CN113755535B - Biosynthesis method from formic acid to formaldehyde and/or methanol - Google Patents
Biosynthesis method from formic acid to formaldehyde and/or methanol Download PDFInfo
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- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 title claims abstract description 165
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 102
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 title claims abstract description 82
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 235000019253 formic acid Nutrition 0.000 title claims abstract description 41
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 80
- 239000002994 raw material Substances 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims description 100
- 108090000790 Enzymes Proteins 0.000 claims description 100
- 239000002253 acid Substances 0.000 claims description 23
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 14
- 238000006555 catalytic reaction Methods 0.000 claims description 14
- 229940044170 formate Drugs 0.000 claims description 14
- SXMOKYXNAPLNCW-GORZOVPNSA-N formyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 SXMOKYXNAPLNCW-GORZOVPNSA-N 0.000 claims description 14
- -1 alkali metal salt Chemical class 0.000 claims description 11
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 11
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 11
- 239000004280 Sodium formate Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical group [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims description 8
- 235000019254 sodium formate Nutrition 0.000 claims description 8
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 6
- 230000008929 regeneration Effects 0.000 claims description 6
- 238000011069 regeneration method Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- CBOCVOKPQGJKKJ-UHFFFAOYSA-L Calcium formate Chemical compound [Ca+2].[O-]C=O.[O-]C=O CBOCVOKPQGJKKJ-UHFFFAOYSA-L 0.000 claims description 4
- 229910052783 alkali metal Inorganic materials 0.000 claims description 4
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims description 4
- 239000004281 calcium formate Substances 0.000 claims description 4
- 235000019255 calcium formate Nutrition 0.000 claims description 4
- 229940044172 calcium formate Drugs 0.000 claims description 4
- 150000004675 formic acid derivatives Chemical class 0.000 claims description 4
- TVISEJUYYBUVNV-UHFFFAOYSA-N formyl dihydrogen phosphate Chemical compound OP(O)(=O)OC=O TVISEJUYYBUVNV-UHFFFAOYSA-N 0.000 claims description 4
- XKPJKVVZOOEMPK-UHFFFAOYSA-M lithium;formate Chemical compound [Li+].[O-]C=O XKPJKVVZOOEMPK-UHFFFAOYSA-M 0.000 claims description 4
- GMDNUWQNDQDBNQ-UHFFFAOYSA-L magnesium;diformate Chemical compound [Mg+2].[O-]C=O.[O-]C=O GMDNUWQNDQDBNQ-UHFFFAOYSA-L 0.000 claims description 4
- WFIZEGIEIOHZCP-UHFFFAOYSA-M potassium formate Chemical compound [K+].[O-]C=O WFIZEGIEIOHZCP-UHFFFAOYSA-M 0.000 claims description 4
- 230000037361 pathway Effects 0.000 abstract description 24
- 238000003786 synthesis reaction Methods 0.000 abstract description 10
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 5
- 230000002194 synthesizing effect Effects 0.000 abstract description 5
- 238000013459 approach Methods 0.000 abstract description 4
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 abstract description 4
- 239000001569 carbon dioxide Substances 0.000 abstract description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000002028 Biomass Substances 0.000 abstract description 2
- 239000000446 fuel Substances 0.000 abstract description 2
- 239000003345 natural gas Substances 0.000 abstract description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000003054 catalyst Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 108010092060 Acetate kinase Proteins 0.000 description 4
- 102000008146 Acetate-CoA ligase Human genes 0.000 description 4
- 108010049926 Acetate-CoA ligase Proteins 0.000 description 4
- 101710174135 Acyl-coenzyme A dehydrogenase Proteins 0.000 description 4
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 4
- 101710181816 Pyruvate-formate-lyase deactivase Proteins 0.000 description 4
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 4
- 239000011777 magnesium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 3
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 3
- 108010081577 aldehyde dehydrogenase (NAD(P)+) Proteins 0.000 description 3
- 238000005580 one pot reaction Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 108030006766 Cyclohexanol dehydrogenases Proteins 0.000 description 2
- 108030007102 Formate kinases Proteins 0.000 description 2
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 description 2
- 108010043075 L-threonine 3-dehydrogenase Proteins 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108010048916 alcohol dehydrogenase (acceptor) Proteins 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000003541 multi-stage reaction Methods 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 102100039702 Alcohol dehydrogenase class-3 Human genes 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 108020004652 Aspartate-Semialdehyde Dehydrogenase Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108700035271 EC 1.1.1.2 Proteins 0.000 description 1
- 108700035017 EC 1.1.1.71 Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- YPZRHBJKEMOYQH-UYBVJOGSSA-N FADH2 Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1COP(O)(=O)OP(O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C(NC(=O)NC2=O)=C2NC2=C1C=C(C)C(C)=C2 YPZRHBJKEMOYQH-UYBVJOGSSA-N 0.000 description 1
- 241000224466 Giardia Species 0.000 description 1
- 102100030764 Inactive L-threonine 3-dehydrogenase, mitochondrial Human genes 0.000 description 1
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 description 1
- 102100027050 Inorganic pyrophosphatase Human genes 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 108030006769 Methanol dehydrogenases Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000204652 Thermotoga Species 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 108010051015 glutathione-independent formaldehyde dehydrogenase Proteins 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
-
- 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
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a new way for synthesizing formaldehyde and/or methanol from formic acid, which realizes the efficient synthesis of the formaldehyde and/or methanol from the formic acid through the combination of different chemical reactions. The novel pathway of the present invention requires less energy (1 mole of ATP to ADP is consumed per 1 mole of formaldehyde produced) and is more efficient, producing 2mM formaldehyde or 4.83mM methanol in 1 hour. The new approach of the invention can be integrated with the existing approaches to realize the purpose of synthesizing complex compounds and liquid fuels by using raw materials such as carbon dioxide, biomass, natural gas and the like.
Description
Technical Field
The invention belongs to the technical field of biosynthesis, and particularly relates to a biosynthesis method for producing formaldehyde and/or methanol from formic acid.
Background
Formic acid, formaldehyde, methanol are products of carbon dioxide reduction to varying degrees, which are important one-carbon feedstocks that can be used to synthesize a variety of compounds. Formaldehyde and methanol have higher chemical activity than formic acid, and are more favorable for subsequent conversion and application.
The methods reported so far mainly achieve the conversion of formic acid to formaldehyde or methanol by formaldehyde dehydrogenase or methanol dehydrogenase in the natural pathway. However, this method requires a large amount of reducing power to obtain a small accumulation of formaldehyde or alcohol due to the disadvantageous free energy.
In addition to the natural pathway, the artificial pathway consisting of acetyl-coa synthase and acetaldehyde dehydrogenase achieves the reduction of formate to formaldehyde, but requires more energy to be expended (1 mole ATP to AMP to generate 1 mole of formaldehyde).
In addition, the artificial pathway using acetate kinase and aspartate semialdehyde dehydrogenase consumes less energy (1 mole of ATP to ADP is required for every 1 mole of formaldehyde production), but the formaldehyde yield is lower, only 0.0163mM.
Therefore, there is a need in the art for a process for the biosynthesis of formic acid to formaldehyde and/or methanol that consumes little energy and yields high yields.
Disclosure of Invention
In order to solve the problems in the prior art, in one aspect, the invention provides a biosynthesis method from formic acid to formaldehyde, comprising the following steps:
step (1): formic acid or salts thereof are used as raw materials and are converted into formylphosphoric acid under the catalysis of enzyme;
step (2): the formyl phosphate obtained in the step (1) is converted into formyl coenzyme A under the catalysis of enzyme; and
step (3): the formyl-CoA obtained in the step (2) is converted into formaldehyde under the catalysis of enzyme.
According to an embodiment of the present invention, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formylphosphoric acid.
According to an embodiment of the invention, the formate is an alkali metal salt or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate, calcium formate, and the like.
According to an embodiment of the present invention, the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA.
According to an embodiment of the invention, the enzyme used in step (3) is an enzyme having the function of catalyzing the conversion of formyl-coa to formaldehyde.
According to an embodiment of the present invention, the steps (1) to (3) may be performed stepwise, or any adjacent two or three steps may be performed simultaneously.
According to an embodiment of the present invention, steps (1) to (3) are carried out simultaneously, for example, the reaction system comprises a substrate formic acid or formate salt, an enzyme having a function of catalyzing the conversion of formic acid to formylphosphoric acid, an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA and an enzyme having a function of catalyzing the conversion of formylCoA to formaldehyde.
According to an embodiment of the invention, the reaction system further comprises NaCl, mg 2+ 、Zn 2+ ATP, NADH, coA, mercaptoethanol, and the like.
According to an embodiment of the invention, the reaction system has a pH of 6.5-8.5, e.g. 7-8, e.g. the pH environment is provided in Hepes buffer.
According to an embodiment of the present invention, wherein the reaction system of step (3) or the reaction system in which steps (1) to (3) are carried out simultaneously may optionally further comprise an auxiliary enzyme for assisting NADH regeneration, such as Formate Dehydrogenase (FDH).
In another aspect, the present invention also provides a method for biosynthesis of formic acid to methanol, comprising the steps of:
step (1): formic acid or salts thereof are used as raw materials and are converted into formylphosphoric acid under the catalysis of enzyme;
step (2): the formyl phosphate obtained in the step (1) is converted into formyl coenzyme A under the catalysis of enzyme;
step (3): the formyl coenzyme A obtained in the step (2) is converted into formaldehyde under the catalysis of enzyme; and
step (4): the formaldehyde obtained in the step (3) is converted into methanol under the catalysis of enzyme.
According to an embodiment of the present invention, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formylphosphoric acid.
According to an embodiment of the invention, the formate is an alkali metal salt or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate, calcium formate, and the like.
According to an embodiment of the present invention, the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA.
According to an embodiment of the invention, the enzyme used in step (3) is an enzyme having the function of catalyzing the conversion of formyl-coa to formaldehyde.
According to an embodiment of the present invention, the enzyme used in step (4) is an enzyme having a function of catalyzing the conversion of formaldehyde into methanol.
According to an embodiment of the present invention, the steps (1) to (4) may be performed stepwise, or any adjacent two, three or four steps may be performed simultaneously.
According to an embodiment of the present invention, steps (1) to (4) are performed simultaneously, and the reaction system comprises a substrate formic acid or formate salt, an enzyme having a function of catalyzing the conversion of formic acid to formylphosphoric acid, an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA, an enzyme having a function of catalyzing the conversion of formylCoA to formaldehyde, and an enzyme having a function of catalyzing the conversion of formaldehyde to methanol.
According to an embodiment of the invention, the reaction system further comprises NaCl, mg 2+ 、Zn 2+ ATP, NADH, coA, mercaptoethanol, and the like.
According to an embodiment of the invention, the reaction system has a pH of 6.5-8.5, e.g. 7-8, e.g. the pH environment is provided in Hepes buffer.
According to an embodiment of the present invention, wherein the reaction system of step (3) or step (4) or the reaction system in which steps (1) to (4) are carried out simultaneously may optionally further comprise an auxiliary enzyme for assisting NADH regeneration, such as Formate Dehydrogenase (FDH).
In the context of the present invention, when the expression "an enzyme having a function of catalyzing the conversion of substance A into substance B" is used, it means that the enzyme may catalyze the conversion of substance A into substance B, the reaction may be a one-step reaction or a multi-step reaction, and the enzyme may be an enzyme required for any one-step reaction of substance A into substance B, and thus, the enzyme may be a single enzyme catalyzing the one-step reaction or a combination of enzymes catalyzing one or more steps of the multi-step reaction. The amino acid sequence and source of the enzyme having the catalytic function are not particularly limited as long as they can perform the catalytic function. In particular, "enzymes having the function of catalyzing the conversion of formate to formylphosphoric acid" refers to enzymes that can catalyze the phosphorylation of formate, including but not limited to acetate kinase (ACKA, EC 2.7.2.1) and formate kinase (EC 2.7.2.6), which can be derived from, but not limited to, different species such as E.coli, salmonella, clostridium, methanoamphaeum, and the like. "enzyme having the function of catalyzing the conversion of formylphosphoric acid to formylCoA" refers to an enzyme that catalyzes the conversion of formylphosphoric acid to formylCoA, including but not limited to phosphoacetyltransferase (PTA, EC 2.3.1.8), which may be derived from, but not limited to, different species such as E.coli, clostridium, thermotoga, methanoamphoaceticus, etc. "enzyme having the function of catalyzing the conversion of formyl-CoA to formaldehyde" refers to an enzyme that catalyzes the conversion of formyl-CoA to formaldehyde, including but not limited to acetaldehyde dehydrogenase (ACDH, EC 1.2.1.10), which may be derived from, but not limited to, listeria, pseudomonas, acinetobacter, giardia, and the like, of different species. "enzymes having the function of catalyzing the conversion of formaldehyde to methanol" refers to enzymes that can catalyze the reaction of formaldehyde to methanol, including but not limited to alcohol dehydrogenase (ADH, EC 1.1.1.1;EC 1.1.1.2;EC 1.1.1.71;EC 1.1.2.8), methanol dehydrogenase (EC 1.1.1.244;EC 1.1.2.7;EC 1.1.2.B2), L-threonine-3-dehydrogenase (EC 1.1.1.103), cyclohexanol dehydrogenase (EC 1.1.1.245), and n-butanol dehydrogenase (EC 1.1.2.9), which can be derived from but not limited to different species of Pichia pastoris, candida, streptomyces, corynebacterium glutamicum, escherichia coli, rhodococcus, dow, and the like.
The enzyme used in the context of the present invention may be in the form of crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme or whole cells. The crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme or whole cell is commercially available or prepared according to a method known in the literature or a conventional method in the art; for example, the crude enzyme liquid freeze-dried powder and the pure enzyme are prepared according to the following steps: expressing the enzyme in a host cell to obtain a recombinant cell; cracking the recombinant cells to obtain the crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme; the whole cells are prepared according to the following steps: expressing the enzyme in a host cell, and obtaining a recombinant cell which is the whole cell.
In the context of the present invention, the term "stepwise" means that after the end of the previous reaction, the purification of the product is performed or not, and then the enzyme or the desired component catalyzing the subsequent reaction is added to perform the subsequent reaction; the term "simultaneously carried out" means that at the beginning of the reaction, enzymes catalyzing the respective reactions involved are put into the reactor together with the substrate to carry out the reaction. The term "adjacent" step means that the product of the previous step can be used as a reactant for the subsequent step, and the two steps can be referred to as "adjacent".
Advantageous effects
The novel way for synthesizing formaldehyde and/or methanol by using formic acid or the salt thereof realizes the efficient synthesis of the formic acid or the salt thereof to the formaldehyde and/or the methanol through the combination of different chemical reactions. The novel pathway of the present invention requires less energy (1 mole of ATP to ADP is consumed per 1 mole of formaldehyde produced) and is more efficient, producing 2mM formaldehyde or 4.83mM methanol in 1 hour. The new approach of the invention can be integrated with the existing approaches to realize the purpose of synthesizing complex compounds and liquid fuels by using raw materials such as carbon dioxide, biomass, natural gas and the like.
Drawings
FIG. 1 shows a comparison of formaldehyde production from the novel pathway of the invention for formaldehyde synthesis from formate with the native pathway and the acetyl-CoA synthetase pathway.
Figure 2 shows the methanol yield of the novel pathway of the invention for methanol synthesis from formic acid.
Detailed Description
The method of synthesizing formaldehyde and methanol from formic acid according to the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
EXAMPLE 1 novel pathway for the Synthesis of formaldehyde from formic acid
The new route for formaldehyde synthesis from formic acid is shown below:
first, a catalyst (i.e., enzyme) that can catalyze each chemical reaction in the pathway is selected (see table 1), but is not limited to the catalysts listed in table 1. Then, different catalysts are combined to establish a corresponding reaction system, and after a period of reaction, the formaldehyde yield is detected.
The information on the enzymes used in example 1 are listed in Table 1, acetate kinase, phosphoacetyl transferase, acetaldehyde dehydrogenase, formate dehydrogenase were obtained by PCR or gene synthesis, and the corresponding expression vectors pET28a-ACKA, pET28a-PTA, pET21b-ACDH, pET20b-FDH were obtained by the method of Simple Cloning (Young, C., et al (2012), "Simple Cloning via Direct Transformation of PCR Product (DNAMultimer) to Escherichia coli and Bacillus subtilis.," appl. Environment. Microbiol.78 (5): 1593-1595.) respectively, into pET20b, pET21b and pET28a vectors (Novagen, madison, wis.). All four plasmids were transformed into E.coli expression bacterium BL21 (DE 3) (Invitrogen, carlsbad, calif.), and protein expression and purification were performed.
TABLE 1 catalyst for novel chemical reactions of formic acid to formaldehyde
Formaldehyde production was measured as follows: to 200. Mu.L of water, 50. Mu.L of a properly diluted solution to be tested was added, followed by 25. Mu.L of an acetylacetone solution (100 mL of acetylacetone solution containing 0.5mL of acetylacetone, 50g of ammonium acetate and 6mL of glacial acetic acid), and after reaction at 60℃for 15min, 200. Mu.L of the supernatant was centrifuged to detect the OD414 value, and the amount of formaldehyde produced by the new route was calculated from the formaldehyde standard curve.
In addition, the natural pathway and the disclosed artificial pathway of acetyl-CoA synthetase were tested for their ability to convert formate to formaldehyde as comparative examples 1-1 and comparative examples 1-2, respectively.
New pathway 1-2-3 of example 1: the reaction system was Hepes buffer 100mM,NaCl 100mM,Mg at pH7.5 2+ 5mM，Zn 2+ 10. Mu.M, 50mM sodium formate, 10mM ATP,0.5mM NADH,0.1mM CoA,2mM mercaptoethanol, 0.24mg/mL ACKA,1.2mg/mL PTA,0.2mg/mL ACDH,0.024mg/mL FDH (formate dehydrogenase, aiding NADH regeneration). The reaction was carried out for 1 hour with a formaldehyde yield of 2mM.
The natural route of comparative example 1-1: the reaction system is Hepes buffer 10 at pH7.50mM，NaCl 100mM，Mg 2+ 5mM，Zn 2+ 10. Mu.M, FADH 4mg/mL, NADH 100mM, sodium formate 250mM. The reaction was carried out for 3 hours with a formaldehyde yield of 0.1mM.
The acetyl-CoA synthetase pathway of comparative examples 1-2: the reaction system was Hepes buffer 100mM,NaCl 100mM,Mg at pH7.5 2+ 5mM，Zn 2+ 10μM，ATP 2mM，NAD + 1.5mM,CoA 0.1mM,ACS 3.7mg/mL, ACDH 0.2mg/mL, FDH 0.024mg/mL (formate dehydrogenase, auxiliary enzyme for regenerating NADH), PPase 0.1mg/mL (inorganic pyrophosphatase, auxiliary enzyme), sodium formate 50mM. The reaction was carried out for 1 hour, and the formaldehyde yield was 0.6mM.
A comparison of formaldehyde production from the novel pathway of example 1 with the synthetic pathways of comparative examples 1-1 and comparative examples 1-2 is shown in FIG. 1. As can be seen from FIG. 1, the novel pathway for formaldehyde synthesis from formate of the present invention achieves significantly higher formaldehyde yields in a shorter period of time than the natural pathway and the acetyl-CoA synthetase pathway.
Example 2 novel pathway for methanol synthesis from formic acid
The new pathway for methanol synthesis from formic acid is shown below:
first, catalysts are selected that catalyze the various chemical reactions in the pathway (see table 2), but are not limited to the catalysts listed in table 2. Then, different catalysts are combined to establish a corresponding reaction system, and after a period of reaction, the yield of methanol is detected.
The information for the enzymes used in example 2 is given in Table 2, the alcohol dehydrogenases being purchased from Sigma company (https:// www.sigmaaldrich.com/china-main land. Html). The remaining enzymes were constructed and expressed in the same manner as in example 1.
TABLE 2 catalyst for novel chemical reactions of formic acid to methanol
The methanol yield was measured as follows: the amount of methanol produced by the new way was calculated according to the standard curve using an Agilent 7890A chromatograph (containing FID detector), with nitrogen as carrier gas, 18kPa (0.4 mL/min) pressure, HP-FFAP (25 m.times.0.32 mm.times.0.5 μm) column temperature at 75℃for 10min, syringe and detector temperatures at 150℃and 300℃respectively, and sample injection amount at 1. Mu.L/needle.
The reaction system was Hepes buffer 100mM,NaCl 100mM,Mg at pH7.5 2+ 5mM，Zn 2+ 10. Mu.M, 100mM sodium formate, 10mM ATP,0.5mM NADH,0.1mM CoA,2mM mercaptoethanol, 0.24mg/mL ACKA,1.2mg/mL PTA,0.2mg/mL ACDH,0.024mg/mL FDH (formate dehydrogenase, co-NADH regeneration), 0.15kU/mLADH; the reaction time was 1 hour, and the methanol yield was 4.83mM. The methanol yield is shown in FIG. 2.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A method of biosynthesis from formic acid to formaldehyde comprising the steps of:
step (1): formic acid or salts thereof are used as raw materials and are converted into formylphosphoric acid under the catalysis of enzyme;
step (2): the formyl phosphate obtained in the step (1) is converted into formyl coenzyme A under the catalysis of enzyme; and
step (3): the formyl coenzyme A obtained in the step (2) is converted into formaldehyde under the catalysis of enzyme;
wherein the enzyme used in the step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formylphosphoric acid; the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA; the enzyme used in step (3) is an enzyme having a function of catalyzing the conversion of formyl-CoA to formaldehyde.
2. The process according to claim 1, wherein the formate is an alkali metal salt or alkaline earth metal salt of formic acid.
3. The process according to claim 2, wherein the formate is selected from sodium formate, potassium formate, lithium formate, magnesium formate or calcium formate.
4. A process according to any one of claims 1 to 3, wherein steps (1) to (3) are carried out in steps, or wherein any adjacent two or three steps are carried out simultaneously.
5. The method according to claim 4, wherein steps (1) to (3) are performed simultaneously.
6. The method according to claim 5, wherein the reaction system comprises a substrate formate or formate salt, an enzyme having a function of catalyzing the conversion of formate to formylphosphoric acid, an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA, and an enzyme having a function of catalyzing the conversion of formylCoA to formaldehyde.
7. A process according to any one of claims 1 to 3, wherein the reaction system of step (3) or the reaction system in which steps (1) to (3) are carried out simultaneously, optionally comprises an auxiliary enzyme for assisting the regeneration of NADH.
8. The method of claim 7, wherein the accessory enzyme is Formate Dehydrogenase (FDH).
9. A method of biosynthesis from formic acid to methanol comprising the steps of:
step (1): formic acid or salts thereof are used as raw materials and are converted into formylphosphoric acid under the catalysis of enzyme;
step (2): the formyl phosphate obtained in the step (1) is converted into formyl coenzyme A under the catalysis of enzyme;
step (3): the formyl coenzyme A obtained in the step (2) is converted into formaldehyde under the catalysis of enzyme; and
step (4): converting formaldehyde obtained in the step (3) into methanol under the catalysis of enzyme;
wherein the enzyme used in the step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formylphosphoric acid; the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA; the enzyme used in step (3) is an enzyme having a function of catalyzing the conversion of formyl-coa to formaldehyde; the enzyme used in the step (4) is an enzyme having a function of catalyzing the conversion of formaldehyde into methanol.
10. The process according to claim 9, wherein the formate is an alkali metal salt or alkaline earth metal salt of formic acid.
11. The process according to claim 10, wherein the formate is selected from sodium formate, potassium formate, lithium formate, magnesium formate or calcium formate.
12. The method according to any one of claims 9-11, wherein steps (1) to (4) are performed stepwise, or wherein any adjacent two, three or four steps are performed simultaneously.
13. The method of claim 12, wherein steps (1) through (4) are performed simultaneously.
14. The method according to claim 13, wherein the reaction system comprises a substrate formic acid or formate salt, an enzyme having a function of catalyzing the conversion of formic acid to formylphosphoric acid, an enzyme having a function of catalyzing the conversion of formylphosphoric acid to formylCoA, an enzyme having a function of catalyzing the conversion of formylCoA to formaldehyde, and an enzyme having a function of catalyzing the conversion of formaldehyde to methanol.
15. The process according to any one of claims 9 to 11, wherein the reaction system of step (3) or step (4) or the reaction system in which steps (1) to (4) are carried out simultaneously may optionally comprise an auxiliary enzyme for assisting the regeneration of NADH.
16. The method of claim 15, wherein the accessory enzyme is Formate Dehydrogenase (FDH).
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| CN101978042A (en) * | 2008-01-22 | 2011-02-16 | 基因组股份公司 | Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol |
| CN102666862A (en) * | 2009-08-21 | 2012-09-12 | 马斯科马公司 | Production of propanols, alcohols, and polyols in consolidated bioprocessing organisms |
| CN107208118A (en) * | 2014-09-18 | 2017-09-26 | 基因组股份公司 | The non-natural microorganism of energy efficiency with raising |
| CN109609426A (en) * | 2019-01-04 | 2019-04-12 | 北京化工大学 | A method of 1,3- propylene glycol is produced using methanol/formaldehyde and glucose as cosubstrate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101978042A (en) * | 2008-01-22 | 2011-02-16 | 基因组股份公司 | Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol |
| CN102666862A (en) * | 2009-08-21 | 2012-09-12 | 马斯科马公司 | Production of propanols, alcohols, and polyols in consolidated bioprocessing organisms |
| CN107208118A (en) * | 2014-09-18 | 2017-09-26 | 基因组股份公司 | The non-natural microorganism of energy efficiency with raising |
| CN109609426A (en) * | 2019-01-04 | 2019-04-12 | 北京化工大学 | A method of 1,3- propylene glycol is produced using methanol/formaldehyde and glucose as cosubstrate |
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