins:
• the NH 2 -terminal region of the AdhE protein is highly homologous to aldehyde :NAD oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe -dependent ethanol:NAD oxidoreductases (Membrillo-Hernandez et al., (2000) J. Biol. Chem. 275: 33869-33875).
• the E. coli AdhE is subject to metal-catalyzed oxidation and therefore oxygen-sensitive (Tamarit et al. (1998) J. Biol. Chem. 273:3027-32).
• Clostridium beijerinckii NRRL B593 (Toth et al. (1999) Appl. Environ. Microbiol. 65: 4973- 4980, accession no: AAD31841 ).
• Ethanolamine can be utilized both as carbon and nitrogen source by many enterobacteria (Stojiljkovic et al. (1995) J. Bacteriol. 177: 1357-1366). Ethanolamine is first converted by ethanolamine ammonia lyase to ammonia and acetaldehyde, subsequently, acetaldehyde is converted by ADA to acetyl-CoA.
• ADA acetyl-CoA.
• An example of this type of ADA is the EutE protein in Salmonella typhimurium (Stojiljkovic et al. (1995) J. Bacteriol. Ill: 1357-1366, accession no:
• E. coli is also able to utilize ethanolamine (Scarlett et al. (1976) J. Gen. Microbiol. 95: 173-176) and has an EutE protein (accession no: AAG57564; see also EU897722.1) which is homologous to the EutE protein in S. typhimurium.
• E. coli has a homologous MphF protein (Ferrandez et al. (1997) J. Bacteriol. 179: 2573-2581 , accession no:
• an ADA (or nucleic acid sequence encoding such activity) useful for the compositions and methods described herein is selected from the group consisting of Escherichia coli adhE, Entamoeba histolytica adh2, Staphylococcus aureus adhE, Piromyces sp.E2 adhE, Clostridium kluyveri (EDK33116), Lactobacillus plantarum acdH, and Pseudomonas putida (YP 001268189), as described in International Publication No. WO 2009/013159, the contents of which are incorporated by reference in their entirety.
• the ADA is selected from the group consisting of Clostridium botulinum eutE (FR745875.1), Desulfotalea psychrophila eutE (CR522870.1), Acinetobacter sp. HBS-2 eutE (ABQ44511.2), Caldithrix abyssi eutE (ZP 09549576), and Halorubrum lacusprofundi ATCC 49239 (YP_002565337.1).
• the ADA useful for the compositions and methods provided herein is eutE from Dickeya zeae.
• a representative eutE nucleotide sequence of Dickey a zeae includes accession number NC 012912.1 : 1110476..1111855, and SEQ ID NO: 9 as provided herein.
• a representative eutE protein sequence of Dickeya zeae includes accession number YP 003003316, and SEQ ID NO: 10 as provided herein.
• AD As also useful in the compositions and methods provided herein include those molecules which are said to be “derivatives” of any of the AD As described herein. Such a “derivative” has the following characteristics: (1) it shares substantial homology with any of the AD As described herein; and (2) is capable of catalyzing the conversion of acetaldehyde to acetyl-CoA. A derivative of an ADA is said to share "substantial homology" with ADA if the amino acid sequences of the derivative is at least 80%, at least 85% and more preferably at least 90%, and most preferably at least 95%, the same as that of any of the AD As described herein.
• Acetyl-CoA can be formed in the mitochondria by oxidative decarboxylation of pyruvate catalyzed by the PDH complex.
• the PDH bypass has an essential role in providing acetyl-CoA in the cytosolic compartment, and provides an alternative route to the PDH reaction for the conversion of pyruvate to acetyl-CoA.
• the PDH bypass involves the enzymes pyruvate decarboxylase (PDC; EC 4.1.1.1), acetaldehyde dehydrogenase (ACDH; EC 1.2.1.5 and EC 1.2.1.4), and acetyl-CoA synthetase (ACS; EC 6.2.1.1).
• Pyruvate decarboxylase catalyzes the decarboxylation of pyruvate to acetaldehyde and carbon dioxide.
• Acetaldehyde dehydrogenase oxidizes acetaldehyde to acetic acid.
• S. cerevisiae the family of aldehyde dehydrogenases contains five members.
• ALD2 (YMR170c), ALD3 (YMR169c), and ALD6 (YPL061w) correspond to the cytosolic isoforms
• ALD4 (YOPv374w) and ALD5 (YER073w) encode the mitochondrial enzyme.
• the main cytosolic acetaldehyde dehydrogenase isoform is encoded by ALD6.
• the formation of acetyl-CoA from acetate is catalyzed by ACS and involves hydrolysis of ATP.
• the genetically modified host cell provided herein further comprises a functional disruption in one or more genes of the PDH-bypass pathway.
• disruption of the one or more genes of the PDH-bypass of the host cell results in a genetically modified microbial cell that is impaired in its ability to catalyze one or more of the following reactions: (1) the decarboxylation of pyruvate into acetaldehyde by pyruvate decarboxylase; (2) the conversion of acetaldehyde into acetate by acetaldehyde dehydrogenase; and (3) the synthesis of acetyl-CoA from acetate and CoA by acetyl-CoA synthetase.
• a host cell comprises a functional disruption in one or more genes of the PDH-bypass pathway, wherein the activity of the reduced-function or non-functional PDH-bypass pathway alone or in combination with a weak ADA is not sufficient to support host cell growth, viability, and/or health.
• the activity or expression of one or more endogenous proteins of the PDH-bypass is reduced by at least about 50%. In another embodiment, the activity or expression of one or more endogenous proteins of the PDH-bypass is reduced by at least about 60%>, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, or by at least about 99% as compared to a recombinant microorganism not comprising a reduction or deletion of the activity or expression of one or more endogenous proteins of the PDH-bypass.
• one or more genes encoding aldehyde dehydrogenase are provided.
• the aldehyde dehydrogenase is encoded by a gene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, and homologs and variants thereof.
• the genetically modified host cell comprises a functional disruption of ALD4. Representative ALD4 nucleotide sequences of
• Saccharomyces cerevisiae include accession number NM_001183794, and SEQ ID NO: 11 as provided herein.
• Representative Ald4 protein sequences of Saccharomyces cerevisiae include accession number NP O 15019.1, and SEQ ID NO: 12 as provided herein.
• the genetically modified host cell comprises a functional disruption of cytosolic aldehyde dehydrogenase (ALD6).
• ALD6p functions in the native PDH-bypass to convert acetaldehyde to acetate.
• Representative ALD6 nucleotide sequences of Saccharomyces cerevisiae include accession number SCU56604, and SEQ ID NO: 13 as provided herein.
• Representative Ald6 protein sequences of Saccharomyces cerevisiae include accession number AAB01219, and SEQ ID NO: 14 as provided herein.
• the activity or expression of more than one aldehyde dehydrogenase can be reduced or eliminated.
• the activity or expression of ALD4 and ALD6 or homologs or variants thereof is reduced or eliminated.
• the activity or expression of ALD5 and ALD6 or homologs or variants thereof is reduced or eliminated.
• the activity or expression of ALD4, ALD5, and ALD6 or homologs or variants thereof is reduced or eliminated.
• the activity or expression of the cytosolically localized aldehyde dehydrogenases ALD2, ALD3, and ALD6 or homologs or variants thereof is reduced or eliminated.
• the activity or expression of the mitochondrially localized aldehyde dehydrogenases, ALD4 and ALD5 or homologs or variants thereof is reduced or eliminated.
• ACS activity are functionally disrupted in the host cell.
• the acetyl- CoA synthetase is encoded by a gene selected from the group consisting of ACS1, ACS2, and homologs and variants thereof.
• one or more genes encoding acetyl-CoA synthetase are encoding acetyl-CoA synthetase
• ACS activity is functionally disrupted in the host cell.
• ACS1 and ACS2 are both acetyl- CoA synthetases that can convert acetate to acetyl-CoA.
• ACS1 is expressed only under respiratory conditions, whereas ACS2 is expressed constitutively.
• ACS2 is knocked out, strains are able to grow on respiratory conditions (e.g. ethanol, glycerol, or acetate media), but die on fermentable carbon sources (e.g. sucrose, glucose).
• the genetically modified host cell comprises a functional disruption of ACS 1.
• the sequence of the ACSl gene of S. cerevisiae has been previously described. See, e.g., Nagasu et ah, Gene 37 (l-3):247-253 (1985).
• Representative ACSl nucleotide sequences of Saccharomyces cerevisiae include accession number X66425, and SEQ ID NO: 15 as provided herein.
• Saccharomyces cerevisiae include accession number AAC04979, and SEQ ID NO: 16 as provided herein.
• the genetically modified host cell comprises a functional disruption of ACS2.
• the sequence of the ACS2 gene of S. cerevisiae has been previously described. See, e.g., Van den Berg et ah, Eur. J. Biochem. 231(3):704-713 (1995).
• Representative ACS2 nucleotide sequences of Saccharomyces cerevisiae include accession number S79456, and SEQ ID NO: 17 as provided herein.
• Representative Acs2 protein sequences of Saccharomyces cerevisiae include accession number CAA97725, and SEQ ID NO: 18 as provided herein.
• CoA synthetase in yeast other than S. cerevisiae can similarly be inactivated using the methods described herein.
• the host cell comprises a cytosolic acetyl-coA synthetase activity that can convert acetate to acetyl-CoA under respiratory conditions (i.e., when the host cell is grown in the presence of e.g. ethanol, glycerol, or acetate).
• the host cell is a yeast cell that comprises ACSl activity.
• the host cell compared to a parent cell comprises no or reduced endogenous acetyl-CoA synthetase activity under respiratory conditions.
• the host cell is a yeast cell that compared to a parent cell comprises no or reduced ACSl activity.
• the host cell comprises a cytosolic acetyl-coA synthetase activity that can convert acetate to acetyl-CoA under non-respiratory conditions (i.e., when the host cell is grown in the presence of fermentable carbon sources (e.g. sucrose, glucose)).
• the host cell is a yeast cell that comprises ACS2 activity.
• the host cell compared to a parent cell comprises no or reduced endogenous acetyl-CoA synthetase activity under non-respiratory conditions.
• the host cell is a yeast cell that compared to a parent cell comprises no or reduced ACS2 activity.
• the host cell comprises a heterologous PK and a cytosolic acetyl-coA synthetase activity (e.g, ACS1 and/or ACS2).
• PK produces acetyl phosphate in the host cell.
• the intact cytosolic ACS activity can convert acetate that accumulates as a result of RHR2 and/or HOR2-catalyzed acetyl phosphate hydrolysis into acetyl-CoA.
• the genetically modified host cell provided herein comprises one or more heterologous enzymes of the MEV pathway.
• the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetyl- CoA with malonyl-CoA to form acetoacetyl-CoA.
• the one or more enzymes of the MEV pathway comprise an enzyme that condenses two molecules of acetyl- CoA to form acetoacetyl-CoA.
• the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA.
• the one or more enzymes of the MEV pathway comprise an enzyme that converts HMG-CoA to mevalonate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-phosphate to mevalonate 5- pyrophosphate. In some embodiments, the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5 -pyrophosphate to isopentenyl
• the one or more enzymes of the MEV pathway are selected from the group consisting of acetyl-CoA thiolase, acetoacetyl-CoA synthetase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase.
• the genetically modified host cell comprises either an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA, e.g., acetyl-CoA thiolase; or an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA, e.g., acetoacetyl-CoA synthase.
• the genetically modified host cell comprises both an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA, e.g., acetyl-CoA thiolase; and an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA, e.g., acetoacetyl-CoA synthase.
• the host cell comprises one or more heterologous nucleotide sequences encoding more than one enzyme of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding two enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding an enzyme that can convert HMG- CoA into mevalonate and an enzyme that can convert mevalonate into mevalonate 5- phosphate. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding three enzymes of the MEV pathway.
• the host cell comprises one or more heterologous nucleotide sequences encoding four enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding six enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding seven enzymes of the MEV pathway. In some embodiments, the host cell comprises a plurality of heterologous nucleic acids encoding all of the enzymes of the MEV pathway.
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).
• the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can condense IPP and/or DMAPP molecules to form a polyprenyl compound.
• the genetically modified host cell further comprise a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.
• the genetically modified host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-coenzyme A to form acetoacetyl-CoA, e.g., an acetyl-CoA thiolase.
• nucleotide sequences encoding such an enzyme include, but are not limited to: (NC_000913 REGION: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).
• Acetyl-CoA thiolase catalyzes the reversible condensation of two molecules of acetyl-CoA to yield acetoacetyl-CoA, but this reaction is thermodynamically unfavorable; acetoacetyl-CoA thiolysis is favored over acetoacetyl-CoA synthesis.
• Acetoacetyl-CoA synthase AACS (alternately referred to as acetyl-CoA:malonyl-CoA acyltransferase; EC 2.3.1.194) condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA.
• AACS -catalyzed acetoacetyl-CoA synthesis is essentially an energy- favored reaction, due to the associated decarboxylation of malonyl-CoA.
• AACS exhibits no thiolysis activity against acetoacetyl-CoA, and thus the reaction is irreversible.
• acetyl-CoA thiolase In host cells comprising acetyl-CoA thiolase and a heterologous ADA and/or phosphotransacetylase (PTA), the reversible reaction catalyzed by acetyl-CoA thiolase, which favors acetoacetyl-CoA thiolysis, may result in a large acetyl-CoA pool. In view of the reversible activity of ADA, this acetyl-CoA pool may in turn drive ADA towards the reverse reaction of converting acetyl-CoA to acetaldehyde, thereby diminishing the benefits provided by ADA towards acetyl-CoA production.
• PTA phosphotransacetylase
• the activity of PTA is reversible, and thus, a large acetyl-CoA pool may drive PTA towards the reverse reaction of converting acetyl-CoA to acetyl phosphate. Therefore, in some embodiments, in order to provide a strong pull on acetyl-CoA to drive the forward reaction of ADA and PTA, the MEV pathway of the genetically modified host cell provided herein utilizes an acetoacetyl- CoA synthase to form acetoacetyl-CoA from acetyl-CoA and malonyl-CoA.
• the AACS is from Streptomyces sp. strain CL190
• Representative AACS protein sequences of Streptomyces sp. strain CL190 include accession numbers D7URV0,
• acetoacetyl-CoA synthases useful for the compositions and methods provided herein include, but are not limited to,
• Streptomyces sp. (AB183750; KO-3988 BAD86806); S anulatus strain 9663 (FN178498; CAX48662); Streptomyces sp. KO-3988 (AB212624; BAE78983); Actinoplanes sp. A40644 (AB113568; BAD07381); Streptomyces sp. C (NZ ACEW010000640; ZP 05511702);
• Nocardiopsis rougevillei DSM 43111 (NZ ABUI01000023; ZP_04335288);
• Mycobacterium ulcerans Agy99 NC_008611; YP_907152); Mycobacterium marinum M (NC 010612; YP 001851502); Streptomyces sp. Mgl (NZ DS570501; ZP 05002626); Streptomyces sp. AA4 (NZ ACEV01000037; ZP 05478992); S. roseosporus NRRL 15998 (NZ ABYBO 1000295; ZP 04696763); Streptomyces sp. ACTE (NZ ADFDO 1000030;
• acetoacetyl-CoA synthases include those described in U.S. Patent Application Publication Nos. 2010/0285549 and 2011/0281315, the contents of which are incorporated by reference in their entireties.
• Acetoacetyl-CoA synthases also useful in the compositions and methods provided herein include those molecules which are said to be “derivatives" of any of the acetoacetyl-CoA synthases described herein. Such a “derivative” has the following characteristics: (1) it shares substantial homology with any of the acetoacetyl-CoA synthases described herein; and (2) is capable of catalyzing the irreversible condensation of acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA.
• a derivative of an acetoacetyl-CoA synthase is said to share "substantial homology" with acetoacetyl-CoA synthase if the amino acid sequences of the derivative is at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of acetoacetyl-CoA synthase.
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), e.g., a HMG-CoA synthase.
• HMG-CoA 3-hydroxy-3-methylglutaryl-CoA
• nucleotide sequences encoding such an enzyme include, but are not limited to: (NC OOl 145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907;
• Kitasatospora griseola (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GenelD 1122571; Staphylococcus aureus).
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA into mevalonate, e.g., a HMG- CoA reductase.
• HMG-CoA reductase is an NADH-using
• HMG-CoA reductases (EC 1.1.1.34; EC 1.1.1.88) catalyze the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate, and can be categorized into two classes, class I and class II HMGrs.
• Class I includes the enzymes from eukaryotes and most archaea
• class II includes the HMG-CoA reductases of certain prokaryotes and archaea.
• the enzymes of the two classes also differ with regard to their cofactor specificity.
• class II HMG-CoA reductases Unlike the class I enzymes, which utilize NADPH exclusively, the class II HMG-CoA reductases vary in the ability to discriminate between NADPH and NADH. See, e.g., Hedl et al, Journal of Bacteriology 186 (7): 1927-1932 (2004). Co-factor specificities for select class II HMG-CoA reductases are provided below.
• HMG-CoA reductases for the compositions and methods provided herein include HMG-CoA reductases that are capable of utilizing NADH as a cofactor, e.g., HMG-CoA reductase from P. mevalonii, A. fulgidus or S. aureus.
• the HMG-CoA reductase is capable of only utilizing NADH as a cofactor, e.g., HMG-CoA reductase from P. mevalonii , S. pomeroyi or D. acidovorans.
• the NADH-using HMG-CoA reductase is from
• Pseudomonas mevalonii The sequence of the wild-type mvaA gene of Pseudomonas mevalonii, which encodes HMG-CoA reductase (EC 1.1.1.88), has been previously described. See Beach and Rodwell, J. Bacteriol. 171 :2994-3001 (1989).
• Representative mvaA nucleotide sequences of Pseudomonas mevalonii include accession number M24015, and SEQ ID NO: 21 as provided herein.
• Representative HMG-CoA reductase protein sequences of Pseudomonas mevalonii include accession numbers AAA25837, P13702, MVAA PSEMV, and SEQ ID NO: 22 as provided herein.
• the NADH-using HMG-CoA reductase is from
• Silicibacter pomeroyi include accession number NC 006569.1, and SEQ ID NO: 23 as provided herein.
• Representative HMG-CoA reductase protein sequences of Silicibacter pomeroyi include accession number YP 164994, and SEQ ID NO: 24 as provided herein.
• the NADH-using HMG-CoA reductase is from Delftia acidovorans.
• a representative HMG-CoA reductase nucleotide sequences of Delftia acidovorans includes NC 010002 REGION: complement(319980..321269), and SEQ ID NO: 25 as provided herein.
• Representative HMG-CoA reductase protein sequences of Delftia acidovorans include accession number YP 001561318, and SEQ ID NO: 26 as provided herein.
• the NADH-using HMG-CoA reductases is from
• Solanum tuberosum (Crane et ah, J. Plant Physiol. 159: 1301-1307 (2002)).
• NADH-using HMG-CoA reductases also useful in the compositions and methods provided herein include those molecules which are said to be "derivatives" of any of the NADH-using HMG-CoA reductases described herein, e.g., from P. mevalonii, S.
• Such a “derivative” has the following characteristics: (1) it shares substantial homology with any of the NADH-using HMG-CoA reductases described herein; and (2) is capable of catalyzing the reductive deacylation of (S)-HMG-CoA to (R)- mevalonate while preferentially using NADH as a cofactor.
• a derivative of an NADH-using HMG-CoA reductase is said to share "substantial homology" with NADH-using HMG-CoA reductase if the amino acid sequences of the derivative is at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of NADH-using HMG-CoA reductase.
• NADH-using means that the NADH-using
• HMG-CoA reductase is selective for NADH over NADPH as a cofactor, for example, by demonstrating a higher specific activity for NADH than for NADPH.
• selectivity for NADH as a cofactor is expressed as a kca ratio.
• the NADH-using HMG-CoA reductase has a k cat / k c NAO ratio of at least 5, 10, 15, 20, 25 or greater than 25.
• the NADH-using HMG- CoA reductase uses NADH exclusively.
• an NADH-using HMG-CoA reductase that uses NADH exclusively displays some activity with NADH supplied as the sole cofactor in vitro, and displays no detectable activity when NADPH is supplied as the sole cofactor.
• Any method for determining cofactor specificity known in the art can be utilized to identify HMG-CoA reductases having a preference for NADH as cofactor, including those described by Kim et al, Protein Science 9: 1226-1234 (2000); and Wilding et al, J. Bacteriol.
• the NADH-using HMG-CoA reductase is engineered to be selective for NADH over NAPDH, for example, through site-directed mutagenesis of the cofactor-binding pocket.
• Methods for engineering NADH-selectivity are described in Watanabe et ah, Microbiology 153:3044-3054 (2007), and methods for determining the co factor specificity of HMG-CoA reductases are described in Kim et ah, Protein Sci. 9: 1226- 1234 (2000), the contents of which are hereby incorporated by reference in their entireties.
• the NADH-using HMG-CoA reductase is derived from a host species that natively comprises a mevalonate degradative pathway, for example, a host species that catabolizes mevalonate as its sole carbon source.
• the NADH-using HMG-CoA reductase which normally catalyzes the oxidative acylation of internalized (R)-mevalonate to (S)-HMG-CoA within its native host cell, is utilized to catalyze the reverse reaction, that is, the reductive deacylation of (S)-HMG-CoA to (R)- mevalonate, in a genetically modified host cell comprising a mevalonate biosynthetic pathway.
• Prokaryotes capable of growth on mevalonate as their sole carbon source have been described by: Anderson et ah, J. Bacteriol, 171(12):6468-6472 (1989); Beach et ah, J.
• the host cell comprises both a NADH-using HMGr and an NADPH-using HMG-CoA reductase.
• nucleotide sequences encoding an NADPH-using HMG-CoA reductase include, but are not limited to: (NM_206548; Drosophila melanogaster),
• NC_002758 Locus tag SAV2545, GenelD 1122570; Staphylococcus aureus), (AB015627; Streptomyces sp. KO 3988), (AX128213, providing the sequence encoding a truncated HMG- CoA reductase; Saccharomyces cerevisiae), and (NC_001145: complement (115734.118898; Saccharomyces cerevisiae).
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate into mevalonate 5 -phosphate, e.g., a mevalonate kinase.
• an enzyme that can convert mevalonate into mevalonate 5 -phosphate, e.g., a mevalonate kinase.
• nucleotide sequences encoding such an enzyme include, but are not limited to: (L77688; Arabidopsis thaliana), and (X55875;
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5 -phosphate into mevalonate 5- pyrophosphate, e.g., a phosphomevalonate kinase.
• an enzyme that can convert mevalonate 5 -phosphate into mevalonate 5- pyrophosphate, e.g., a phosphomevalonate kinase.
• nucleotide sequences encoding such an enzyme include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. complement 712315.713670; Saccharomyces cerevisiae).
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5 -pyrophosphate into isopentenyl diphosphate (IPP), e.g., a mevalonate pyrophosphate decarboxylase.
• IPP isopentenyl diphosphate
• nucleotide sequences encoding such an enzyme include, but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens).
• the host cell further comprises a heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the MEV pathway into dimethylallyl pyrophosphate (DMAPP), e.g., an IPP isomerase.
• DMAPP dimethylallyl pyrophosphate
• nucleotide sequences encoding such an enzyme include, but are not limited to: (NC_000913, 3031087.3031635; Escherichia coli), and (AF082326; Haematococcus pluvialis).
• the host cell further comprises a heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and/or DMAPP molecules to form polyprenyl compounds containing more than five carbons.
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense one molecule of IPP with one molecule of DMAPP to form one molecule of geranyl pyrophosphate ("GPP"), e.g., a GPP synthase.
• GPP geranyl pyrophosphate
• nucleotide sequences encoding such an enzyme include, but are not limited to: (AF513111; Abies grandis), (AF5131 12; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsis thaliand), (AE016877, Locus API 1092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ips pini),
• the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of IPP with one molecule of DMAPP, or add a molecule of IPP to a molecule of GPP, to form a molecule of farnesyl pyrophosphate ("FPP"), e.g., a FPP synthase.
• FPP farnesyl pyrophosphate
• nucleotide sequences that encode such an enzyme include, but are not limited to: (ATU80605; Arabidopsis thaliand), (ATHFPS2R; Arabidopsis thaliand), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, Locus AAL95523;
• Streptococcus pyogenes (CP000017, Locus AAZ51849; Streptococcus pyogenes),
• NC_008022 Locus YP_598856; Streptococcus pyogenes MGAS 10270
• NC_008023 Locus YP 600845; Streptococcus pyogenes MGAS2096
• NC 008024 Locus YP 602832; Streptococcus pyogenes MGAS10750
• MZEFPS Zea mays
• NP_873754 Haemophilus ducreyi 35000HP
• L42023 Locus AAC23087; Haemophilus influenzae Rd KW20
• J05262 Homo sapiens
• YP_395294 Lactobacillus sakei subsp. sakei 23K
• NC_005823 Locus YP_000273; Leptospira interrogans serovar Copenhageni str.
• Fiocruz Ll-130 (AB003187; Micrococcus luteus), (NC_002946, Locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp. NGR234), (J05091; Saccharomyces cerevisae), (CP000031, Locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890; Streptococcus pneumoniae R6), and (NC 004556, Locus NP 779706; Xylella fastidiosa Temeculal).
• the host cell further comprises a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate ("GGPP").
• GGPP geranylgeranyl pyrophosphate
• nucleotide sequences that encode such an enzyme include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845; Arabidopsis thaliana), (NZ AAJMO 1000380, Locus ZP_00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sql563), (CRGGPPS; Catharanthus roseus),
• the host cell further comprises a heterologous nucleotide sequence encoding an enzyme that can modify a polyprenyl to form a
• hemiterpene a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified isoprenoid compound.
• the heterologous nucleotide encodes a carene synthase.
• suitable nucleotide sequences include, but are not limited to:
• the heterologous nucleotide encodes a geraniol synthase.
• suitable nucleotide sequences include, but are not limited to: (AJ457070; Cinnamomum tenuipilum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora).
• the heterologous nucleotide encodes a linalool synthase.
• a suitable nucleotide sequence include, but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana),
• Lycopersicon esculentum (DQ263741; Lavandula angustifolia), (AY083653; Mentha citrate), (AY693647; Ocimum basilicum), (XM_463918; Oryza sativa), (AP004078, Locus BAD07605; Oryza sativa), (XM_463918, Locus XP_463918; Oryza sativa), (AY917193; Perilla citriodora), (AF271259; Perilla frutescens), (AY473623; Picea abies), (DQ195274; Picea sitchensis), and (AF444798; Perilla frutescens var. crispa cultivar No. 79).
• the heterologous nucleotide encodes a limonene synthase.
• suitable nucleotide sequences include, but are not limited to: (+)-limonene synthases (AF514287, REGION: 47.1867; Citrus limon) and (AY055214, REGION: 48.1889; Agastache rugosa) and (-)-limonene synthases (DQ195275, REGION: 1.1905; Picea sitchensis), (AF006193, REGION: 73.1986; Abies grandis), and (MHC4SLSP, REGION: 29.1828; Mentha spicata).
• the heterologous nucleotide encodes a myrcene synthase.
• suitable nucleotide sequences include, but are not limited to: (U87908; Abies grandis), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (NM_127982; Arabidopsis thaliana TPS10), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercus ilex).
• the heterologous nucleotide encodes a ocimene synthase.
• suitable nucleotide sequences include, but are not limited to: (AY195607; Antirrhinum majus), (AY195609; Antirrhinum majus), (AY195608;
• Antirrhinum majus (AK221024; Arabidopsis thaliana), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (NM_117775; Arabidopsis thaliana ATTPS03), (NM_001036574; Arabidopsis thaliana ATTPS03), (NMJ27982; Arabidopsis thaliana TPS 10), (AB 110642; Citrus unshiu CitMTSL4), and (AY575970; Lotus corniculatus var. japonicus).
• the heterologous nucleotide encodes an a-pinene synthase.
• suitable nucleotide sequences include, but are not limited to: (+) a-pinene synthase (AF543530, REGION: 1.1887; Pinus taeda), (-)a-pinene synthase (AF543527, REGION: 32.1921; Pinus taeda), and (+)/(-)a-pinene synthase (AGU87909, REGION: 6111892; Abies grandis).
• the heterologous nucleotide encodes a ⁇ -pinene synthase.
• suitable nucleotide sequences include, but are not limited to: (-) ⁇ -pinene synthases (AF276072, REGION: 1.1749; Artemisia annua) and (AF514288, REGION: 26.1834; Citrus Union).
• the heterologous nucleotide encodes a sabinene synthase.
• An illustrative example of a suitable nucleotide sequence includes but is not limited to AF051901, REGION: 26.1798 from Salvia officinalis.
• the heterologous nucleotide encodes a ⁇ -terpinene synthase.
• suitable nucleotide sequences include: (AF514286,
• the heterologous nucleotide encodes a terpinolene synthase.
• a suitable nucleotide sequence include, but are not limited to: (AY693650 from Oscimum basilicum) and (AY906866, REGION: 10.1887 from
• the heterologous nucleotide encodes an amorphadiene synthase.
• An illustrative example of a suitable nucleotide sequence is SEQ ID NO. 37 of U.S. Patent Publication No. 2004/0005678.
• the heterologous nucleotide encodes a a-farnesene synthase.
• suitable nucleotide sequences include, but are not limited to DQ309034 from Pyrus communis cultivar dAnjou (pear; gene name AFSl) and
• the heterologous nucleotide encodes a ⁇ -farnesene synthase.
• suitable nucleotide sequences include but is not limited to accession number AF024615 from Mentha x piperita (peppermint; gene Tspal 1), and AY835398 from Artemisia annua. Picaud et al., Phytochemistry 66(9): 961-967 (2005).
• the heterologous nucleotide encodes a farnesol synthase.
• suitable nucleotide sequences include, but are not limited to accession number AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128: 149-158 (2006).
• the heterologous nucleotide encodes a nerolidol synthase.
• An illustrative example of a suitable nucleotide sequence includes, but is not limited to AF529266 from Zea mays (maize; gene tpsl).
• the heterologous nucleotide encodes a patchouliol synthase.
• suitable nucleotide sequences include, but are not limited to AY508730 REGION: 1.1659 from Pogostemon cablin.
• the heterologous nucleotide encodes a nootkatone synthase.
• suitable nucleotide sequences include, but are not limited to AF441 124 REGION: 1.1647 from Citrus sinensis and AY917195 REGION: 1.1653 from Per ilia frutescens.
• the heterologous nucleotide encodes an abietadiene synthase.
• suitable nucleotide sequences include, but are not limited to: (U50768; Abies grandis) and (AY473621; Picea abies).
• the host cell produces a C 5 isoprenoid.
• These compounds are derived from one isoprene unit and are also called hemiterpenes.
• An illustrative example of a hemiterpene is isoprene.
• the isoprenoid is a Cio isoprenoid.
• monoterpenes are limonene, citranellol, geraniol, menthol, perillyl alcohol, linalool, thujone, and myrcene.
• the isoprenoid is a C 15 isoprenoid.
• These compounds are derived from three isoprene units and are also called sesquiterpenes.
• Illustrative examples of sesquiterpenes are periplanone B, gingkolide B, amorphadiene, artemisinin, artemisinic acid, valencene, nootkatone, epi-cedrol, epi-aristolochene, farnesol, gossypol, sanonin, periplanone, forskolin, and patchoulol (which is also known as patchouli alcohol).
• the isoprenoid is a C 2 o isoprenoid.
• diterpenes are casbene, eleutherobin, paclitaxel, prostratin, pseudopterosin, and taxadiene.
• the isoprenoid is a C 2 o + isoprenoid.
• These compounds are derived from more than four isoprene units and include: triterpenes (C 30 isoprenoid compounds derived from 6 isoprene units) such as arbrusideE, bruceantin, testosterone, progesterone, cortisone, digitoxin, and squalene; tetraterpenes (C 40 isoprenoid compounds derived from 8 isoprenoids) such as ⁇ -carotene; and polyterpenes (C 40+ isoprenoid compounds derived from more than 8 isoprene units) such as polyisoprene.
• triterpenes C 30 isoprenoid compounds derived from 6 isoprene units
• tetraterpenes C 40 isoprenoid compounds derived from 8 isoprenoids
• polyterpenes C 40+ isoprenoid compounds derived from more than 8 isoprene units
• the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, a-farnesene, ⁇ -farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, ⁇ -pinene, sabinene, ⁇ -terpinene, terpinolene and valencene.
• Isoprenoid compounds also include, but are not limited to, carotenoids (such as lycopene, a- and ⁇ -carotene, a- and ⁇ -cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein), steroid compounds, and compounds that are composed of isoprenoids modified by other chemical groups, such as mixed terpene-alkaloids, and coenzyme Q-10.
• carotenoids such as lycopene, a- and ⁇ -carotene, a- and ⁇ -cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein
• steroid compounds and compounds that are composed of isoprenoids modified by other chemical groups, such as mixed terpene-alkaloids, and coenzyme Q-10.
• a method for the production of an isoprenoid comprising the steps of: (a) culturing a population of any of the genetically modified host cells described herein that are capable of producing an isoprenoid in a medium with a carbon source under conditions suitable for making an isoprenoid compound; and (b) recovering said isoprenoid compound from the medium.
• the genetically modified host cell comprises one or more modifications selected from the group consisting of: heterologous expression of a phosphoketolase, heterologous expression of a phosphotransacetylase, heterologous expression of one or more enzymes of the mevalonate pathway; and optionally, heterologous expression of an ADA, heterologous expression of an NADH-using HMG-CoA reductase, and heterologous expression of an AACS; and the genetically modified host cell produces an increased amount of the isoprenoid compound compared to a parent cell not comprising the one or more modifications, or a parent cell comprising only a subset of the one or more modifications of the genetically modified host cell, but is otherwise genetically identical.
• the increased amount is at least 1%, 5%, 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%), as measured, for example, in yield, production, productivity, in grams per liter of cell culture, milligrams per gram of dry cell weight, on a per unit volume of cell culture basis, on a per unit dry cell weight basis, on a per unit volume of cell culture per unit time basis, or on a per unit dry cell weight per unit time basis.
• the host cell produces an elevated level of isoprenoid that is greater than about 10 grams per liter of fermentation medium.
• the isoprenoid is produced in an amount from about 10 to about 50 grams, more than about 15 grams, more than about 20 grams, more than about 25 grams, or more than about 30 grams per liter of cell culture.
• the host cell produces an elevated level of isoprenoid that is greater than about 50 milligrams per gram of dry cell weight.
• the isoprenoid is produced in an amount from about 50 to about 1500 milligrams, more than about 100 milligrams, more than about 150 milligrams, more than about 200 milligrams, more than about 250 milligrams, more than about 500 milligrams, more than about 750 milligrams, or more than about 1000 milligrams per gram of dry cell weight.
• the host cell produces an elevated level of isoprenoid that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%o, at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 60%>, at least about 70%>, at least about 80%>, at least about 90%>, at least about 2- fold, at least about 2.
• the host cell produces an elevated level of isoprenoid that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%o, at least about 35%, at least about 40%>, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2- fold, at least about 2.
• the host cell produces an elevated level of an isoprenoid that is at least about 10%, at least about 15%, at least about 20%, at least about 25%), at least about 30%>, at least about 35%, at least about 40%>, at least about 45%, at least about 50%), at least about 60%>, at least about 70%>, at least about 80%>, at least about 90%>, at least about 2-fold, at least about 2.
• the host cell produces an elevated isoprenoid that is at least about 10%>, at least about 15%, at least about 20%>, at least about 25%, at least about 30%), at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%), at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.
• the production of the elevated level of isoprenoid by the host cell is inducible by an inducing compound.
• an inducing compound is then added to induce the production of the elevated level of isoprenoid by the host cell.
• production of the elevated level of isoprenoid by the host cell is inducible by changing culture conditions, such as, for example, the growth temperature, media
• the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products.
• Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof.
• strains can be grown in a fermentor as described in detail by Kosaric, et al, in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley- VCH Verlag GmbH & Co. KDaA, Weinheim, Germany.
• the culture medium is any culture medium in which a genetically modified microorganism capable of producing an isoprenoid can subsist, i.e., maintain growth and viability.
• the culture medium is an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients.
• the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation media, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass.
• Suitable conditions and suitable media for culturing microorganisms are well known in the art.
• the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer ⁇ e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor ⁇ e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent ⁇ e.g., an antibiotic to select for microorganisms comprising the genetic modifications).
• an inducer e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter
• a repressor e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter
• a selection agent e.g.
• the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof.
• suitable monosaccharides include glucose, galactose, mannose, fructose, xylose, ribose, and combinations thereof.
• suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.
• suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof.
• suitable non-fermentable carbon sources include acetate and glycerol.
• the concentration of a carbon source, such as glucose, in the culture medium should promote cell growth, but not be so high as to repress growth of the microorganism used.
• a carbon source such as glucose
• the concentration of a carbon source, such as glucose, in the culture medium is greater than about 1 g/L, preferably greater than about 2 g/L, and more preferably greater than about 5 g/L.
• the concentration of a carbon source, such as glucose, in the culture medium is typically less than about 100 g/L, preferably less than about 50 g/L, and more preferably less than about 20 g/L. It should be noted that references to culture component concentrations can refer to both initial and/or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during culture.
• Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources, in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1.0 g/L. Beyond certain
• the concentration of the nitrogen sources, in the culture medium is less than about 20 g/L, preferably less than about 10 g/L and more preferably less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culture.
• the effective culture medium can contain other compounds such as inorganic salts, vitamins, trace metals or growth promoters. Such other compounds can also be present in carbon, nitrogen or mineral sources in the effective medium or can be added specifically to the medium.
• the culture medium can also contain a suitable phosphate source.
• phosphate sources include both inorganic and organic phosphate sources.
• Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate and mixtures thereof.
• the concentration of phosphate in the culture medium is greater than about 1.0 g/L, preferably greater than about 2.0 g/L and more preferably greater than about 5.0 g/L.
• the concentration of phosphate in the culture medium is typically less than about 20 g/L, preferably less than about 15 g/L and more preferably less than about 10 g/L.
• a suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
• a source of magnesium preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
• the concentration of magnesium in the culture medium is greater than about 0.5 g/L, preferably greater than about 1.0 g/L, and more preferably greater than about 2.0 g/L. Beyond certain concentrations, however, the addition of magnesium to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of magnesium in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 3 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of a physiologically acceptable
• the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate.
• a biologically acceptable chelating agent such as the dihydrate of trisodium citrate.
• the concentration of a chelating agent in the culture medium is greater than about 0.2 g/L, preferably greater than about 0.5 g/L, and more preferably greater than about 1 g/L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of a chelating agent in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 2 g/L.
• the culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium.
• Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and mixtures thereof.
• Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.
• the culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride.
• a biologically acceptable calcium source including, but not limited to, calcium chloride.
• concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 mg/L, preferably within the range of from about 20 mg/L to about 1000 mg/L, and more preferably in the range of from about 50 mg/L to about 500 mg/L.
• the culture medium can also include sodium chloride.
• the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, preferably within the range of from about 1 g/L to about 4 g/L, and more preferably in the range of from about 2 g/L to about 4 g/L.
• the culture medium can also include trace metals.
• trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium.
• the amount of such a trace metals solution added to the culture medium is greater than about 1 ml/L, preferably greater than about 5 mL/L, and more preferably greater than about 10 mL/L. Beyond certain concentrations, however, the addition of a trace metals to the culture medium is not advantageous for the growth of the microorganisms.
• the amount of such a trace metals solution added to the culture medium is typically less than about 100 mL/L, preferably less than about 50 mL/L, and more preferably less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.
• the culture media can include other vitamins, such as pantothenate, biotin, calcium, pantothenate, inositol, pyridoxine-HCl, and thiamine-HCl.
• vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of the microorganisms .
• the fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous and semi-continuous.
• the fermentation is carried out in fed-batch mode.
• some of the components of the medium are depleted during culture, including pantothenate during the production stage of the fermentation.
• the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and/or isoprenoid production is supported for a period of time before additions are required.
• the preferred ranges of these components are maintained throughout the culture by making additions as levels are depleted by culture.
• Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations.
• additions can be made at timed intervals corresponding to known levels at particular times throughout the culture.
• the rate of consumption of nutrient increases during culture as the cell density of the medium increases.
• addition is performed using aseptic addition methods, as are known in the art.
• a small amount of anti- foaming agent may be added during the culture.
• the temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and/or production of isoprenoids.
• the culture medium prior to inoculation of the culture medium with an inoculum, can be brought to and maintained at a temperature in the range of from about 20°C to about 45°C, preferably to a temperature in the range of from about 25°C to about 40°C, and more preferably in the range of from about 28°C to about 32°C.
• the pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium.
• the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.
• the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture.
• Glucose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium.
• the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L, and can be determined readily by trial.
• the glucose when glucose is used as a carbon source the glucose is preferably fed to the fermentor and maintained below detection limits.
• the glucose concentration in the culture medium is maintained in the range of from about 1 g/L to about 100 g/L, more preferably in the range of from about 2 g/L to about 50 g/L, and yet more preferably in the range of from about 5 g/L to about 20 g/L.
• the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium. The use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium (e.g. the nitrogen and phosphate sources) can be maintained simultaneously.
• the trace metals concentrations can be maintained in the culture medium by addition of aliquots of the trace metals solution.
• an organic phase comprising the isoprenoid is separated from the fermentation by centrifugation.
• an organic phase comprising the isoprenoid separates from the fermentation spontaneously.
• an organic phase comprising the isoprenoid is separated from the fermentation by adding a demulsifier and/or a nucleating agent into the fermentation reaction.
• demulsifiers include flocculants and coagulants.
• nucleating agents include droplets of the isoprenoid itself and organic solvents such as dodecane, isopropyl myristrate, and methyl oleate.
• the isoprenoid produced in these cells may be present in the culture supernatant and/or associated with the host cells.
• the recovery of the isoprenoid may comprise a method of permeabilizing or lysing the cells.
• the isoprenoid in the culture medium can be recovered using a recovery process including, but not limited to, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization.
• the isoprenoid is separated from other products that may be present in the organic phase. In some embodiments, separation is achieved using adsorption, distillation, gas-liquid extraction (stripping), liquid-liquid extraction (solvent extraction), ultrafiltration, and standard chromatographic techniques.
• the genetically modified host cell provided herein is capable of producing a polyketide from acetyl-CoA.
• Polyketides are synthesized by sequential reactions catalyzed by a collection of enzyme activities called polyketide synthases (PKSs), which are large multi-enzyme protein complexes that contain a coordinated group of active sites.
• PKSs polyketide synthases
• Polyketide biosynthesis proceeds stepwise starting from simple 2-, 3-, 4-carbon building blocks such as acetyl-CoA, propionyl CoA, butyryl-CoA and their activated derivatives, malonyl-, methylmalonyl- and ethylmalonyl-CoA, primarily through
• Type I polyketide synthases are large, highly modular proteins subdivided into two classes: 1) iterative PKSs, which reuse domains in a cyclic fashion and 2) modular PKSs, which contain a sequence of separate modules and do not repeat domains.
• Type II polyketide synthases are aggregates of monofunctional proteins, and Type III polyketide synthases do not use acyl carrier protein domains.
• Component domains or separate enzyme functionalities active in this biosynthesis include acyl-transferases for the loading of starter, extender and intermediate acyl units; acyl carrier proteins which hold the growing macrolide as a thiol ester; ⁇ -keto-acyl synthases which catalyze chain extension; ⁇ -keto reductases responsible for the first reduction to an alcohol functionality; dehydratases which eliminate water to give an unsaturated thiolester; enoyl reductases which catalyze the final reduction to full saturation; and thiolesterases which catalyze macrolide release and cyclization.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can condense at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein, e.g. an acyl- transferase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can condense a first reactant selected from the group consisting of acetyl-CoA and malonyl-CoA with a second reactant selected from the group consisting of malonyl-CoA or methylmalonyl-CoA to form a polyketide product, e.g. a ⁇ -keto-acyl synthase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can reduce a ⁇ -keto chemical group on a polyketide compound to a ⁇ -hydroxy group, e.g. a ⁇ -keto reductase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can dehydrate an alkane chemical group in a polyketide compound to produce an ⁇ - ⁇ -unsaturated alkene, e.g. a dehydratase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can reduce an ⁇ - ⁇ -double-bond in a polyketide compound to a saturated alkane, e.g. an enoyl-reductase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can hydrolyze a polyketide compound from an acyl carrier protein, e.g. a thioesterase.
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic region. In some embodiments, the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an AT catalytic region. In some embodiments, the polyketide producing cell comprises more than one heterologous nucleotide sequence encoding an enzyme comprising an AT catalytic region. In some embodiments, the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a CLF catalytic region.
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an ACP activity. In some embodiments, the polyketide producing cell comprises more than one heterologous nucleotide sequence encoding an enzyme comprising an ACP activity.
• the polyketide producing cell comprises a minimal aromatic PKS system, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic region, an enzyme comprising an AT catalytic region, an enzyme comprising a CLF catalytic region, and an enzyme comprising an ACP activity, respectively.
• the polyketide producing cell comprises a minimal modular PKS system, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic region, an enzyme comprising an AT catalytic region, and an enzyme comprising an ACP activity, respectively.
• the polyketide producing cell comprises a modular aromatic PKS system for de novo polyketide synthesis, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic region, one or more enzymes comprising an AT catalytic region, and one or more enzymes comprising an ACP activity, respectively.
• a modular aromatic PKS system for de novo polyketide synthesis e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic region, one or more enzymes comprising an AT catalytic region, and one or more enzymes comprising an ACP activity, respectively.
• the polyketide producing cell comprising a minimal
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a cyclase (CYC) catalytic region, which facilitates the cyclization of the nascent polyketide backbone.
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a ketoreductase (KR) catalytic region.
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an aromatase (ARO) catalytic region. In some embodiments, the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an enoylreductase (ER) catalytic region. In some embodiments, the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a thioesterase (TE) catalytic region. In some embodiments, the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a holo ACP synthase activity, which effects pantetheinylation of the ACP.
• ARO aromatase
• the polyketide producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an enoylreductase (ER) cata
• the polyketide producing cell further comprises one or more heterologous nucleotide sequences conferring a postsynthesis polyketide modifying activity. In some embodiments, the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a glycosylase activity, which effects postsynthesis modifications of polyketides, for example, where polyketides having antibiotic activity are desired. In some embodiments, the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a hydroxylase activity. In some embodiments, the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an epoxidase activity. In some embodiments, the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a methylase activity.
• the polyketide producing cell further comprises one or more heterologous nucleotide sequences encoding a biosynthetic enzyme including, but not limited to, at least one polyketide synthesis pathway enzyme, and enzymes that can modify an acetyl-CoA compound to form a polyketide product such as a macrolide, an antibiotic, an antifungal, a cytostatic compound, an anticholesterolemic compound, an antiparasitic compound, a coccidiostatic compound, an animal growth promoter or an insecticide.
• the HACD compound is a polyene.
• the HACD compound is a cyclic lactone.
• the HACD compound comprises a 14, 15, or 16-membered lactone ring.
• the HACD compound is a polyketide selected from the group consisting of a polyketide macrolide, antibiotic, antifungal, cytostatic, anticholesterolemic, antiparasitic, a coccidiostatic, animal growth promoter and insecticide.
• the polyketide producing cell comprises heterologous nucleotide sequences, for example sequences encoding PKS enzymes and polyketide modification enzymes, capable of producing a polyketide selected from, but not limited to, the following polyketides: Avermectin (see, e.g., U.S. Pat. No. 5,252,474; U.S. Pat. No. 4,703,009; EP Pub. No. 118,367; MacNeil et al, 1993, "Industrial Microorganisms: Basic and Applied Molecular Genetics"; Baltz, Hegeman, & Skatrud, eds. (ASM), pp.
• ASM Address Translation
• FK-506 see, e.g., Motamedi et al, 1998; Eur. J Biochem. 256: 528-534; and Motamedi et al, 1997, Eur. J Biochem. 244: 74-80
• FK-520 see, e.g., PCT Pub. No. 00/020601; and Nielsen et al, 1991, Biochem. 30:5789-96
• Griseusin see, e.g., Yu et al, J Bacteriol.
• Lovastatin see, e.g., U.S. Pat. No. 5,744,350
• Frenolycin see, e.g., Khosla et al, Bacteriol. 1993 Apr;175(8):2197-204; and Bibb et al, Gene 1994 May 3;142(l):31-9
• Granaticin see, e.g., Sherman et al, EMBO J. 1989 Sep;8(9):2717-25; and Bechtold et al, Mol Gen Genet.
• Medermycin see, e.g., Ichinose et al, Microbiology 2003 Jul;149(Pt 7): 1633-45
• Monensin see, e.g., Arrowsmith et al, Mol Gen Genet. 1992 Aug;234(2):254-64
• Nonactin see, e.g., FEMS Microbiol Lett. 2000 Feb l;183(l): 171-5
• Nanaomycin see, e.g., Kitao et al, J Antibiot (Tokyo).
• Nemadectin see, e.g., MacNeil et al, 1993, supra
• Niddamycin see, e.g., PCT Pub. No. 98/51695; and Kakavas et al, 1997, J. Bacteriol. 179: 7515-7522
• Nemadectin see, e.g., MacNeil et al, 1993, supra
• Niddamycin see, e.g., PCT Pub. No. 98/51695; and Kakavas et al, 1997, J. Bacteriol. 179: 7515-7522
• Oleandomycin see e.g., Swan et al, 1994, Mol. Gen. Genet. 242: 358-362; PCT Pub. No. 00/026349; Olano et al, 1998, Mol. Gen. Genet. 259(3): 299-308; and PCT Pat. App. Pub. No. WO 99/05283); Oxytetracycline (see, e.g., Kim et al, Gene. 1994 Apr 8;141(1): 141-2); Picromycin (see, e.g., PCT Pub. No. 99/61599; PCT Pub. No.
• the genetically modified host cell provided herein is capable of producing a fatty acid from acetyl-CoA.
• Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acetyl-CoA and malonyl-CoA catalyzed by fatty acid synthases. Similar to polyketide synthases, fatty acid synthases are not a single enzyme but an enzymatic system composed of 272 kDa multifunctional polypeptide in which substrates are handed from one functional domain to the next. Two principal classes of fatty acid synthases have been characterized: Type I fatty acid synthases are single,
• Type II synthases found in archaeabacteria and eubacteria, are a series of discrete, monofunctional enzymes that participate in the synthesis of fatty acids. The mechanisms fatty acid elongation and reduction is the same in the two classes of synthases, as the enzyme domains responsible for these catalytic events are largely homologous amongst the two classes.
• the ⁇ -keto group is reduced to a fully saturated carbon chain by the sequential action of a ketoreductase, a dehydratase, and an enol reductase.
• the growing fatty acid chain moves between these active sites attached to an acyl carrier protein and is ultimately released by the action of a thioesterase upon reaching a carbon chain length of 16 (palmitidic acid).
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding a biosynthetic enzyme including, but not limited to, at least one fatty acid synthesis pathway enzyme, and enzymes that can modify an acetyl-CoA compound to form a fatty acid product such as a palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
• a biosynthetic enzyme including, but not limited to, at least one fatty acid synthesis pathway enzyme, and enzymes that can modify an acetyl-CoA compound to form a fatty acid product such as a palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, lino
• the HACD compound is a fatty acid selected from the group consisting of palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can covalently link at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein, e.g. an acyltransferase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can condense acetyl chemical moiety and a malonyl chemical moiety, each bound to an acyl carrier protein (ACP), to form acetoacetyl-ACP, e.g. a ⁇ -Ketoacyl-ACP synthase.
• ACP acyl carrier protein
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can reduce the double bond in acetoacetyl-ACP with NADPH to form a hydroxyl group in D-3- hydroxybutyryl hydroxylase-ACP, e.g. a ⁇ -Ketoacyl-ACP reductase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can dehydrate D-3-Hydroxybutyryl hydroxylase-ACP to create a double bond between the beta- and gamma-carbons forming crotonyl-ACP, e.g. a ⁇ -hydroxyacyl-ACP dehydrase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can reduce crotonyl ACP with NADPH to form butyryl-ACP, e.g. an enoyl ACP reductase.
• the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can hydrolyze a C16 acyl compound from an acyl carrier protein to form palmitate, e.g. a thioesterase.
• the fatty acid producing cell comprises one or more heterologous nucleotide sequences encoding acetyl-CoA synthase and/or malonyl-CoA synthase, to effect increased production of one or more fatty acids as compared to a genetically unmodified parent cell.
• one or more of the following genes can be expressed in the cell: pdh, panK, aceEF (encoding the EIp dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2- oxoglutarate dehydrogenase complexes), fabH,fabD, fab G, acpP, and fabF.
• nucleotide sequences encoding such enzymes include, but are not limited to: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC76952), aceEF (AAC73227, AAC73226),/a£H (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178),/a£ (AAC74179).
• increased fatty acid levels can be effected in the cell by attenuating or knocking out genes encoding proteins involved in fatty acid degradation.
• the expression levels of fadE, gpsA, idhA, pflb, adhE, pta, poxB, ackA, and/or ackB can be attenuated or knocked-out in an engineered host cell using techniques known in the art.
• nucleotide sequences encoding such proteins include, but are not limited to: fa dE (AAC73325), gspA (AAC76632), IdhA (AAC74462), pflb (AAC73989), adhE (AAC74323), /?ta (AAC '5357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430).
• the resulting host cells will have increased acetyl-CoA production levels when grown in an appropriate environment.
• the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert acetyl-CoA into malonyl-CoA, e.g., the multisubunit AccABCD protein.
• a suitable nucleotide sequence encoding AccABCD includes but is not limited to accession number AAC73296, EC 6.4.1.2.
• the fatty acid producing cell comprises a heterologous nucleotide sequence encoding a lipase.
• suitable nucleotide sequences encoding a lipase include, but are not limited to accession numbers CAA89087 and
• increased fatty acid levels can be effected in the cell by inhibiting PlsB, which can lead to an increase in the levels of long chain acyl-ACP, which will inhibit early steps in the fatty acid biosynthesis pathway (e.g., accABCD,fabH, and fabl).
• the expression level of PlsB can be attenuated or knocked-out in an engineered host cell using techniques known in the art.
• An illustrative example of a suitable nucleotide sequence encoding PlsB includes but is not limited to accession number AAC77011.
• the plsB D31 IE mutation can be used to increase the amount of available acyl- CoA in the cell.
• increased production of monounsaturated fatty acids can be effected in the cell by overexpressing an sfa gene, which would result in suppression of fab A.
• An illustrative example of a suitable nucleotide sequence encoding sfa includes but is not limited to accession number AAN79592.
• increased fatty acid levels can be effected in the cell by modulating the expression of an enzyme which controls the chain length of a fatty acid substrate, e.g., a thioesterase.
• the fatty acid producing cell has been modified to overexpress a tes or fat gene.
• Suitable tes nucleotide sequences include but are not limited to accession numbers: ⁇ tes A: AAC73596, from E. coli, capable of producing C 18:1 fatty acids) and (tesB AAC73555 from E. coli).
• suitable fat nucleotide sequences include but are not limited to: (fatB: Q41635 and AAA34215, from Umbellularia California, capable of producing Ci 2 :o fatty acids), (fatB2: Q39513 and AAC49269, from Cuphea hookeriana, capable of producing Cs : o - Cio : o fatty acids), (fatB3: AAC49269 and AAC72881, from Cuphea hookeriana, capable of producing Ci4:o - Ci6:o fatty acids), (fatB: Q39473 and AAC49151, from Cinnamonum camphorum, capable of producing Ci4:o fatty acids ), (fatB [M141TJ: CAA85388, from mArabidopsis thaliana, capable of producing Ci 6: i fatty acids ), (fatA : NP 189147 and NP 193041, from Arabidopsis thaliana, capable of producing C 18:1 fatty acids ), (fatB [M141
• increased levels of C 10 fatty acids can be effected in the cell by attenuating the expression or activity of thioesterase C 18 using techniques known in the art.
• Illustrative examples of suitable nucleotide sequences encoding thioesterase C 18 include, but are not limited to accession numbers AAC73596 and P0ADA1.
• increased levels of C 10 fatty acids can be effected in the cell by increasing the expression or activity of thioesterase C 10 using techniques known in the art.
• An illustrative example of a suitable nucleotide sequence encoding thioesterase C 10 includes, but is not limited to accession number Q39513.
• increased levels of C 14 fatty acids can be effected in the cell by attenuating the expression or activity of endogenous thioesterases that produce non- Ci4 fatty acids, using techniques known in the art.
• increased levels of Ci4 fatty acids can be effected in the cell by increasing the expression or activity of thioesterases that use the substrate C14-ACP, using techniques known in the art.
• An illustrative example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to accession number Q39473.
• increased levels of C 12 fatty acids can be effected in the cell by attenuating the expression or activity of endogenous thioesterases that produce non- Co fatty acids, using techniques known in the art.
• increased levels of Ci2 fatty acids can be effected in the cell by increasing the expression or activity of thioesterases that use the substrate C12-ACP, using techniques known in the art.
• An illustrative example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to accession number Q41635.
• the genetically modified host cell provided herein ⁇ e.g., a host cell comprising PK/PTA and a functional disruption of a polypeptide encoding acetyl phosphatase activity, e.g., RHR2, HOR2, or homologues thereof) is engineered for the expression of biosynthetic pathways that initiate with cellular pyruvate to produce, for example, 2,3-butanediol, 2-butanol, 2-butanone, valine, leucine, lactic acid, malate, isoamyl alcohol, and isobutanol, as described in U.S. Patent Application Publication No.
• PDC-KO recombinant host cells can be used to produce the products of pyruvate- utilizing biosynthetic pathways
• PDC-KO recombinant host cells require exogenous carbon substrate supplementation (e.g., ethanol or acetate) for their growth.
• two exogenous carbon substrates are needed: one of which is converted to a desired product, the other fully or partly converted into acetyl-CoA by recombinant host cells requiring such supplementation for growth.
• expression of a heterologous phosphoketolase pathway reduces or eliminates the need for providing these exogenous carbon substrates for their growth compared to PDC-KO cells not heterologously PK/PTA.
• the additional functional disruption of RHR2, HOR2, or homologues thereof capable of catalzying the hydrolysis of acetyl phosphate to acetate is expected to further improve the ability of PK/PTA to increase the supply of acetyl-CoA available as a substrate for cellular growth in these cells.
• Expression of a heterologous enzyme in a host cell can be accomplished by introducing into the host cells a nucleic acid comprising a nucleotide sequence encoding the enzyme under the control of regulatory elements that permit expression in the host cell.
• the nucleic acid is an
• the nucleic acid is a chromosomal integration vector that can integrate the nucleotide sequence into the chromosome of the host cell.
• Nucleic acids encoding these proteins can be introduced into the host cell by any method known to one of skill in the art without limitation (see, for example, Hinnen et al. (1978) Proc. Natl. Acad. Set USA 75: 1292-3; Cregg et al. (1985) Mol. Cell. Biol. 5:3376- 3385; Goeddel et al. eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc. , CA; Krieger, 1990, Gene Transfer and Expression— A Laboratory Manual, Stockton Press, NY; Sambrook et al. , 1989, Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al.
• Exemplary techniques include, but are not limited to, spheroplasting, electroporation, PEG 1000 mediated transformation, and lithium acetate or lithium chloride mediated transformation.
• the copy number of an enzyme in a host cell may be altered by modifying the transcription of the gene that encodes the enzyme. This can be achieved for example by modifying the copy number of the nucleotide sequence encoding the enzyme ⁇ e.g., by using a higher or lower copy number expression vector comprising the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the genome of the host cell or by deleting or disrupting the nucleotide sequence in the genome of the host cell), by changing the order of coding sequences on a polycistronic mRNA of an operon or breaking up an operon into individual genes each with its own control elements, or by increasing the strength of the promoter or operator to which the nucleotide sequence is operably linked.
• the copy number of an enzyme in a host cell may be altered by modifying the level of translation of an mRNA that encodes the enzyme. This can be achieved for example by modifying the stability of the mRNA, modifying the sequence of the ribosome binding site, modifying the distance or sequence between the ribosome binding site and the start codon of the enzyme coding sequence, modifying the entire intercistronic region located "upstream of or adjacent to the 5' side of the start codon of the enzyme coding region, stabilizing the 3 '-end of the mRNA transcript using hairpins and specialized sequences, modifying the codon usage of enzyme, altering expression of rare codon tRNAs used in the biosynthesis of the enzyme, and/or increasing the stability of the enzyme, as, for example, via mutation of its coding sequence.
• the activity of an enzyme in a host cell can be altered in a number of ways, including, but not limited to, expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme that lacks a domain through which the activity of the enzyme is inhibited, expressing a modified form of the enzyme that has a higher or lower Kcat or a lower or higher Km for the substrate, or expressing an altered form of the enzyme that is more or less affected by feedback or feed- forward regulation by another molecule in the pathway.
• a nucleic acid used to genetically modify a host cell comprises one or more selectable markers useful for the selection of transformed host cells and for placing selective pressure on the host cell to maintain the foreign DNA.
• the selectable marker is an antibiotic resistance marker.
• antibiotic resistance markers include, but are not limited to, the BLA, NAT1, PAT, AUR1-C, PDR4, SMR1, CAT, mouse dhfr, HPH, DSD A, KAN , and SHBLE gene products.
• the BLA gene product from E. coli confers resistance to beta-lactam antibiotics ⁇ e.g. , narrow-spectrum cephalosporins, cephamycins, and carbapenems
• Tu94 confers resistance to bialophos
• the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA)
• the PDR4 gene product confers resistance to cerulenin
• the SMR1 gene product confers resistance to sulfometuron methyl
• the CAT gene product from Tn9 transposon confers resistance to chloramphenicol
• the mouse dhfr gene product confers resistance to methotrexate
• the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B
• the DSDA gene product of E confers resistance to bialophos
• the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA)
• the PDR4 gene product confers resistance to cerulenin
• the SMR1 gene product confers resistance to sulfometuron methyl
• the CAT gene product from Tn9 transposon confer
• the antibiotic resistance marker is deleted after the genetically modified host cell disclosed herein is isolated.
• the selectable marker rescues an auxotrophy ⁇ e.g., a nutritional auxotrophy) in the genetically modified microorganism.
• a parent microorganism comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway and that when non-functional renders a parent cell incapable of growing in media without supplementation with one or more nutrients.
• gene products include, but are not limited to, the HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast.
• the auxotrophic phenotype can then be rescued by transforming the parent cell with an expression vector or
• chromosomal integration construct encoding a functional copy of the disrupted gene product, and the genetically modified host cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent cell.
• Utilization of the URA3, TRP1, and LYS2 genes as selectable markers has a marked advantage because both positive and negative selections are possible.
• Positive selection is carried out by auxotrophic complementation of the URA3, TRP1, and LYS2 mutations, whereas negative selection is based on specific inhibitors, i.e., 5- fluoro-orotic acid (FOA), 5-fluoroanthranilic acid, and aminoadipic acid (aAA), respectively, that prevent growth of the prototrophic strains but allows growth of the URA3, TRP1, and LYS2 mutants, respectively.
• the selectable marker rescues other non- lethal deficiencies or phenotypes that can be identified by a known selection method.
• genes and proteins useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary.
• changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutations and silent mutations.
• modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
• Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization” or "controlling for species codon bias.”
• Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant R A transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
• Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S.
• DNA molecules differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure.
• the native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
• a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
• the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
• the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
• homo logs of enzymes useful for the compositions and methods provided herein are encompassed by the disclosure.
• two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%), 96%o, 97%), 98%), or 99% identity.
• the sequences are aligned for optimal comparison purposes ⁇ e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%>, typically at least 40%>, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%), 100%) of the length of the reference sequence.
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
• amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W. R., 1994, Methods in Mol Biol 25: 365-89).
• the following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
• Sequence homology for polypeptides is typically measured using sequence analysis software.
• a typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences.
• any of the genes encoding the foregoing enzymes may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
• genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway.
• a variety of organisms could serve as sources for these enzymes, including, but not limited to,
• Saccharomyces spp. including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S.
• Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
• Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia, coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Cory neb acterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
• Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
• analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities.
• Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes.
• techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest.
• Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity ⁇ e.g. as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence.
• analogous genes and/or analogous enzymes or proteins techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC.
• the candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.
• Y967 and Y968 are wildtype prototrophic Saccharomyces cerevisiae
• CEN.PK2 Y967 is MatA
• Y968 is Matalpha.
• Y 12869 was generated through three successive integrations into Y003.
• the gene ACS2 was deleted by introducing an integration construct ( ⁇ 2235; SEQ ID NO:27) consisting of the native S. cerevisiae LEU2 gene, flanked by sequences consisting of upstream and downstream nucleotide sequences of the ACS2 locus.
• this construct can integrate by homologous recombination into the ACS2 locus of the genome, functionally disrupting ACS2 by replacing the ACS2 coding sequence with its integrating sequence.
• Transformants were plated onto CSM -leu plates containing 2% EtOH as the sole carbon source, and were confirmed by PCR amplification. The resulting strain was Y4940.
• ALD6 was deleted and Dickeya zeae eutE was introduced in Y4940 with the integration construct ( ⁇ 74804; SEQ ID NO:28) pictured below.
• This integration construct comprises a selectable marker (TRP1), as well as two copies a yeast-codon-optimized sequence encoding the gene eutE from Dickeya zeae (NCBI Reference Sequence: YP 003003316.1) under control of the TDH3 promoter (840 basepairs upstream of the native S. cerevisiae TDH3 coding region), and the TEF2 terminator (508 basepairs downstream of the native S. cerevisiae TEF2 coding region). These components are flanked by upstream and downstream nucleotide sequences of the ALD6 locus. Upon introduction into a host cell, this construct integrates by homologous
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982.
• the construct was transformed into Y4940, and transformants were selected on CSM-TRP plates with 2% glucose and confirmed by PCR amplification.
• the resulting strain was yl2602.
• Y 12747 was transformed with a PCR product amplified from the native
• URA3 sequence This sequence restores the ura3-52 mutation. See Rose and Winston, Mol Gen Genet 193:557-560 (1984). Transformants were plated onto CSM-ura plates containing 2% glucose as the sole carbon source, and were confirmed by PCR amplification. The resulting strain was Y12869.
• Y12745 was generated through three successive integrations into Y4940.
• Y4940 was transformed with the integration construct ( ⁇ 73830; SEQ ID NO:30)
• This integration construct comprises a selectable marker (URA3); a yeast codon-optimized version of phosphoketolase from Leuconostoc mes enter oides (NCBI Reference Sequence YP 819405.1) under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); a yeast codon-optimized version of Clostridium kluyveri phosphotransacetylase (NCBI Reference Sequence: YP 001394780.1) under control of the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and the PGKl terminator (259 bp downstream of the PGKl coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the S.
• UAA3 selectable marker
• YP 819405.1 under the TDH3 promoter (870 bp upstream of
• this construct Upon introduction into a host cell, this construct integrates by homologous recombination into the host cell genome, functionally disrupting BUD9 by replacing the BUD9 coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982. Transformants were selected on CSM-URA plates with 2% glucose. The resulting construct
• This construct comprising a selectable marker (TRPl); two copies of phosphoketolase from Leuconostoc mes enter oides under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the ALD6 locus.
• TRPl selectable marker
• two copies of phosphoketolase from Leuconostoc mes enter oides under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the ALD6 locus.
• TRPl selectable marker
• Transformants were selected on CSM-URA plates with 2% glucose and confirmed by PCR amplification.
• ACS 1 was deleted in by introducing an integration construct ( ⁇ 76220;
• SEQ ID NO:29 consisting of the upstream and downstream nucleotide sequences of ACS1, flanking the native S. cerevisiae HIS3 gene under its own promoter and terminator.
• Transformants were plated onto CSM -his plates containing 2% glucose as the sole carbon source, and were confirmed by PCR amplification.
• Y12746 was generated through three successive integrations into Y4940.
• Y4940 was transformed with the integration construct ( ⁇ 73830; SEQ ID NO:30) pictured below.
• This integration construct comprises a selectable marker (URA3); a yeast codon-optimized version of phosphoketolase from Leuconostoc mes enter oides (NCBI Reference Sequence YP 819405.1) under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); a yeast codon-optimized version of Clostridium kluyveri phosphotransacetylase (NCBI Reference Sequence: YP 001394780.1) under control of the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and the PGKl terminator (259 bp downstream of the PGKl coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the S.
• UAA3 selectable marker
• YP 819405.1 under the TDH3 promoter (870 bp upstream of
• this construct Upon introduction into a host cell, this construct integrates by homologous recombination into the host cell genome, functionally disrupting BUD9 by replacing the BUD9 coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982. Transformants were selected on CSM-URA plates with 2% glucose.
• This construct comprising a selectable marker (TRPl); two copies of phosphoketolase from Leuconostoc mes enter oides under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the ALD6 locus.
• TRPl selectable marker
• two copies of phosphoketolase from Leuconostoc mes enter oides under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the ALD6 locus.
• TRPl selectable marker
• Transformants were selected on CSM-URA plates with 2% glucose and confirmed by PCR amplification.
• This construct comprises a selectable marker (HIS3); as well as two copies a yeast-codon-optimized sequence encoding the gene eutE from Dickeya Zeae (NCBI).
• HIS3 selectable marker
• NCBI yeast-codon-optimized sequence encoding the gene eutE from Dickeya Zeae
• Y19390 is a direct descendant of Y12869.
• Y12869 was transformed with the integration construct MS49253 (SEQ ID NO:36) shown below:
• This integration construct comprises a selectable marker (URA3); two copies of a yeast codon-optimized version of phosphoketolase from Leuconostoc mes enter oides (NCBI Reference Sequence YP 819405.1) under the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and TDH3 terminator (259 bp downstream of the TDH3 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the S. cerevisiae BUD9 locus.
• UAA3 selectable marker
• this construct Upon introduction into a host cell, this construct integrates by homologous recombination into the host cell genome, functionally disrupting BUD9 by replacing the BUD9 coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982.
• Transformants were selected on CSM-URA plates with 2% glucose.
• Y19391 is a direct descendant of Y12869.
• This integration construct comprises a selectable marker (URA3); two copies of a yeast codon-optimized version of phosphotransacetylase from Clostridium kluyveri (NCBI Reference Sequence: YP 001394780.1) under control of the TDH3 promoter (870 bp upstream of the TDH3 coding sequence) and the PGK1 terminator (259 bp downstream of the PGK1 coding sequence); flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of the S. cerevisiae BUD9 locus.
• UAA3 selectable marker
• this construct Upon introduction into a host cell, this construct integrates by homologous recombination into the host cell genome, functionally disrupting BUD9 by replacing the BUD9 coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No.
• the precultures were then inoculated into a 125 ml flask carrying 25 ml of seed media with 50 mM succinate pH 5.0, and 40 g/L sucrose to an initial OD600 of 0.1, and grown at 30C and 200rpm.
• FIG. 3B shows that wildtype Cen.PK2, Y967, produces acetate during growth in batch defined sucrose shakeflask cultures.
• Y 12869 comprising a deletion of the PDH- bypass (acslA acs2 ⁇ ald6A) and heterologously expressing acetaldehyde dehydrogenase acylating (Dz.eutE), produces far less acetate than the wildtype control which uses the PDH- bypass, likely due to the deletion of ALD6, the cytosolic acetaldehyde dehydrogenase that converts acetaldehyde to acetate.
• strain Y 12746 comprising a deletion of the PDH- bypass (acsl acs2 A ald6A) and heterologously expressing acetaldehyde dehydrogenase acylating (Dz.eutE) as well as phosphoketolase (Lm.PK) and phosphotransacetylase
• phosphoketolase and phosphotransacetylase produces acetyl phosphate. Therefore, acetate accumulation may arise from spontaneous or catalyzed hydrolysis of acetyl phosphate in Y12746.
• Y 12746 we transformed a strain which uses only ADA to provide cytosolic AcCoA (Y 12869, comprising a deletion of the PDH-bypass (acslA acs2 A ald6A) and heterologously expressing acetaldehyde dehydrogenase acylating ⁇ Dz.eutE)) with either (1) an integration construct encoding two overexpressed copies of PK driven by the strong promoter P TDH3 , resulting in Y 19390, or (2) an integration construct encoding two overexpressed copies of PTA driven by the strong promoter P TDH3 , resulting in Y19391. As shown in FIG.
• This example describes the identification of an enzyme capable of hydro lyzing acetyl phosphate in yeast.
• the tubes were then placed back in the bead beater again for 1 minute at 6 M/S and returned to the ice bath for 5 minutes. Tubes were spun at a minimum of 16000 x g for 20 minutes to pellet cell debris. The supernatant was then transferred to a new cold tube. Protein concentration was measured using the classic Bradford assay for proteins (Bradford MM A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72, 248-254 (1976)).
• Acetyl phosphatase activity assays were carried out at 30 °C in reaction buffer consisting of 100 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM MgCl 2+ . Acetyl phosphate was added to a starting concentration of either 5 mM or 10 mM as indicated. The reaction was initiated by the addition of cell free extract in the amounts indicated. To test for phosphatase inhibition, sodium fluoride was added to select wells at 30 mM concentration. The reactions were carried out in a sealed 96 well plate and total reaction volume of 250 ⁇ . Acetylphosphate concentration was measured by the method developed by Lipmann and Tuttle (Lipmann F, Tuttle LC, J.
• Peptone media with 2% dextrose (YPD) as an overnight starter culture.
• YPD dextrose
• the flasks were incubated at 30 °C by shaking at 160 RPM for 24 hours.
• the culture was harvested by centrifugation at 4000x g for 5 minutes.
• the cell pellet was washed with 500mL sterile water and centrifuged at 4000x g for 5 minutes.
• the cell pellet was then resuspended in 50 mL ice cold lysis buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM DTT, 1 EDTA free protease inhibitor tablet (Roche) per lOmL).
• Cell suspension was split into six 15 mL falcon tubes filled with 5 mL disruption beads
• Tubes were then placed in a bead beater for 45 seconds at 6 M/S. The tubes were immediately placed in an ice water bath for at least 5 minutes. Bead beating was repeated 3 additional times with at least 5 minutes in an ice water bath in between each disruption segment. Tubes were spun for 30 minutes at 16,000 rpm (30,966 x g) in a Beckman centrifuge J-E in a JA-20 rotor chilled to 4 °C to pellet cell debris. Cell lysate was additionally clarified by the selective flocculation method described by Salt et al. (Selective flocculation of cellular contaminants from soluble proteins using
• polyethyleneimine A study of several organisms and polymer molecular weights. Enzyme and Microbial Technology 17, 107-113(1995)) as follows: cell free lysate was adjusted to pH 7.4 by addition of 5mM NaOH stock solution. Then equal volume of PEI/Borax solution (0.5M NaCl 0.25% PEI, lOOmM Borax) was added to the cell lysate and mixed well.
• Dialyzed sample was centrifuged 16000 x g for 10 minutes to pellet precipitated protein. Protein concentration was measured using the classic Bradford assay for proteins (Bradford MM, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72, 248- 254 (1976)). 20 mg protein was loaded onto a Source 15Q 4.6/100 PE anion exchange column on a GE AKTAexplorer FPLC. Protein was eluted with a 0-100% gradient of buffer B (20 mM Tris-Cl pH 7, 1M NaCl, 10% glycerol) over 30 column volumes at a flow rate of 0.5 mL/minute and 1 mL samples were collected.
• buffer B (20 mM Tris-Cl pH 7, 1M NaCl, 10% glycerol
• each fraction was added to 8 mM ACP in a 250 reaction containing lOOmM Tris-Cl pH 7, 150 mM NaCl, 1 mM MgC12 and assayed as described above.
• the active fraction from this separation was again dialyzed against buffer A overnight.
• the entire sample was then loaded onto the same a Source 15Q 4.6/100 PE anion exchange column and eluted with a gradient of 0-45% buffer B over 30 column volumes at a flow rate of 0.5 mL/minute and 1 mL samples were collected. Samples were assayed for activity as above.
• Protein fractions were analyzed using a Criterion gel electrophoresis system.
• the reaction was quenched with 0.1% formic acid and injected onto an Ascentis Peptide express column (5cmx2.1mm ID, 2.1 um particle size), and separated over a 90 minute gradient from low acetonitrile to high acetonitrile, with 0.1% formic acid as a modifier.
• the LC pumps were two Shimadzu LC20AD's operated by a Shimadzu CBM20A LC Controller.
• a QTPvAP 4000 hybrid triple-quadrupole linear ion tram mass spectrometer was used to identify peptides being eluted from the column.
• IDA parameters were as follows: Select ions from 350 to 1300 da; ER Scan used for charge state determination; 1+ ions rejected, unknowns allowed; Rolling collision energy: yes (AB SCIEX standard for qtrap 4000); Max fill time for each MS/MS: 950 ms.
• a version of Y968 lacking a functional URA3 gene was transformed with either ms59858 to knock out RHR2 or ms59971 to knock out HOR2.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982. Transformants were selected on CSM-URA plates with 2% glucose and confirmed by PCR amplification.
• the URA 3 marker in this construct is flanked by direct repeats, facilitating its recycling.
• To recycle the URA3 marker cells were grown in YPD overnight, then plated on 5'FOA. The loopout of URA3 was confirmed by PCR amplification and inability to grow on CSM-URA plates.
• the ura- version of Y968.ms59858 was then transformed with ms59971 to generate a double RHR2 and HOR2 knockout strain Y968.ms59858.ms59971
• FIG. 5A shows that nearly all of the phosphatase activity was concentrated in one fraction, and the remaining activity in adjacent fractions. This indicates that the enzyme responsible for this activity in the cell free extract is either a single protein or proteins with similar ionic interactions which co-elute when separated by anion exchange chromatography.
• the active fraction #10 from FPLC anion exchange purification was purified a second time using a more shallow gradient 0-45% buffer B.
• the most active fraction from this purification, # 14, shown in FIG. 6A was analyzed by mass spectrometry to determine the identity of the proteins in the fraction.
• Rhr2 and its homolog Hor2 which cannot be distinguished by mass spectrometry due to significant sequence similarity, were the only proteins on the list identified as phosphatases by the SGD database.
• Rhr2 is a glycerol- 1 -phosphatase that is expressed constitutively at high levels.
• Hor2 catalyzes the identical reaction but is expressed only at low levels under normal conditions and is induced by osmotic stress (Norbeck et. ah, Purification and Characterization of Two Isoenzymes of DL-Glycerol-3 -phosphatase from Saccharomyces cerevisiae, J. Biol. Chem., 271, 13875-13881 (1996)).
• Acetyl phosphate is not a metabolite that is native to yeast, therefore it is expected that the hydrolysis is caused by a promiscuous reaction of an enzyme that targets a similar substrate.
• Rhr2/Hor2 were top candidates for this reaction since their native substrate, glycerol- 1 -phosphate, is also a low molecular weight phosphorylated compound similar to acetyl phosphate, as shown below.
• This construct comprises nucleotide sequences that encode a selectable marker (URA3); a copy of the native yeast GAL4 transcription factor under its own promoter; two native yeast enzymes of the mevalonate pathway (ERG 10 which encodes Acetoacetyl-CoA thiolase, and ERG13, which encodes HMG-CoA synthase), as well as two copies of a yeast codon- optimized version of Silicibacter pomeroyi HMG-CoA reductase, all under galactose- inducible promoters (promoters of the S. cerevisiae genes GAL1 and GAL 10, flanked by homologous sequences consisting of upstream and downstream nucleotide sequences of the S.
• UAA3 selectable marker
• ERG13 which encodes HMG-CoA synthase
• the ms63907 construct integrates by homologous integration into the host cell genome, functionally disrupting HO by replacing the HO coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982. Transformants were selected on CSM-URA plates with 2% glucose and confirmed by PCR amplification.
• the URA3 marker in this construct is flanked by direct repeats, facilitating its recycling. To recycle the URA3 marker, cells were grown in YPD overnight, then plated on 5 TO A. The loopout of URA3 was confirmed by PCR amplification and inability to grow on CSM-URA plates.
• the ms63909 integration construct ( ⁇ 84026; SEQ ID NO:34) is identical to ms63907, with one exception: the sequences encoding S. pomeroyi HMG-CoA reductase are replaced by tHMGr, the truncated HMG1 coding sequence which encodes the native S. cerevisiae HMG-CoA reductase.
• This construct comprises nucleotide sequences that encode a selectable marker (URA3); five native yeast enzymes of the ergosterol pathway (ERG12 which encodes mevalonate kinase, ERG8 which encodes phosphomevalonate kinase, ERG19 which encodes mevalonate pyrophosphate decarboxylase, IDIl which encodes dimethylallyl diphosphate isomerase, and ERG20 which encodes farnesyl pyrophosphate synthetase), as well as an evolved, yeast codon-optimized version of Artemisia annua farnesene synthase, all under galactose- inducible promoters (Promoters of the S.
• UAA3 selectable marker
• ERG8 which encodes phosphomevalonate kinase
• ERG19 which encodes mevalonate pyrophosphate decarboxylase
• IDIl which encodes dimethylallyl diphosphate isomerase
• GAL1, GAL 10, and GAL7 cerevisiae genes GAL1, GAL 10, and GAL7). These sequences are flanked by homologous sequences consisting of the upstream and downstream nucleotide sequences of GAL80.
• the ms64472 construct integrates by homologous integration into the host cell genome, functionally disrupting GAL80 by replacing the GAL80 coding sequence with its integrating sequence.
• the construct was assembled using the methods described in U.S. Patent No. 8,221,982. Transformants were selected on CSM-URA plates with 2% glucose and confirmed by PCR amplification.
• the URA3 marker in this construct is flanked by direct repeats, facilitating its recycling. To recycle the URA3 marker, cells were grown in YPD overnight, then plated on 5 TO A. The loopout of URA3 was confirmed by PCR amplification and inability to grow on CSM-URA plates.
• ura- versions of Y968.ms63907.ms64472, Y12869.ms63907.ms64472, and Y12747.ms63907.ms64472 were transformed with ms59858 to knock out the RHR2 ORF.
• This integration construct consists of the upstream and downstream nucleotide sequences of RHR2, flanking the native S. cerevisiae URA3 gene under its own promoter and terminator. Transformants were plated onto CSM -his plates containing 2% glucose as the sole carbon source, and were confirmed by PCR amplification. 6.3.1.2 Culture conditions
• Acetate and glycerol were quantitated by transferring 1 ml of whole cell broth to a 1.5 ml eppendorf tubes, and spinning at 13,000 RPM for 1 minute using a tabletop centrifuge to clarify the supernatant. The supernatant was then diluted (1 : 1 v/v) in 8mM sulfuric acid, vortexed, and recentrifuged before transferring to a 1.8ml vial. Samples were analyzed with an Agilent 1200 HPLC, with variable wavelength and refractive index detection, using a BioRad Aminex HPX-87H 300mm x 7.8mm column. The mobile phase was 4mM sulfuric acid, column temperature was 40C, and the flow rate was 0.5 ml/min.
• FIG. 8 A shows that strain Yl 2746. ms63909. ms64472, comprising a deletion of the PDH-bypass (acslA acs2 ⁇ ald6A), heterologously expressing acetaldehyde dehydrogenase aceylating (Dz.eutE) as well as phosphoketolase (Lm.PK) and
• Rhr2 which was responsible for the acetyl phosphate phosphatase activity in cell free extract, is also the primary cause behind the hydrolysis of acetyl phosphate to acetate in vivo.
• FIG. 8D shows that strain Y 12746, comprising a deletion of the PDH-bypass (acslA acs2 ⁇ ald6A), heterologously expressing acetaldehyde
• phosphotransacetylase (Ck.PTA), secretes more acetate than a version of Y 12746 in which the RHR2 gene has been deleted.
• Ck.PTA phosphotransacetylase
• FIG. 8E the substantially reduced levels of acetate in Yl 2746. ms63909. ms64472 rhr2 A are not due to reduced cell growth, as cell densities are similar for both RHR2+ and rhr2 A populations.
• FIG. 9 shows that the deletion of rhr2 improves farnesene production in
• Y12746.ms63907.ms64472 by 2.1-fold, and in Y12745.ms63907.ms64472 by 1.4-fold (In each strain background, the RHR2+ parent is normalized to 1). Moreover, deletion of rhr2 improves the final optical density of Y12746.ms63907.ms64472 at carbon exhaustion. Both Y12745.ms63907.64472 and Y12746.ms63907.ms64472 use phosphoketolase and phosphotransacetylase, and thus acetyl phosphate as a pathway intermediate, to produce cytosolic acetyl-CoA, which is used for synthesis of farnesene. Strains
• Y968.ms63907.ms64472 and Y12869.ms63907.ms64472 do not express phosphoketolase or phosphotransacetylase, and do not use acetyl phosphate as a pathway intermediate. Deletion of rhr2 in these strain backgrounds has no effect on farnesene production or optical density in either strain background. This indicates that the benefit of knocking out rhr2 specifically applies to strains which use acetyl phosphate as an intermediate metabolite, e.g., strains comprising heterologous PK and/or PTA.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)
• Mycology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Publications (2)

Family

ID=50391556

Family Applications (1)

Country Status (13)

Cited By (14)

Families Citing this family (37)

Citations (34)

Family Cites Families (5)

• 2014
  • 2014-03-14 MY MYPI2015703178A patent/MY171958A/en unknown
  • 2014-03-14 WO PCT/US2014/028421 patent/WO2014144135A2/en not_active Ceased
  • 2014-03-14 KR KR1020157028637A patent/KR102159691B1/ko active Active
  • 2014-03-14 CN CN201480025977.7A patent/CN105189772B/zh active Active
  • 2014-03-14 ES ES14714149T patent/ES2721920T3/es active Active
  • 2014-03-14 BR BR112015023089A patent/BR112015023089A2/pt not_active Application Discontinuation
  • 2014-03-14 US US14/214,062 patent/US9410214B2/en active Active
  • 2014-03-14 MX MX2015012365A patent/MX364748B/es active IP Right Grant
  • 2014-03-14 AU AU2014227811A patent/AU2014227811C1/en active Active
  • 2014-03-14 CA CA2903053A patent/CA2903053C/en active Active
  • 2014-03-14 EP EP14714149.3A patent/EP2971027B1/en active Active
  • 2014-03-14 JP JP2016502782A patent/JP6595449B2/ja active Active
• 2015
  • 2015-09-01 ZA ZA2015/06406A patent/ZA201506406B/en unknown
• 2019
  • 2019-09-26 JP JP2019175333A patent/JP2019213558A/ja active Pending

Patent Citations (37)

Non-Patent Citations (104)

Also Published As

Similar Documents

Legal Events