WO2015031504A1 - Voie et organismes recombinés pour la synthèse de la malonyl-coenzyme a - Google Patents

Voie et organismes recombinés pour la synthèse de la malonyl-coenzyme a Download PDF

Info

Publication number
WO2015031504A1
WO2015031504A1 PCT/US2014/052960 US2014052960W WO2015031504A1 WO 2015031504 A1 WO2015031504 A1 WO 2015031504A1 US 2014052960 W US2014052960 W US 2014052960W WO 2015031504 A1 WO2015031504 A1 WO 2015031504A1
Authority
WO
WIPO (PCT)
Prior art keywords
coa
malonyl
converting
recombinant microorganism
aspartate
Prior art date
Application number
PCT/US2014/052960
Other languages
English (en)
Inventor
James C. Liao
Ethan I. LAN
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2015031504A1 publication Critical patent/WO2015031504A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01075—Malonyl CoA reductase (malonate semialdehyde-forming)(1.2.1.75)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01001—Aspartate transaminase (2.6.1.1), i.e. aspartate-aminotransferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01002—Alanine transaminase (2.6.1.2), i.e. alanine-aminotransferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01011—Aspartate 1-decarboxylase (4.1.1.11)

Definitions

  • the disclosure provides both cell-free systems and recombinant microorganism for the production of malonyl-CoA.
  • the disclosure provides a recombinant microorganism that produces malonyl-CoA at levels greater than a parental organism comprising a pathway selected from the group consisting of: (a) phosphoenolpyruvate to malonate semialdehyde ; and (b) pyruvate to malonate semialdehyde; wherein the pathway comprises a malonyl-CoA reductase.
  • the recombinant microorganism is engineered to express an acetyl-CoA carboxylase and a malonyl-CoA reductase.
  • the recombinant microorganism is engineered to expresses or overexpresses one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to Glyceraldehyde-3P; (ii) converting
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to oxaloacetate ; (ii) converting oxaloacetate to malonate semialdehyde; and (iii) converting malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to oxaloacetate ; (ii) converting oxaloacetate to aspartate, (iii) converting aspartate to beta-alanine ; (iv) converting beta-alanine to malonate semialdehyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • a metabolic function selected from the group consisting of (i) converting PEP to oxaloacetate ; (ii) converting oxaloacetate to aspartate, (iii) converting aspartate to beta-alanine ; (iv) converting beta-alanine to malonate semialdehyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) optionally converting PEP to pyruvate; (ii) converting pyruvate to alanine; (iii) converting alanine to bet-alanine; (iv) converting beta-alanine to malonate semialdhyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • a metabolic function selected from the group consisting of (i) optionally converting PEP to pyruvate; (ii) converting pyruvate to alanine; (iii) converting alanine to bet-alanine; (iv) converting beta-alanine to malonate semialdhyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to pyruvate; (ii) converting pyruvate to lactate; (iii) converting lactate to lactoyl-CoA; (iv) converting lactoyl-CoA to acrylyl-CoA; (v) converting acrylyl-CoA to 3-hydroxyproprionyl-CoA; (vi) 3-hdroxypropionyl-CoA to 3- hydroxyproprionate ; (vii) converting 3-hydroxyproprionate to malonate semialdehyde; and (viii) converting malonate semialdehyde to malonyl-coA.
  • the recombinant that carries out a metabolic function selected from the group consisting of (i) converting PEP to pyruvate; (ii) converting pyruvate to lactate; (
  • microorganism is engineered to express or overexpress one or more enzyme selected from the group consisting of trios phosphate isomerase, glycerol-3-phosphate dehydrogenase, glycerol-3- phosphatase, glycerol dehydratase, glycerol dehydratase reactivase, aldehyde dehydrogenase, malonate semialdehyde dehydrogenase and malonyl-CoA reductase.
  • one or more enzyme selected from the group consisting of trios phosphate isomerase, glycerol-3-phosphate dehydrogenase, glycerol-3- phosphatase, glycerol dehydratase, glycerol dehydratase reactivase, aldehyde dehydrogenase, malonate semialdehyde dehydrogenase and malonyl-CoA reductase.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes selected from the group consisting of a phosphoenolpyruvate carboxylase, a oxaloacetate 1-decarboxylase and malonyl-CoA
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes selected from the group consisting of a phosphoenolpyruvate carboxylase, an aspartate aminotransferase, an aspartate 1- decarboxylase or a PLP-dependent aspartate 1-decarboxylase, a beta- alanine aminotransferase and a malonyl-CoA reductase.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes selected from the group consisting of a pyruvate kinase, an alanine aminotransferase, an alanine aminomutase and a malonyl-CoA reductase.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes selected from the group consisting of a pyruvate kinase, an alanine aminotransferase, an alanine aminomutase and a malonyl-CoA reductase.
  • the recombinant microorganism is engineered to express or overexpress one or more enzymes selected from the group
  • a lactoyl-CoA consisting of a pyruvate kinase, a lactate dehydrogenase, a lactoyl- CoA transferase, a propionyl-CoA synthase, a lactoyl-CoA
  • the recombinant microorganism comprises an enzyme or homologs thereof selected from the group consisting of Tpi, GpsA, GPP, DhaB123/GdrAB, PuuC, Msr and Mcr.
  • the recombinant microorganism comprises enzyme or homologs thereof selected from the group consisting of Ppc, Oad and Mcr.
  • the recombinant microorganism comprises enzyme or homologs thereof selected from the group consisting of Ppc, AspC, PanD or AeADC, SkPYD4 and Mcr. In yet another embodiment, the recombinant
  • microorganism comprises enzyme or homologs thereof selected from the group consisting of Pyk, Aat, Aam and Mcr.
  • enzyme or homologs thereof selected from the group consisting of Pyk, Aat, Aam and Mcr.
  • the recombinant microorganism comprises enzyme or homologs thereof selected from the group consisting of Pyk, Ldh, Pet (and Pes), Led, Hpd, Hph, Msr and Mcr.
  • the aspartate amino transferase can comprise a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:2 and having aspartate aminotransferase activity
  • the aspartate 1-decarboxylase can comprise a sequences having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 4 and having aspartate 1-decarboxylase activity
  • the PLP-dependent aspartate 1-decarboxylase can comprise a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 6 and having PLP-dependent aspartate 1-decarboxylase;
  • beta-alanine aminotransferase can comprise a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 8 and having beta-alanine aminotransferase.
  • the malonyl-CoA reductase can comprise a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:10 and having malonyl-CoA reductase.
  • the disclosure also provides an in vitro metabolic pathway for producing malonyl-CoA comprising the enzymes trios phosphate isomerase, glycerol-3-phosphate dehydrogenase, glycerol-3- phosphatase, glycerol dehydratase, glycerol dehydratase reactivase, aldehyde dehydrogenase, malonate semialdehyde dehydrogenase and malonyl-CoA reductase.
  • the in vitro metabolic pathway comprises an enzyme or homologs thereof selected from the group consisting of Tpi, GpsA, GPP, DhaB123/GdrAB, PuuC, Msr and Mcr .
  • the disclosure also provides an in vitro metabolic pathway for producing malonyl-CoA comprising the enzymes
  • the in vitro metabolic pathway comprises enzyme or homologs thereof selected from the group consisting of Ppc, Oad and Mcr.
  • the disclosure also provides an in vitro metabolic pathway for producing malonyl-CoA comprising the enzymes
  • the in vitro metabolic pathway comprises enzyme or homologs thereof selected from the group consisting of Ppc, AspC, PanD or AeADC, SkPYD4 and Mcr.
  • the disclosure also provides an in vitro metabolic pathway for producing malonyl-CoA comprising the enzymes pyruvate kinase, alanine aminotransferase, alanine aminomutase and malonyl- CoA reductase.
  • the in vitro metabolic pathway comprises an enzyme or homologs thereof selected from the group consisting of Pyk, Aat, Aam and Mcr.
  • the disclosure provides an in vitro metabolic pathway for producing malonyl-CoA comprising the enzymes pyruvate kinase, lactate dehydrogenase, lactoyl-CoA transferase, propionyl-CoA synthase, lactoyl-CoA dehydratase, hydroxypropionyl-CoA dehydratase, hydroxypropionyl-CoA hydrolase, malonate semialdehyde dehydrogenase and malonyl-CoA reductase.
  • the in vitro metabolic pathway comprises an enzyme or homologs thereof selected from the group consisting of Pyk, Ldh, Pet (and Pes) , Led, Hpd, Hph, Msr and Mcr .
  • the disclosure also provides a method for producing a chemical or malonyl-CoA, the method comprising: (a) providing a recombinant microorganism as described herein; (b) culturing the microorganism ( s ) of (a) in the presence of a carbon substrate under conditions suitable for the conversion of the substrate to the chemical or malonyl-CoA; and (c) purifying the chemical or malonyl- CoA.
  • the disclosure also provides a method for producing a chemical or malonyl-CoA, the method comprising: (a) providing an in vitro metabolic pathway as described herein; (b) incubating the enzymes of the in vitro metabolic pathway in (a) in the presence of a carbon substrate under conditions suitable for the conversion of the substrate to the chemical or malonyl-CoA; and (c) purifying the chemical or malonyl-CoA.
  • the disclosure provides a recombinant microorganism that produces malonyl-CoA at levels greater than a parental organism comprising a pathway selected from the group consisting of: (a) phosphoenolpyruvate to malonate semialdehyde ; and (b) pyruvate to malonate semialdehyde; wherein the pathway comprises a malonyl-CoA reductase.
  • the organism comprises expression or elevated expression of an enzyme that converts (i) PEP to
  • Glyceraldehyde-3P (ii) Glyceraldehyde-3P to DHAP; (iii) DHAP to glycerol-3P; (iv) glycerol-3P to glycerol; (v) glycerol to 3-HPA;
  • the organism comprises expression or elevated expression of an enzyme that converts (i) PEP to oxaloacetate ; (ii) oxaloacetate to malonate semialdehyde; and (iii) malonate semialdehyde to malonyl-coA.
  • the organism comprises expression or elevated expression of an enzyme that converts (i) PEP to oxaloacetate; (ii) oxaloacetate to aspartate, (iii) aspartate to beta-alanine ; (iv) beta-alanine to malonate semialdehyde; and (v) malonate semialdehyde to malonyl-coA.
  • the organism comprises expression or elevated expression of an enzyme that converts (i) PEP to pyruvate; (ii) pyruvate to alanine; (iii) alanine to bet-alanine; (iv) beta-alanine to malonate semialdehyde; and (v) malonate semialdehyde to malonyl-coA.
  • an enzyme that converts (i) PEP to pyruvate; (ii) pyruvate to alanine; (iii) alanine to bet-alanine; (iv) beta-alanine to malonate semialdehyde; and (v) malonate semialdehyde to malonyl-coA.
  • the organism comprises expression or elevated expression of an enzyme that converts (i) PEP to pyruvate; (ii) pyruvate to lactate; (iii) lactate to lactoyl-CoA; (iv) lactoyl-CoA to acrylyl- CoA; (v) acrylyl-CoA to 3-hydroxyproprionyl-CoA; (vi) 3- hdroxypropionyl-CoA to 3-hydroxyproprionate ; (vii) 3- hydroxyproprionate to malonate semialdehyde; and (viii) malonate semialdehyde to malonyl-coA.
  • an enzyme that converts (i) PEP to pyruvate; (ii) pyruvate to lactate; (iii) lactate to lactoyl-CoA; (iv) lactoyl-CoA to acrylyl- CoA; (v) acrylyl-CoA to 3-hydroxyproprionyl-CoA; (vi)
  • the microorganism comprises an enzyme or homologs thereof selected from the group consisting of Tpi, GpsA, GPP, DhaB123/GdrAB, PuuC, Msr and Mcr. In one embodiment, the microorganism comprises an or homologs thereof selected from the group consisting of Ppc, Oad and Mcr. In one embodiment, the microorganism comprises an enzyme or homologs thereof selected from the group consisting of Ppc, AspC, PanD or AeADC, SkPYD4 and Mcr. In one embodiment, the microorganism comprises an enzyme or homologs thereof selected from the group consisting of Pyk, Aat, Aam and Mcr.
  • the microorganism comprises an enzyme or homologs thereof selected from the group consisting of Pyk, Ldh, Pet (and Pes) , Led, Hpd, Hph, Msr and Mcr.
  • the microorganism is engineered to express or overexpress (a) an aspartate aminotransferase (EC 2.6.1.1); (b) an aspartate 1-decarboxylase or a PLP-dependent aspartate 1-decarboxylase (EC 4.1.1.11); (c) a beta-alanine aminotransferase (EC 2.6.1.19); and (d) a malonyl-CoA reductase (Mcr, EC 1.2.1.75) .
  • the aspartate amino transferase comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 2 and having aspartate
  • the aspartate 1-decarboxylase comprises a sequences having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 4 and having aspartate 1-decarboxylase activity;
  • the PLP-dependent aspartate 1-decarboxylase comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 6 and having PLP-dependent aspartate 1- decarboxylase ;
  • the beta-alanine aminotransferase comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 8 and having beta-alanine aminotransferase.
  • the malonyl-CoA reductase comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 10 and having malonyl-CoA reductase.
  • the disclosure also provides a method for producing a chemical or malonyl-CoA, the method comprising (a) providing a recombinant microorganism as described above; (b) culturing the microorganism ( s ) of (a) in the presence of a carbon substrate under conditions suitable for the conversion of the substrate to the chemical or malonyl-CoA; and (c) purifying the chemical or malonyl- CoA.
  • Figure 1A-E is a schematics malonyl-CoA biosynthesis.
  • Natural malonyl-CoA biosynthesis via acetyl-CoA Natural malonyl-CoA biosynthesis via acetyl-CoA.
  • Synthetic malonyl- CoA biosynthesis pathway via malonate semialdehyde which can be produced by B) sequential carboxylation and decarboxylation of PEP, C) functionalization of a-keto to a-amino followed by a-amino transfer and regeneration of keto moiety, D) reduction of pyruvate to lactate in which net hydroxyl transfer can be achieved by sequential dehydration and hydration followed by re-oxidation of ⁇ - hydroxyl to ⁇ -keto, E) hydration of PEP to 2-phosphoglycerate and reduction to glycerol followed by dehydration of hydroxyl on carbon 2 of glycerol to 3-hydroxypropionaldehyde which can be oxidized to malonate semialdehyde.
  • Figure 4 shows a further detailed schematic of the isomerization via amino transfer for production of malonyl-CoA.
  • Figure 5 shows a further detailed schematic of the isomerization via net hydroxyl transfer (dehydration first followed by hydration) in the production of malonyl-CoA.
  • FIG. 6 shows a further detailed schematic of
  • Figure 7 shows precursors to malonate semialdehyde .
  • Figure 8 shows the thermodynamics of malonyl-CoA
  • Figure 9 shows the aspartate shunt to bypass oxaloacetate
  • FIG. 10A-F shows biosynthesis of malonyl-CoA from ⁇ - alanine .
  • Figure 11A-E shows one pot biosynthesis of malonyl-CoA from oxaloacetate.
  • Reaction mixture contained at 1 mM of NADP+, 1 mM of CoA, 20 mM oxaloacetate, 15 mM a-ketoglutarate, 15 mM glutamate, 100 ⁇ of PLP.
  • FIG. 12A-D shows titration of individual enzymes in malonyl-CoA biosynthesis with PLP-dependent aspartate decarboxylase. Titration of individual enzymes: B) ADC, C) AspC, D) SkPYD4, and E) Mcr. Mcr. Reaction mixture contained at 1 mM of NADP+, 1 mM of CoA, 20 mM oxaloacetate , 15 mM a-ketoglutarate, 15 mM glutamate, and 100 ⁇ of PLP. Except the enzyme being titrated, concentrations of other enzymes were kept constant at 1 ⁇ .
  • Figure 13 shows a pathway schematic useful for
  • Tpi triose phosphate isomerase
  • GpsA glycerol-3-phosphate dehydrogenase
  • GPP glycerols-phosphatase
  • DhaB123 glycerol dehydratase
  • GdrAB glycerol dehydratase reactivase
  • PuuC aldehyde dehydrogenase
  • Msr malonate semialdehyde dehydrogenase
  • Ppc phosphoenolpyruvate carboxylase
  • AspC aspartate aminotransferase
  • PanD aspartate 1-decarboxylase
  • AeADC PLP-dependent aspartate 1-decarboxylase
  • SkPYD4 ⁇ -alanine aminotransferase
  • Oad oxaloacetate 1-decarboxylase
  • AccDA acetyl-CoA carboxyltransferase
  • AccC biotin carboxylase
  • Ldh lactate dehydrogenase
  • Pet lactoyl-CoA
  • Hpd hydroxypropionyl-CoA dehydratase
  • Hph hydroxypropionyl-CoA dehydratase
  • Malonyl-CoA is an essential metabolite that serves as the common building block for fatty acids, polyketides, and flavonoids .
  • Malonyl-CoA is synthesized via carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (Acc) (Fig. 1A) , which typically is a four subunit enzyme complex or a muti-domain enzyme.
  • Biotin-carboxyl carrier protein (BCCP) domain is first biotinylated into active carboxyl-carrier .
  • Biotin carboxylase then carboxylates BCCP-biotin complex while consuming ATP.
  • Acc-dependent malonyl-CoA synthesis has also been used in an engineered pathway for acetoacetyl-CoA synthesis that leads to n-butanol production. Because Acc catalyzes the first step for directing acetyl-CoA flux towards secondary metabolite biosynthesis instead of amino acid biosynthesis or energy production, Acc expression is tightly regulated at all levels including: transcriptional
  • the disclosure provides methods and compositions for the production of malonyl-CoA using a culture of microorganisms that utilizes oxaloacetate, malonate semialdehyde or malonate
  • the disclosure can used photoautotrophic organisms, photoheterotrophic organisms or combinations thereof.
  • the recombinant organism can be any organism that produces pyruvate or phosphoenolpyruvate (PEP) as an intermediate .
  • malonate, oxaloacetate, malonate semialdehyde-CoA, and malonate semialdehyde are the possible direct precursors to malonyl-CoA (Fig. 7) .
  • malonate semialdehyde is more biologically feasible.
  • Malonate semialdehyde is a naturally occurring metabolite found in 3- hydroxypropionate dependent CO2 fixation pathway, indicating its intracellular stability.
  • PEP was chosen as a starting reference point for thermodynamics analysis because it is the common intermediate for all synthetic pathways for malonyl-CoA biosynthesis.
  • thermodynamics of these pathways were calculated based on
  • Group 1 pathways has an ATP yield of zero from either PEP or glucose and include Natural Acc dependent pathway (Fig. 1A) , oxaloacetate (Fig. IB), and aspartate (Fig. 9) dependent pathways.
  • Group 2 pathway has ATP yield greater than zero and contains only
  • Fig. 1C alanine pathway
  • Group 3 pathways have ATP yield less than one, indicating extra energy cost, and include both the lactate (Fig. ID) and glycerol (Fig. IE) pathways.
  • the disclosure shows the in vitro conversion of oxaloacetate to malonyl-CoA in four enzymatic steps.
  • the rate of malonyl-CoA biosynthesis demonstrated is comparable to that of in vitro fatty acid biosynthesis, suggesting compatibility of this synthetic pathway for biofuel production.
  • ⁇ -carboxylate of malonyl-CoA can also be achieved by CoA- acylating oxidation of malonate semialdehyde , a naturally occurring metabolite .
  • the disclosure describes at least four general alternatives (Fig. IB, C, D, E and Fig. 2, 3, 4, 5 and 6) and other variations thereof (Fig. 13 for complete metabolic interconnections between the pathways) to achieve biosynthesis of malonyl-CoA via malonate semialdehyde, a structural isomer of pyruvate.
  • the four general alternatives are: sequential
  • Mcr malonyl-CoA reductase
  • the recombinant microorganism [ 0048 ] In one embodiment, the recombinant microorganism
  • a pathway that includes at least one recombinant enzyme that converts phosphoenolpyruvate (PEP) to malonyl-CoA, wherein the pathway includes converting: (i) PEP to Glyceraldehyde-3P; (ii) Glyceraldehyde-3P to dihydroxyacetone phosphate (DHAP) ; (iii) DHAP to glycerol-3P; (iv) glycerol-3P to glycerol; (v) glycerol to 3- hydroxypropionaldehyde (3-HPA) ; (vi) 3HPA to 3-hydoxypropionate
  • PEP phosphoenolpyruvate
  • DHAP dihydroxyacetone phosphate
  • DHAP dihydroxyacetone phosphate
  • DHAP dihydroxyacetone phosphate
  • DHAP dihydroxyacetone phosphate
  • DHAP dihydroxyacetone phosphate
  • DHAP dihydroxyacetone phosphate
  • the recombinant microorganism comprises a pathway having enzyme or homologs thereof selected from the group consisting of a triose phosphate isomerase (e.g., Tpi) , a glycerol-3-phosphate dehydrogenase (e.g., GpsA) , a glycerol-3- phosphatase (e.g., GPP), aglycerol dehydratase (e.g., DhaB123) /a glycerol dehydratase reactivase (e.g., GdrAB) , an aldehyde
  • a triose phosphate isomerase e.g., Tpi
  • GpsA glycerol-3-phosphate dehydrogenase
  • GPP glycerol-3- phosphatase
  • aglycerol dehydratase e.g., DhaB123
  • dehydrogenase e.g., PuuC
  • PuuC a malonate semialdehyde dehydrogenase
  • At least one of the foregoing enzymes or homologs thereof is recombinantly engineered into the microorganism to produce malonyl-coA from PEP.
  • This pathway uses gluconeogenesis .
  • PEP is first hydrated at ⁇ -position to 2-phosphoglycerate .
  • glycerol is synthesized by reduction of dihydroxyacetone-phosphate using glycerol-3-phosphate dehydrogenase followed by
  • Gdh glycerol dehydratase
  • Klebsiella Gdh is composed of three subunits and utilizes coenzyme B12 as a prosthetic group. However, Klebsiella Gdh is irreversibly inactivated upon reaction with glycerol. A reactivating factor is necessary for subsequent glycerol dehydration.
  • Glycerol dehydratase reactivating factor utilizes energy from ATP hydrolysis to reactivate Gdh, increasing additional ATP cost and contributing to the net ATP deficiency of this synthetic pathway.
  • Clostridium Gdh on the other hand does not require Coenzyme B12 for catalysis. Instead, Clostridium Gdh requires an activation enzyme to introduce a glycyl radical required for catalysis. Similar to pyruvate : formate lyase, Clostridium Gdh is oxygen sensitive, prohibiting its function in some aerobic
  • the disclosure can use either a combination of the glycerol dehydratase reactivating factor (GdrAB) and glycerol dehydratase (DhaB123) or an oxygen sensitive Gdh.
  • GdrAB glycerol dehydratase reactivating factor
  • DhaB123 glycerol dehydratase
  • Gdh oxygen sensitive Gdh
  • the recombinant microorganism comprises a pathway that includes at least one recombinant enzyme that converts PEP to malonyl-CoA, wherein the pathway includes the steps: (i) PEP to oxaloacetate ; (ii) oxaloacetate to malonate semialdehyde ; and (iii) malonate semialdehyde to malonyl-coA (see, Figure IB) .
  • the recombinant microorganism comprises a pathway having enzyme or homologs thereof selected from the group consisting of a phosphoenolpyruvate carboxylase (e.g., Ppc, or homolog thereof), an oxaloacetate 1-decarboxylase (e.g., Oad, or homolog thereof) and a malonyl-CoA reductase (e.g., Mcr or homolog thereof) .
  • a phosphoenolpyruvate carboxylase e.g., Ppc, or homolog thereof
  • an oxaloacetate 1-decarboxylase e.g., Oad, or homolog thereof
  • a malonyl-CoA reductase e.g., Mcr or homolog thereof
  • a-decarboxylation occurs naturally in many biological processes such as pyruvate
  • the recombinant microorganism includes an aspartate shunt, wherein in this embodiment an
  • oxaloacetate 1-decarboxylase e.g., Oad, or homolog thereof
  • the recombinant microorganism comprises an aspartate shunt
  • the recombinant microorganism comprises a pathway that includes at least one recombinant enzyme that converts PEP to malonyl-CoA, wherein the pathway includes the steps: (i) PEP to oxaloacetate; (ii)
  • oxaloacetate to aspartate (iii) aspartate to beta-alanine ; (iv) beta-alanine to malonate semialdehyde ; and (v) malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism comprises a pathway having enzyme or homologs thereof selected from the group consisting of a phosphoenolpyruvase (e.g., Ppc or homolog thereof), an aspartate aminotransferase (e.g., AspC, or homolog thereof), an aspartate 1-decarboxylase (e.g., PanD, or homolog thereof) and/or a PLP-dependent aspartate 1-decarboxylase (e.g., AeADC, or homolog thereof), a beta-alanine aminotransferase (e.g., SkPYD4, or homolog thereof) and a malonyl-CoA reductase (e.g., Mcr, or homolog thereof) .
  • at least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP
  • the disclosure uses the decarboxylation of aspartate which is readily available in nature.
  • the disclosure describes a shunt utilizing aspartate decarboxylase.
  • the limiting step, cofactor independent aspartate decarboxylase was further solved by recruiting a PLP-dependent aspartate decarboxylase.
  • PLP-dependent aspartate decarboxylase was further solved by recruiting a PLP-dependent aspartate decarboxylase.
  • the steady state kinetics of this synthetic pathway and effects of substrate concentration were analyzed.
  • a-keto group of oxaloacetate is transaminated into amino group to facilitate the a-decarboxylation into ⁇ -alanine . Subsequent transamination returns keto functionality and forms malonate semialdehyde .
  • the disclosure demonstrates that conversion of oxaloacetate into malonate semialdehyde. This synthetic pathway has the same ATP cost as the natural Acc dependent pathway.
  • oxaloacetate is transaminated into aspartate by aspartate aminotransferase (AspC) using glutamate as the amino donor.
  • Aspartate then undergoes a- decarboxylation using aspartate 1-decarboxylase into ⁇ -alanine, a metabolite in panthothenate biosynthesis.
  • ⁇ -alanine is formed, amino-group can then be transaminated back into keto functionality using a-ketoglutarate as the amino receptor to produce malonate semialdehyde.
  • transamination of ⁇ -alanine is catalyzed by a ⁇ -Alanine aminotransferase (SkPYD4) found in
  • PanD Aspartate a-decarboxylase
  • PanD is an unusual enzyme using pyruvate as a prosthetic group. PanD is translated as an inactive proenzyme, which after self-proteolysis forms two functional subunits and the pyruvyl-group . Decarboxylation of aspartate provides an irreversible trap for carbon flux, which serves as a driving force for this synthetic malonyl-CoA pathway.
  • the recombinant microorganism comprises a pathway that includes at least one recombinant enzyme that converts PEP (or pyruvate) to malonyl-CoA, wherein the pathway includes the steps: (ia) PEP to pyruvate and/or (ib) pyruvate to alanine; (ii) alanine to bet-alanine ; (iii) beta-alanine to malonate semialdhyde ; and (iv) malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism comprises a pathway having enzyme or homologs thereof selected from the group consisting of an optional pyruvate kinase (e.g., Pyk, or homolog thereof), an alanine aminotransferase (e.g., Aat, or homolog thereof), an alanine aminomutase (e.g., Aam, or homolog thereof) and a malonyl-CoA reductase (e.g., Mcr, or homolog thereof) .
  • at least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • Pyruvate is first transaminated into alanine, effectively converting ⁇ -keto to a-amino which is converted into ⁇ -amino by an alanine aminomutase. Subsequent transamination of ⁇ -alanine returns keto group by using either pyruvate or a-ketoglutarate as an amino receptor. This synthetic pathway has a net positive ATP yield.
  • this synthetic pathway uses alanine aminomutase.
  • Enzymatic functional relatives of alanine aminomutase such as lysine 2,3- aminomutase and glutamate 2
  • 3-aminomutase contains iron sulfur clusters for generating radical SAM, which facilitates amino transfer .
  • the recombinant microorganism comprises a pathway that includes at least one recombinant enzyme that converts PEP (or pyruvate) to malonyl-CoA, wherein the pathway includes the steps: (ia) PEP to pyruvate and/or (ib) pyruvate to lactate; (ii) lactate to lactoyl-CoA; (iii) lactoyl-CoA to acrylyl- CoA; (iv) acrylyl-CoA to 3-hydroxyproprionyl-CoA; (v) 3- hdroxypropionyl-CoA to 3-hydroxyproprionate ; (vi) 3- hydroxyproprionate to malonate semialdehyde; and (vii) malonate semialdehyde to malonyl-coA.
  • the recombinant microorganism comprises a pathway having enzyme or homologs thereof selected from the group consisting of an optional pyruvate kinase (e.g., Pyk, or homolog thereof), a lactate dehydrogenase (e.g., Ldh, or homolog thereof), a lactoyl-CoA transferase (e.g., Pet, or homolog thereof), a propionyl-CoA synthase (e.g., Pes, or homolog thereof), a lactoyl- CoA dehydratase (e.g., Led, or homolog thereof), a hydroxypropionyl- CoA dehydratase (e.g., Hpd, or homolog thereof), a hydroxypropionyl- CoA hydrolase (e.g., Hph, or homolog thereof), a malonate
  • an optional pyruvate kinase e.g., Pyk, or homolog thereof
  • At least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • lactate in which its a-hydroxyl can be removed by dehydration, resulting in formation of acrylate (Figs. ID and 5) .
  • Hydration at ⁇ -position of acrylate followed by oxidation returns keto functionality at the ⁇ - position, forming malonate semialdehyde.
  • lactate is first activated into lactoyl-CoA using propionyl-CoA as CoA donor.
  • Propionyl-CoA is synthesized from propionate, CoA, and ATP using AMP forming propionyl-CoA synthase.
  • ATP Two ATP are used to convert AMP back into ATP, which increases ATP cost of this pathway and contributes to the net ATP deficiency of this pathway.
  • Lactoyl-CoA dehydrates into acryloyl-CoA which is then rehydrated at the ⁇ -position, forming 3-hydroxypropionyl-CoA .
  • 3- hydroxypropionyl-CoA is then hydrolyzed to 3-hydroxypropionate , structural isomer of lactate. Subsequent re-oxidation of ⁇ -hydroxyl group of 3-hydroxypropionate yields malonate semialdehyde.
  • 3-hydroxypropionyl-CoA can be converted directly to malonyl-CoA In two reduction steps.
  • the ATP demand of this pathway and the number of enzymes required is larger than the aspartate dependent pathway (described above) .
  • the disclosure provides microorganisms that comprise an artificially engineered pathway to produce malonyl-CoA and may further include additional enzymes to produced fatty acids, and other chemicals from malonyl-CoA.
  • the disclosure demonstrates recombinant pathways for the production of malonyl-CoA from a suitable carbon source (e.g., via pyruvate or PEP) .
  • the malonyl-CoA produced by the recombinant microorganism described herein can also be used for CoA-dependent chain
  • CoA dependent chain elongation includes the Clostridium pathway for 1-butanol production and mevalonate pathway for production of diverse downstream isoprenoids-based compounds. Incorporation of the synthetic malonyl-CoA pathway into in vivo systems would enhance production of these diverse malonyl- CoA derived products.
  • biosynthetic pathway also referred to as
  • metabolic pathway refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another.
  • Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
  • metabolic engineering involves rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired metabolite, such as an acetoacetyl-CoA or higher alcohol, in a microorganism.
  • a desired metabolite such as an acetoacetyl-CoA or higher alcohol
  • Methodabolically engineered can further include optimization of metabolic flux by regulation and optimization of transcription, translation, protein stability, reducing agents and protein functionality using genetic engineering and appropriate culture condition including the reduction of, disruption, or knocking out of, a competing metabolic pathway that competes with an intermediate or use of a cofactor or energy source, leading to a desired pathway.
  • a biosynthetic gene can be heterologous to the host microorganism, either by virtue of being foreign to the host, or being modified by mutagenesis, recombination, and/or association with a heterologous expression control sequence in an endogenous host cell.
  • the polynucleotide is
  • the polynucleotide can be codon optimized .
  • Microorganisms provided herein are modified to produce metabolites in quantities not available in the parental
  • a “metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., malonate semialdehyde) in, or an end product
  • Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
  • substrate or “suitable substrate” refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
  • the term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term
  • substrate encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as CO2, or any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein.
  • Recombinant microorganisms provided herein can express a plurality of target enzymes involved in pathways described above and herein for the production of malonyl-CoA from a suitable carbon substrate and may further include pathways for the synthesis of fatty acids, polyketides and flavanoids .
  • metabolically “engineered” or “modified” microorganisms are recombinant microorganisms produced via the introduction of genetic material into a host or parental
  • the microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism.
  • the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, a metabolite.
  • the introduction of genetic material into a parental microorganism results in a new or modified ability to produce malonyl-CoA.
  • the genetic material introduced into the parental microorganism contains gene (s) , or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of malonyl-CoA or an intermediate or downstream product thereof and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.
  • the recombinant microorganisms comprises at least one recombinant metabolic pathway that comprises a target enzyme and may further include a reduction in activity or reduction in expression of an enzyme in a competitive biosynthetic pathway.
  • the pathway acts to modify a substrate or metabolic intermediate in the production of malonyl-CoA.
  • the target enzyme is encoded by, and expressed from, a polynucleotide derived from a suitable biological source .
  • the polynucleotide comprises a gene derived from a bacterial or yeast source and recombinantly
  • the disclosure provides a recombinant microorganism comprising elevated expression of at least one target enzyme as compared to a parental microorganism or encodes an enzyme not found in the parental organism.
  • a microorganism is engineered to express or overexpress one or more polypeptides that convert pyruvate to malonyl-CoA or PEP to malonyl- CoA.
  • a plurality of pathways can be engineered to provide for metabolic pathways to produce malonyl-CoA.
  • a recombinant microorganism is engineered to express an acetyl-CoA carboxylase.
  • a recombinant microorganism can be engineered to expresses or overexpresses one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to Glyceraldehyde-3P; (ii) converting Glyceraldehyde- 3P to DHAP; (iii) converting DHAP to glycerol-3P; (iv) converting glycerol-3P to glycerol; (v) converting glycerol to 3-HPA; (vi) converting 3HPA to 3HP; (vii) converting 3HP to malonate
  • a recombinant microorganism can be engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to oxaloacetate ; (ii) converting oxaloacetate to malonate semialdehyde; and (iii) converting malonate semialdehyde to malonyl-coA.
  • a recombinant microorganism can be engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to oxaloacetate; (ii) converting oxaloacetate to aspartate, (iii) converting aspartate to beta-alanine ; (iv) converting beta-alanine to malonate semialdehyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • a recombinant microorganism can be engineered to express or
  • a metabolic function selected from the group consisting of (i) optionally converting PEP to pyruvate; (ii) converting pyruvate to alanine; (iii) converting alanine to bet-alanine; (iv) converting beta- alanine to malonate semialdhyde; and (v) converting malonate semialdehyde to malonyl-coA.
  • a recombinant microorganism can be engineered to express or overexpress one or more enzymes that carries out a metabolic function selected from the group consisting of (i) converting PEP to pyruvate; (ii) converting pyruvate to lactate; (iii) converting lactate to lactoyl-CoA; (iv) converting lactoyl-CoA to acrylyl-CoA; (v) converting acrylyl-CoA to 3-hydroxyproprionyl-CoA; (vi) 3-hdroxypropionyl-CoA to 3- hydroxyproprionate ; (vii) converting 3-hydroxyproprionate to malonate semialdehyde; and (viii) converting malonate semialdehyde to malonyl-coA.
  • the microorganism comprises a photoautotrophic, photoheterotrophic, chemotrophic, or autotrophic organism that is engineered to express or overexpress a trios phosphate isomerase (e.g., Tpi, or homolog thereof), glycerol- 3-phosphate dehydrogenase (e.g., GpsA, or homolog thereof), glycerol-3-phosphatase (e.g., GPP, or homolog thereof), glycerol dehydratase (e.g., DhaB123, or homolog thereof), glycerol
  • a trios phosphate isomerase e.g., Tpi, or homolog thereof
  • glycerol- 3-phosphate dehydrogenase e.g., GpsA, or homolog thereof
  • glycerol-3-phosphatase e.g., GPP, or homolog thereof
  • glycerol dehydratase e.g
  • dehydratase reactivase GdrAB, or homolog thereof
  • aldehyde dehydrogenase e.g., PuuC, or homolog thereof
  • At least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • the recombinant microorganism is engineered to express or over express a
  • phosphoenolpyruvate carboxylase e.g., Ppc, or homolog thereof
  • an oxaloacetate 1-decarboxylase e.g., Oad, or homolog thereof
  • malonyl-CoA reductase e.g., Mcr or homolog thereof
  • at least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • the recombinant microorganism is engineered to express or overexpress a
  • phosphoenolpyruvase e.g., Ppc or homolog thereof
  • an aspartate aminotransferase e.g., AspC, or homolog thereof
  • an aspartate 1- decarboxylase e.g., PanD, or homolog thereof
  • a PLP-dependent aspartate 1-decarboxylase e.g., AeADC, or homolog thereof
  • a beta- alanine aminotransferase e.g., SkPYD4, or homolog thereof
  • a malonyl-CoA reductase e.g., Mcr, or homolog thereof
  • at least one of the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • the foregoing enzymes or homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • recombinant microorganism is engineered to express or overexpress a pyruvate kinase (e.g., Pyk, or homolog thereof), an alanine aminotransferase (e.g., Aat, or homolog thereof), an alanine aminomutase (e.g., Aam, or homolog thereof) and a malonyl-CoA reductase (e.g., Mcr, or homolog thereof) .
  • a pyruvate kinase e.g., Pyk, or homolog thereof
  • an alanine aminotransferase e.g., Aat, or homolog thereof
  • an alanine aminomutase e.g., Aam, or homolog thereof
  • Mcr malonyl-CoA reductase
  • the recombinant microorganism is engineered to express or overexpress a pyruvate kinase (e.g., Pyk, or homolog thereof), a lactate dehydrogenase (e.g., Ldh, or homolog thereof), a lactoyl-CoA transferase (e.g., Pet, or homolog thereof), a propionyl-CoA synthase (e.g., Pes, or homolog thereof), a lactoyl- CoA dehydratase (e.g., Led, or homolog thereof), a hydroxypropionyl- CoA dehydratase (e.g., Hpd, or homolog thereof), a hydroxypropionyl- CoA hydrolase (e.g., Hph, or homolog thereof), a malonate
  • a pyruvate kinase e.g., Pyk, or homolog thereof
  • a lactate dehydrogenase
  • homologs thereof is recombinant engineered into the microorganism to produce malonyl-coA from PEP.
  • Tables A-G provide information regarding the various enzymes and pathways described above and elsewhere herein including substrate specificity, products, and co-factors.
  • Table D Enzymes for a-alanine dependent pathway.
  • Table E Enzymes for lactate dependent pathway.
  • Table G Enzymes for aspartate dependent pathway.
  • the microorganism comprises a reduction, disruption or knockout of at least one gene encoding an enzyme that competes with a metabolite necessary for the production of a malonly-CoA or a downstream product of malonyl-CoA metabolism .
  • a recombinant microorganism of the disclosure comprise expression of a
  • heterologous aspartate aminotransferase or elevated expression of an endogenous aspartate aminotransferase means an enzyme that catalyzes the conversion of
  • aspartate aminotransferase is derived from E. coli.
  • an aspartate aminotransferase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO:l, which encodes the amino acid sequence of SEQ ID NO : 2.
  • an aspartate amino transferase can include polypeptides having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:2 and having aspartate aminotransferase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous aspartate 1-decarboxylase or elevated expression of an endogenous aspartate 1-decarboxylase.
  • aspartate 1-decarboxylase aspartate 1-decarboxylase (PanD, EC 4.1.1.11)) means an enzyme that catalyzes the conversion of aspartate to beta-alanine and CO2.
  • aspartate 1- decarboxylase is derived from Corynebacterium glutamicum .
  • an aspartate 1-decarboxylase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 3, which encodes the amino acid sequence of SEQ ID NO : 4.
  • an aspartate 1-decarboxylase can include polypeptides having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:4 and having aspartate 1-decarboxylase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous PLP-dependent aspartate 1-decarboxylase or elevated expression of an endogenous PLP-dependent aspartate 1-decarboxylase.
  • PLP-dependent aspartate 1-decarboxylase means an enzyme that catalyzes the conversion of aspartate to beta-alanine and CO2.
  • PLP-dependent aspartate 1-decarboxylase is derived from Aedes aegypti.
  • a PLP-dependent aspartate 1-decarboxylase can comprise a codon optimized nucleotide sequence of SEQ ID NO: 5, which encodes the amino acid sequence of SEQ ID NO : 6.
  • a PLP-dependent aspartate 1- decarboxylase can include polypeptides having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO : 6 and having PLP- dependent aspartate 1-decarboxylase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous beta-alanine aminotransferase or elevated expression of an endogenous beta-alanine aminotransferase.
  • beta-alanine aminotransferase (beta- alanine aminotransferase (SkPYD4, EC 2.6.1.19)) means an enzyme that catalyzes the conversion of beta-alanine and alphaketoglutarate to malonate semialdehyde and glutamate .
  • beta-alanine aminotransferase is derived from Saccharomyces kluyveri.
  • a beta-alanine aminotransferase can comprise a codon optimized nucleotide sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO : 8.
  • a beta- alanine aminotransferase can include polypeptides having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 8 and having beta-alanine aminotransferase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous malonyl-CoA reductase or elevated expression of an endogenous malonyl-CoA reductase.
  • malonyl-CoA reductase means the enzyme that catalyzes the conversion of malonate semialdehyde and CoA and NADP + to malonyl-CoA and NADPH.
  • malonyl-CoA reductase is derived from Sulfolobus tokodaii.
  • a malonyl-CoA reductase can comprise a codon optimized nucleotide sequence of SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10.
  • a malonyl-CoA reductase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 10 and having malonyl-CoA reductase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous phosphoenolpyruvate carboxylase or elevated expression of an endogenous phosphoenolpyruvate
  • phosphoenolpyruvate carboxylase As used herein, the term "phosphoenolpyruvate carboxylase” (phosphoenolpyruvate carboxylase (Ppc, EC 4.1.1.31)) means the enzyme that catalyzes the conversion of
  • phosphoenolpyruvate carboxylase is derived from E. coli.
  • a phosphoenolpyruvate carboxylase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12.
  • a phosphoenolpyruvate carboxylase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:12 and having phosphoenolpyruvate carboxylase activity.
  • PEP carboxykinase An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP.
  • PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP.
  • S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes- Hevia et al . , FEBS Lett. 258:313-316 (1989).
  • coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K m for bicarbonate of PEP carboxykinase (Kim et al . , Appl . Environ. Microbiol. 70:1238-1241
  • PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP.
  • PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al . , Biotechnol . Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum succiniciproducens (Laivenieks et al . , Appl . Environ. Microbiol.
  • the PEPCK enzyme from Megathyrsus maximus has a low K m for CO2, a substrate thought to be rate-limiting in the E. coli enzyme (Chen et al . , Plant Physiol 128:160-164 (2002); Cotelesage et al . , Int. J Biochem. Cell Biol. 39:1204-1210 (2007)).
  • carboxykinase enzyme of Haemophilus influenza is effective at forming oxaloacetate from PEP.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous pyruvate kinase or elevated expression of an endogenous pyruvate kinase.
  • a heterologous pyruvate kinase or elevated expression of an endogenous pyruvate kinase.
  • pyruvate kinase (pyruvate kinase (Pyk, EC 2.7.1.40)) means the enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP.
  • pyruvate kinase is derived from E. coli.
  • a pyruvate kinase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 13, which encodes the amino acid sequence of SEQ ID NO: 14.
  • a pyruvate kinase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and having pyruvate kinase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous beta alanine aminotransferase or elevated expression of an endogenous beta alanine aminotransferase.
  • beta alanine aminotransferase (beta alanine aminotransferase (Baat, EC 2.6.1.18)) means the enzyme that catalyzes the conversion of pyruvate and beta-alanine to alanine and malonate semialdehyde .
  • a beta alanine aminotransferase (beta alanine aminotransferase (Baat, EC 2.6.1.18)) means the enzyme that catalyzes the conversion of pyruvate and beta-alanine to alanine and malonate semialdehyde .
  • a beta alanine aminotransferase (beta alanine aminotransferase (Baat, EC 2.6.
  • a beta alanine aminotransferase is derived from Streptomyces avermitilis .
  • a beta alanine aminotransferase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16.
  • a beta alanine aminotransferase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:16 and having beta alanine aminotransferase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous alanine aminomutase or elevated expression of an endogenous alanine aminomutase.
  • alanine aminomutase (alanine aminomutase (Aam) means the enzyme that catalyzes the conversion of alanine to beta-alanine.
  • Various alanine aminomutases are known in the art and include engineered alanine aminomutases (see, e.g., U.S. Patent Publication No. 20120040437A1, which is incorporated herein by reference) .
  • a recombinant microorganism of the disclosure comprise expression of a heterologous lactate dehydrogenase or elevated expression of an endogenous lactate dehydrogenase.
  • lactate dehydrogenase lactate dehydrogenase (Ldh, EC 1.1.1.27)) means the enzyme that catalyzes the conversion of pyruvate and NADH to lactate.
  • Lactate Dehydrogenase also referred to as D-lactate dehydrogenase and fermentive dehydrogenase
  • ldhA the NADH-dependent
  • D-lactate dehydrogenase D-lactate dehydrogenase
  • Framentative lactate dehydrogenase Fermentative lactate dehydrogenase
  • a recombinant microorganism of the disclosure comprise expression of a heterologous triose phosphate isomerase or elevated expression of an endogenous triose phosphate isomerase.
  • triose phosphate isomerase or “Tpi” refer to proteins that are capable of catalyzing the formation of dihydroxyacetone phosphate from glyceraldehyde-3-phosphate , and which share at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence similarity, as calculated by NCBI BLAST, using default parameters, to SEQ ID NO: 18. Additional homologs include: Rattus norvegicus AAA42278.1 having 45% identity to SEQ ID NO:18; Homo sapiens AAH17917.1 having 45% identity to SEQ ID NO: 18;
  • the sequences associated with the foregoing accession numbers are incorporated herein by reference.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous glycerol-3-phosphate dehydrogenase or elevated expression of an endogenous glycerol-3-phosphate
  • glycerol-3-phosphate dehydrogenase or “GpsA” referse to an enzyme that converts DHAP and NADH to glycerol-3-phophosphate .
  • the gpsA gene is of prokaryotic origin.
  • the gpsA gene is of bacterial origin.
  • Exemplary embodiments of gpsA genes include, but are not limited to, those originating from E. coli (SEQ ID NO: 19) .
  • a glycerol-3-phosphate dehydrogenase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 19, which encodes the amino acid sequence of SEQ ID NO: 20.
  • a glycerol-3-phosphate dehydrogenase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:20 and having glycerol-3-phosphate activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous glycerol-3-phosphatase or elevated expression of an endogenous glycerol-3-phosphatase .
  • glycerol-3-phosphatase or “GPP” refers to an enzyme that converts glycerol-3-phosphate to glycerol.
  • a "glycerol-3- phosphatase” or “sn-glycerol-3-phosphatase” or “d, 1-glycerol phosphatase” or “G3P phosphatase” refer to the polypeptide (s) responsible for an enzyme activity that is capable of catalyzing the conversion of glycerol-3-phosphate to glycerol.
  • a glycerol-3-phosphatase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 21, which encodes the amino acid sequence of SEQ ID NO: 22.
  • a glycerol-3- phosphatase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 22 and having glycerol-3- phosphatase activity.
  • a recombinant microorganism of the disclosure comprise expression of a heterologous aldehyde dehydrogenase or elevated expression of an endogenous aldehyde dehydrogenase.
  • aldehyde dehydrogenase aldehyde dehydrogenase (PUUC, EC 1.2.1.-)) means the enzyme that catalyzes the conversion of 3- hydroxypropionatealdehyde to 3-hydroxypropionate .
  • aldehyde dehydrogenase is derived from E. coli .
  • an aldehyde dehydrogenase can comprise a nucleotide sequence or codon optimized sequence of SEQ ID NO: 23, which encodes the amino acid sequence of SEQ ID NO: 24.
  • a nucleotide sequence or codon optimized sequence of SEQ ID NO: 23 which encodes the amino acid sequence of SEQ ID NO: 24.
  • a nucleotide sequence or codon optimized sequence of SEQ ID NO: 23 which
  • phosphoenolpyruvate carboxylase can include polypeptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:24 and having aldehyde dehydrogenase activity.
  • yield refers to the molar yield. For example, the yield equals 100% when one mole of glucose is converted to one mole of malonyl-CoA.
  • yield is defined as the mole of product obtained per mole of carbon source monomer and may be expressed as percent. Unless otherwise noted, yield is expressed as a percentage of the
  • Theoretical yield is defined as the maximum moles of product that can be generated per a given mole of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. In one embodiment, the yield is at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12 or more. In another example, the yield of a recombinant microorganism can be from 5% to 90% of theoretical.
  • the disclosure provides a culture of recombinant microorganisms of the disclosure comprising a population that is substantially homogenous (e.g., from about 70- 100% homogenous) .
  • a culture can comprise a combination of microorganism each having distinct biosynthetic pathways that produced metabolites that can be used by at least one other microorganism in culture leading to the production of malonyl- CoA or a product derived therefrom.
  • at least one "population" of recombinant microorganism comprises the ability to make more malonyl-CoA compared to the same microorganism that has not been recombinantly engineered.
  • the disclosure also provides an in vitro method of producing malonyl-CoA and chemicals and biofuels that use malonyl- CoA as a substrate.
  • an in vitro metabolic pathway can be obtained in a number of ways.
  • the enzymes of the any of the pathways described herein can be obtained in a number of ways.
  • the enzyme can be bound to a support or expressed in a phage display or other surface expression system and, for example, fixed in a fluid pathway corresponding to points in the metabolic pathway.
  • a pathway is partially cloned and expressed in a recombinant microorganism of the disclosure and then the cells are disrupted and the necessary addition enzymes and substrates are added.
  • the full pathways of any of the malonyl-CoA production pathways described herein are cloned into a recombinant microorganism.
  • the microorganism is then cultured to express the polypeptides and the cells are disrupted.
  • the disrupted milieu can then be used directly or the polypeptide for the pathway additionally purified.
  • accession numbers for various genes, homologs and variants (and EC enzyme reference numbers) useful in the generation of recombinant microorganism described herein can be identified using the information in Tables A-G. It is to be understood that homologs and variants described herein are exemplary and non- limiting. Additional homologs, variants and sequences are available to those of skill in the art using various databases including, for example, the National Center for Biotechnology Information (NCBI) access to which is available on the World-Wide-Web.
  • NCBI National Center for Biotechnology Information
  • the disclosure identifies genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary. For example, 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 mutation and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme activity using methods known in the art .
  • RNA 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. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al . (1996) Nucl. Acids Res. 24: 216-218) .
  • Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein.
  • disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as they modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
  • homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
  • the term "homologs" used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
  • a protein has "homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences) .
  • 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%, 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid
  • 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.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, hist
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG) , University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid
  • GCG contains programs such as "Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.
  • BLAST Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997) .
  • Typical parameters for BLASTp are:
  • polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference) .
  • percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix) , as provided in GCG Version 6.1, hereby incorporated herein by
  • microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of e.g., malonyl-CoA or products derived therefrom. It is also understood that various microorganisms can act as "sources” for genetic material encoding target enzymes suitable for use in a recombinant microorganism provided herein.
  • microorganism includes prokaryotic and eukaryotic photosynthetic microbial species.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • Bacteria refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes ,
  • Mycoplasmas (2) Proteobacteria, e.g., Purple photosynthetic +non- photosynthetic Gram-negative bacteria (includes most "common” Gram- negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs;
  • Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium .
  • Gram positive bacteria include cocci, nonsporulating rods, and sporulating rods.
  • the genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium,
  • Photoautotrophic bacteria are typically Gram-negative rods which obtain their energy from sunlight through the processes of photosynthesis. In this process, sunlight energy is used in the synthesis of carbohydrates, which in recombinant photoautotrophs can be further used as intermediates in the synthesis of biofuels. In other embodiment, the photoautotrophs serve as a source of
  • anoxygenic photoautotrophs grow only under anaerobic conditions and neither use water as a source of hydrogen nor produce oxygen from
  • photoautotrophic bacteria are oxygenic photoautotrophs. These bacteria are typically cyanobacteria . They use chlorophyll pigments and photosynthesis in photosynthetic processes resembling those in algae and complex plants. During the process, they use water as a source of hydrogen and produce oxygen as a product of photosynthesis.
  • Cyanobacteria include various types of bacterial rods and cocci, as well as certain filamentous forms.
  • the cells contain thylakoids, which are cytoplasmic, platelike membranes containing chlorophyll.
  • the organisms produce heterocysts, which are
  • microorganisms that have been genetically modified to express or over-express endogenous nucleic acid sequences, or to express non- endogenous sequences, such as those included in a vector.
  • the nucleic acid sequence generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite as described above.
  • recombinant microorganisms described herein have been genetically engineered to express or over-express target enzymes not previously expressed or over-expressed by a parental microorganism. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism.
  • a "parental microorganism” refers to a cell used to generate a recombinant microorganism. The term "parental microorganism"
  • microorganism describes a cell that occurs in nature, i.e. a "wild- type” cell that has not been genetically modified.
  • parental microorganism also describes a cell that has been genetically modified but which does not express or over-express a target enzyme e.g., an enzyme involved in the biosynthetic pathway for the production of a desired metabolite such as malonyl-CoA.
  • a target enzyme e.g., an enzyme involved in the biosynthetic pathway for the production of a desired metabolite such as malonyl-CoA.
  • a wild-type microorganism can be genetically modified to express or over express a first target enzyme such as
  • This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or over-express a second target enzyme e.g., aspartate aminotransferase.
  • a second target enzyme e.g., aspartate aminotransferase.
  • the microorganism modified to express or over express e.g., aspartate 1-decarboxylase, which can be modified to express or over express a yet another target enzyme e.g., beta alanine aminotransferase.
  • a parental microorganism functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or over-expression of a target enzyme.
  • the term “facilitates” encompasses the activation of endogenous nucleic acid sequences encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of exogenous nucleic acid sequences encoding a target enzyme in to a parental microorganism.
  • a method of producing a recombinant microorganism that converts a suitable carbon substrate to e.g., malonyl-CoA includes transforming a microorganism with one or more recombinant nucleic acid sequences as described above and elsewhere herein (see, e.g., Figures 1-6) .
  • Nucleic acid sequences that encode enzymes useful for generating metabolites including homologs, variants, fragments, related fusion proteins, or functional equivalents thereof, are used in recombinant nucleic acid molecules that direct the expression of such
  • polypeptides in appropriate host cells such as bacterial or yeast cells (such enzymes are described in Tables A-G) . It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a nonfunctional or non-coding sequence, is a conservative variation of the basic nucleic acid.
  • the "activity" of an enzyme is a measure of its ability to catalyze a reaction resulting in a metabolite, i.e., to "function", and may be expressed as the rate at which the metabolite of the reaction is produced. For example, enzyme activity can be represented as the amount of metabolite produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
  • a "protein” or “polypeptide”, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
  • An “enzyme” means any substance, composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.
  • the term “enzyme” can also refer to a catalytic polynucleotide (e.g., RNA or DNA) .
  • a "native” or “wild- type” protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature .
  • nucleic acid sequences described above include “genes” and that the nucleic acid molecules described above include “vectors” or “plasmids . " For example, a nucleic acid sequence encoding a keto thiolase can be encoded by an atoB gene or homolog thereof, or a fadA gene or homolog thereof. Accordingly, the term “gene”, also called a “structural gene” refers to a nucleic acid sequence that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
  • the transcribed region of the gene may include untranslated regions, including introns, 5'- untranslated region (UTR) , and 3 ' -UTR, as well as the coding sequence.
  • the term "nucleic acid” or “recombinant nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) , and, where appropriate, ribonucleic acid (RNA) .
  • expression with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence .
  • operon refers two or more genes which are transcribed as a single transcriptional unit from a common promoter.
  • the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter.
  • any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide.
  • the modification can result in an increase in the activity of the encoded polypeptide.
  • the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions .
  • a "vector” is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes) , BACs (bacterial artificial chromosomes) , and PLACs (plant artificial chromosomes) , and the like, that are “episomes, " that is, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a
  • polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conj ugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium .
  • Transformation refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection) , can be achieved by any one of a number of means including electroporation, microinjection, biolistics (or particle bombardment-mediated delivery) , or agrobacterium mediated
  • nucleic acid molecules in the form of recombinant DNA expression vectors or plasmids, as described in more detail below, that encode one or more target enzymes.
  • such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector
  • the disclosure provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified
  • expression vector refers to a nucleic acid that can be introduced into a host microorganism or cell-free transcription and translation system.
  • An expression vector can be maintained permanently or transiently in a microorganism, whether as part of the chromosomal or other DNA in the microorganism or in any cellular compartment, such as a replicating vector in the cytoplasm.
  • An expression vector also comprises a promoter that drives
  • RNA which typically is translated into a
  • the expression vector for efficient translation of RNA into protein, the expression vector also serves as a promoter for efficient translation of RNA into protein.
  • ribosome-binding site sequence typically contains a ribosome-binding site sequence positioned upstream of the start codon of the coding sequence of the gene to be expressed.
  • Other elements such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes by which host microorganisms containing the vector can be identified and/or selected, may also be present in an expression vector.
  • Selectable markers i.e., genes that confer antibiotic resistance or sensitivity, are used and confer a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • an expression vector can vary widely, depending on the intended use of the vector and the host cell (s) in which the vector is intended to replicate or drive expression.
  • Expression vector components suitable for the expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces, and other commonly used cells are widely known and commercially available.
  • suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of
  • promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan ( trp) , beta-lactamase (bla) , bacteriophage lambda PL, and T5 promoters.
  • synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used.
  • E. coli expression vectors it is useful to include an E. coli origin of replication, such as from pUC, plP, pi, and pBR.
  • recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a biosynthetic gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells.
  • the host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome .
  • the term "host cell” is used interchangeably with the term “recombinant microorganism” and includes any cell type which is suitable for producing e.g., malonyl-CoA and susceptible to transformation with a nucleic acid construct such as a vector or plasmid.
  • a nucleic acid of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and those procedures described in the Examples section below.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • nucleic acid molecule encoding a polypeptide homologous to the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the nucleic acid sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a non-conservative amino acid
  • substitutions in some positions it is preferable to make conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a method for producing e.g., malonyl-CoA includes culturing a
  • microorganism or culture comprising an autotroph, photoautotroph or photoheterotroph and a recombinant non-photosynthetic or photoheterotroph microorganism as provided herein in the presence of a suitable substrate and under conditions suitable for the
  • the malonyl-CoA produced by a microorganism or culture provided herein can be detected by any method known to the skilled artisan.
  • Culture conditions suitable for the growth and maintenance of a recombinant microorganism provided herein are described in the Examples below. The skilled artisan will recognize that such conditions can be modified to accommodate the requirements of each microorganism.
  • RNA polymerase mediated techniques e.g., NASBA
  • RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.
  • Plasmid constructions A list of plasmids used in this work is presented in Table 1. All plasmids were constructed by isothermal DNA assembly method. Plasmids were propagated and stored in E. coli strain XL-1 blue. Promoter and enzyme coding regions of all plasmids were verified by DNA sequencing performed by Genewiz
  • eQuilibrator with either standard condition (AG'°) or physiological relevant condition (AG' at 1 mM substrate and product concentration, pH 7.0, and ionic strength of 0.1 M) .
  • Product elution was carried out using gradient program with 50 mM sodium acetate pH 3.7 as aqueous solvent and acetonitrile as organic solvent with flow rate of 1 mL/min.
  • the gradient program used is summarized in Table 2.
  • Product elution was monitored at 254 nm using diode array detector. Injection volume was 20 L . column temperature was kept at 30 °C.
  • thermodynamics analysis suggests the reversibility of Mcr, Mcr has not been demonstrated to function in the oxidative direction. Since malonate semialdehyde is not commercially available, a coupled reaction using SkPYD4 and Mcr (Fig. 10) was used to convert ⁇ - alanine to malonyl-CoA. SkPYD4 and Mcr were individually purified using poly-His-tag . The results are shown in HPLC chromatogram (Fig. 10B) . Malonyl-CoA is synthesized upon presence of the components in the reaction, indicating both the reversibility of Mcr and the feasibility of malonyl-CoA biosynthesis from ⁇ -alanine.
  • PLP-dependent aspartate a-decarboxylase enhanced rate of malonyl-CoA biosynthesis.
  • PanD used to reach optimum rate of malonyl-CoA biosynthesis
  • an alternative candidate enzyme for aspartate decarboxylation was examined.
  • Most amino acid a-decarboxylases such as glutamate decarboxylase and lysine decarboxylase use pyridoxal 5-phosphate (PLP) for catalysis.
  • PLP pyridoxal 5-phosphate
  • Turnover number of glutamate decarboxylase (24.85 s "1 ) and lysine decarboxylase (30 s "1 ) are both higher than that of PanD (0.65 s "1 ) in E.
  • ADC Aspartate decarboxylase
  • AspC :ADC : SkPYD4 : Mcr is 1:8:4:8, which may be easier to implement in vivo .

Landscapes

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

Abstract

L'invention concerne des micro-organismes modifiés métaboliquement, utiles dans la production de malonyl-coenzyme A ou de produits chimiques issus de celle-ci. L'invention concerne également cinq voies distinctes de synthèse de la malonyl-coenzyme A à partir de métabolites centraux tels que le phosphoenolpyruvate et le pyruvate. L'invention concerne encore un micro-organisme recombiné produisant la malonyl-coenzyme A à des niveaux supérieurs à ceux d'un organisme parental comprenant une voie sélectionnée dans le groupe constitué par : (a) phosphoenolpyruvate à malonate-semialdéhyde ; et (b) pyruvate à malonate-semialdéhyde ; ladite voie contenant une réductase de malonyl-coenzyme A.
PCT/US2014/052960 2013-08-27 2014-08-27 Voie et organismes recombinés pour la synthèse de la malonyl-coenzyme a WO2015031504A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361870488P 2013-08-27 2013-08-27
US61/870,488 2013-08-27

Publications (1)

Publication Number Publication Date
WO2015031504A1 true WO2015031504A1 (fr) 2015-03-05

Family

ID=52587297

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/052960 WO2015031504A1 (fr) 2013-08-27 2014-08-27 Voie et organismes recombinés pour la synthèse de la malonyl-coenzyme a

Country Status (1)

Country Link
WO (1) WO2015031504A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109072172A (zh) * 2015-11-12 2018-12-21 利戈斯股份有限公司 用于厌氧产生L-天冬氨酸和β-丙氨酸的重组宿主细胞和方法
CN113039281A (zh) * 2018-06-25 2021-06-25 利戈斯股份有限公司 生产天冬氨酸和β-丙氨酸的重组宿主细胞和方法
WO2022019950A1 (fr) * 2020-07-23 2022-01-27 Cargill, Incorporated Enzymes et procédés de production d'acide malonique et de dérivés correspondants
WO2022019949A1 (fr) * 2020-07-23 2022-01-27 Cargill, Incorporated Enzymes et procédés de production de l'acide malonique et de ses dérivés

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122887A1 (en) * 2003-12-18 2007-05-31 Basf Aktiengesellschaft Gene variants coding for proteins from the metabolic pathway of fine chemicals
US20120135481A1 (en) * 2010-11-22 2012-05-31 Novozymes, Inc. Compositions and methods for 3-hydroxypropionic acid production
US20120244586A1 (en) * 2000-11-20 2012-09-27 Cargill, Incorporated 3-hydroxypropionic acid and other organic compounds
US20130122541A1 (en) * 2010-01-27 2013-05-16 The Regents Of The University Of Colorado Microorganism production of high-value chemical products, and related compositions, methods and systems

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120244586A1 (en) * 2000-11-20 2012-09-27 Cargill, Incorporated 3-hydroxypropionic acid and other organic compounds
US20070122887A1 (en) * 2003-12-18 2007-05-31 Basf Aktiengesellschaft Gene variants coding for proteins from the metabolic pathway of fine chemicals
US20130122541A1 (en) * 2010-01-27 2013-05-16 The Regents Of The University Of Colorado Microorganism production of high-value chemical products, and related compositions, methods and systems
US20120135481A1 (en) * 2010-11-22 2012-05-31 Novozymes, Inc. Compositions and methods for 3-hydroxypropionic acid production

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIAO ET AL.: "Structure of Glycerol Dehydratase Reactivase: A New Type of Molecular Chaperone", STRUCTURE, vol. 11, 1 January 2003 (2003-01-01), pages 109 - 119 *
NENE ET AL.: "Genome sequence of Aedes aegypti, a major arbovirus vector", SCIENCE, vol. 316, 22 June 2007 (2007-06-22), pages 1718 - 1723 *
Q171 SO: "Glutamate decarboxylase", 2005, pages 1 - 2., Retrieved from the Internet <URL:http://www.uniprot.org/uniprot/Q171S0> [retrieved on 20141206] *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109072172A (zh) * 2015-11-12 2018-12-21 利戈斯股份有限公司 用于厌氧产生L-天冬氨酸和β-丙氨酸的重组宿主细胞和方法
EP3374505A4 (fr) * 2015-11-12 2019-05-01 Lygos, Inc. Cellules hôtes recombinantes et procédés de production anaérobie de l-aspartate et de bêta-alanine
CN113039281A (zh) * 2018-06-25 2021-06-25 利戈斯股份有限公司 生产天冬氨酸和β-丙氨酸的重组宿主细胞和方法
WO2022019950A1 (fr) * 2020-07-23 2022-01-27 Cargill, Incorporated Enzymes et procédés de production d'acide malonique et de dérivés correspondants
WO2022019949A1 (fr) * 2020-07-23 2022-01-27 Cargill, Incorporated Enzymes et procédés de production de l'acide malonique et de ses dérivés

Similar Documents

Publication Publication Date Title
US9121041B2 (en) Method for the preparation of diols
US11613768B2 (en) Microbial production of 2-phenylethanol from renewable substrates
US20100221800A1 (en) Microorganism engineered to produce isopropanol
AU2015360786B2 (en) Recombinant microorganisms exhibiting increased flux through a fermentation pathway
JP2011510611A (ja) 組換え微生物によるバイオ燃料の生成
JP2013544516A (ja) タンパク質性バイオマスの発酵を介した組換え微生物によるバイオ燃料および化学物質の生成
US9145542B2 (en) Method for producing aldehyde from CO2
US11377671B2 (en) Co-production pathway for 3-HPA and acetyl-CoA derivatives from malonate semialdehyde
US9193965B2 (en) Acetolactate synthase having 2-ketoisovalerate decarboxylase activity and uses thereof
WO2015031859A1 (fr) Bactéries modifiées pour la production d&#39;ester
WO2013126855A1 (fr) Fabrication photosynthétique directe dirigée par l&#39;atp de combustibles et de produits chimiques
EP2925873B1 (fr) Production d&#39;acide 3-hydroxypropionique par des levures recombinantes
CN113710813A (zh) 通过逆向乙醛酸支路生产乙醇酸和甘氨酸的微生物和方法
Matsakas et al. New trends in microbial production of 3-hydroxypropionic acid
WO2015031504A1 (fr) Voie et organismes recombinés pour la synthèse de la malonyl-coenzyme a
US20140134690A1 (en) Microbes and methods for producing 1-propanol
US8846354B1 (en) Microorganisms for producing organic acids
US20140296571A1 (en) Microorganisms And Methods For Producing Propionic Acid
WO2014087184A1 (fr) Production par fermentation de néopentyl glycol par un micro-organisme recombinant
JP2023071865A (ja) メチオニン生産酵母
US8846329B1 (en) Microorganisms for producing organic acids
US20140107377A1 (en) Microorganisms And Methods For Producing Acrylate And Other Products From Propionyl-CoA
US20140329275A1 (en) Biocatalysis cells and methods
WO2022244809A1 (fr) Microorganisme recombiné ayant la capacité de produire de la diamine et procédé de fabrication de diamine
EP3652322A1 (fr) Levure produisant de la thréonine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14841105

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14841105

Country of ref document: EP

Kind code of ref document: A1