WO2017191483A1 - Enzymatic methods for butanol production - Google Patents

Enzymatic methods for butanol production Download PDF

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Publication number
WO2017191483A1
WO2017191483A1 PCT/IB2016/000903 IB2016000903W WO2017191483A1 WO 2017191483 A1 WO2017191483 A1 WO 2017191483A1 IB 2016000903 W IB2016000903 W IB 2016000903W WO 2017191483 A1 WO2017191483 A1 WO 2017191483A1
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coa
lactate
dehydrogenase
nad
enzyme
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PCT/IB2016/000903
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French (fr)
Inventor
Claudio Garibay ORIJEL
Mónica María Ríos LOZANO
Jessica Valeria Guerrero TORRES
Iván Alejandro de la Peña MIRELES
José Raúl Iván Garza RODRIQUEZ
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Newpek S.A. De C.V.
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Priority to PCT/IB2016/000903 priority Critical patent/WO2017191483A1/en
Priority to EP16901046.9A priority patent/EP3452606A4/en
Priority to CA3021033A priority patent/CA3021033A1/en
Priority to US16/098,614 priority patent/US11692207B2/en
Publication of WO2017191483A1 publication Critical patent/WO2017191483A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
    • 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)
    • C12Y102/01057—Butanal dehydrogenase (1.2.1.57)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01044—Trans-2-enoyl-CoA reductase (NAD+) (1.3.1.44)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01086—Crotonyl-CoA reductase (1.3.1.86)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
    • 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)
    • C12Y102/01051—Pyruvate dehydrogenase (NADP+) (1.2.1.51)
    • 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)
    • C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
    • C12Y102/04001—Pyruvate dehydrogenase (acetyl-transferring) (1.2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01004—Dihydrolipoyl dehydrogenase (1.8.1.4), i.e. lipoamide-dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01012—Dihydrolipoyllysine-residue acetyltransferase (2.3.1.12)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the field of biotechnology, more particularly to a method for the enzymatic conversion of lactic acid or lactate to butanol coupled to a regeneration system of NAD (P) + / NAD (P) H and regeneration of acetyl-Co A / CoA.
  • the 4 carbon alcohols such as n-butanol (or simply butanol) and isobutanol are important industrial chemicals, useful as fuel additives, raw materials in the plastics industry, and as extracting agents in the food grade.
  • n-butanol or simply butanol
  • isobutanol are important industrial chemicals, useful as fuel additives, raw materials in the plastics industry, and as extracting agents in the food grade.
  • butanol can be produced chemically by the hydroformylation of propylene, a process wherein propylene is contacted with a catalyst containing rhodium, that allows the hydroformylation of propylene to form butyraldehyde, then the aldehyde is hydrogenated to form butanol, as described in European patent EP1733003B1.
  • butanol can be produced biologically, by a metabolic pathway known as fermentation ABE (Jones and Woods, 1986 widely metabolic pathway; and others, 2012). This fermentation pathway is widely used in industry, using the microorganism Clostridium Acetobutylicum.
  • fermentation ABE Jones and Woods, 1986 widely metabolic pathway; and others, 2012
  • the European patent EP2204453B 1 claims butanoi production enzymatically. However, to carry out this process glucose is used as raw material. This means that at least 5 enzymes are required to convert glucose into pyruvate. In addition to involving various enzymes to produce pyruvate, the EP2204453B1 patent describes in its examples that the operating temperature of the system should be greater than 50 ° C.
  • EP2700714A1 mentions a very similar scheme to EP2204453B1, but using at least 13 enzymes to carry out the process.
  • an object of the present invention is to provide an enzymatic method for producing butanol from lactate, wherein the production of butanol is coupled with
  • a further object of the present invention is to provide a method in which the production of butanol from lactate coupled with a regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA, can be performed in a controlled
  • Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA is coupled with the production of butanol from lactate in a batch process by using free enzymes or immobilized enzymes.
  • Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyi-CoA / CoA is coupled with the production of butanol from lactate in a semicontinuous process by using free enzymes or immobilized enzymes.
  • Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyi-CoA / CoA is coupled with the production of butanol from lactate in a continuous process by using immobilized or free enzymes.
  • Another object of the present invention is to provide a biofuel or biofuel precursor made by the process of the present invention, and an automotive engine fuel comprising a mixture of hydrocarbons, and the biofuel or biofuel precursor.
  • reaction mixture A) mixing water, lactate, an enzyme mixture comprising at least one enzyme, at least one cofactor and at least one coenzyme, to prepare a reaction mixture:
  • Figure 1 shows a schematic of a regeneration system NAD (P) + / NAD (P) H coupled with butanol production starting from lactate,
  • Figure 2 shows a graph illustrating the behavior of the batch production method of butanol.
  • Figure 3 shows a graph illustrating the behavior of the method of continuous production of butanol in a reactor CSTR.
  • Figure 4 shows a graph illustrating the behavior of the method of continuous production of butanol (along z axis) of a reactor PBR.
  • lactic acid refers to the same molecule, wherein the said molecule has three carbons and holds the molecular formula F C-CHGH-COOH (C 3 H 6 O 3 ).
  • lactic acid refers to any of the isomers or mixture of isomers reported in the international databases with identification numbers CAS 50-21 -5, 79-33-4, 10326-41-7, 598-82-3, which can be L-lactate or D-iactate or a mixUire of both in any proportion.
  • lactate is equivalent to lactic acid in dissolution conditions and depending on pH lactic acid can be present in its ionic form. Lactate can be obtained in different ways, whether biologically or chemically. Biologically, lactate can be obtained, for example, by the fermentation of organic compounds. Some of the lactate-producing organisms are Escherichia coli, Lactobacil lus casei, Lactobacillus delhrueckii, Lactococcus lactis, etc.
  • the lactate can be obtained, for example from ethanol, sodium cyanide and sulfuric acid, wherein the process ends with a cyanide nucleophilic attack of the carbonyl group of the aldehyde by forming the nitriie of lactic acid in a racemic form.
  • the nitriie is saponified in the presence of water and excess sulfuric acid to give the free lactic acid.
  • pyruvate refers to the same molecule, wherein the said molecule has three carbons and holds the molecular formula CH3COCOOH (C 3 H 4 O 3 , CAS: 127-17-3).
  • coenzymes and “Coenzyme” refers to organic compounds, non-protein, necessary for the action of enzymes that are required, for example flavin adenine dinucleotide (FAD), thiamine pyrophosphate (THPP), flavin mononucleotide (FMN) , Coenzyme A, etc..
  • FAD flavin adenine dinucleotide
  • THPP thiamine pyrophosphate
  • FMN flavin mononucleotide
  • Coenzyme A etc.
  • Coenzyme A refers to one molecule widely known in the art, wherein the molecule has twenty one carbon atoms (C21H36N7O16P3S, CAS: 85-61 -0) .
  • the CoA is used by cells in ail realms and domains, as described in the prior technique, to help carry out enzymatic reactions.
  • acetyi-CoA refers to the same molecule, wherein the said molecule has twenty three carbons and holds the molecular formula CH 3 COSC0A (C23H38N7O17P3S, CAS: 72-89-9).
  • acetoacetyl-CoA refers to a molecule wherein said molecule has twenty five carbons and holds the molecular formula CH 3 COCH 2 COSCoA (C25H40 7O 18P3S, CAS: 1420-36-6).
  • crotonyl-CoA refers to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula CH 3 (CH) 2 COSCoA ( C ; ;i ⁇ , ⁇ - ⁇ ⁇ ⁇ , ⁇ , CAS: 992-67-6).
  • butanoyl-CoA refers to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula CH 3 (CH 2 ) 2 COSCoA ((Vi l !2 : () i ;P ; S,CAS: 2140-48-9).
  • butanal refers to a molecule, wherein said molecule has four carbons and holds the molecular formula CH 3 (CH 2 ) 2 COH (C 4 H g O, CAS: 123-72-8).
  • acetolactic-acid 2 refers to a molecule, wherein said molecule has five carbons and holds the molecular formula
  • ketoisovalerate 3-methyl-2-oxo-butanoic acid
  • 2-Oxo isovalerate 2-Oxoisopentanoate
  • 2-cetovaline refers to a molecule, Wherein Said molecule has five carbons and holds the molecular formula (CH 3 ) 2 CHCOCOOH (C 5 H 8 0 3; CAS: 759-05-7).
  • isobutyryl-CoA refers to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula (CH 3 ) 2 CHCOSCoA iC 25 H 42 N 7 O i7 P 3 S, CAS: 15621-60-0).
  • NADH Nicotinamide adenine dinucleotide reduced
  • NAD + nicotinamide adenine dinucleotide
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • NADP + nicotinamide adenine dinucleotide phosphate
  • NAD (P) + is equivalent to the terms “NAD + and/or NADP + and/or mixture of both”
  • NAD (P) H is equivalent to the terms” NADH and/or NADPH and/or mixture of both ", ie, when in a chemical reaction the term NAD (P) + is recited, the reaction may be carried out either by NAD + or NADP + or a mixture of both in an indiscriminate manner.
  • NAD (P) H when a chemical reaction recites the term NAD (P) H, the reaction can be carried out either NADH or NADPH or a mixture of both in an indiscriminated way.
  • the term "theoretical yield” refers to the maximum amount of product that can be obtained by a reaction, and is calculated by a stoichiometric equation. This performance will be used as a basis for comparing an experimental reaction with a theoretical amount based on the stoichiometry of the reaction.
  • experimental performance refers to the amount of product that is obtained experimentally by a chemical reaction on the amount of substrate consumed.
  • conversion efficiency refers to the percentage obtained from the ratio between the experimental and theoretical yield performance, such that the values range from O to 100%.
  • redox and “redox reaction” refer to a biochemical reaction that is mediated through the action of an enzyme, wherein a compound is reduced and another is oxidized. Generally, these reactions occur in the cells due to the presence of NAD (P) H (oxidizing agents) and NAD (P) + (reducing agents).
  • polypeptide and "enzyme” refer to a biological molecule consisting of amino acids that is capable of performing a transformation reaction of the starting compound to a final compound, wherein these two compounds are different spatially or moiecuiariy.
  • gene refers to biological molecules containing adenine, guanine, cytosine and thymine nucleotide bases or nitrogen compounds. Genes are molecules that transmit information in a ceil for the biological synthesis of enzymes.
  • reactor refers to a physical space constructed of a suitable material wherein in a controlled manner, a chemical, biochemical, or biological reaction or combinations of these, can be carried out.
  • CSTR continuous stirred tank reactors
  • PFR plug flow reactors
  • FBR Fiuidized bed reactors
  • PBR packed bed reactors
  • Some characteristics of the reactors are: a) corrosion resistance depending on the reaction being carried out; b) its ability to monitor and control operating variables, such as temperature, agitation, pH, dissolved gas concentration, pressure, etc.; c) operating mode, which can be continuous, semicontmuous or batch, the differences between which are readily understood in the art; d) the ability to use different types of catalysts which carry out the reaction, for example, the catalysts may be dissolved or may be trapped or immobilized.
  • the term "cofactor” refers to inorganic compounds, which are necessary for the action of enzymes required, for example Mg 2 1 , Fe 2 , Zn" + , Na ' , K + , Co 2 , Ni 2 1 , Mn * + , etc., depending on the particular enzyme.
  • substrate refers to the molecule on which an enzyme acts. The enzyme can be related and selective for the substrate.
  • enzyme mix refers to the set of enzymes found in the same solution, allowing the production of butanol from lactate.
  • reaction mixture refers to the group of chemical compounds in aqueous, oily, gaseous or solid phase allowing the catalyzed reactions of a polypeptide or a mixture of polypeptides that can be performed. It comprises “enzyme mix”, “cofactors”, “coenzymes”, “NAD (P) + / NAD (P) H” and "lactate”
  • the term "sequential” refers to the orderly transformation of pyruvate to lactate by lactate dehydrogenase enzymes (EC 1.1.1.27 and / or EC 1.1.1.28), of pyruvate to acetyl-CoA by pyruvate dehydrogenase complex (EC 1.2.4.1 , EC 2.3.1.12 EC 1.8.1.4, and EC 1.2.1.51), of acetyl-CoA a acetoacetyi-CoA by the enzyme acetoacetyl-CoA thiolase (EC 2.3.1.9), of acetoacetyl-CoA to (S)-3-hydroxybutanoyi-CoA by the enzyme 3-hydroxybutyryI-CoA dehydrogenase (EC 1.1.1.35 and/or EC 1.1.1.157), of (S)-3 ⁇ hydroxybutanoyi-CoA to crotonyl-CoA by the enzyme (S)-3-hydroxybutan
  • the term "sequential” also refers to the orderly transformation of lactate to pyruvate by using lactate dehydrogenase enzymes (EC 1.1.1.27 and/or EC 1.1.1.28), of pyruvate to formate and acetyl CoA by the enzyme formate acetyl transferase (EC 2.3.1 .54), of the generation of NADH by enzyme formate oxidoreductase (1.2.1.2 and/or 1.2.1.4), of acetyl- CoA to acetoacetyl-CoA by the enzyme acetoacetyl-CoA thiolase (EC 2,3.1.9), of acetoacetyf-CoA to (S)-3-hydroxybutanoyl-CoA by the enzyme hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 and/or EC 1.1.1.157), of (S)-3-hydroxybutanoyi-CoA to crotonyl-CoA
  • the term "sequential” also refers to orderly transformation of lactate to pyruvate by lactate dehydrogenase enzymes (EC 1.1.1 .27 and or EC 1.1.1 .28), of pyruvate to 2- acetolactate by the enzyme acetolactate synthase (EC 2.2.1.6), of 2 -aceto lactate to 2,3- hydroxyvalerate by the enzyme keto acid reductoisomerase (EC 1.1.1.86), of 2,3- dihydroxyvaierate to ketoisovaierate by the enzyme dihydroxyacid dehydratase (EC 4.2.1.9), of ketoisovaierate to isobutyryl-CoA by the enzyme 2-oxoisovalerate dehydrogenase ( EC 1.2.1.25 and/or EC 1.2.4.4), of isobutyryl-CoA to butyryl-CoA by the enzyme isobutyryl- CoA mutase (EC 5.4.99.13), of butyry i-Co A to
  • multienzymatic system refers to a group of enzymes that sequentially transform the lactate into butanol.
  • the terrn “elimination of genes” refers to a cleavable procedure of a region of DNA coding for a protein.
  • the term “exogeneous gene” refers to a region of ADN coding for a protein that is foreign to the organism.
  • endogeneous gene refers to a region of ADN coding for a protein found within the organism.
  • the terrn “overexpression” refers to increased expression levels of a protein encoded by an endogeneous gene or an exogeneous gene.
  • the term "regeneration of NAD(P)7NAD(P)H” refers to the transformation of molecules of NAD(P) + by molecules of NAD(P)H due to the action of any enzyme that may have this activity, as well as the transformation of molecules of NAD(P)H of molecules of NAD(P) T due to the action of any enzyme that may have this activity. Such transformations can be found in a single reaction system or several reactions, depending on the process.
  • the term "regeneration of acetyl-Co A/CoA” refers to the transformation of molecules of acetyl-CoA in molecules CoA due to the action of any enzyme that may have this activity, as well as the transformation of molecules of CoA in molecules acetyl-CoA due to the action of any enzyme that may have this activity. Such transformations can be found in a single reaction system or several reactions, depending on the process.
  • free enzyme refers to an enzyme distributed in a solution.
  • free enzymes refers to a group of enzymes distributed in a solution.
  • support refers to a solid or semisolid, inert matrix, which preferably does not modify the protein structure, for example activated carbon, zeolite, calcium alginate, silica gel, etc..
  • immobilized enzyme refers to the enzyme attached, trapped, absorbed, adsorbed, bound, etc., by any physical or chemical method to a support.
  • the terrn "immobilized enzymes” refers to a group of enzymes attached, trapped, absorbed, adsorbed, bound, etc., by any physical or chemical method to a support.
  • the term “L-lactate dehydrogenase” (EC 1.1.1.27) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform a compound of L-lactate into pyruvate using NAD(P) " .
  • NAD(P) NAD
  • Examples of enzymes that can carry out the conversion reaction of L-lactate to pymvate are described in Table 1.
  • the enzymes described in Table 1 are shown for reference only, since there are databases which can be found more examples of these enzymes, such as in GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc.
  • Table 1 Examples of L-iactate dehydrogenase that can be used to transform the L- actate to pyruvate.
  • D-lactate dehydrogenase (EC 1 A .1.28) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform a compound of D-lactate into pyruvate using NAD(P) 1 .
  • NAD(P) 1 NAD(P) 1
  • Table 2 Examples of D-lactate dehydrogenase that can be used to transform the D- lactate to pyruvate.
  • pyruvate dehydrogenase complex refers to a group of polypeptides (EC
  • Enzymes described in Table 3 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc.
  • Table 3 Examples of pyruvate dehydrogenase complex that can be used to transform pyruvate to acetyl-CoA.
  • acetoacetyS-CoA thiolase (EC 2.3.1.9) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform 2 molecules of acetyl-CoA in a molecule of acetoacetyl-CoA.
  • acetoacetyS-CoA thiolase (EC 2.3.1.9) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform 2 molecules of acetyl-CoA in a molecule of acetoacetyl-CoA.
  • there may be another enzyme that is not cataloged in this set of enzymes that carry out the reaction described so that enzyme will be considered as an analogue of acetoacetyl-CoA thiolase.
  • Examples of enzymes that can carry out the conversion reaction of acetyl-CoA to acetoacetyl-CoA are described in Table 4.
  • the enzymes disclosed in Table 4 are shown for reference only,
  • Table 4 Examples of acetoacetyl-CoA thiolase of which can be used to transform two molecules of acetyl-CoA in 1 molecule of acetoacetyl-CoA.
  • STM14__3646 enterica serovar typhimurium
  • 3-hydroxyacyl-CoA dehydrogenase or "3-hidroxibutyryl-CoA dehydrogenase” (EC 1.1.1.35 and/or EC 1.1.1.157) refers to a polypeptide having catalytic activity, where the catalytic activity is to convert acetoacetyl-CoA compound to (S) -3- hydroxybutanoyl-CoA using NAD (P) H.
  • P NAD
  • Examples of enzymes that can carry out the conversion reaction of acetoacetyl-CoA to (S) -3-hydroxybutanoyl-CoA are described in Table 5.
  • the enzymes described in Table 5 are shown for reference only, since there many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
  • Table 5 Examples of 3-hydroxyacyl CoA dehydrogenase that can be used to transform acetoacetyl-CoA to(S)-3-hydroxybutanoyl-CoA.
  • (S)-3-hydroxybutanoyl-CoA hydro-lyase (EC 4.1 ,1 .55) and "enoyl-CoA hydratase” (EC 4.2.1.17) refers to polypeptides having catalytic activity, where the catalytic activity is to transform a compound of (S)-3-hydroxybutanoyl-CoA into crotonyl-CoA.
  • an enzyme that is not cataloged in this set of enzymes that carry out the reaction described, so that enzyme will be considered as an analogue of "(S) -3- hydroxybutanoyl-CoA hydro-lyase and / or enoyl-CoA hydratase.
  • dehydrogenase or "crotonyl Coenzyme A reductase” (EC 1.3.1.86 and/or 1.3.8.1) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of crotonyl-CoA into butanoyl-CoA using NAD(P)H.
  • this catalytic activity is to transform a compound of crotonyl-CoA into butanoyl-CoA using NAD(P)H.
  • NAD(P)H NAD(P)H
  • Table 7 Examples of trans-2-enoyl-CoA reductase and butyryl-CoA dehydrogenase which can be used to transform crotonyl-CoA to butanoyl-CoA.
  • dehydrogenase (EC 1.2.1.10) refers to a polypeptide having catalytic activity, where this catalytic activity is to transforai a compound of butanoyl-CoA into butanal using NAD(P)H.
  • this catalytic activity is to transforai a compound of butanoyl-CoA into butanal using NAD(P)H.
  • NAD(P)H NAD(P)H
  • Examples of enzymes that can carry out the conversion reaction of butanoyl-CoA to butanal are described in Table 8. The enzymes described in Table 8 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
  • Table 8 Examples of butanal dehydrogenase and/or acetaldehyde dehydrogenase which can be used to transform el butanoyl-CoA to butanal.
  • alcohol dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of butanal into butanol using NAD(P)H.
  • this catalytic activity is to transform a compound of butanal into butanol using NAD(P)H.
  • NAD(P)H NAD(P)H
  • Table 9 Examples of alcohol dehydrogenase which can be used to transform butanai to butanol.
  • pyruvate formate-lyase or "formate acetyl transferase” refers to a polypeptide (EC 2.3.1.54) having catalytic activity, where this catalytic activity is to transform a compound of pyruvate into acetyl-CoA and formate.
  • a polypeptide EC 2.3.1.54
  • catalytic activity is to transform a compound of pyruvate into acetyl-CoA and formate.
  • Table 10 Examples of the enzyme pyruvate formate-lyase which can be used to transform pyruvate to acetyl-CoA.
  • formate dehydrogenase refers to a polypeptide (EC 1.2.1.43) having catalytic activity where this catalytic activity is to transform a compound of formate to C0 2 , using a molecule of NAD(P) + ,
  • Examples of enzymes that can carry out the conversion reaction of formate to C0 2 are described in Table 1 1 , The enzymes described in Table 1 1 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
  • Table 1 Examples of the enzyme formate dehydrogenase which can be used to transform formate to C0 2 .
  • acetolactate synthase (EC 2.2.1.6) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of pymvate into 2- acetolactate.
  • acetolactate synthase there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of acetolactate synthase. Examples of enzymes that can carry out the conversion reaction of pyruvate to 2-aeeto!aetate are described in Table 12.
  • Table 12 The enzymes described in Table 12 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc. [0094] Table 12. Examples of acetolactate synthase which can be used to transform pyruvate to 2-acetolactate.
  • ketoacid reductoisomerase (EC 1.1.1 .86) refers to a polypeptide having catalytic activity, where this catalytic activity is to transfomi a compound of acetolactate into 2,3-dihydroxyvalerate.
  • this catalytic activity is to transfomi a compound of acetolactate into 2,3-dihydroxyvalerate.
  • Examples of enzymes that can cany out the conversion reaction of 2-acetolactate to 2,3-dihydroxyvalerate are described in Table 13. The enzymes described in Table 13 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
  • dihydroxyacid dehydratase (EC 4.2, 1.9) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of 2,3- dihydroxyvalerate into ketoisovalerate.
  • this catalytic activity is to transform a compound of 2,3- dihydroxyvalerate into ketoisovalerate.
  • another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of dihydroxyacid dehydratase. Examples of enzymes that can cany out the conversion reaction of 2, 3 -dihydroxy valerate to ketoisovalerate are described in Table 14.
  • 2-oxoisovalerate dehydrogenase (EC 1.2.1 ,25), t '3-methy]-2-oxobutanate dehydrogenase” or “ketoacid-dehydrogenase” (EC 1.2.4.4) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of ketoisovalerate into isobutyryi-CoA, using a molecule of NAD(P) .
  • thai is not listed in this group of enzymes that cam' out the described reaction, so that enzyme will be considered as an analogue of 2-oxoisovalerate dehydrogenase.
  • Examples of enzymes that can cany out the conversion reaction of ketoisovalerate to isobutyryl-CoA are described in Table 15.
  • the enzymes described in Table 15 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
  • Table 16 Examples of isobutyryl-CoA mutase which can be used to transform isobutyry]-CoA to butyryl-CoA.
  • the present invention refers to a method wherein a multi-enzyme system produces butanol starting from lactate sequentially coupled with a regeneration system of NAD (P) + / NAD (P) E and / or regeneration of acetyl-CoA / Co A ( Figure 1).
  • This transformation can be performed in a container or reactor where the whole process can be carried out continuously, semicontinuously or in batch.
  • the present invention overcomes the deficiencies of the prior art by providing polypeptides that, with an experimental performance equal or lower than the theoretical yield, transform lactate sequentially to butanol.
  • the present invention can use smaller amounts than stoichiometrically required of NAD (P) +, NAD (P) H and Co A to perform the process described above, because the method of the present invention allows the regeneration of NAD(P) + /NAD(P)H and / or the regeneration of acetyl -CoA/CoA in three different ways: a) during the conversion of lactate to pyruvate, of pyruvate to acetyl-CoA, acetoacetyl-CoA to (S) -3- hydroxybutanoyl-CoA, of crotonyl-CoA to butanoyl-CoA, of butanoyl-CoA to butanal and butanal to butanol; b) during the conversion of lactate to pyruvate, formate to CG 2 , of acetoacetyl-CoA to (S)-3-hydroxybutanoyl-CoA, of croton
  • the method of the present invention may employ unitary operations recirculating to system NAD (P) + and / ' or NAD (P) H and / ' or CoA, allowing lower amounts than those established by the stoichiometry amounts to be transformed to larger amounts of lactate to butanol
  • the present invention may employ the enzymes lactate dehydrogenase (EC 1.1 , 1 .27 and/or EC 1.1.1.28), pyruvate dehydrogenase (EC 1.2.4.1 , EC 2.3.1.12, EC 1.8.1.4 and EC 1.2.1.51), acetoacetyl-CoA thiolase (EC 2.3.1.9), hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 and or EC 1.1.1.157), (S)-3-hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and or enoyl-CoA hydratase (EC 4.2.1 .17), trans-2-enoyl-CoA reductases (EC 1 .3.1.44) and or butyryl-CoA dehydrogenase (EC 1.3.1.86 and or EC 1.3.8.1), butanal dehydrogenase (EC 1.2.1.57) and or acetalde
  • the present invention may also employ the enzymes lactate dehydrogenase (EC 1.1.1.27 and or EC 1.1.1.28), pyruvate formate-iyase (EC 2.3.1.54), formate dehydrogenase (EC 1.2.1.43), acetoacetyl-CoA thiolase (EC 2.3.1.9),
  • hydroxybutyryl-CoA dehydrogenase (EC 1.1 .1.35 and/or EC 1 .1 .1.157), (S)-3- hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and/or enoyl-CoA hydratase (EC 4.2.1.17), trans-2-enoyl-CoA reductase (EC 1.3.1.44) and/or hutyryl-CoA dehydrogenase (EC 1 .3.1 ,86 and/or EC 1.3.8.1 ), butanal dehydrogenase (EC 1.2.1.57) and/or acetaidehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2) and/or its analogs to transform lactate into butanol, wherein the amount of NAD (P) + added to the system may be less than the amount set by the stoichiometry
  • the present invention may employ the enzymes lactate dehydrogenase (EC 1 .1.1 .27 and/or EC 1 .1.1 .28), acetolactate synthase (EC 2.2.1.6), ketoacid reductoisomerase (EC 1.1.1.86), dihydroxyacid dehydratase (EC 4.2.1.9), 2-oxoisovalerato dehydrogenase (EC 1.2.1.25) and/or ketolacid dehydrogenase (EC 1.2.4.4), isobutyryl-Coenzyme A mutase (EC 5.4.99.13), butanal dehydrogenase (EC 1 .2.1.57) and/or acetaidehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1 .1 .1 and/or EC 1 .1 .1.2) and/or its analogs to transform lactate into butanol, wherein the quantity of NAD(P) " added
  • the enzyme mixture may be any of those described above. This method comprises different stages which are described below:
  • a mixture is prepared continuously starting from water, lactate, the enzyme mixture, NAD(P) 7NAD(P)H, CoA and the cofactors and coenzymes that uses the enzymes to perform catalysis.
  • the cofactors and coenzymes depend on the nature of each enzyme.
  • Table 17 are shown some of the cofactors and coenzymes used by the different enzymes in the present invention. The cofactors and coenzymes described in Table 17 are examples, this does not exempt other cofactors or coenzymes to be used by enzymes to perform catalysis.
  • stage II The flow obtained from stage I is passed continuously through a reactor that stably maintains the conditions of reaction of pH between 4 and 10, preferably between 6 and 8 and at a temperature between 15°C and 40°C, preferably between 25°C and 37°C .
  • this current enters the reactor, it carries out the production of butanol from lactate with a conversion efficiency equal to or less than 100%.
  • the separation system can be: a membrane system (reverse osmosis, nanofiStration, ultrafiltration, etc.), distillation, evaporation or any other system that allows the separation of molecules either by size or by any of its physiochemical properties.
  • the mixture of immobilized enzymes can be any of those described above.
  • the immobilization of enzymes on the support can be carried out by any method that is appropriate.
  • Table 18 shows some of the supports used to immobil ize enzymes. The supports described in Table 18 are examples of some supports, this does not exclude other materials that are suitable for the immobilization of enzymes and which are not in Table 18. This method comprises different stages which are described below:
  • a mixture of the enzymes are immobilized on a support.
  • the support can also be multiple supports of the same type with different numbers of enzymes, or may be supports of different types, sizes or chemical compositions and each support containing one or more enzymes, such that in the reactor are all enzymes either on the same support or on different supports. Coenzymes and cofactors may also be present on the support. Once the enzymes are immobilized, these immobilized enzymes and their supports will be added to the reactor.
  • a mixture is prepared continuously from water, lactate and NAD (P) T / '
  • NAD (P) H Cofactors and coenzymes employed by each enzyme to perform catalysis depend on the nature of each enzyme. Some of the cofactors and coenzymes employed by the different enzymes used in the present invention are shown in Table 17. Cofactors and coenzymes described in Table 17 are examples, this does not exclude other cofactors or coenzymes that could be used by enzymes to perform catalysis.
  • the stream exiting stage II is passed continuously through the reactor containing the immobilized enzymes, so that this flow is continuously in contact with said enzymes, stably maintaining the conditions of reaction at pH between 4 and 10, preferably between 6 and 8 and at a temperature between 15 ° C and 40 ° C, preferably between 25 ° C and 37 ° C.
  • this stream is contacted with enzymes, the production of butanol from lactate is carried out with a conversion efficiency equal to or less than 100%.
  • the support is maintained within the reactor, although the output of the reactor support does not imply that this support can not be recirculated to the reactor.
  • stage III enriched and depleted butanol lactate
  • stage III enriched and depleted butanol lactate
  • Coenzymes and cofactors form a concentrated stream which can be recycled to stage II, or to the enzyme reactor.
  • stage III whether or not coenzymes and cofactors have been separated, one can separate the fraction of butanol in another separation system. This system generates two streams, a butanol water stream and another stream,
  • Separation systems mentioned in stages IV and V can be: membrane system (reverse osmosis, nanofiltration, ultrafiltration, etc.), distillation, evaporation or any other system which al lows the separation of molecules, either by their size or for some of its
  • Example 1 Quantification of enzyme activity
  • Lactate dehydrogenase (EC 1 .1.1.27 and / or EC 1 .1.1.28)
  • Lactate dehydrogenase can transform lactate to pynivate using NAD (P) " so the test was conducted by varying the initial concentrations of lactate, NAD (P) + ' pH and temperature following the protocols described in the literature (CetineS et al ., 2013).Four enzymes of various microorganisms were used (Escherichia coli APEC 01, Escherichia coli PMV-1, Lactobacillus lactis subsp. lactis IL1403 and Streptococcus pneumoniae AP200). The conditions tested are shown in Table 19.
  • the pyruvate dehydrogenase complex transforms pynivate into acetyl-CoA, using a molecule of NAD (P) ' so the test was conducted by varying the initial concentrations of pyruvate, NAD (P) + ' CoA, pH and temperature following the protocols described in the literature (Nemeria et al., 2001). Tliree enzymes from different microorganisms were used (Escherichia coli K-12 MG1655, Corynebacterium sp. ATCC 6931 and Lactobacillus reuleri). The conditions tested are shown in Table 20.
  • Acetyl-CoA thiolase enzyme converts two molecules of acetyl-CoA in a molecule of acetoacetyl-CoA, so the test was conducted by varying the initial concentrations of acetyl- CoA, pH and temperature following the protocols described in the literature (Huth et al., 1975). Three enzymes from different microorganisms were used (.: H38 042 Xanthomonas campestris pv campestris ATCC 33913, Shigella sonnei and Escherichia coli 044 Ss046). The conditions tested are shown in Table 21.
  • Acetyl -CoA (g/L) 1, 25 and 75
  • 3-hydroxybutyryl-CoA dehydrogenase (EC 1 , 1.1.35 and / or 1.1.1.157)
  • 3-hydroxybutyryl-CoA dehydrogenase converts acetoacetyl-CoA to 3- hydroxybutyryl-CoA, using a molecule of NAD (P) H, so the test was conducted by varying the initial concentrations of acetoacetyl-CoA, NAD (P) H , pH and temperature following the protocols described in the literature (Madan et ai., 1973). Three enzymes from different microorganisms were used (: H2 12009, Xanthomonas albilineans, Escherichia coli 0103 Aspergillus niger). The conditions tested are shown in Table 22.
  • the enoyl-CoA hydratase can transform 3-hydroxybutyryl-CoA to crotonyl-CoA, so the test was conducted by varying the initial concentrations of 3-hydroxybutyryl-CoA, pH and temperature following the protocols described in the literature (Fukui et al., 1998). Three enzymes from different microorganisms were used (Acinetobacter baumannii, and
  • the butyryl-CoA dehydrogenase enzyme converts crotonyl-CoA to butanoyi-CoA using a molecule of NAD (P) H, so the test was conducted by varying the initial
  • Dehydrogenase butanal enzyme transforms butanoyl-CoA to butanal using a molecule of NAD (P) H, so the test was conducted by varying the initial concentrations of butanoyl-CoA, NAD (P) H, pH and temperature following the protocols described in the literature (Palosaari and Rogers, 1988). Three enzymes from different microorganisms were used. (: Closiridhim acetobutylicum DSM SE15 H5 1731 , Escherichia coli and Salmonella enterica 0150). The conditions tested are shown in Table 25.
  • Alcohol dehydrogenase can transform isobutyraldehyde into isobutanol using NAD (P) H, so the test was conducted by varying the initial concentrations of isobutyraldehyde, NAD (P) H, pH and temperature following the protocols described in the literature (Atsumi et at., 2010), Three enzymes from different microorganisms were used (Escherichia coli K-l 2 MDS42, Bacillus cereus and Clostridium acetobutylicum DSM B4264 1731). The conditions tested are shown in Table 26.
  • the enzynie formate dehydrogenase transforms the formate into CO 2 using a molecule of NAD (P) + ' so the test was conducted by varying the initial concentrations of formate, NAD (P) + ' pH and temperature following the protocols described in literature (Hatrongjit and Packdibamrung, 2010). Three enzymes from different microorganisms were used (Wolinella succinogenes, Su!furicurvum kujiense and Geohacter sulfurreducens PC A). The conditions tested are shown in Table 28.
  • Acetolactate synthase converts pyruvate into 2-acetolactate, so the test was conducted by varying the initial concentrations of pyruvate, pH and temperature fol lowing the protocols described in the literature (Holtzclaw and Chapman, 1975; Barak et al, 1987; Atsumi et to the. 2009). Three enzymes from different microorganisms were used (Escherichia coli were used K-12 MG1655, Bacillus suhtilis subsp. suhtilis sir. 168 and Saccharomyces cerevisiae S288c). The conditions tested are shown in Table 29.
  • the keto acid reductoisomerase 2-acetolactate transforms 2,3- dihydroxy valerate while dihydroxyacid dehydratase transforms hydroxy valerate into ketoisovaierate.
  • the 2-acetoiactate is not a commercial compound as 2,3- dihydroxy valerate is an unstable compound
  • the determination of the activity of these two enzymes was measured indirectly by coupling a test acetolactate synthase with keto acid reductoisomerase and dihydroxy acid dehydratase. This was done by varying the initial concentrations of pyruvate, NAD (P) H, pH and temperaUire using as a basis the protocols described in the literature (Flint et al . , 1993; Bastian et al, 201 1 ;.. Li et al, 201 1)
  • isobutyryl-CoA using a molecule of NAD (P) T ' so the test was conducted by varying the initial concentrations of ketoisovalerate, NAD (P) f ' pH and temperature following the protocols described in the literature (Hakozaki et al., 2002). Three enzymes from different organisms were used (Streptomyces coelicolor and Thermoplasma acidophilum and
  • Example 2 Enzymatic production of butanol from lactate, coupled with a regeneration system of NAD (P) T / NAD (P) H and / or regeneration of acetyl-CoA / CoA in a batch process.
  • Lactate dehydrogenase (EC 1.1.1.27 1-10
  • NAD (P) + / NAD (P) H and / or acetyl-CoA / CoA 0.1 g of NAD (P) + alone was required and O. lg of CoA to transform 20 g lactate into 8,22 g butanol.
  • Example 3 Enzymatic production of butanol from lactate, coupled to the regeneration system NAD (P) + / NAD (P) H and / or acetyl-CoA / ' CoA in a continuous process.
  • This example is intended to demonstrate the concept of regeneration of NAD (P) T / NAD (P) H and / ' or regeneration of acetyl-CoA / CoA in a continuous process, when the enzymes lactate dehydrogenase (EC 1.1.
  • Lactate dehydrogenase (EC 1.1,1.27 1-10
  • Table 36 Reactor operating conditions for the production of butanol from lactate.
  • the output stream of the reactor was coupled to a membrane system that separated enzymes, cofactors and coenzymes, and butanol. Stream of enzymes, cofactors and coenzymes was recirculated to the reactor. [00240] In all the conditions listed in Tables 35 and 36 the evolution of reaction intermediates in the output stream of the reactor was monitored. The evolution of NAD (P) H was measured on a Cary-60 spectrophotometer at a wavelength of 340 nm. Lactate and butanoi were measured by HPLC with refractive index detector using a Rezex ROA-organic acids H + column.
  • Example 4 Enzymatic production of butanoi from lactate, coupled to the
  • This example is intended to demonstrate the concept of regeneration of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA in a continuous process using immobilized enzymes, where the enzymes lactate dehydrogenase are used ( EC 1.1.1 .27 and / or EC 1.1.1 .28), acetolactate synthase (EC 2.2.1 ,6), keto acid reductoisomerase (EC 1.1 .1 .86), dihydroxy acid dehydratase (EC 4.2.1.9), 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 and / or EC 1 .2.4.4), isobutyryl-CoA mutase (EC 5.4.99.13), butanal dehydrogenase (EC 1.2.1.57 and / or EC 1.2.1.10), alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1.1.1.2) , or its analogs,
  • the overall stoichiometric equation multi-enzymatic system has no loss or gain of NAD (P) + ' NAD (P) H, CoA and / or acetyl-CoA, so that the overall reaction results in the use of two molecules of lactate to produce a molecule of butanol, obtaining an efficiency of 100% conversion according to the following reaction:
  • Butanol production in continuous form from lactate was carried out in a reactor immobilizing each enzyme or enzyme mixture (Table 37) in different media (Table 18), varying the amounts of immobilized protein.
  • the operating conditions which were used are shown in Table 38.
  • the reaction mixture was formulated by mixing coenzymes (at the concentrations described in the prior art) immobilized enzymes (Table 38), cofactors and lactic acid and NAD (P) +.
  • Table 37 Enzymes used to formulate the enzyme mixture to produce butanol from lactate in a continuous process with immobilized enzymes.
  • CoA (g/L) 0.01-10 The output current of the reactor was coupled to a reverse osmosis system that can recirculate a mixture of cofactors and coenzymes to the reactor and / or mixing tank.
  • the initial concentration of NAD (P) T was 0.1 g / L, while the lactate concentration to the reactor inlet was varied according to Table 38. In all the conditions listed in Tables 37 and 38, the evolution of reaction intermediates along the tubular reactor was monitored. Changes in the NAD (P) H were measured on a Cary-60 spectrophotometer at a wavelength of 340 nm. Lactate and butanol were measured by HPLC with refractive index detector using a Rezex ROA-organic acids H 1 column.
  • reaction began when the mixture of cofactors, coenzymes, lactate and NAD (P) ' entered the packed reactor.

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Abstract

A process for producing butanol is provided, involving: A) mixing water, lactate, an enzyme mixture comprising at least one enzyme, at least one cofactor and at least one coenzyme, to prepare a reaction mixture; B) catalytically reacting the reaction mixture for an amount of time sufficient to cause conversion of lactate into butanol; and wherein the conversion of lactate into butanol in B) is associated with a regeneration system of NAD (P) + / NAD (P) H and / or acetyl-CoA / CoA.

Description

TITLE OF THE INVENTION
ENZYMATIC METHODS FOR BUTANOL PRODUCTION
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
[0001] The present invention relates to the field of biotechnology, more particularly to a method for the enzymatic conversion of lactic acid or lactate to butanol coupled to a regeneration system of NAD (P) + / NAD (P) H and regeneration of acetyl-Co A / CoA.
DESCRIPTION OF THE RELATED ART
[0002] The 4 carbon alcohols such as n-butanol (or simply butanol) and isobutanol are important industrial chemicals, useful as fuel additives, raw materials in the plastics industry, and as extracting agents in the food grade. Each year, because of increased demand, large amounts of these alcohols are produced in the petrochemical industry.
[0003] Typically these alcohols can be produced by chemical synthesis or by biological processes. Butanol can be produced chemically by the hydroformylation of propylene, a process wherein propylene is contacted with a catalyst containing rhodium, that allows the hydroformylation of propylene to form butyraldehyde, then the aldehyde is hydrogenated to form butanol, as described in European patent EP1733003B1. Further butanol can be produced biologically, by a metabolic pathway known as fermentation ABE (Jones and Woods, 1986 widely metabolic pathway; and others, 2012). This fermentation pathway is widely used in industry, using the microorganism Clostridium Acetobutylicum. However, the production of biological butanol has not yet reached production yields that are truly competitive against chemical processes.
[0004] Various documents have been found describing the production of butanol by fermentation that use wild-type or genetically modified material which increases the production of butanol, such as Clostridium acetobutyl icum, Saccharomyces, Escherichia coli and Pseudomonas; see for example the following patents or patent applications,
US20090155869A1 , US20120149080A1 , US201 10236941 Al , US9096872B2,
CN101952430B, WO2012033334A3, and US9005953B2. The raw material that is described in these patents is generally a carbohydrate, e.g. glucose, sucrose or fructose, as is highlighted in the patents US20120149080A1 and US 20140377825A1. While this technology has been developed, it is important to note that there are deficiencies in the techniques associated with the use of genetically modified organisms to produce butanol, such as:
[0005] 1 ,- There must be many living cells to carry out the process quickly; if there is little biomass, the fermentation process becomes very slow. This fact is widely known in the prior technique.
[0006] 2 - Introducing an exogeneous metabolic pathway to an organism involves competing with the metabolism of the organism itself, because the carbon flux is divided between microbial growth and the production of butanol. This prevents the process from reaching values close to the theoretical yields (for example, for the case of glucose, 0.41 1 grams of n- butanol per gram of glucose). Therefore, to achieve acceptable yields, it is not only enough to express the metabolic pathway for the production of butanol, but the metabolic pathways that compete with the production of butanol must also be modified by removing genes. For example, the genes encoding for enzymes acetate kinase, lactate dehydrogenase and butyrate kinase, among others, have been deleted as described in patents or patent applications WO2013128230A1 , US20100136640A1 and CA2665102C. [0007] 3. -It is not enough to eliminate genes, sometimes you have to overexpress endogeneous genes and/or exogeneous genes that establish the biochemical pathway of interest and increase yields in the production of butanoi. For example, overexpression of the genes crt, bed and hbd allows greater activity of the enzymes invol ved in the synthesis of butanoi, as described in patent application WO2014135633A1.
[0008] 4.- It is common and known in the art to remove and/or overexpress genes in many cases back to the metabolically unstable organisms.
[0009] Because of this, it is desirable to have a process where there is no interaction or competition or substrates difference and wherein no growth of any microorganism is associated with the process.
[0010] In reference to this, the European patent EP2204453B 1 claims butanoi production enzymatically. However, to carry out this process glucose is used as raw material. This means that at least 5 enzymes are required to convert glucose into pyruvate. In addition to involving various enzymes to produce pyruvate, the EP2204453B1 patent describes in its examples that the operating temperature of the system should be greater than 50 ° C. This is because the enzymes that generate pyruvate from glucose do not work efficiently at lower temperatures and, additionally, some of the enzymes that convert pyruvate to butanoi that operate efficiently at temperatures of 20 to 37 ° C, generate the enzymes in an incompatible system and, also, some of them lose their activity quickly, as mentioned in different examples throughout the patent, particularly in example 10.
[0011] Furthermore EP2700714A1 mentions a very similar scheme to EP2204453B1, but using at least 13 enzymes to carry out the process.
[0012] Accordingly, there is needed a process where the production of butanoi is made from lactate, a carbon source that is widely available, where the process is carried out
enzymatically, in a process where the action of these enzymes regenerates the acceptor molecules and electron donors, so that the process can be continuous and stable for extended periods of time.
SUMMARY OF THE INVENTION
[0013] Therefore, an object of the present invention is to provide an enzymatic method for producing butanol from lactate, wherein the production of butanol is coupled with
regeneration of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA and where this process is not associated with the growth of a microorganism.
[0014] A further object of the present invention is to provide a method in which the production of butanol from lactate coupled with a regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA, can be performed in a controlled
environment, where any component of the reaction mixture can be recirculated to the process.
[0015] Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA is coupled with the production of butanol from lactate in a batch process by using free enzymes or immobilized enzymes.
[0016] Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyi-CoA / CoA is coupled with the production of butanol from lactate in a semicontinuous process by using free enzymes or immobilized enzymes.
[0017] Another object of the present invention is to develop a method wherein the regeneration system of NAD (P) + / NAD (P) H and / or regeneration of acetyi-CoA / CoA is coupled with the production of butanol from lactate in a continuous process by using immobilized or free enzymes. [0018] Another object of the present invention is to provide a biofuel or biofuel precursor made by the process of the present invention, and an automotive engine fuel comprising a mixture of hydrocarbons, and the biofuel or biofuel precursor.
[0019] These and other objects of the present invention, alone or in combinations, have been satisfied by the discovery of a process for producing butanol, comprising:
A) mixing water, lactate, an enzyme mixture comprising at least one enzyme, at least one cofactor and at least one coenzyme, to prepare a reaction mixture:
B) catalytically reacting the reaction mixture for an amount of time sufficient to cause conversion of lactate into butanol; and
wherein the conversion of lactate into butanol in B) is associated with a regeneration system of NAD (P) + / NAD (P) H and / or acetyi-CoA / Co A.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
[0021] Figure 1 shows a schematic of a regeneration system NAD (P) + / NAD (P) H coupled with butanol production starting from lactate,
[0022] Figure 2 shows a graph illustrating the behavior of the batch production method of butanol.
[0023] Figure 3 shows a graph illustrating the behavior of the method of continuous production of butanol in a reactor CSTR.
[0024] Figure 4 shows a graph illustrating the behavior of the method of continuous production of butanol (along z axis) of a reactor PBR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] For a clearer understanding of the object of the present invention, the following definitions and abbreviations are established.
[0026] The terms "lactic acid", "lactate", "2-hydroxy-propanoic acid " and "a- hydroxypropanoic acid" refers to the same molecule, wherein the said molecule has three carbons and holds the molecular formula F C-CHGH-COOH (C3H6O3). For purposes of the present invention, the term lactic acid refers to any of the isomers or mixture of isomers reported in the international databases with identification numbers CAS 50-21 -5, 79-33-4, 10326-41-7, 598-82-3, which can be L-lactate or D-iactate or a mixUire of both in any proportion. Also, for purposes of the present invention, the term "lactate" is equivalent to lactic acid in dissolution conditions and depending on pH lactic acid can be present in its ionic form. Lactate can be obtained in different ways, whether biologically or chemically. Biologically, lactate can be obtained, for example, by the fermentation of organic compounds. Some of the lactate-producing organisms are Escherichia coli, Lactobacil lus casei, Lactobacillus delhrueckii, Lactococcus lactis, etc. Chemically, the lactate can be obtained, for example from ethanol, sodium cyanide and sulfuric acid, wherein the process ends with a cyanide nucleophilic attack of the carbonyl group of the aldehyde by forming the nitriie of lactic acid in a racemic form. The nitriie is saponified in the presence of water and excess sulfuric acid to give the free lactic acid.
[0027] The terms "pyruvate", "pyruvic acid", "2-oxopropanoic acid", "propionic a-keto acid", "pyroracemic acid" and "acetylformic acid" refers to the same molecule, wherein the said molecule has three carbons and holds the molecular formula CH3COCOOH (C3H4O3, CAS: 127-17-3). [0028] The terms "coenzymes" and "Coenzyme" refers to organic compounds, non-protein, necessary for the action of enzymes that are required, for example flavin adenine dinucleotide (FAD), thiamine pyrophosphate (THPP), flavin mononucleotide (FMN) , Coenzyme A, etc..
[0029] The terms "coenzyme A", "CoA" and "CoA-SH" refers to one molecule widely known in the art, wherein the molecule has twenty one carbon atoms (C21H36N7O16P3S, CAS: 85-61 -0) . The CoA is used by cells in ail realms and domains, as described in the prior technique, to help carry out enzymatic reactions.
[0030] The terms "acetyi-CoA", "acetyl coenzyme A", "ac-CoA", "coenzyme A-acetyl A", "acetyl-S-CoA" and "ac-S-CoA" refers to the same molecule, wherein the said molecule has twenty three carbons and holds the molecular formula CH3COSC0A (C23H38N7O17P3S, CAS: 72-89-9).
[0031] The terms "acetoacetyl-CoA", "acetoacetyi coenzyme A", "3-acetoacetyl-CoA", "acetoacetyi-S-CoA", "S-acetoacetyl-CoA" and "S-acetoacetylcoenzyme A" refers to a molecule wherein said molecule has twenty five carbons and holds the molecular formula CH3COCH2COSCoA (C25H40 7O 18P3S, CAS: 1420-36-6).
[0032] The terms "(S) -3-hydroxybutanoyl-CoA", "(S) -3-hydroxybutyryl-CoA", "(3S) -3- hydroxybutanoyl-CoA", "L-3-hydroxybutyryl-CoA", "L (+) - beta-hydroxybutyroyi-CoA "," p-hydroxybuiyryi-CoA "and 3-hydroxybutyiyl-coenzyme A" refer to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula
CH3CHOHCH2COSC0A (C25H42N7O18P3S, CAS : 2871 -66-1 ) .
[0033] The terms "crotonyl-CoA", "crotonyi-coenzyme A", "2-butenoyl-CoA", "trans-but-2- enoyl-CoA", "but-2-enoyi-CoA", "(E)-but-2-enoyl-CoA" and "crotonyl-S-CoA" refer to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula CH3(CH)2COSCoA ( C;;;i Ι ,Ο -ΓΟ ^ Ρ ,Η , CAS: 992-67-6). [0034] The terms "butanoyl-CoA", "butyryl-CoA" and "butyryl-coenzyme A" refer to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula CH3(CH2)2COSCoA ((Vi l !2 : () i ;P ;S,CAS: 2140-48-9).
[0035] The terms "butanal", "butyraldehyde", "1 -butanal", "n-butyraldehyde" and "butyric aldehyde" refer to a molecule, wherein said molecule has four carbons and holds the molecular formula CH3(CH2)2COH (C4HgO, CAS: 123-72-8).
[0036] The terms "formic acid", "formate", "methanoic acid", "hydroxycarboxylic acid" refer to a molecule, wherein said molecule has one carbon and holds the molecular formula HCOOH(CH202, CAS: 123-72-8).
[0037] The terms "acetolactic-acid 2", "2-acetoiactate", "2-Hydroxy-2-methyl-3- oxobutanoic acid", "acetolactate", "acetolactic-acid" and "2-acetyl lactic acid " refer to a molecule, wherein said molecule has five carbons and holds the molecular formula
CH3COC(CH3)OHCOOH (C5H804, CAS: 7169-08-3).
[0038] The terms "2,3-dihydroxyvalerate", "2,3-dihydroxy-3-methyl butanate," "2,3- dihydroxy-isovalerate," "2,3-dihydroxy-isovaleric acid" refer to a molecule, Wherein Said molecule has five carbons and holds the molecular formula (CH3)2COHCHOHCOOH (C«H„,()., CAS: 1756-18-9).
[0039] The terms "ketoisovalerate", "3-methyl-2-oxo-butanoic acid", "2-Oxo isovalerate", "2-Oxoisopentanoate" and "2-cetovaline" refer to a molecule, Wherein Said molecule has five carbons and holds the molecular formula (CH3)2CHCOCOOH (C5H803; CAS: 759-05-7).
[0040] The terms "isobutyryl-CoA", "2-methylpropanol-CoA" and "2-methylpropionyl- CoA" refer to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula (CH3)2CHCOSCoA iC25H42N7Oi7P3S, CAS: 15621-60-0).
[0041 ] The terms "1-butanol", "n-butanol", "n-butyl alcohol", "butyl alcohol", "butanoi", "1- hydro xybutane", "butyric alcohol", "butyryi alcohol" and "butan-l-ol" refer to a molecule, wherein said molecule has four carbons and holds the molecular formula
CH3(CH2)3OH (C4H10O, CAS: 71-36-3).
[0042] The terms "Nicotinamide adenine dinucleotide reduced (NADH)" and "nicotinamide adenine dinucleotide (NAD +)" refers to molecules of the cellular metabolism and are responsible for carrying out reactions of oxidation-reduction or redox.
[0043] The terms "reduced nicotinamide adenine dinucleotide phosphate (NADPH)" and "nicotinamide adenine dinucleotide phosphate (NADP +)" refers to molecules of the cellular metabolism and are responsible for carrying out oxidation-reduction reactions or redox.
[0044] For purposes of the present invention, the use of the term "NAD (P) +" is equivalent to the terms "NAD + and/or NADP + and/or mixture of both," Likewise, the term "NAD (P) H "is equivalent to the terms" NADH and/or NADPH and/or mixture of both ", ie, when in a chemical reaction the term NAD (P) + is recited, the reaction may be carried out either by NAD + or NADP + or a mixture of both in an indiscriminate manner. Similarly, when a chemical reaction recites the term NAD (P) H, the reaction can be carried out either NADH or NADPH or a mixture of both in an indiscriminated way.
[0045] The term "theoretical yield" refers to the maximum amount of product that can be obtained by a reaction, and is calculated by a stoichiometric equation. This performance will be used as a basis for comparing an experimental reaction with a theoretical amount based on the stoichiometry of the reaction.
[0046] The term "experimental performance" refers to the amount of product that is obtained experimentally by a chemical reaction on the amount of substrate consumed.
[0047] The term "conversion efficiency" refers to the percentage obtained from the ratio between the experimental and theoretical yield performance, such that the values range from O to 100%. [0048] The terms "redox" and "redox reaction" refer to a biochemical reaction that is mediated through the action of an enzyme, wherein a compound is reduced and another is oxidized. Generally, these reactions occur in the cells due to the presence of NAD (P) H (oxidizing agents) and NAD (P) + (reducing agents).
[0049] The terms "polypeptide" and "enzyme" refer to a biological molecule consisting of amino acids that is capable of performing a transformation reaction of the starting compound to a final compound, wherein these two compounds are different spatially or moiecuiariy.
[0050] The terms "gene" or "genes" refers to biological molecules containing adenine, guanine, cytosine and thymine nucleotide bases or nitrogen compounds. Genes are molecules that transmit information in a ceil for the biological synthesis of enzymes.
[0051] The term "reactor" refers to a physical space constructed of a suitable material wherein in a controlled manner, a chemical, biochemical, or biological reaction or combinations of these, can be carried out. Different types of reactors can be found in the art. As an example are mentioned continuous stirred tank reactors (CSTR), plug flow reactors (PFR), Fiuidized bed reactors (FBR) and packed bed reactors (PBR). Some characteristics of the reactors are: a) corrosion resistance depending on the reaction being carried out; b) its ability to monitor and control operating variables, such as temperature, agitation, pH, dissolved gas concentration, pressure, etc.; c) operating mode, which can be continuous, semicontmuous or batch, the differences between which are readily understood in the art; d) the ability to use different types of catalysts which carry out the reaction, for example, the catalysts may be dissolved or may be trapped or immobilized.
[0052] The term "cofactor" refers to inorganic compounds, which are necessary for the action of enzymes required, for example Mg 2 1 , Fe2 , Zn" +, Na ' , K +, Co2 , Ni2 1 , Mn* +, etc., depending on the particular enzyme. [0053] The term "substrate" refers to the molecule on which an enzyme acts. The enzyme can be related and selective for the substrate.
[0054] The term "enzyme mix" refers to the set of enzymes found in the same solution, allowing the production of butanol from lactate.
[0055] The term "reaction mixture" refers to the group of chemical compounds in aqueous, oily, gaseous or solid phase allowing the catalyzed reactions of a polypeptide or a mixture of polypeptides that can be performed. It comprises "enzyme mix", "cofactors", "coenzymes", "NAD (P) + / NAD (P) H" and "lactate"
[0056] The term "sequential" refers to the orderly transformation of pyruvate to lactate by lactate dehydrogenase enzymes (EC 1.1.1.27 and / or EC 1.1.1.28), of pyruvate to acetyl-CoA by pyruvate dehydrogenase complex (EC 1.2.4.1 , EC 2.3.1.12 EC 1.8.1.4, and EC 1.2.1.51), of acetyl-CoA a acetoacetyi-CoA by the enzyme acetoacetyl-CoA thiolase (EC 2.3.1.9), of acetoacetyl-CoA to (S)-3-hydroxybutanoyi-CoA by the enzyme 3-hydroxybutyryI-CoA dehydrogenase (EC 1.1.1.35 and/or EC 1.1.1.157), of (S)-3~hydroxybutanoyi-CoA to crotonyl-CoA by the enzyme (S)-3-hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and/or enoyl-CoA hydratase (EC 4.2.1 .17), of crotonyi-CoA to butanoyl-CoA by the enzyme trans- 2-enoyl-CoA reductase (EC 1.3.1.44) and/or butyryl-CoA dehydrogenase (EC 1.3.1.86), of butanoyl-CoA to butanai by the enzyme butanai dehydrogenase (EC 1.2.1.57) and/or acetaSdehyde dehydrogenase (EC 1.2.1.10) and of butanai to butanol by the enzyme alcohol dehydrogenase (EC 1.1 .1.1 and/or EC 1.1 .1.2), using the ABE fermentation metabol ic pathway via pyruvate dehydrogenase complex.
[0057] The term "sequential" also refers to the orderly transformation of lactate to pyruvate by using lactate dehydrogenase enzymes (EC 1.1.1.27 and/or EC 1.1.1.28), of pyruvate to formate and acetyl CoA by the enzyme formate acetyl transferase (EC 2.3.1 .54), of the generation of NADH by enzyme formate oxidoreductase (1.2.1.2 and/or 1.2.1.4), of acetyl- CoA to acetoacetyl-CoA by the enzyme acetoacetyl-CoA thiolase (EC 2,3.1.9), of acetoacetyf-CoA to (S)-3-hydroxybutanoyl-CoA by the enzyme hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 and/or EC 1.1.1.157), of (S)-3-hydroxybutanoyi-CoA to crotonyl-CoA by the enzyme (S)-3-hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and/or enoyl-CoA hydratase (EC 4.2.1.17), of crotonyl-CoA to butanoyl-CoA by the enzymes trans- 2-enoyl-CoA reductase (EC 1.3.1.44) and/or butyryl-CoA dehydrogenase (EC 1.3.1.86), of butanoyl-CoA to butanal by the enzyme butanal dehydrogenase (EC 1.2.1.57) and/or acetaldehyde dehydrogenase (EC 1.2.1.10) and of butanal to butanol by the enzyme alcohol dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2), using the ABE fermentation metabolic pathway via oxidoreductase format.
[0058] The term "sequential" also refers to orderly transformation of lactate to pyruvate by lactate dehydrogenase enzymes (EC 1.1.1 .27 and or EC 1.1.1 .28), of pyruvate to 2- acetolactate by the enzyme acetolactate synthase (EC 2.2.1.6), of 2 -aceto lactate to 2,3- hydroxyvalerate by the enzyme keto acid reductoisomerase (EC 1.1.1.86), of 2,3- dihydroxyvaierate to ketoisovaierate by the enzyme dihydroxyacid dehydratase (EC 4.2.1.9), of ketoisovaierate to isobutyryl-CoA by the enzyme 2-oxoisovalerate dehydrogenase ( EC 1.2.1.25 and/or EC 1.2.4.4), of isobutyryl-CoA to butyryl-CoA by the enzyme isobutyryl- CoA mutase (EC 5.4.99.13), of butyry i-Co A to butanal by the enzyme butanal dehydrogenase (EC 1.2.1.57) and/or acetaldehyde dehydrogenase (EC 1.2.1.10) and of butanal to butanol by the enzyme alcohol dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2), using the metabolic pathway of ketoacids.
[0059] The term "multienzymatic system" refers to a group of enzymes that sequentially transform the lactate into butanol.
[0060] The terrn "elimination of genes" refers to a cleavable procedure of a region of DNA coding for a protein. [0061] The term "exogeneous gene" refers to a region of ADN coding for a protein that is foreign to the organism.
[0062] The term "endogeneous gene" refers to a region of ADN coding for a protein found within the organism.
[0063] The terrn "overexpression" refers to increased expression levels of a protein encoded by an endogeneous gene or an exogeneous gene.
[0064] The term "regeneration of NAD(P)7NAD(P)H" refers to the transformation of molecules of NAD(P)+ by molecules of NAD(P)H due to the action of any enzyme that may have this activity, as well as the transformation of molecules of NAD(P)H of molecules of NAD(P)T due to the action of any enzyme that may have this activity. Such transformations can be found in a single reaction system or several reactions, depending on the process.
[0065] The term "regeneration of acetyl-Co A/CoA" refers to the transformation of molecules of acetyl-CoA in molecules CoA due to the action of any enzyme that may have this activity, as well as the transformation of molecules of CoA in molecules acetyl-CoA due to the action of any enzyme that may have this activity. Such transformations can be found in a single reaction system or several reactions, depending on the process.
[0066] The term "free enzyme" refers to an enzyme distributed in a solution.
[0067] The term "free enzymes" refers to a group of enzymes distributed in a solution.
[0068] The term "support" refers to a solid or semisolid, inert matrix, which preferably does not modify the protein structure, for example activated carbon, zeolite, calcium alginate, silica gel, etc..
[0069] The term "immobilized enzyme" refers to the enzyme attached, trapped, absorbed, adsorbed, bound, etc., by any physical or chemical method to a support.
[0070] The terrn "immobilized enzymes" refers to a group of enzymes attached, trapped, absorbed, adsorbed, bound, etc., by any physical or chemical method to a support. [0071] The term "L-lactate dehydrogenase" (EC 1.1.1.27) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform a compound of L-lactate into pyruvate using NAD(P)". However, there may be another enzyme that is not listed by this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of L-lactate dehydrogenase. Examples of enzymes that can carry out the conversion reaction of L-lactate to pymvate are described in Table 1. The enzymes described in Table 1 are shown for reference only, since there are databases which can be found more examples of these enzymes, such as in GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc.
[0072] Table 1. Examples of L-iactate dehydrogenase that can be used to transform the L- actate to pyruvate.
Name of the gene Organism
L-Lactate dehydrogenase Escherichia coli APEC 01
APECOl 2404
L-Lactate dehydrogenase Escherichia coli 045: Kl : H7 S88 ECS88 4540
L- lactate dehydrogenase Escherichia coli IHE3034
ECOK1 4554
L-lactate dehydrogenase Mus musculus
Ldhal6b
L- lactate dehydrogenase Cricetulus griseus
Ldha
L-lactate dehydrogenase Pongo abelii
Ldhb
L-lactate dehydrogenase Canis familiaris
LDHAL6B
L-lactate dehydrogenase Staphylococcus aureus subsp . COL 1DH1 aureus (MRSA)
L-lactate dehydrogenase Ldh Corynebacterium glutamicum K051
L-Lactate dehydrogenase Sorangium cellulosum So ce 56
L-lactate dehydrogenase Bacillus subtilis subsp. subtilis ldh 168
Lactate dehydrogenase L- Lactococcus lactis subsp. lactis
111403
L- lactate dehydrogenase Aspergillus fumigatus
AFUA 5G14800 L- lactate dehydrogenase Enterobacter sp . 638
Erit638 2002
L- lactate dehydrogenase Streptococcus pneumoniae AP200 SPAP 1246
[0073] The term "D-lactate dehydrogenase" (EC 1 A .1.28) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform a compound of D-lactate into pyruvate using NAD(P) 1. However, there may be another enzyme that is not listed by this group of enzymes that cany out the described reaction, so that enzyme will be considered as an analogue of D-lactate dehydrogenase. Examples of enzymes that can cany out the conversion reaction of D-lactate to pyruvate are described in Table 2. The enzymes described in Table 2 are shown for reference only, since there are databases in which can be found more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc.
[0074] Table 2. Examples of D-lactate dehydrogenase that can be used to transform the D- lactate to pyruvate.
Gene name Organism
D-Lactate dehydrogenase Escherichia coli K-12 MG1655 ldha
D-Lactate dehydrogenase Escherichia coli 026: Hll 11368 ldha (EHEC)
D-Lactate dehydrogenase Escherichia coli PMV-1
1dha
D-Lactate dehydrogenase Escherichia coli 0145: H28 RM13514 1dha (EHEC)
D-Lactate dehydrogenase Shigella boydii Sb227
did
Lactate dehydrogenase D- Shewanella pealeana
Scea 0742
D-Lactate dehydrogenase Treponema pallidum Fribourg-Blanc 1dha
D-Lactate dehydrogenase RP73 Pseudomonas aeruginosa
M062 04545
D-Lactate dehydrogenase Acinetobacter sp . ADP1
did
D-Lactate dehydrogenase Pectobacterium carotovorum subsp. PCI 2294 carotovoru PCI
D-Lactate dehydrogenase WUE 2594 Neisseria meningitidis did (serogroup A)
D-Lactate dehydrogenase Cytophaga hutchinsonii
ldha
D-Lactate dehydrogenase Planctomyces brasiliensis
Plabr 4649
D-Lactate dehydrogenase sphaerobacter thermophilics
Sthe 3421
D-Lactate dehydrogenase Aiistipes finegoldii
Alfi 3240
0075] The term "pyruvate dehydrogenase complex" refers to a group of polypeptides (EC
1.2.1.51 , EC 1.2.4.1 , 2.3.1.12 and 1.8.1.4) that holds catalytic activity, where this catalytic activity is to transform the a compound of pyruvate into acetyi-CoA using NAD(P)+. However, there may be another enzyme that is not cataloged in this set of enzymes that carries out the reaction described, so that enzyme will be considered as an analogue of pyruvate dehydrogenase complex. Examples of enzymes that can carry out the conversion reaction of pyruvate to acetyl-CoA are described in Table 3. Enzymes described in Table 3 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc.
[0076] Table 3. Examples of pyruvate dehydrogenase complex that can be used to transform pyruvate to acetyl-CoA.
Name of the gene Organism
Pyruvate dehydrogenase Escherichia coli K-12 MG1655 b011
Pyruvate dehydrogenase Salmonella enterica subsp. serovar TY21A 00815 Typhi Ty21a enterica
Pyruvate dehydrogenase Corynebacteriu sp.ATCC 6931
DR71 379
Pyruvate dehydrogenase Chlamydomonas reinhardtii
CHLREDRAFT 139515
Pyruvate dehydrogenase Escherichia coli O103: H2
ECO103 0115
Pyruvate dehydrogenase Yersinia enterocolitica
LC20 04470
Pyruvate dehydrogenase Pseudomonas aeruginosa
N297 5189 Pyruvate dehydrogenase Escherichia coli K-12 MG1655
b0116
Pyruvate dehydrogenase Lactobacillus reuteri
HMPREF0538_21877
[0077] The term "acetoacetyS-CoA thiolase" (EC 2.3.1.9) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform 2 molecules of acetyl-CoA in a molecule of acetoacetyl-CoA. However, there may be another enzyme that is not cataloged in this set of enzymes that carry out the reaction described, so that enzyme will be considered as an analogue of acetoacetyl-CoA thiolase. Examples of enzymes that can carry out the conversion reaction of acetyl-CoA to acetoacetyl-CoA are described in Table 4. The enzymes disclosed in Table 4 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
(http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0078] Table 4. Examples of acetoacetyl-CoA thiolase of which can be used to transform two molecules of acetyl-CoA in 1 molecule of acetoacetyl-CoA.
Name of the genee organism
Acetyl-CoA thiolase Haemophilus influenzae R2866 R2866 1623
Acetyl-CoA thiolase Escherichia coli 044: H18 042 EC042 2465
Acetyl-CoA thiolase XCC1297 Xanthomonas campestris pv.
campestris ATCC 33913
Acetyl-CoA thiolase Cronohacter sakazakii ES15
ES15 0718
Acetyl-CoA thiolase Shigella sonnei Ss046
SSON 2283
Acetyl-CoA thiolase Helicobacter pylori83
HMPREF0462 0751
Acetyl-CoA thiolase Acinetobacter sp.ADPl
ACIAD2516
Acetyl-CoA thiolase Escherichia coli 017: K52 : H18 ECUMN 2562 UMNO26 Acetyl -CoA thiolase Salmonella enterica subsp.
STM14__3646 enterica serovar typhimurium
14028s
Acetyl -CoA thiolase PP 2051 Pseudomonas putida KT2440
[0079] The term "3-hydroxyacyl-CoA dehydrogenase" or "3-hidroxibutyryl-CoA dehydrogenase" (EC 1.1.1.35 and/or EC 1.1.1.157) refers to a polypeptide having catalytic activity, where the catalytic activity is to convert acetoacetyl-CoA compound to (S) -3- hydroxybutanoyl-CoA using NAD (P) H. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue-3-hydroxyacyl CoA dehydrogenase. Examples of enzymes that can carry out the conversion reaction of acetoacetyl-CoA to (S) -3-hydroxybutanoyl-CoA are described in Table 5. The enzymes described in Table 5 are shown for reference only, since there many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0080] Table 5. Examples of 3-hydroxyacyl CoA dehydrogenase that can be used to transform acetoacetyl-CoA to(S)-3-hydroxybutanoyl-CoA.
Name of the gene Organism
3 -hydroxyacyl -CoA dehydrogenase Salmonella enterica
SEEH1578_05995 subsp. enterica serovar
Heidelberg 41578
3 -hydroxyacyl -CoA dehydrogenase Escherichia coli O103: ECO103 2805 H2 12009
3 -hydroxyacyl -CoA dehydrogenase Xantho onas albilineans XALc 1594
3 - ydroxyacyl -CoA dehydrogenase Aspergillus niger
A I 1 165104
3 -hydroxyacyl -CoA dehydrogenase Corynebacteriu jeikeium jk0159
3 -hydroxyacyl -CoA dehydrogenase Enterobacter cloacae A3UG 16295 subsp . Dissolve s
3 -hydroxyacyl -CoA dehydrogenase Serratia piymuthica AS9 SerAS9 0217 3 -hydroxyacyl -CoA dehydrogenase Acinetobacter sp .
ACIAD1690
3 -hydroxyacyl -CoA dehydrogenase Klebsiella pneumoniae KPN 01476
3 -hydroxyacyl -CoA dehydrogenase Escherichia coli K-12 bl395 MG1655
[0081 ] The term "(S)-3-hydroxybutanoyl-CoA hydro-lyase" (EC 4.1 ,1 .55) and "enoyl-CoA hydratase" (EC 4.2.1.17) refers to polypeptides having catalytic activity, where the catalytic activity is to transform a compound of (S)-3-hydroxybutanoyl-CoA into crotonyl-CoA. However, there may be another enzyme that is not cataloged in this set of enzymes that carry out the reaction described, so that enzyme will be considered as an analogue of "(S) -3- hydroxybutanoyl-CoA hydro-lyase and / or enoyl-CoA hydratase. Examples of enzymes that can carry out the conversion reaction of (S) -3-hydroxybutanoyl CoA to crotonyl-CoA are described in Table 6. The enzymes described in Table 6 are shown for reference, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc. [0082] Table 6. Examples of (S)-3-hydroxyhutanoyl-CoA hydro-lyase and/or enoyl-CoA hydratase which can be used to transform the (S)-3-hydroxybutanoyl-CoA to crotonyl-CoA.
Name of the. gene Organism
(S) -3 -hydroxybutanoyl - CoA hydro- Acinetobacter
lyase M3Cj__289 baumannii
(S) -3 -hydroxybutanoyl -CoA hydro- Vibrio parahaemolyticus lyase M634_03020
(S) -3 -hydroxybutanoyl -CoA hydro- Ralstonia eutropha lyase Reut C6351
(S) -3 -hydroxybutanoyl - CoA hydro- Xanthobacter
lyase Xaut 2482 autotrophicus
(S) -3 -hydroxybutanoyl - CoA hydro- Azospirillum sp . B510 lyase AZh 006240
erioyl CoA hydratase-AT3GO6860 Arabidopsis thaliana enoyl CoA hydratase-ECMDS42 1912 Escherichia coli K-12
MDS42
enovl CoA hydratase-TY21A 02420 Salmonella enterica enoyl CoA hydratase-YP 2417 Yersinia pestis
enoyl CoA hydratase-APA22 22590 Acetobacter [0083] The terms "trans-2-enoyl-CoA reductase" (EC 1.3, 1 .44) and "butyryl-CoA
dehydrogenase" or "crotonyl Coenzyme A reductase" (EC 1.3.1.86 and/or 1.3.8.1) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of crotonyl-CoA into butanoyl-CoA using NAD(P)H. However, there may be another enzyme that is not listed in this group of enzymes that cany out the described reaction, so that enzyme will be considered as an analogue of trans-2-enoyl-CoA reductases and'Or butyryl- CoA dehydrogenase. Examples of enzymes that can carry out the conversion reaction of crotonyl-CoA to butanoyl-CoA using NAD(P)H are described in Table 7. The enzymes described in Table 7 are shown for reference only, since there are many databases where oen can find more examples of these enzymes, such as the GeneBank
(http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0084] Table 7. Examples of trans-2-enoyl-CoA reductase and butyryl-CoA dehydrogenase which can be used to transform crotonyl-CoA to butanoyl-CoA.
Name of the gene Organism
trans-2-enoyl-CoA reductase Yersinia pestis
YPDSF 3930
trans-2-enoyl-CoA reductase. Serratia marcescens
SMWW4_vlcl9670
trans-2-enoyl-CoA reductase Clostridium
SMB G0472 acetobutylicumOS 1731 trans-2-enoyl-CoA reductase Clostridium
CA C0462 acetobutyli cum ATCC 824 trans-2-enoyl-CoA reductase. Clostridium perfringenes CPE2074 13
butyryl - CoA Dehydrogenase Strepto yces griseus SGR 1170
butyryl- CoA Dehydrogenase Streptomyces collinus B446 01590
butyryl -CoA dehydrogenase Amycolatopsis
AMES 1564 edi terranei S699
butyryl -CoA dehydrogenase Kitasatospora setae
KSE 56510
butyryl -CoA dehydrogenase Acidimicrobium Afer 0105 ferrooxidans
[0085] The terms "butanal dehydrogenase" (EC 1.2.1.57) and/or "acetaldehyde
dehydrogenase (EC 1.2.1.10) refers to a polypeptide having catalytic activity, where this catalytic activity is to transforai a compound of butanoyl-CoA into butanal using NAD(P)H. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of butanal dehydrogenase and/or acetaldehyde dehydrogenase. Examples of enzymes that can carry out the conversion reaction of butanoyl-CoA to butanal are described in Table 8. The enzymes described in Table 8 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
(http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0086] Table 8. Examples of butanal dehydrogenase and/or acetaldehyde dehydrogenase which can be used to transform el butanoyl-CoA to butanal.
Figure imgf000023_0001
[0087] The term "alcohol dehydrogenase" (EC 1.1.1.1 and/or EC 1.1.1.2) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of butanal into butanol using NAD(P)H. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of alcohol dehydrogenase. Examples of enzymes that can carry out the conversion reaction of isobutyraldehyde to butanol using NAD(P)H are described in Table 9. The e zymes described in Table 9 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0088] Table 9. Examples of alcohol dehydrogenase which can be used to transform butanai to butanol.
Figure imgf000024_0001
[0089] The term "pyruvate formate-lyase" or "formate acetyl transferase" refers to a polypeptide (EC 2.3.1.54) having catalytic activity, where this catalytic activity is to transform a compound of pyruvate into acetyl-CoA and formate. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of the enzyme pyruvate formate- lyase. Examples of enzymes that can cany out the conversion reaction of pyruvate to acetyl- CoA are described in Table 10. The enzymes described in Table 10 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0090] Table 10. Examples of the enzyme pyruvate formate-lyase which can be used to transform pyruvate to acetyl-CoA.
Figure imgf000025_0001
[0091] The term "formate dehydrogenase" refers to a polypeptide (EC 1.2.1.43) having catalytic activity where this catalytic activity is to transform a compound of formate to C02, using a molecule of NAD(P)+, However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of the enzyme formate dehydrogenase. Examples of enzymes that can carry out the conversion reaction of formate to C02 are described in Table 1 1 , The enzymes described in Table 1 1 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
(http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
Table 1 1. Examples of the enzyme formate dehydrogenase which can be used to transform formate to C02.
Figure imgf000026_0001
[0093] The term "acetolactate synthase" (EC 2.2.1.6) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of pymvate into 2- acetolactate. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of acetolactate synthase. Examples of enzymes that can carry out the conversion reaction of pyruvate to 2-aeeto!aetate are described in Table 12. The enzymes described in Table 12 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda-enzymes.org), etc. [0094] Table 12. Examples of acetolactate synthase which can be used to transform pyruvate to 2-acetolactate.
Figure imgf000027_0001
[0095] The term "ketoacid reductoisomerase" (EC 1.1.1 .86) refers to a polypeptide having catalytic activity, where this catalytic activity is to transfomi a compound of acetolactate into 2,3-dihydroxyvalerate. However, there may be another enzyme that is not listed in this group of enzymes that cany out the described reaction, so that enzyme will be considered as an analogue of ketoacid reductoisomerase. Examples of enzymes that can cany out the conversion reaction of 2-acetolactate to 2,3-dihydroxyvalerate are described in Table 13. The enzymes described in Table 13 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank
(http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[0096] Table 13. Examples of ketoacid reductoisomerase which can be used to transform 2- acetolactate to 2,3-dihydroxyvalerate. Name of the gene Organism
Keto acid Escherichia coli str. K-12 substr. reductoisomerase ilvC MG1655
Keto acid Escherichia coli str. K-12 substr. reductoisomerase ilvC VI3110
Keto acid Corynebacterium glutamicum ATCC 13032 reductoisomerase ilvC
Keto acid Corynebacterium glutamicum K051 reductoisomerase ilvC
Keto acid Salmonella enterica subsp. serovar reductoisomerase ilvC Typhimurium str.L 2
Keto acid Saccharomyces cerevisiae chromosome reductoisomerase ilv5 XII S288c
Keto acid Campylobacter jejuni RM1221
reductoisomerase ilvC
Keto acid Methylococcus capsulatus str. Bath reductoisomerase ilvC
Keto acid Shewanella oneidensis MR-1
reductoisomerase ilvC
Keto acid Dehalococcoides ethenogenes 195 reductoisomerase ilvC
Keto acid Carboxydothermus hydrogeneoformans Z- reductoisomerase ilvC 2901
Keto acid Listeria monocytogenes serotype 4b reductoisomerase ilvC str. F2365
Keto acid Geobacter sulfurreducens PCA
reductoisomerase ilvC
Keto acid Streptomyces avermitilis MA-4680 reductoisomerase ilvC
Keto acid Pseudomonas aeruginosa PAOl
reductoisomerase ilvC
[0097] The term "dihydroxyacid dehydratase" (EC 4.2, 1.9) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of 2,3- dihydroxyvalerate into ketoisovalerate. However, there may be another enzyme that is not listed in this group of enzymes that carry out the described reaction, so that enzyme will be considered as an analogue of dihydroxyacid dehydratase. Examples of enzymes that can cany out the conversion reaction of 2, 3 -dihydroxy valerate to ketoisovalerate are described in Table 14. The enzymes described in Table 14 are shown for reference only, since there are many- databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
Table 14. Examples of dihydroxyacid dehydratase which can be used to transfomi
2,3-dihydroxyvalerate to ketoisovalerate.
Figure imgf000029_0001
[0099] The term "2-oxoisovalerate dehydrogenase" (EC 1.2.1 ,25), t'3-methy]-2-oxobutanate dehydrogenase" or "ketoacid-dehydrogenase" (EC 1.2.4.4) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of ketoisovalerate into isobutyryi-CoA, using a molecule of NAD(P) . However, there may be another enzyme thai is not listed in this group of enzymes that cam' out the described reaction, so that enzyme will be considered as an analogue of 2-oxoisovalerate dehydrogenase. Examples of enzymes that can cany out the conversion reaction of ketoisovalerate to isobutyryl-CoA are described in Table 15. The enzymes described in Table 15 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes
(http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
00100] Table 15. Examples of 2-oxoisovalerate dehydrogenase which can be used to transform ketoisovalerate to isobutyryl-CoA.
Figure imgf000030_0001
[00101] The terms "isobutyryl-CoA mutase" and "isobutyryl-Coenzyme A mutase" (EC
5.4.99.13), refers to a polypeptide having catalytic activity, where this catalytic activity' is to transform a compound of isobutyryl-CoA into butyryl-CoA. However, there may be another enzyme that is not listed in this group of enzymes that cam' out the described reaction, so that enzyme will be considered as an analogue of isobutyryl-CoA mutase. Examples of enzymes that can cany out the conversion reaction of isobutyryl-CoA to butyryl-CoA are described in Table 16. The enzymes described in Table 6 are shown for reference only, since there are many databases where one can find more examples of these enzymes, such as the GeneBank (http://www.ncbi.nlm.nih.gov), the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp), the Braunschweig Enzyme Database (http://www.brenda- enzymes.org), etc.
[00102] Table 16. Examples of isobutyryl-CoA mutase which can be used to transform isobutyry]-CoA to butyryl-CoA.
Figure imgf000031_0001
[00103] The present invention refers to a method wherein a multi-enzyme system produces butanol starting from lactate sequentially coupled with a regeneration system of NAD (P) + / NAD (P) E and / or regeneration of acetyl-CoA / Co A (Figure 1). This transformation can be performed in a container or reactor where the whole process can be carried out continuously, semicontinuously or in batch.
[00104] Furthermore, the present invention overcomes the deficiencies of the prior art by providing polypeptides that, with an experimental performance equal or lower than the theoretical yield, transform lactate sequentially to butanol.
[00105] Furthermore, the present invention can use smaller amounts than stoichiometrically required of NAD (P) +, NAD (P) H and Co A to perform the process described above, because the method of the present invention allows the regeneration of NAD(P)+/NAD(P)H and / or the regeneration of acetyl -CoA/CoA in three different ways: a) during the conversion of lactate to pyruvate, of pyruvate to acetyl-CoA, acetoacetyl-CoA to (S) -3- hydroxybutanoyl-CoA, of crotonyl-CoA to butanoyl-CoA, of butanoyl-CoA to butanal and butanal to butanol; b) during the conversion of lactate to pyruvate, formate to CG2, of acetoacetyl-CoA to (S)-3-hydroxybutanoyl-CoA, of crotonyl-CoA to butanoyl-CoA, of butanoyl-CoA to butanal and of butanal to butanol; c) during the conversion of lactate to pyruvate, of acetoiactate to hydroxy valerate, of ketoisovalerate to isobutyryl-CoA, of butanoyl-CoA to butanal and of butanal to butanol ,
[00106] In rum, the method of the present invention may employ unitary operations recirculating to system NAD (P) + and /' or NAD (P) H and /' or CoA, allowing lower amounts than those established by the stoichiometry amounts to be transformed to larger amounts of lactate to butanol
[00107] The present invention may employ the enzymes lactate dehydrogenase (EC 1.1 , 1 .27 and/or EC 1.1.1.28), pyruvate dehydrogenase (EC 1.2.4.1 , EC 2.3.1.12, EC 1.8.1.4 and EC 1.2.1.51), acetoacetyl-CoA thiolase (EC 2.3.1.9), hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 and or EC 1.1.1.157), (S)-3-hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and or enoyl-CoA hydratase (EC 4.2.1 .17), trans-2-enoyl-CoA reductases (EC 1 .3.1.44) and or butyryl-CoA dehydrogenase (EC 1.3.1.86 and or EC 1.3.8.1), butanal dehydrogenase (EC 1.2.1.57) and or acetaldehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1 .1.1 and/or EC 1 .1.1.2) and or its analogs to transform lactate into butanol , wherein the amount of NAD (P) + added to the system may be less than the amount set by the
stoichiometry for the conversion of lactate into butanol, and wherein the experimental yield obtained in the transformation of lactate into butanol is equal or lower than theoretical material usage (0.41 1 grams of butanol per gram of lactate).
[00108] At the same time, the present invention may also employ the enzymes lactate dehydrogenase (EC 1.1.1.27 and or EC 1.1.1.28), pyruvate formate-iyase (EC 2.3.1.54), formate dehydrogenase (EC 1.2.1.43), acetoacetyl-CoA thiolase (EC 2.3.1.9),
hydroxybutyryl-CoA dehydrogenase (EC 1.1 .1.35 and/or EC 1 .1 .1.157), (S)-3- hydroxybutanoyl-CoA hydro-lyase (EC 4.2.1.55) and/or enoyl-CoA hydratase (EC 4.2.1.17), trans-2-enoyl-CoA reductase (EC 1.3.1.44) and/or hutyryl-CoA dehydrogenase (EC 1 .3.1 ,86 and/or EC 1.3.8.1 ), butanal dehydrogenase (EC 1.2.1.57) and/or acetaidehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2) and/or its analogs to transform lactate into butanol, wherein the amount of NAD (P) + added to the system may be less than the amount set by the stoichiometry for conversion of lactate into butanol, and wherein the experimental yield obtained in the transformation of lactate to butanol is equal or lower than theoretical material usage (0.41 1 grams of butanol per gram of lactate).
[00109] Also, the present invention may employ the enzymes lactate dehydrogenase (EC 1 .1.1 .27 and/or EC 1 .1.1 .28), acetolactate synthase (EC 2.2.1.6), ketoacid reductoisomerase (EC 1.1.1.86), dihydroxyacid dehydratase (EC 4.2.1.9), 2-oxoisovalerato dehydrogenase (EC 1.2.1.25) and/or ketolacid dehydrogenase (EC 1.2.4.4), isobutyryl-Coenzyme A mutase (EC 5.4.99.13), butanal dehydrogenase (EC 1 .2.1.57) and/or acetaidehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1 .1 .1 and/or EC 1 .1 .1.2) and/or its analogs to transform lactate into butanol, wherein the quantity of NAD(P)" added to the system may be less than the amount set by the stoichiometry for the conversion of lactate into butanol, and wherein the experimental yield obtained in the transformation of lactate to butanol is equal or lower than theoretical material usage (0.41 1 gr of butanol for a gram of lactate)
[00110] In another embodiment of the present invention, there is provided a method for the production of butanol from lactate, engaged with a system of regeneration of
NAD(P) 7NAD(P)H and the regeneration of acetyl-CoA/CoA, wherein the operation mode is continued using free enzymes. The enzyme mixture may be any of those described above. This method comprises different stages which are described below:
[00111] I. A mixture is prepared continuously starting from water, lactate, the enzyme mixture, NAD(P) 7NAD(P)H, CoA and the cofactors and coenzymes that uses the enzymes to perform catalysis. The cofactors and coenzymes depend on the nature of each enzyme. In Table 17 are shown some of the cofactors and coenzymes used by the different enzymes in the present invention. The cofactors and coenzymes described in Table 17 are examples, this does not exempt other cofactors or coenzymes to be used by enzymes to perform catalysis.
[00112] II. The flow obtained from stage I is passed continuously through a reactor that stably maintains the conditions of reaction of pH between 4 and 10, preferably between 6 and 8 and at a temperature between 15°C and 40°C, preferably between 25°C and 37°C . When this current enters the reactor, it carries out the production of butanol from lactate with a conversion efficiency equal to or less than 100%.
[00113] III. At the outflow of the reactor enriched with butanol and depleted of lactate, one can separate the cofactors, the coenzymes and enzymes by passing it through a separation system. The enzymes, coenzymes and cofactors form a concentrated stream which can be recycled to step I or else to the reactor.
[00114] IV. At the outflow of the reactor enriched with butanol and depleted of lactate, one can separate the cofactors, the coenzymes and enzymes by passing it through a separation system. The separation system can be: a membrane system (reverse osmosis, nanofiStration, ultrafiltration, etc.), distillation, evaporation or any other system that allows the separation of molecules either by size or by any of its physiochemical properties.
[00115] Table 17. Coenzymes and cofactors used by the enzymes to form the enzyme mixture
Enzyme name Source cofactors coenzymes
Pyruvate Escherichia coli Mg 2+ THPP, FAD dehydrogenase
Pyruvate Saccharomyces THPP, FAD dehydrogenase cerevisiae
Pyruvate Azotobacter Ma 2+ THPP dehydrogeriase vinelandii
Pyruvate Salmonella enterica THPP dehydrogenase subsp enterica
serovar Typhimurium
Pyruvate Enterobacter cloacae THPP dehydrogenase
Figure imgf000035_0001
dehydrogenase cerevisiae
[00116] In another embodiment of the present invention, another method is provided for the production of butanol out of lactate, coupled with a system of regeneration of
NAD(P)7NAD(P)H and the regeneration of acetyl-CoA/CoA, wherein the mode of operation is continued using immobilized enzymes. The mixture of immobilized enzymes can be any of those described above. The immobilization of enzymes on the support can be carried out by any method that is appropriate. Table 18 shows some of the supports used to immobil ize enzymes. The supports described in Table 18 are examples of some supports, this does not exclude other materials that are suitable for the immobilization of enzymes and which are not in Table 18. This method comprises different stages which are described below:
[00117] I. A mixture of the enzymes are immobilized on a support. The support can also be multiple supports of the same type with different numbers of enzymes, or may be supports of different types, sizes or chemical compositions and each support containing one or more enzymes, such that in the reactor are all enzymes either on the same support or on different supports. Coenzymes and cofactors may also be present on the support. Once the enzymes are immobilized, these immobilized enzymes and their supports will be added to the reactor.
[00118] II. A mixture is prepared continuously from water, lactate and NAD (P) T /'
NAD (P) H. Cofactors and coenzymes employed by each enzyme to perform catalysis depend on the nature of each enzyme. Some of the cofactors and coenzymes employed by the different enzymes used in the present invention are shown in Table 17. Cofactors and coenzymes described in Table 17 are examples, this does not exclude other cofactors or coenzymes that could be used by enzymes to perform catalysis.
[00119] III. The stream exiting stage II, is passed continuously through the reactor containing the immobilized enzymes, so that this flow is continuously in contact with said enzymes, stably maintaining the conditions of reaction at pH between 4 and 10, preferably between 6 and 8 and at a temperature between 15 ° C and 40 ° C, preferably between 25 ° C and 37 ° C. When this stream is contacted with enzymes, the production of butanol from lactate is carried out with a conversion efficiency equal to or less than 100%. Preferably the support is maintained within the reactor, although the output of the reactor support does not imply that this support can not be recirculated to the reactor.
[00120] IV. The outflow of stage III, enriched and depleted butanol lactate, can go through a separation system, where the co - enzymes and cofactors butanol and water are separated. Coenzymes and cofactors form a concentrated stream which can be recycled to stage II, or to the enzyme reactor.
[00121] V. Regarding the outflow of stage III, whether or not coenzymes and cofactors have been separated, one can separate the fraction of butanol in another separation system. This system generates two streams, a butanol water stream and another stream,
[00122] Separation systems mentioned in stages IV and V can be: membrane system (reverse osmosis, nanofiltration, ultrafiltration, etc.), distillation, evaporation or any other system which al lows the separation of molecules, either by their size or for some of its
physicochemical properties.
[00123] Table 18. Supports used to immobilize enzymes.
Figure imgf000037_0001
[00124] EXAMPLES
[00125] The following examples are intended to clarify the present invention. It should be understood that the fol lowing examples do not constitute a limitation on the scope of the present invention. From the description of the invention and the following examples, a person skilled in the field of the invention can make some modifications which anyway remain within the scope protected by the present invention.
[00126] Example 1 . Quantification of enzyme activity
[00127] To determine the enzymatic activity of different enzymes, first the different genes of the enzymes were cloned into commercial expression vectors, such as the DUET (Merck, USA) series, following the protocols described in Green and Sarabrook, 2010. Subsequently enzymes were purified according to protocols described in Sambrook and Green, 2010.
[00128] Enzyme assays and results are described below:
[00129] a) Lactate dehydrogenase (EC 1 .1.1.27 and / or EC 1 .1.1.28)
[00130] Lactate dehydrogenase can transform lactate to pynivate using NAD (P) " so the test was conducted by varying the initial concentrations of lactate, NAD (P) +' pH and temperature following the protocols described in the literature (CetineS et al ., 2013).Four enzymes of various microorganisms were used (Escherichia coli APEC 01, Escherichia coli PMV-1, Lactobacillus lactis subsp. lactis IL1403 and Streptococcus pneumoniae AP200). The conditions tested are shown in Table 19.
[00131 ] Table 19, Reaction conditions tested for lactate dehydrogenase enzyme (EC 1.1.1 .27 and / or EC 1.1.1.28).
Figure imgf000038_0001
[00132] In all tests performed conversion of lactate to pynivate production was observed as NAD (P) H after 1 hour.
[00133] ) Pyruvate dehydrogenase complex (EC 1 .2.1.51 , EC 1 .2.4.1 , EC 2.3.1 .12 and
EC 1.8.1.4)
[00134] The pyruvate dehydrogenase complex transforms pynivate into acetyl-CoA, using a molecule of NAD (P) ' so the test was conducted by varying the initial concentrations of pyruvate, NAD (P) +' CoA, pH and temperature following the protocols described in the literature (Nemeria et al., 2001). Tliree enzymes from different microorganisms were used (Escherichia coli K-12 MG1655, Corynebacterium sp. ATCC 6931 and Lactobacillus reuleri). The conditions tested are shown in Table 20.
[00135] Table 20.Reaction conditions tested for pyruvate dehydrogenase complex (EC 1.2.4.1 , EC 2.3.1.12, EC 1.8.1.4 and EC 1.2.1.51).
Figure imgf000039_0001
[00136] In all trials consumption both NAD (P) was observed as pyruvate after one hour reaction.
[00137] c) Acetyl-CoA thiolase (2.3.1.9)
[00138] Acetyl-CoA thiolase enzyme converts two molecules of acetyl-CoA in a molecule of acetoacetyl-CoA, so the test was conducted by varying the initial concentrations of acetyl- CoA, pH and temperature following the protocols described in the literature (Huth et al., 1975). Three enzymes from different microorganisms were used (.: H38 042 Xanthomonas campestris pv campestris ATCC 33913, Shigella sonnei and Escherichia coli 044 Ss046). The conditions tested are shown in Table 21.
[00139] Table 21. Reaction conditions tested for enzyme acetyl-CoA thiolase (2.3.3.9). Variable test conditions
PH 5, 7, and 10
Temperature (°C) 5, 25 and 55
Acetyl -CoA (g/L) 1, 25 and 75
[00140] In all trials consumption of acetyl CoA it was observed after one hour reaction.
[00141] d) 3-hydroxybutyryl-CoA dehydrogenase (EC 1 , 1.1.35 and / or 1.1.1.157) [00142] 3-hydroxybutyryl-CoA dehydrogenase converts acetoacetyl-CoA to 3- hydroxybutyryl-CoA, using a molecule of NAD (P) H, so the test was conducted by varying the initial concentrations of acetoacetyl-CoA, NAD (P) H , pH and temperature following the protocols described in the literature (Madan et ai., 1973). Three enzymes from different microorganisms were used (: H2 12009, Xanthomonas albilineans, Escherichia coli 0103 Aspergillus niger). The conditions tested are shown in Table 22.
[00143]
[00144] Table 22.Reaction conditions tested for hydroxybutyryl-3-CoA dehydrogenase (EC 1.1.1.35).
Figure imgf000040_0001
[00145] In all trials consumption NAD (P) H after 30 minutes reaction was observed. [00146] e) Enoyi CoA hydratase (EC 4.2.1.17)
[00147] The enoyl-CoA hydratase can transform 3-hydroxybutyryl-CoA to crotonyl-CoA, so the test was conducted by varying the initial concentrations of 3-hydroxybutyryl-CoA, pH and temperature following the protocols described in the literature (Fukui et al., 1998). Three enzymes from different microorganisms were used (Acinetobacter baumannii, and
Arabidopsis lhaliana Escherichia coli K-12 MDS42). The conditions tested are shown in Table 23.
[00148] Table 23.Reaction conditions tested for enzyme enoyl-CoA hydratase (EC 4.2.1.17).
Figure imgf000041_0001
[00149] In all trials consumption hydroxybutyryl-CoA was observed after 30 minutes of reaction.
[00150] f) Butyryl-CoA dehydrogenase (EC 1.3.1.44, 1.3.1.86 and / or 1.3.8.1)
[00151] The butyryl-CoA dehydrogenase enzyme converts crotonyl-CoA to butanoyi-CoA using a molecule of NAD (P) H, so the test was conducted by varying the initial
concentrations of crotonyl-CoA, NAD (P) H, pH and temperature following the protocols described in the literature (Hu et ai., 2013). Three enzymes from different microorganisms were used (Clostridium acetobutylicum DSM 1731 , Clostridium perfringenes Streptomyces griseus and 13). The conditions tested are shown in Table 24.
[00152] Table 24, Reaction conditions tested for butanoyl-CoA oxidoreductase (EC
1.3.1.44).
Variable test conditions
pH 2, 5, 7, 10 and 12
Temperature. (°C) 5, 15, 25, 35, 45 and 55 Crotonyl-CoA (g/L) 1 , 25 , 50 and 75
NAD(P)H (g/L) 1 , 5, and 10
[00153] In all trials consumption NAD (P) H after 30 minutes reaction was observed.
[00154] h) Butyraidehyde dehydrogenase (EC 1 .2.1.57 and / or EC 1 .2.1.10)
[00155] Dehydrogenase butanal enzyme transforms butanoyl-CoA to butanal using a molecule of NAD (P) H, so the test was conducted by varying the initial concentrations of butanoyl-CoA, NAD (P) H, pH and temperature following the protocols described in the literature (Palosaari and Rogers, 1988). Three enzymes from different microorganisms were used. (: Closiridhim acetobutylicum DSM SE15 H5 1731 , Escherichia coli and Salmonella enterica 0150). The conditions tested are shown in Table 25.
[00156] Table 25.Reaction conditions tested for butanal dehydrogenase enzyme (EC
1.2.1.57).
Figure imgf000042_0001
[00157] In all trials consumption NAD (P) H after 30 minutes reaction was observed.
[00158] i) Alcohol dehydrogenase (EC 1.1 .1.1 and / or EC 1.1 .1.2)
[00159] Alcohol dehydrogenase can transform isobutyraldehyde into isobutanol using NAD (P) H, so the test was conducted by varying the initial concentrations of isobutyraldehyde, NAD (P) H, pH and temperature following the protocols described in the literature (Atsumi et at., 2010), Three enzymes from different microorganisms were used (Escherichia coli K-l 2 MDS42, Bacillus cereus and Clostridium acetobutylicum DSM B4264 1731). The conditions tested are shown in Table 26.
[00160] Table 26, Reaction conditions tested for the enzyme alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1.1.1.2)
Figure imgf000043_0001
[00161 ] In all tests performed both isobutyraldehyde to isobutanol conversion was observed after 15 minutes as reaction consuming NAD (P) H.
[00162] j) Pyruvate formate -lyase (EC 2.3.1.54)
[00163] The enzynie pyruvate formate lyase converts pyruvate to Acetyl-CoA and C02, so the test was conducted by varying the initial concentrations of pymvate, pH and temperature following the protocols described in the literature (Takahashi, et al., 1982) . Three enzymes from different microorganisms were used (lucimarinus Ostreococcus, Baihycoccus Prasinos and Escherichia coli K-l 2 MG1655), The conditions tested are shown in Table 27.
[00164] Table 27.Reaction conditions tested for enzyme pyruvate formate lyase (EC 2.3.1.54)
Variable test conditions
PH 5, 7 and 10
Temperature (°C) 5 , 35 and 55
Pyruvate (g/L) 1, 50 and 100 [00165] In all tests performed pyruvate consumption was observed after one hour of reaction.
[00166] k) Formate dehydrogenase (EC 1 .2.1 ,43)
[00167] The enzynie formate dehydrogenase transforms the formate into CO 2 using a molecule of NAD (P) +' so the test was conducted by varying the initial concentrations of formate, NAD (P) +' pH and temperature following the protocols described in literature (Hatrongjit and Packdibamrung, 2010). Three enzymes from different microorganisms were used (Wolinella succinogenes, Su!furicurvum kujiense and Geohacter sulfurreducens PC A). The conditions tested are shown in Table 28.
[00168] Table 28.Reaction conditions tested for the enzyme fomiate dehydrogenase (EC 1.2.1.43).
Figure imgf000044_0001
[00169] In all tests conducted formate consumption and NAD (P) T was observed after 2 hours of reaction.
[00170] 1) Acetolactate synthase (EC 2.2.1.6)
[00171] Acetolactate synthase converts pyruvate into 2-acetolactate, so the test was conducted by varying the initial concentrations of pyruvate, pH and temperature fol lowing the protocols described in the literature (Holtzclaw and Chapman, 1975; Barak et al, 1987; Atsumi et to the. 2009). Three enzymes from different microorganisms were used (Escherichia coli were used K-12 MG1655, Bacillus suhtilis subsp. suhtilis sir. 168 and Saccharomyces cerevisiae S288c). The conditions tested are shown in Table 29.
[00172] Table 29.Reaction conditions tested for the enzyme acetoSactate synthase (EC 2,2.1.6),
Figure imgf000045_0001
[00173] In all tests performed pyruvate consumption was observed after 20 minutes of reaction,
[00174] m) Keto acid reductoisomerase (EC 1.1.1.86) and dihydroxy acid dehydratase (EC 4.2.1.9)
[00175] On one hand, the keto acid reductoisomerase 2-acetolactate transforms 2,3- dihydroxy valerate while dihydroxyacid dehydratase transforms hydroxy valerate into ketoisovaierate. Because the 2-acetoiactate is not a commercial compound as 2,3- dihydroxy valerate is an unstable compound, the determination of the activity of these two enzymes was measured indirectly by coupling a test acetolactate synthase with keto acid reductoisomerase and dihydroxy acid dehydratase. This was done by varying the initial concentrations of pyruvate, NAD (P) H, pH and temperaUire using as a basis the protocols described in the literature (Flint et al . , 1993; Bastian et al, 201 1 ;.. Li et al, 201 1)
.Reductoisomerase combination of two enzymes and two dihydroxy keto acid dehydratase enzymes from different microorganisms were used (Escherichia coli K-12 MG1655, Escherichia coli UTI89, Staphylococcus aureus, Corynebacterium glutamicum A TCC 13032). The conditions tested are shown in Table 30. [00176] Table 30.Reaction conditions tested for ketoacid reductoisomerase (EC 1.1.1.86) and dihydroxy acid dehydratase (EC 4.2.1.9).
Figure imgf000046_0001
[00177] In all trials consumption pyruvate and NAD (P) H was observed after 2 hours of reaction.
[00178] n) 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 and / or EC 3.2.4.4)
[00179] The enzyme 2-oxoisovalerate dehydrogenase transforms ketoisovalerate to
isobutyryl-CoA, using a molecule of NAD (P) T' so the test was conducted by varying the initial concentrations of ketoisovalerate, NAD (P) f ' pH and temperature following the protocols described in the literature (Hakozaki et al., 2002). Three enzymes from different organisms were used (Streptomyces coelicolor and Thermoplasma acidophilum and
Oncorhynchns mykiss). The test conditions are shown in Table 31.
[00180] Table 31. Reaction conditions tested for the enzyme 2-oxoisovalerato dehydrogenase (EC 1.2.1.25 and / or EC 1.2.4.4).
Figure imgf000046_0002
[00181] In all tests we performed ketoisovalerate consumption and NAD (P) + was observed after one hour of reaction. [00182] o) Isobutyryl-CoA mutase (EC 5.4.99.13)
[00183] The isobutyryl-CoA mutase transforms isobutyryl-CoA to butyryl-CoA, so the test was conducted by varying the initial concentrations of isobutyryl-CoA, pH and temperature following the protocols described in the literature (Ratnatilleke et al., 1999) . 2 enzymes from different organisms were used (Streptomyces coelicolor A3 (2) and Streptomyces cinnamonensis) . The conditions tested are shown in Table 32.
[00184] Table 32. Reaction conditions tested for isobutyryl-CoA mutase (EC 5.4.99.13).
Figure imgf000047_0001
[00185] In all trials consumption isobutyryl-CoA was observed after 45 minutes of reaction.
[00186] Example 2. Enzymatic production of butanol from lactate, coupled with a regeneration system of NAD (P) T / NAD (P) H and / or regeneration of acetyl-CoA / CoA in a batch process.
[00187] This example is intended to demonstrate the concept of regeneration of NAD (P) T / NAD (P) H and /' or regeneration of acetyl-CoA / CoA in a batch process when enzyme lactate dehydrogenase (EC 1.1.1.27 and / or EC 1.1.1.28), pyruvate dehydrogenase (EC 1.2.1.51 , EC 1.2.4.1, EC 2.3.1 .12 and / or EC 1.8.1.4), acetoacetyi-CoA thiolase (EC 2.3.1.9 ), 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1 .35 and / or EC 1.1.1 .157), enoyl-CoA hydratase (EC 4.1.1.55 and /' or EC 4.2.1.17), butyryl-CoA dehydrogenase (EC 1.3.1.44, EC 1.3.1.86 and / or 1.3.8.1), butanal dehydrogenase (EC 1.2.1.57 and / or EC 1.2.1.10), alcohol dehydrogenase (EC 1.1 .1.1 and / or EC 1.1 .1.2), or its analogs, according to the following reactions:
[00188] - Conversion reaction of lactate to pyruvate. In this reaction two molecules of NAD (P) H are formed from two molecules of NAD (P) + with the transformation of two molecules of lactate to two pyruvate molecules , this reaction can be performed by the enzyme lactate dehydrogenase ( EC 1.1.1.27 and / or EC 1.1.1.28) or any of its analogs:
[00189] 2C3H603 + 2NAD (P) + => 2C3H403 + 2NAD(P)H + 2H + (1 )
[00190] - Conversion reaction of pyruvate to acetyl-CoA. In this reaction two molecules of NAD (P) H are formed from two molecules of NAD (P) with the transformation of two molecules of pyruvate into two molecules of acetyl-CoA, this reaction can be carried out using pyruvate complex dehydrogenase (EC 1.2.1.51 , EC 1.2.4.1 , EC 1.8.1.4 and EC
2,3,1 ,12) or its analogs:
[00191] 2C3H 403 + 2NAD (P) + + 2CoA => 2C23H38 7Oi7P3S + 2NAD(P)H+2 H+ + 2C02 (2)
[00192] - Transformation reaction of acetyl-CoA into acetoacetyl-CoA. In this reaction two molecules of acetyl-CoA are converted into a molecule of acetoacetyl-CoA, this reaction can be carried out by acetoacetyl-CoA thiolase enzyme (EC 2.3.1.9) or its analogues:
[00193] 2C23H38N7Oi7P3S => C25H4oN7Oi8p3S + CoA (3) [00194] - Transformation reaction of acetoacetyl-CoA to 3-hydroxybutyryl-in CoA, In this reaction one molecule of NAD (P) + is formed from one moiecuie of NAD (P) H, with the transformation of a moiecuie of acetoacetyl-CoA in a moiecuie of 3-hydroxybutyryl-CoA, this reaction can be performed using hydroxybutyryl-3-CoA dehydrogenase (EC 1.1.1 .35 and / or EC 1 .1.1.1 57) or its analogs:
[00195] C25H40N7O18P3S + NAD(P)H + H :> C25H42 7O18P3S + NAD (P)
(4)
[00196] - Transformation reaction of 3-hydroxybutyryl-CoA to crotonyl-CoA. In this reaction one molecule of 3-hydroxybutyryl-CoA becomes a molecule of crotonyl-CoA and water, this reaction can be carried out using enoyl-CoA hydratase enzyme (EC 4.1.1.55 and / or EC 4.2, 1 .17 ) or its analogs:
00197] C25H42 7O18P3S > C25H40N7O17P3S (5)
[00198] - Transformation reaction of crotonyl-CoA to butanoyl-CoA. In this reaction one moiecuie of NAD (P) + is formed from one molecule of NAD (P) H, with the transformation of a molecule of crotonyl-CoA in a moiecuie of butanoyl-CoA, this reaction can be earned out using butyryl-CoA dehydrogenase (EC 1.3, 1 .44, EC 1.3, 1 .86 and / or 1 .3.8.1 ) or any of its analogs:
00199] C
Figure imgf000049_0001
+ NAD(P) (6) [00200] - Transformation reaction of hutanoy!-CoA into butanal. In this reaction molecule of NAD (P) + is formed from one moiecuie of NAD (P) H, with the transformation of a molecule butanoyl-CoA in a moiecuie butanal, this reaction can be carried out using butanal dehydrogenase enzyme (EC 1 ,2.1.57 and /' or EC 1.2.1.10) or any of its analogs:
)201] C25H42N7O17P3S + NAD(P)H + H + ==> C4HgO + NAD(P) + + Co A
[00202] - Transformation reaction of butanal into butanol . In this reaction one molecul e of NAD (P) + is formed from one molecule of NAD (P) H, with the formation of one moiecuie of butanol from a molecule butanal, this reaction can be performed using enzyme Alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1.1.1.2):
[00203] C4H80 + NAD(P)H + H => C4H 10O + NAD(P) (8) i] From the above equations, the overall stoichiometric equation of multi-enzymatic system has no loss or gain of NAD (P) +' NAD (P) H, CoA and / or acetyl-CoA, so that the overall reaction results in the use of two molecules of lactate to produce a molecule of butanol, obtaining an efficiency of 100% conversion according to the following reaction:
[00205] 2C :. H ,,() :; => C4H10O + 2CO 2 + H20 (9)
[00206] To attach the regeneration of NAD (P) f / NAD (P) H and / or regeneration of acetyl-CoA / CoA with the production of butanol from lactate, a batch system was developed for use in different operating conditions (Table 33). The reaction mixture was formulated with the enzyme mixture (Table 34), cofactors and coenzymes (at the concentrations described in the prior art), lactic acid and NAD (P) +. In Figure 2 the result of one of the conditions made to the batch process is shown. In this particular operating condition a volume of 1 L with an initial concentration of L-iactate 20 g / L and NAD (P) * 0.1 g / L was used. The concentration of each of the enzymes in the reaction mixture was adjusted to 1 g / L. Significantly, for the different conditions shown in Tables 33 and 34, similar behaviors were obtained.
[00207] In all cases, the reaction was initiated with the addition of lactate. From this point, the reaction medium was continuously sampled to determine the progress of the reaction. The variation in time of NADH and / or NADPH was measured on a Cary-60 spectrophotometer at a wavelength of 340 ran. Lactate and butanol were monitored by HPLC with refractive index detector using a Rezex ROA-organic acids H T column.
[00208] Table 33, Reaction conditions for the production of butanol from lactate in batch.
Figure imgf000051_0001
[00209] Table 34. Enzymes used to formulate the enzyme mixture to produce butanol from lactate.
Enzyme. Concentration in the enzyme mixture (g / L)
Lactate dehydrogenase (EC 1.1.1.27 1-10
and / or EC 1.1.1.28)
Pyruvate dehydrogenase (EC 1.2.1.51, 1-10 EC 1.2.4.1, EC 2,3,1,12 and / or EC
1.8.1.4)
Acetoacetyl-CoA thiolase (EC 1-10
2.3.1.9)
3 -Hydroxybutyryl -CoA dehydrogenase 1-10
(EC 1.1.1.35 and / or EC 1.1.1.157)
Enoyl-CoA hydratase (EC 4.1.1.55 and 1-10
/ or EC 4.2.1.17)
Butyryl-CoA dehydrogenase (EC 1-10
1.3.1.44 EC 1.3.1.86 and / or
1.3.8.1)
Butyraldehyde dehydrogenase (EC 1-10
1.2.1.57 and / or EC 1.2.1.10)
Alcohol dehydrogenase (EC 1.1.1.1 1-10
and / or 1.1.1.2)
[00210] Considering a system without regeneration of NADH, the theoretical stoichiometric balance indicates that to transform 19.55 g of pvmvate (equivalent to 20 g of lactate) to 8.22 g of butanol, 147.8 g of NADH and 170.5 g CoA would be needed. However, by
implementing a regeneration system of NAD (P) + / NAD (P) H and / or acetyl-CoA / CoA, as proposed in the present invention, 0.1 g of NAD (P) + alone was required and O. lg of CoA to transform 20 g lactate into 8,22 g butanol.
[00211] This shows that butanol production from lactate in a batch process, coupling the regeneration system NAD (P) + / NAD (P) H and / or acetyl-CoA / CoA is possible.
[00212] Example 3. Enzymatic production of butanol from lactate, coupled to the regeneration system NAD (P) + / NAD (P) H and / or acetyl-CoA /' CoA in a continuous process. [00213] This example is intended to demonstrate the concept of regeneration of NAD (P) T / NAD (P) H and /' or regeneration of acetyl-CoA / CoA in a continuous process, when the enzymes lactate dehydrogenase (EC 1.1. 1.27 and / or EC 1.1.1.28) pyruvate formate- lyase (EC 2.3.1.54), formate dehydrogenase (EC 1.2.1 .43), acetoacetyl-CoA thiolase (EC 2.3. 3.9), 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1 .1.35 and / or EC 1.1 .1.157), enoyl-CoA hydratase (EC 4.1.1.55 and /' or EC 4.2.1.17), butyryl-CoA dehydrogenase (EC 1.3.1.44, EC 1.3.1.86 and / or 1.3.8.1 ) , butanal dehydrogenase (EC 1.2.1.57 and / or EC 1.2.1.10), alcohol dehydrogenase (EC 1 . 3.1.1 and / or EC 1 .1.1.2) or any of its analogs are used, according to the following reactions:
[00214] - Conversion reaction of lactate to pyruvate. In this reaction two molecules of NAD (P) H are formed from two mo3ecules of NAD (P) with the transformation of two molecules of lactate into two molecules of pyruvate, this reaction can be performed using enzymes lactate dehydrogenase (EC 1.1.1.27 and / or EC 1.1.1.28) or any of its analogs:
[00215] 2C3H603 + 2NAD (P) + : < 2C3H4O3 + 2NAD (P) 1 ! · 21 1 (10)
[00216] - Conversion reaction of pyruvate to acetyl-CoA. In this reaction two pyruvate molecules are transformed into two molecules of acetyl-CoA, this reaction can be carried out using pyruvate formate-lyase enzyme (EC 2.3.1.54) or its analogues:
[00217] 2C3H4O3 + 2C0A • 2C23H38N7Oi7P3S + 2CH202 (1 1 ) [00218] - Conversion reaction of formate to C02, In this reaction two formate molecules are transformed into two molecules of C02 using two molecules of NAD (P) +' this reaction can be carried out using the enzyme formate dehydrogenase (EC 1.2.1.43) or its analogues:
+
[00219] 2 CH2O + 2NAD (?) T ==> 2CO, + 2NAD (?) H + 2H (12)
[00220] - Conversion reaction of acetyl-CoA into acetoacetyl-CoA. In this reaction two molecules of acetyl-CoA are converted in a molecule of acetoacetyl-CoA, this reaction can be carried out using acetoacetyl-CoA thiolase enzyme (EC 2.3.1.9) or its analogues:
[00221] 2C23H38 7O17P3S ==> C25H40 7O18P3S + CoA (13)
[00222] - Conversion reaction of acetoacetyl-CoA to 3-hydroxybutyryl-in CoA. in this reaction one molecule of NAD (P) + is formed from one molecule of NAD (P) H, with the transformation of a molecule of acetoacetyl-CoA in a molecule of 3-hydroxybutyryl-CoA, this reaction can be carried out using hydroxybutyryl-3-CoA dehydrogenase (EC 1.1.1.35 and / or EC 1.1.1.157) or its analogs:
00223] C25H4oN7GssP3S + NAD(P)H + H > C-H i2 7OisP.:S + NAD(P) (14)
[00224] - Conversion reaction of 3-hydroxybutyryl-CoA to crotonyl-CoA. In this reaction one molecule of 3-hydroxybutyryl-CoA becomes a molecule of crotonyl-CoA and water, this reaction can be carried out using enoyl-CoA hydratase enzyme (EC 4.1.1.55 and / or EC 4,2.1.17 ) or its analogs: [00225] C25H42N70i8P3S => CzsPL^OnPsS (15)
[00226] - Conversion reaction of crotonyl-CoA to butanoyi-CoA. In this reaction one molecule of NAD (P) 1 is formed from one molecule of NAD (P) H, with the transformation of a molecule of crotonyl-CoA in a molecule butanoyl-CoA, this reaction can be carried out using butyryl-CoA dehydrogenase (EC 1.3.1.44, EC 1.3.1.86 and / or 1.3.8.1) or any of its analogs:
[00227] C25H40N7O17P3S + N AD(P)H + H + => C25H42N7O17P3S + NAD(P) + (16)
[00228] - Conversion reaction of butanoyl-CoA to butanal. In this reaction a molecule of NAD(P)+ is formed from one molecule of NAD(P)H, with the transformation of a molecule butanoyl-CoA in a molecule of butanal, this reaction can be carried out using enzynie butanal dehydrogenase (EC 1.2.1.57 and / or EC 1.2.1.10) or any of its analogs:
[00229] C25H42N7O17P3S + NAD (P)H + H+==> G¾H80 + +NAD (P) + + CoA (17)
[00230] - Conversion reaction of butanal to butanol. In this reaction one molecule of NAD (P) is foraied from one molecule of NAD (P) H, with the formation of one molecule of butanol from a molecule butanal, this reaction can be performed using enzyme Alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1.1.1.2):
00231] C i! lsO + NAD(P)H + H 1 · C¾Hi0O + NAD(P) (18) [00232] From the above equations, the overall stoichiometric equation multi-enzymatic system has no loss or gain of NAD (P) +' NAD (P) H, CoA and / or acetyl-CoA, so that the overall reaction results in the use of two molecules of lactate to produce a molecule of butanol, obtaining an efficiency of 100% conversion according to the following reaction:
[00233] 2C3H603 => C4H10O + 2C02 + H20 (19)
[00234] Transforming lactate into butanol was carried out in a continuous reactor using free enzymes. The reaction mixture was formulated with the enzyme mixture (Table 35), cofactors and coenzymes (at the concentrations described in the prior art), lactic acid and NAD (P) +. The operating conditions of the reactor are shown in Table 36. The reactor inlet stream and reactor outlet stream were operated at the same flow rate, to have a continuous process.
[00235] In Figure 3 some results from different conditions carried out in mechanically stirred reactor (CSTR) for the continuous process, corresponding to an initial lactate concentration 200 g / L and NAD (P) + lg / L . The concentration of each of the enzymes in the reaction mixture was adjusted to 1 g / L. For all these operating conditions a volume 50 L was used, varying flow conditions.
[00236] Table 35. Enzymes used to formulate the enzyme mixture to produce butanol from lactate in a continuous process.
Enzyme Concentration in the enzyme mixture (g / L)
Lactate dehydrogenase (EC 1.1,1.27 1-10
and / or EC 1.1.1.28) Pyruvate formate- lyase (EC 2.3.1.54) 1-10
Formate dehydrogenase (EC 1.2.1.43) 1-10
Acetoacetyl-CoA thiolase (EC 1-10
2.3.1.9)
3 -Hydroxybutyryl -CoA dehydrogenase 1-10
(EC 1.1.1.35 and / or EC 1.1.1.157)
Enoyl-CoA hydratase (EC 4.1.1.55 and 1-10
/ or EC 4.2.1.17)
Butyryl-CoA dehydrogenase (EC 1-10
1.3.1.44 EC 1.3.1.86 and / or
1.3.8.1)
Butyraldehyde dehydrogenase (EC 1-10
1.2.1.57 and / or EC 1.2.1.10)
Alcohol dehydrogenase (EC 1.1.1.1 1-10
and / or 1.1.1.2)
[00237] Table 36, Reactor operating conditions for the production of butanol from lactate.
Figure imgf000057_0001
[00238] The reaction was initiated in the same way as in the batch process (see Example 2), immediately after the addition and removal began, continuously, from the reaction medium,
[00239] The output stream of the reactor was coupled to a membrane system that separated enzymes, cofactors and coenzymes, and butanol. Stream of enzymes, cofactors and coenzymes was recirculated to the reactor. [00240] In all the conditions listed in Tables 35 and 36 the evolution of reaction intermediates in the output stream of the reactor was monitored. The evolution of NAD (P) H was measured on a Cary-60 spectrophotometer at a wavelength of 340 nm. Lactate and butanoi were measured by HPLC with refractive index detector using a Rezex ROA-organic acids H + column.
[00241] As shown in Figure 3, the conversion efficiency did not change relative to the flow condition and was close to 100%. In the same manner as in Example 2, it was demonstrated that the enzymatic production of butanoi from lactate, coupled with the regeneration system of NAD (P) + / NAD (P) H and / or acetyl-CoA / Co A is possible, in the case of this example, using only 1 g /' L NADP + and 1 g / L CoA to transform 200 g /' L lactate with a continuous process.
[00242] It is noteworthy that for other conditions shown in Tables 35 and 36 very similar conversion efficiencies were obtained.
[00243] Example 4. Enzymatic production of butanoi from lactate, coupled to the
regeneration system NAD (P) / NADH and / or acetyl-CoA / CoA in a continuous process using immobilized enzymes.
[00244] This example is intended to demonstrate the concept of regeneration of NAD (P) + / NAD (P) H and / or regeneration of acetyl-CoA / CoA in a continuous process using immobilized enzymes, where the enzymes lactate dehydrogenase are used ( EC 1.1.1 .27 and / or EC 1.1.1 .28), acetolactate synthase (EC 2.2.1 ,6), keto acid reductoisomerase (EC 1.1 .1 .86), dihydroxy acid dehydratase (EC 4.2.1.9), 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 and / or EC 1 .2.4.4), isobutyryl-CoA mutase (EC 5.4.99.13), butanal dehydrogenase (EC 1.2.1.57 and / or EC 1.2.1.10), alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1.1.1.2) , or its analogs, according to the following reactions:
[00245] - Conversion reaction of lactate to pymvate. In this reaction two molecules of NAD (P) H are formed from two molecules of NAD (P) T' with the transformation of two molecules of lactate into two molecules of pyruvate, this reaction can be performed using enzymes lactate dehydrogenase (EC 1.1.1.27 and / or EC 1.1.1.28) or any of its analogs:
[00246] 2C3H603 + 2NAD(P) + ==> 2C3H403 + 2NAD(P)H + 2H + (20)
[00247] - Conversion reaction of pyruvate to acetolactate. In this reaction two pyruvate molecules are transformed into a molecule of acetolactate, this reaction can be carried out using ketoiactate synthase enzyme (EC 2.2.1.6) or its analogues:
[00248] 2C3H4O3 => C5H804 + C02 (21)
[00249] - Conversion reaction of acetolactate to 2,3-dihydroxyvalerate.In this reaction a molecule acetolactate becomes one molecule of 2,3 -dihydroxy valerate using a molecule of NAD (P) +' this reaction can be carried out using ketoacid reductoisomerase enzyme (EC 1.1.1.86) or its analogs:
00250] C5H804 + NAD(P)H + H • C5Hio04 + NAD (P) (22) [00251] - Conversion reaction of 2,3-dihydroxyvalerate to ketoisovalerate, In this reaction one molecule of 2,3-dihydroxyvalerate is transformed to ketoisovalerate molecule and a water molecule, this reaction can be carried out using dihydroxyacid dehydratase (EC 4.2.1.9) or its analogues:
00252] C5Hl0O4 C5Hg03 + H20 (23)
[00253] - Conversion reaction of ketoisovalerate to isobutyryl-CoA. In this reaction one molecule of NAD (P) H is formed from one molecule of NAD (P) T' with the transformation of a molecule ketoisovalerate into a molecule of isobutyryi-CoA, this reaction can be performed using enzyme 2-oxoisovaierato dehydrogenase (EC 1.2.1.25 and / or EC 1.2.4.4) or any of its analogs:
[00254] C5H803 + CoA + NAD(P) + ==> C^^O^S + NAD(P)H + H + +C02
(24)
[00255] - Conversion reaction of isobutyryl-CoA to butyryl-CoA. In this reaction one molecule of isobutyryl-CoA is converted into a butyryi-CoA molecule, this reaction can be carried out using isobutyryl-CoA mutase enzyme (EC 5.4.99.33) or its analogues:
56] (CH3)2CHCOSCoA ==> CH3(CH2)2COSCoA (25)
[00257] - Conversion reaction of butyryl-CoA to butanal. In this reaction a molecule of NAD (P) + is formed from one molecule of NA D (P) H, with the transformation of a molecule butanoyl-CoA into a molecule of butanal, this reaction can be carried out using butanal dehydrogenase enzyme (EC 1.2.1.57 and /' or EC 1.2.1.20) or any of its analogs:
58] C25H42N7O .7P3S + NAD(P)H + H 1 · C¾H80 + NAD(P) + + CoA (26)
[00259] - Conversion reaction of butanal to butanol. In this reaction one molecule of NAD (P) T is formed from one molecule of NAD (P) H, with the formation of one molecule of butanol from a molecule of butanal, this reaction can be performed using enzyme A lcohol dehydrogenase (EC 1.1 .1.1 and / or EC 1.1 .1.2):
[00260] C4H80 + N,ADiP)H + H ÷ ==> C4H|oO + NAD(P) + (27)
[00261] From the above equations, the overall stoichiometric equation multi-enzymatic system has no loss or gain of NAD (P) +' NAD (P) H, CoA and / or acetyl-CoA, so that the overall reaction results in the use of two molecules of lactate to produce a molecule of butanol, obtaining an efficiency of 100% conversion according to the following reaction:
[00262] 2C3H603 ==> C4H!0O + 2C02 + H20 (28)
[00263] Butanol production in continuous form from lactate was carried out in a reactor immobilizing each enzyme or enzyme mixture (Table 37) in different media (Table 18), varying the amounts of immobilized protein. The operating conditions which were used are shown in Table 38. The reaction mixture was formulated by mixing coenzymes (at the concentrations described in the prior art) immobilized enzymes (Table 38), cofactors and lactic acid and NAD (P) +. [00264] Table 37. Enzymes used to formulate the enzyme mixture to produce butanol from lactate in a continuous process with immobilized enzymes.
Figure imgf000062_0001
[00265] Table 38, Operating conditions of the continuous reactor with immobilized for processing enzymes lactate butanol.
[00266]
Reactor volume (L) 1-100
Amount of immobilized enzyme 0001-0.2
(g/g)■
Lactate (g/L) 1-300
Temperature (°C) 20-37
PH 6-8
NAD(P) + (g/L) 0.01-10
CoA (g/L) 0.01-10 [00267] The output current of the reactor was coupled to a reverse osmosis system that can recirculate a mixture of cofactors and coenzymes to the reactor and / or mixing tank. The initial concentration of NAD (P) T was 0.1 g / L, while the lactate concentration to the reactor inlet was varied according to Table 38. In all the conditions listed in Tables 37 and 38, the evolution of reaction intermediates along the tubular reactor was monitored. Changes in the NAD (P) H were measured on a Cary-60 spectrophotometer at a wavelength of 340 nm. Lactate and butanol were measured by HPLC with refractive index detector using a Rezex ROA-organic acids H 1 column.
[00268] In Figure 4 some results from different conditions made for continuous process using immobilized enzymes packed in a tubular reactor are shown. For all these conditions a trading volume 50 L was used, varying the conditions of feed flow, with a concentration of L- lactate to input 264 g / L and NAD (P) + 0.1 g / L. The amount of each of the enzymes by support amount was adjusted to 0.01 g / g.
[00269] For this particular case, the reaction began when the mixture of cofactors, coenzymes, lactate and NAD (P) ' entered the packed reactor.
[00270] As shown in Figure 4, lactate was transformed in butanol, under pressure through the packed tubular reactor, until 100% conversion efficiency. The same happened with the different operating conditions mentioned in the Tables 37 and 38. In the same way as in Examples 2 and 3, it was shown that the coupling of the en ymatic production of butanol from lactate, with the regeneration system NAD (P) + / NAD (P) H and / or of acetylCoA / CoA is possible using only 0.1 g / L NAD (P) + to convert 264 g / L lactate 108 g / L butanol.
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[00301] Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

CLAIMS:
1. A process for producing butanol, comprising:
A) mixing water, lactate, an enzyme mixture comprising at least one enzyme, at least one cofactor and at least one coenzyme, to prepare a reaction mixture;
B) caialytically reacting the reaction mixture for an amount of time sufficient to cause conversion of lactate into butanol: and
wherein the conversion of lactate into butanol in B) is associated with a regeneration system of NAD (P) + / NAD (P) H and / or acetyi-CoA / Co A,
2. The process of claim 1 , further comprising separating the butanol produced in B) from the reaction mixture.
3. The process of claim 1 wherein, sequentially in B):
the lactate is converted into pyruvate;
the pyruvate is converted into acetyl-CoA;
the acetyi-CoA is converted into acetoacetyl-CoA;
the acetoacetyl-CoA is converted into 3-hydroxybutyryl-CoA; the 3-hydroxyhutyryl-CoA is converted into crotonyl-CoA;
the crotonyl-CoA is converted into butyryi-CoA;
the butyryl-CoA is converted into butanal; and
the butanal is converted into butanol.
4. The process of claim 1 wherein, in B):
the lactate is converted into pyruvate; the pyruvate is converted to acetyl-CoA and formate;
the formate is converted into C02;
the acetyl-CoA is converted into acetoacetyl-CoA;
the acetoacetyl-CoA is converted into 3-hydroxybutyryl-CoA;
the 3-hydroxybutyryl-CoA is converted into crotonyl-CoA; the crotonyl-CoA is converted into butyryl-CoA;
the butyryl-CoA is converted into butanal; and
the butanal is converted into butanol.
5. The process of claim 1 wherein, in B):
the lactate is converted into pyruvate;
the pyruvate is converted into acetolactate;
the acetolactate is converted into 2,3-dihydroxyvalerate;
the 2,3-dihydroxyvalerate is converted into ketoisovalerate; the ketoisovalerate is converted into isobutyryl-CoA;
the isobutyryl-CoA is converted into butyryl-CoA;
the butyryl-CoA is converted into butanal; and
the butanal is converted into butanol.
6. The process of claim 1 wherein the yield of butanol from lactate is 98 to 100%.
7. The process of claim 1 wherein the enzyme mixture is prepared in a container before mixing in A).
8. The process of claim 7 wherein the container is a tube, a tank or a reactor or a combination thereof.
9. The process of claim 1 wherein in A), the mixture is carried out in a suitable container to prepare the reaction mixture.
10. The process of claim 9 wherein the container is a tube, a tank or a reactor or a combination thereof.
11. The process of claim 1 wherein in A), the mixture is carried out by an appropriate method to allow interaction between at least one enzyme and the substrates and / or compounds.
12. The process of claim 1 wherein in A), the mixture is held mechanically, pneumatically, hydrauiicaily or by a combination of any.
13. The process of claim 1 wherein, in B), the catalytic reactions are carried out in a suitable container to promote interactions between enzymes and substrates and / or compounds.
14. The process of claim 13 wherein, in B), the catalytic conversions are carried out in a stined tank, a plug flow reactor a fluidized bed reactor, packed bed reactor, or any combination thereof.
15. The process of claim 1 wherein, in B), the catalytic reactions are carried out in a pH range of 5 to 10.
16. The process of claim 2 wherein the butanol is separated from the reaction mixture by a method that separates molecules based on their physicochemical properties.
17. The process of claim 2 wherein the butanol is separated from the reaction mixture using membranes, distil lation, evaporation, or a combination of the above.
18. The process of claim 17 wherein the membranes can be reverse osmosis, pervaporation, ultrafiltration or nanofiltration.
19. The process of claim 1 wherein the process is carried out continuously, semicontinuously or batch manner.
20. The process of claim 1 wherein the lactate is L-iactate, D-lactate or a mixture of both.
21. The process of claim 1 wherein the lactate concentration in the reaction mixture is at least 1 g / L.
22. The process of claim 1 wherein the enzyme mixture contains at least one enzyme selected from the group consisting of: lactate dehydrogenase (EC 1.1.1.27 and /' or EC 1.1 .1.28), pyruvate dehydrogenase (EC 1.2.1 .51 , EC 1.2.4.1 , EC 2.3.1.12 and / or EC 1.8.1.4), acetoacetyl-CoA thioiase (EC 2.3.1.9), 3-hydroxybutyryl-CoA dehydrogenase (EC
1.1.1 .35 and / or EC 1.1 .1.157), enoyl-CoA hydratase (EC 4.1 .1 .55 and / or EC 4.2.1 .17), butyryl-CoA dehydrogenase (EC 1.3.1.44, EC 1.3.1.86 and / or 1.3.8.3), butanal
dehydrogenase (EC 1.2. 1.57 and / or EC 1.2.1.10), alcohol dehydrogenase (EC 1.1.1.1 and / or EC 1 .1.1.2) pyruvate formate lyase (EC 2.3.1.54), formate dehydrogenase (EC 1.2.1.43), acetolactate synthase (EC 2.2.1.6), ketoacid reductoisomerase (EC 1.1.1.86), dihydroxy acid dehydratase (EC 4.2.1.9), 2-oxoisovalerato dehydrogenase (EC 1.2.1.25 and / or EC 1.2.4.4), isobutyryl-CoA mutase (EC 5.4.99.13 ) or its analogs.
23. The process of claim 1 wherein at least one enzyme may be immobilized, entrapped, embedded, adhered, attached, secured, absorbed, adsorbed or otherwise attached or associated with a support.
24. The process of claim 23 wherein the support is at least one of the following types of media, zeolite, activated carbon, acrylamide, agarose, silica gel, silica, alginate or methacrylate polymers.
25. The process of claim 1 wherein the concentration of enzymes in the enzyme mixture is higher than 0.001 g / L.
26. The process of claim 23 wherein the concentration of enzymes on the support is greater than 0.001 grams of enzyme per gram of support.
27. The process of claim 1 wherein the amount of NAD (P) / NAD (P) II in the regeneration system is less than stoichiometricaliy required.
28. The process of claim 1 wherein the amount of acetyl CoA / CoA in the regeneration system is less than stoichiometrically required.
29. The process of claim 1 wherein the total amount of NAD (P) 1 and NAD (P) H used to convert two moles lactate in a mole of isobutanol is less than 1 mol.
30. The process of claim 1 wherein the total amount of acetyl CoA and CoA used to convert two moles lactate in a mole of isobutanol is less than 1 mol,
31. The process of claim 2 wherein the separation of butanoi from the reaction mixture comprises:
C- 1 ) separating the compounds obtained by the catalytic reactions in a first stream and a second stream, wherein the first stream contains butanoi and water, and the second stream does not contain butanoi; and
C-2) separating the first stream into two output streams, wherein the first output current contains butanoi and second output current contains water.
32. The process of claim 31 , wherein the second stream is recycled or reused by mixing it with the reaction mixture in A), B), or both.
33. The process of claim 31 , wherein, in C-l), the separation is carried out by an appropriate method to separate based on their physicochemicai properties molecules.
34. The process of claim 31 , wherein the separation is carried out by membranes, distillation, evaporation, or a combination of any of the above.
35. The process of claim 31, wherein, in C-2), the separation is carried out by an appropriate method to separate based on their physicochemicai properties molecules.
36. The process of claim 31, wherein the separations are carried out using membranes, distillation, evaporation, or a combination of any of the above.
37. A biofuel or biofuel precursor, prepared by the process of claim 1.
38. The biofuel or biofuel precursor of claim 37, wherein the biofuel or biofuel precursor meets the requirements of ASTM D7862.
39. An automotive engine fuel, comprising a mixture of hydrocarbons and a biofuel or biofuel precursor according to claim 37.
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