Patent Application
20210222209
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-CoA/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. Each year, because of increased demand, large amounts of these alcohols are produced in the petrochemical industry.
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.
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 acetobutylicum, Saccharomyces, Escherichia coli and Pseudomonas; see for example the following patents or patent applications, US20090155869A1, US20120149080A1, US20110236941A1, U.S. Pat. No. 9,096,872B2, CN101952430B, WO2012033334A3, and U.S. Pat. No. 9,005,953B2. 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:
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.
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, 0.411 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.
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 butanol. For example, overexpression of the genes crt, bed and hbd allows greater activity of the enzymes involved in the synthesis of butanol, as described in patent application WO2014135633A1.
4.—It is common and known in the art to remove and/or overexpress genes in many cases back to the metabolically unstable organisms.
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.
In reference to this, the European patent EP2204453B1 claims butanol 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 butanol 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.
Furthermore EP2700714A1 mentions a very similar scheme to EP2204453B1, but using at least 13 enzymes to carry out the process.
Accordingly, there is needed a process where the production of butanol 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.
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.
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.
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 acetyl-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 acetyl-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.
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 acetyl-CoA/CoA.
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:
For a clearer understanding of the object of the present invention, the following definitions and abbreviations are established.
The terms “lactic acid”, “lactate”, “2-hydroxy-propanoic acid” and “α-hydroxypropanoic acid” refers to the same molecule, wherein the said molecule has three carbons and holds the molecular formula H3C—CHOH—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-lactate or a mixture 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, Lactobacillus casei, Lactobacillus delbrueckii, 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 nitrile of lactic acid in a racemic form. The nitrile is saponified in the presence of water and excess sulfuric acid to give the free lactic acid.
The terms “pyruvate”, “pyruvic acid”, “2-oxopropanoic acid”, “propionic α-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).
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.
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 all realms and domains, as described in the prior technique, to help carry out enzymatic reactions.
The terms “acetyl-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 CH3COSCoA (C23H38N7O17P3S, CAS: 72-89-9).
The terms “acetoacetyl-CoA”, “acetoacetyl coenzyme A”, “3-acetoacetyl-CoA”, “acetoacetyl-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 (C25H40N7O18P3S, CAS: 1420-36-6).
The terms “(S)-3-hydroxybutanoyl-CoA”, “(S)-3-hydroxybutyryl-CoA”, “(3S)-3-hydroxybutanoyl-CoA”, “L-3-hydroxybutyryl-CoA”, “L(+)-beta-hydroxybutyroyl-CoA”, “β-hydroxybutyryl-CoA” and 3-hydroxybutyryl-coenzyme A” refer to a molecule, wherein said molecule has twenty five carbons and holds the molecular formula CH3CHOHCH2COSCoA (C25H42N7O18P3S, CAS: 2871-66-1).
The terms “crotonyl-CoA”, “crotonyl-coenzyme A”, “2-butenoyl-CoA”, “trans-but-2-enoyl-CoA”, “but-2-enoyl-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 (C25H40N7O17P3S, CAS: 992-67-6).
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 (C25H42N7O17P3S, CAS: 2140-48-9).
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(C4H8O, CAS: 123-72-8).
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(CH2O2, CAS: 123-72-8).
The terms “acetolactic-acid 2”, “2-acetolactate”, “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(C5H8O4, CAS: 7169-08-3).
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(C5H10O4 CAS: 1756-18-9).
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(C5H8O3, CAS: 759-05-7).
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 (C25H42N7O17P3S. CAS: 15621-60-0).
The terms “1-butanol”, “n-butanol”, “n-butyl alcohol”, “butyl alcohol”, “butanol”, “1-hydroxybutane”, “butyric alcohol”, “butyryl alcohol” and “butan-1-ol” refer to a molecule, wherein said molecule has four carbons and holds the molecular formula CH3(CH2)3OH(C4H10O, CAS: 71-36-3).
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.
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.
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.
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.
The term “experimental performance” refers to the amount of product that is obtained experimentally by a chemical reaction on the amount of substrate consumed.
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 0 to 100%.
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).
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 molecularly.
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 cell for the biological synthesis of enzymes.
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), Fluidized 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, semicontinuous 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 Mg2+, Fe2+, Zn2+, Na+, K+, Co2+, Ni2+, Mn2+, etc., depending on the particular enzyme.
The term “substrate” refers to the molecule on which an enzyme acts. The enzyme can be related and selective for the substrate.
The term “enzyme mix” refers to the set of enzymes found in the same solution, allowing the production of butanol from lactate.
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”
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 acetoacetyl-CoA by the enzyme acetoacetyl-CoA thiolase (EC 2.3.1.9), of acetoacetyl-CoA to (S)-3-hydroxybutanoyl-CoA by the enzyme 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 and/or EC 1.1.1.157), of (S)-3-hydroxybutanoyl-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 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 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 pyruvate dehydrogenase complex.
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 acetyltransferase (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 acetoacetyl-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-hydroxybutanoyl-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.
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-acetolactate to 2,3-hydroxyvalerate by the enzyme keto acid reductoisomerase (EC 1.1.1.86), of 2,3-dihydroxyvalerate to ketoisovalerate by the enzyme dihydroxyacid dehydratase (EC 4.2.1.9), of ketoisovalerate 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 butyryl-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 metabolic pathway of ketoacids.
The term “multienzymatic system” refers to a group of enzymes that sequentially transform the lactate into butanol.
The term “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.
The term “endogeneous gene” refers to a region of ADN coding for a protein found within the organism.
The term “overexpression” refers to increased expression levels of a protein encoded by an endogeneous gene or an exogeneous gene.
The term “regeneration of NAD(P)+/NAD(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)+ 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-CoA/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.
The term “free enzyme” refers to an enzyme distributed in a solution.
The term “free enzymes” refers to a group of enzymes distributed in a solution.
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.
The term “immobilized enzyme” refers to the enzyme attached, trapped, absorbed, adsorbed, bound, etc., by any physical or chemical method to a support.
The term “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)+. 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-lacate dehydrogenase. Examples of enzymes that can carry out the conversion reaction of L-lactate to pyruvate 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.
Escherichia coli APEC 01
Escherichia coli O45: K1: H7 S88
Escherichia coli IHE3034
Mus musculus
Cricetulus griseus
Pongo abelii
Canis familiaris
Staphylococcus aureus subsp. COL
aureus (MRSA)
Corynebacterium glutamicum K051
Sorangium cellulosum So ce 56
Bacillus subtilis subsp. subtilis
Lactococcus lactis subsp. lactis
Aspergillus fumigatus
Enterobacter sp. 638
Streptococcus pneumoniae AP200
The term “D-lactate dehydrogenase” (EC 1.1.1.28) refers to a polypeptide having catalytic activity, where the catalytic activity is to transform a compound of D-lactate into pyruvate using NAD(P)+. 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 D-lactate dehydrogenase. Examples of enzymes that can carry 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.
Escherichia coli K-12 MG1655
Escherichia coli O26: H11 11368
Escherichia coli PMV-1
Escherichia coli O145: H28 RM13514
Shigella boydii Sb227
Shewanella pealeana
Treponema pallidum Fribourg-Blanc
Acinetobacter sp. ADP1
Pectobacterium carotovorum subsp.
carotovorum PC1
Cytophaga hutchinsonii
Planctomyces brasiliensis
sphaerobacter thermophilus
Alistipes finegoldii
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 acetyl-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.
Escherichia coli K-12 MG1655
Salmonella enterica subsp. serovar
Typhi Ty21a enterica
Corynebacterium sp. ATCC 6931
Chlamydomonas reinhardtii
Escherichia coli O103: H2
Yersinia enterocolitica
Pseudomonas aeruginosa
Escherichia coli K-12 MG1655
Lactobacillus reuteri
The term “acetoacetyl-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.
Haemophilus influenzae R2866
Escherichia coli O44: H18 042
Xanthomonas campestris pv.
campestris ATCC 33913
Cronobacter sakazakii ES15
Shigella sonnei Ss046
Helicobacter pylori83
Acinetobacter sp. ADP1
Escherichia coli O17: K52: H18
Salmonella enterica subsp.
enterica serovar typhimurium
Pseudomonas putida KT2440
The term “3-hydroxyacyl-CoA dehydrogenase” or “3-hidroxybutyryl-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.
Salmonella enterica
Heidelberg 41578
Escherichia coli O103:
Xanthomonas albilineans
Aspergillus niger
Corynebacterium jeikeium
Enterobacter cloacae
Serratia plymuthica AS9
Acinetobacter sp.
Klebsiella pneumoniae
Escherichia coli K-12
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.
Acinetobacter
baumannii
Vibrio parahaemolyticus
Ralstonia eutropha
Xanthobacter
autotrophicus
Azospirillum sp. B510
Arabidopsis thaliana
Escherichia coli K-12
Salmonella entérica
Yersinia pestis
Acetobacter
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 carry 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 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.
Yersinia pestis
Serratia marcescens
Clostridium
acetobutylicum DSM 1731
Clostridium
acetobutylicum ATCC 824
Clostridium perfringenes
Streptomyces griseus
Streptomyces collinus
Amycolatopsis
mediterranei S699
Kitasatospora setae
Acidimicrobium
ferrooxidans
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 transform 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.
Escherichia coli K-12 MDS42
Escherichia coli O150: H5 SE15
Salmonella entérica
Clostridium acetobutylicum DSM
Lactobacillus plantarum JDM1
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 enzymes 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.
Mycobacterium bovis
Aspergillus fumigatus
Lactobacillus rhamnosus
Clostridium kluyveri
Clostridium acetobutylicum
Escherichia coli K-12 MDS42
Salmonella enterica subsp.
enterica
Xanthomonas campestris
Bacillus cereus B4264
Clostridium perfringenes 13
The term “pyruvate formate-lyase” or “formate acetyltransferase” 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 carry 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.
Ostreococcus lucimarinus
Ostreococcus tauri
Bathycoccus Prasinos
Escherichia coli K-12 MG1655
Escherichia coli K-12 W3110
Streptococcus mutans strain JC2
Clostridium pasteurianum
Chlamydomonas reinhardtii
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 CO2, 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 CO2 are described in Table 11. The enzymes described in Table 11 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.
Wolinella succinogenes
Sulfurimonas denitrificans
Sulfuricurvum kujiense
Geobacter sulfurreducens PCA
Geobacter sulfurreducens PCA
Burkholderia stabilis 15516
Pelobacter carbinolicus
Desulfomicrobium baculatum
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 pyruvate 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-acetolactate 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.
Escherichia coli str. K-12 substr.
Escherichia coli str. K-12 substr.
Mycobacterium tuberculosis H37Rv
Bacillus subtilis subsp. subtilis
Saccharomyces cerevisiae chromosome
Methanococcus aeolicus Nankai-3
Arabidopsis chromosome 3
The term “ketoacid reductoisomerase” (EC 1.1.1.86) refers to a polypeptide having catalytic activity, where this catalytic activity is to transform a compound of acetolactate into 2,3-dihydroxyvalerate. 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 ketoacid reductoisomerase. Examples of enzymes that can carry 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.
Escherichia coli str. K-12 substr.
Escherichia coli str. K-12 substr.
Corynebacterium glutamicum ATCC 13032
Corynebacterium glutamicum K051
Salmonella enterica subsp. serovar
Typhimurium str. LT2
Saccharomyces cerevisiae chromosome
Campylobacter jejuni RM1221
Methylococcus capsulatus str. Bath
Shewanella oneidensis MR-1
Dehalococcoides ethenogenes 195
Carboxydothermus hydrogeneoformans Z-
Listeria monocytogenes serotype 4b
Geobacter sulfurreducens PCA
Streptomyces avermitilis MA-4680
Pseudomonas aeruginosa PAO1
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 carryout the described reaction, so that enzyme will be considered as an analogue of dihydroxyacid dehydratase. Examples of enzymes that can carry out the conversion reaction of 2,3-dihydroxyvalerate 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.
Saccharomyces cerevisiae S288c
Shewanella oneidensis MR-1
Ruegeria pomeroyi DSS-3
Escherichia coli O157: H7 str.
Escherichia coli UTI89
Escherichia coli CFT073
Escherichia coli BW2952
Campylobacter jejuni RM1221
Dehalococcoides ethenogenes 195
Methylococcus capsulatus str. Bath
Pseudomonas syringae pv. tomato
Geobacter sulfurreducens PCA
Listeria monocytogenes serotype 4b
Staphylococcus aureus subsp. aureus
Yersinia pestis Nepal516
The term “2-oxoisovalerate dehydrogenase” (EC 1.2.1.25), “3-methyl-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 isobutyryl-CoA, 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 2-oxoisovalerate dehydrogenase. Examples of enzymes that can carry 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.
Homo sapiens
Pan troglodytes
Streptomyces coelicolor
Thermoplasma acidophilum
Oncorhynchus mykiss
Gorilla gorilla gorilla
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 carry out the described reaction, so that enzyme will be considered as an analogue of isobutyryl-CoA mutase. Examples of enzymes that can carry out the conversion reaction of isobutyryl-CoA to butyryl-CoA are described in Table 16. The enzymes described in Table 16 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.
Streptomyces coelicolor A3 (2)
Streptomyces cinnamonensis
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) H and/or regeneration of acetyl-CoA/CoA (
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.
Furthermore, the present invention can use smaller amounts than stoichiometrically required of NAD (P)+, NAD (P) H and CoA 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 CO2, 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 acetolactate to hydroxyvalerate, of ketoisovalerate to isobutyryl-CoA, of butanoyl-CoA to butanal and of butanal to butanol.
In turn, 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 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 (0.411 grams of butanol per gram of lactate).
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-lyase (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 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 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 (0.411 grams of butanol per gram of lactate).
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 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 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 (0.411 gr of butanol for a gram of lactate)
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)+/NAD(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:
I. A mixture is prepared continuously starting from water, lactate, the enzyme mixture, NAD(P)+/NAD(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.
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%.
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.
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, nanofiltration, ultrafiltration, etc.), distillation, evaporation or any other system that allows the separation of molecules either by size or by any of its physiochemical properties.
Escherichia coli
Saccharomyces
cerevisiae
Azotobacter
vinelandii
Salmonella enterica
Enterobacter cloacae
Clostridiurn kluyvery
Clostridium
aminobutyricum
Clostridium difficile
Pseudomonas putida
Clostridium kluyveri
Escherichia coli
Acinetobacter sp
Oenococcus oeni
Saccharomyces
cerevisiae
Geobacillus
thermodenitrificans
Acetobacter
pasteurianus SKU1108
Natronomonas
pharaonic
Emericella nidulans
Flavobacterium
frigidimaris KUC-1
Desulfovibrio gigas
Saccharomyces
cerevisiae
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)+/NAD(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 immobilize 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:
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.
II. A mixture is prepared continuously from water, lactate and NAD(P)+/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.
I. 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.
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.
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.
Separation systems mentioned in stages IV and V can be: membrane system (reverse osmosis, nanofiltration, ultrafiltration, etc.), distillation, evaporation or any other system which allows the separation of molecules, either by their size or for some of its physicochemical properties.
The following examples are intended to clarify the present invention. It should be understood that the following 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.
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 Sambrook, 2010. Subsequently enzymes were purified according to protocols described in Sambrook and Green, 2010.
Enzyme assays and results are described below:
a) Lactate Dehydrogenase (EC 1.1.1.27 and/or EC 1.1.1.28)
Lactate dehydrogenase can transform lactate to pyruvate 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 (Cetinel et al., 2013). Four enzymes of various microorganisms were used (Escherichia coli APEC O1, Escherichia coli PMV-1, Laclobacillus lactis subsp. laclis IL1403 and Streptococcus pneumoniae AP200). The conditions tested are shown in Table 19.
In all tests performed conversion of lactate to pyruvate production was observed as NAD (P) H after 1 hour.
) Pyruvate dehydrogenase complex (EC 1.2.1.51, EC 1.2.4.1, EC 2.3.1.12 and EC 1.8.1.4)
The pyruvate dehydrogenase complex transforms pyruvate 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). Three enzymes from different microorganisms were used (Escherichia coli K-12 MG1655. Corynebacterium sp. ATCC 6931 and Lactobacillus reuteri). The conditions tested are shown in Table 20.
In all trials consumption both NAD (P)+ was observed as pyruvate after one hour reaction.
c) Acetyl-CoA Thiolase (2.3.1.9)
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 (:H 18 042 Xanthomonas campestris pv campestris ATCC 33913, Shigella sonnei and Escherichia coli O44 Ss046). The conditions tested are shown in Table 21.
In all trials consumption of acetyl CoA it was observed after one hour reaction.
d) 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 al., 1973). Three enzymes from different microorganisms were used (: H2 12009, Xanthomonas albilineans, Escherichia coli O103 Aspergillus niger). The conditions tested are shown in Table 22.
In all trials consumption NAD (P) H after 30 minutes reaction was observed.
e) Enoyl CoA Hydratase (EC 4.2.1.17)
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 (Acinetobacler baumannii, and Arabidopsis thaliana Escherichia coli K-12 MDS42). The conditions tested are shown in Table 23.
In all trials consumption hydroxybutyryl-CoA was observed after 30 minutes of reaction.
f) Butyryl-CoA Dehydrogenase (EC 1.3.1.44, 1.3.1.86 and/or 1.3.8.1)
The butyryl-CoA dehydrogenase enzyme converts crotonyl-CoA to butanoyl-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 al., 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.
In all trials consumption NAD (P) H after 30 minutes reaction was observed.
h) Butyraldehyde Dehydrogenase (EC 1.2.1.57 and/or EC 1.2.1.10)
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 (: Clostridium acetobutylicum DSM SE15 H5 1731, Escherichia coli and Salmonella enterica O150). The conditions tested are shown in Table 25.
In all trials consumption NAD (P) H after 30 minutes reaction was observed.
i) Alcohol Dehydrogenase (EC 1.1.1.1 and/or EC 1.1.1.2)
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 al., 2010). Three enzymes from different microorganisms were used (Escherichia coli K-12 MDS42, Bacillus cereus and Clostridium acetobutylicum DSM B4264 1731). The conditions tested are shown in Table 26.
In all tests performed both isobutyraldehyde to isobutanol conversion was observed after 15 minutes as reaction consuming NAD (P) H.
j) Pyruvate Formate-Lyase (EC 2.3.1.54)
The enzyme pyruvate formate lyase converts pyruvate to Acetyl-CoA and CO2, so the test was conducted by varying the initial concentrations of pyruvate, pH and temperature following the protocols described in the literature (Takahashi, et al., 1982). Three enzymes from different microorganisms were used (Iucimarinus ostreococcus, Bathycoccus prasinos and Escherichia coli K-12 MG1655). The conditions tested are shown in Table 27.
In all tests performed pyruvate consumption was observed after one hour of reaction.
k) Formate Dehydrogenase (EC 1.2.1.43)
The enzyme formate dehydrogenase transforms the formate into CO2 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, Sulfuricurvum kujiense and Geobacter sulfurreducens PCA). The conditions tested are shown in Table 28.
In all tests conducted formate consumption and NAD (P)+ was observed after 2 hours of reaction.
l) Acetolactate Synthase (EC 2.2.1.6)
Acetolactate synthase converts pyruvate into 2-acetolactate, so the test was conducted by varying the initial concentrations of pyruvate, pH and temperature following 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 subtilis subsp. subtilis str. 168 and Saccharomyces cerevisiae, S288c). The conditions tested are shown in Table 29.
In all tests performed pyruvate consumption was observed after 20 minutes of reaction.
m) Keto Acid Reductoisomerase (EC 1.1.1.86) and Dihydroxy Acid Dehydratase (EC 4.2.1.9)
On one hand, the keto acid reductoisomerase 2-acetolactate transforms 2,3-dihydroxyvalerate while dihydroxyacid dehydratase transforms hydroxyvalerate into ketoisovalerate. Because the 2-acetolactate is not a commercial compound as 2,3-dihydroxyvalerate 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 temperature using as a basis the protocols described in the literature (Flint et al., 1993; Bastian et al, 2011; Li et al, 2011). 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 ATCC 13032). The conditions tested are shown in Table 30.
In all trials consumption pyruvate and NAD (P) H was observed after 2 hours of reaction.
n) 2-oxoisovalerate Dehydrogenase (EC 1.2.1.25 and/or EC 1.2.4.4)
The enzyme 2-oxoisovalerate dehydrogenase transforms ketoisovalerate to isobutyryl-CoA, using a molecule of NAD (P)+′ so the test was conducted by varying the initial concentrations of ketoisovalerate, NAD (P)+′ 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 Oncorhynchus mykiss). The test conditions are shown in Table 31.
In all tests we performed ketoisovalerate consumption and NAD (P)+ was observed after one hour of reaction.
o) Isobutyryl-CoA Mutase (EC 5.4.99.13)
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.
In all trials consumption isobutyryl-CoA was observed after 45 minutes of reaction.
Example 2. Enzymatic production of butanol from lactate, coupled with a regeneration system of NAD (P)+/NAD (P) H and/or regeneration of acetyl-CoA/CoA in a batch process.
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 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), acetoacetyl-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:
2C3H6O3+2NAD(P)+==>2C3H4O3+2NAD(P)H+2H+ (1)
2C3H4O3+2NAD(P)++2CoA=>2C23H38N7O17P3S+2NAD(P)H+2H++2CO2 (2)
2C23H38N7O17P3S==>C25H40N7O18P3S+CoA (3)
C25H40N7O18P3S+NAD(P)H+H+==>C25H42N7O18P3S+NAD(P)+ (4)
C25H42N7O18P3S==>C25H40N7O17P3S+H2O (5)
C25H40N7O17P3S+NAD(P)H+H+==>C25H42N7O17P3S+NAD(P)+ (6)
C25H42N7O17P3S+NAD(P)H+H+==>C4H8O+NAD(P)++CoA (7)
C4H8O+NAD(P)H+H+==>C4H10O+NAD(P)+ (8)
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:
2C3H6O3==>C4H10O+2CO2+H2O (9)
To attach the regeneration of NAD (P)+/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
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 nm. Lactate and butanol were monitored by HPLC with refractive index detector using a Rezex ROA-organic acids H+ column.
Considering a system without regeneration of NADH, the theoretical stoichiometric balance indicates that to transform 19.55 g of pyruvate (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 0.1 g of CoA to transform 20 g lactate into 8.22 g butanol.
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.
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)+/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 2.3.1.9), 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 any of its analogs are used, according to the following reactions:
2C3H6O3+2NAD(P)+==>2C3H4O3+2NAD(P)H+2H+ (10)
2C3H4O3+2CoA==>2C23H38N7O17P3S+2CH2O2 (11)
2CH2O2+2NAD(P)+==>2CO2+2NAD(P)H+2H+ (12)
2C23H38N7O17P3S==>C25H40N7O18P3S+CoA (13)
C25H40N7O18P3S+NAD(P)H+H+==>C25H42N7O18P3S+NAD(P)+ (14)
C25H42N7O18P3S==>C25H40N7O17P3S+H2O (15)
C25H40N7O17P3S+NAD(P)H+H+==>C25H42N7O17P3S+NAD(P)+ (16)
C25H42N7O17P3S+NAD(P)H+H+==>C4H8O++NAD(P)++CoA (17)
C4H8O+NAD(P)H+H+==>C4H10O+NAD(P)+ (18)
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:
2C3H6O3==>C4H10O+2CO2+H2O (19)
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.
In
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.
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.
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 butanol were measured by HPLC with refractive index detector using a Rezex ROA-organic acids H+ column.
As shown in
It is noteworthy that for other conditions shown in Tables 35 and 36 very similar conversion efficiencies were obtained.
Example 4. Enzymatic production of butanol from lactate, coupled to the regeneration system NAD (P)+/NADH and/or acetyl-CoA/CoA in a continuous process using immobilized enzymes.
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:
2C3H6O3+2NAD(P)+==>2C3H4O3+2NAD(P)H+2H+ (20)
2C3H4O3==>C5H8O4+CO2 (21)
C5H8O4+NAD(P)H+H+==>C5H10O4+NAD(P)+ (22)
C5H10O4==>C5H8O3+H2O (23)
C5H8O3+CoA+NAD(P)+==>C25H42N7O17P3S+NAD(P)H+H++CO2 (24)
(CH3)2CHCOSCoA==>CH3(CH2)2COSCoA (25)
C25H42N7O17P3S+NAD(P)H+H+==>C4H8O+NAD(P)++CoA (26)
C4H8O+NAD(P)H+H+==>C4H10O+NAD(P)+ (27)
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:
2C3H6O3==>C4H10O+2CO2+H2O (28)
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)+.
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)+ 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+ column.
In
For this particular case, the reaction began when the mixture of cofactors, coenzymes, lactate and NAD (P)+ entered the packed reactor.
As shown in
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.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2016/000903 | 5/5/2016 | WO | 00 |
