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WO2023198017A1 - 一种制备酮酸的方法及该方法在制备氨基酸或氨基酸衍生物中的应用 - Google Patents

一种制备酮酸的方法及该方法在制备氨基酸或氨基酸衍生物中的应用 Download PDF

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WO2023198017A1
WO2023198017A1 PCT/CN2023/087476 CN2023087476W WO2023198017A1 WO 2023198017 A1 WO2023198017 A1 WO 2023198017A1 CN 2023087476 W CN2023087476 W CN 2023087476W WO 2023198017 A1 WO2023198017 A1 WO 2023198017A1
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gene
seq
ltae
nucleotide sequence
ilva
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French (fr)
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聂梦真
张科春
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Mint Biotechnologies Co Ltd
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Mint Biotechnologies Co Ltd
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Priority to EP23787661.0A priority Critical patent/EP4509610A4/en
Priority to US18/855,803 priority patent/US20250305012A1/en
Publication of WO2023198017A1 publication Critical patent/WO2023198017A1/zh
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Definitions

  • the invention requires a Chinese patent application submitted to the China Patent Office on April 11, 2022, with the application number 202210371436.6 and the invention title "A method for preparing keto acids and the application of the method in preparing amino acids or amino acid derivatives" priority, the entire contents of which are incorporated into this application by reference.
  • the present invention relates to the field of bioengineering technology, and specifically relates to a method for preparing keto acids and the application of the method in preparing amino acids.
  • Amino acids are the basic building blocks of biologically functional macromolecule proteins and play a vital role in the life activities and nutritional health of humans and animals.
  • Amino acids are organic compounds containing a basic amino group (-NH 2 ) and an acidic carboxyl group (-COOH), and the amino group and carboxyl group are connected to the same carbon atom. Its general chemical formula is RCHNH 2 COOH. According to the degree of human need, it can be divided into essential amino acids and non-essential amino acids. In addition to biosynthesized natural amino acids, it also includes artificially synthesized unnatural amino acids.
  • Amino acid products are widely used in medical/health care, food (condiments), feed additives, cosmetics, chemical synthesis and other fields, and have become a major global industry.
  • the main production methods of amino acids in the world include fermentation method, chemical synthesis method, hydrolysis method and chemical synthesis-enzyme method.
  • the fermentation method uses the ability of microorganisms to synthesize various amino acids, and through strain mutagenesis and other treatments, various defective and resistant mutant strains are selected to relieve feedback and inhibition in the metabolic pathway to achieve a certain A production method for the purpose of overproduction of amino acids.
  • the inevitable disadvantages of fermentation method for producing amino acids are that it is difficult to control the production and accumulation of various non-target amino acids, the production cycle is long, the purification is difficult, and the cost is high.
  • Chemical synthesis is a method of producing amino acids by means of complex reactions between various organic substances.
  • the hydrolysis method uses protein-rich materials such as hair as raw materials, hydrolyzes them through acid, alkali or proteolytic enzyme, and then A process for separating and purifying various amino acids.
  • the hydrolysis method has relatively abundant raw materials and is relatively easy to put into production, but its output is low, the cost is high and it causes relatively large environmental pollution.
  • US08153839B2 discloses a method for synthesizing ketoacids or amino acids by hydration of acetylene compounds, which uses hydration of metal salts and transition metal complexes to allow the synthesis of ketoacids (including ketoacids) from acetylene-carboxylic acids under mild conditions. and ketoacid derivatives) without using extremely harmful mercury catalysts, and amino acids (including amino acids) can be readily synthesized from synthetic ketoacids (including ketoacids and ketoacid derivatives) by subsequent reductive amination in the same vessel and amino acid derivatives), however, its operation is complex, the production process is difficult to control, and the yield and conversion rate are low, making it difficult to achieve industrial mass production.
  • the present invention aims to break through technological innovation starting from the preparation of keto acids, comprehensively improve the production of various natural amino acids or unnatural amino acids and derivatives, and improve the market competitiveness of products.
  • One of the technical problems solved by the present invention is to provide a method for preparing keto acids with high substrate utilization, low pollution, high yield and low production cost.
  • the second technical problem solved by the present invention is to provide a method for preparing diol organic compounds with high substrate utilization, low pollution, high yield and low production cost.
  • the third technical problem solved by the present invention is that it can produce a variety of important ketoacids: phenylpyruvic acid, 4-methyl 2-oxopentanoic acid, pyruvic acid, 2-oxobutyric acid, etc., and this approach can be applied to In the production of other keto acids and amino acids.
  • the first aspect provides a method for preparing keto acids, using glycine and alcoholic organic matter as substrates to perform an enzyme-catalyzed reaction.
  • alcoholic organic matter is converted into aldehyde-based organic matter
  • glycine and aldehyde-based organic matter are converted into beta -Hydroxy- ⁇ amino acid
  • ⁇ -hydroxy- ⁇ amino acid is converted into keto acid.
  • the prepared ketoacids include but are not limited to ketoacids with the following general formula:
  • R can be (CH 3 ) 2 CH 2 -, (CH 3 )C-, CH3-, wait.
  • glycine and alcohol organic matter as substrates, using the enzyme produced by overexpression of the first recombinant microorganism containing L-aldolase and the first dehydratase gene and the second recombinant microorganism containing dehydrogenase as the catalyst to carry out enzyme Catalytic reaction; alcoholic organic matter is converted into aldehyde organic matter under the action of dehydrogenase, glycine and aldehyde organic matter are converted into ⁇ -hydroxy- ⁇ amino acid under the sole catalysis of L-aldolase, and ⁇ -hydroxy- ⁇ amino acid is Keto acid is generated under the catalysis of the first dehydratase;
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 Or several; preferably ilvA or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is shown in SEQ ID NO.3 or SEQ ID NO.4;
  • the D-acetalase gene is selected from one or more of A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CH LRE; More preferably, the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7; the racemase gene is selected from one or more of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, and MJ8_44540 ; Preferably ILE2E_LENBU; More preferably, the nucleotide sequence of the ILE2E_LENBU;
  • the first recombinant microorganism also contains an enamine/imine deaminase gene; preferably it is ridA; more preferably, the nucleotide sequence of the ridA gene is as shown in SEQ ID NO: 5.
  • the organic alcohols are selected from the group consisting of benzyl alcohol, 4-imidazolemethanol, 2-methylthioethanol, indole-3-carbinol, 2-hydroxyethyl-methylphosphinate, p-hydroxybenzyl alcohol, 3,4 - One or more of dihydroxybenzyl alcohol, p-methylbenzyl alcohol, phenethyl alcohol, pivalol, isobutanol, and ethanol.
  • a method for preparing ketoacids is provided, using glycine and aldehyde organic matter as substrates for an enzyme-catalyzed reaction.
  • glycine and aldehyde organic matter are converted into ⁇ -hydroxy- ⁇ amino acids, ⁇ -hydroxyl -Alpha amino acids are then converted into keto acids.
  • the prepared ketoacids include but are not limited to ketoacids with the following general formula:
  • R can be (CH 3 ) 2 CH 2 -, (CH 3 )C-, CH3-, wait.
  • glycine and aldehyde organic matter as substrates, and using the enzyme produced by overexpression of the first recombinant microorganism containing L-aldolase and the first dehydratase gene as a catalyst to perform an enzymatic reaction; glycine and aldehyde organic matter are reacted in L -Convert to ⁇ -hydroxy- ⁇ amino acid under the sole catalysis of aldolase, and ⁇ -hydroxy- ⁇ amino acid generates keto acid under the catalysis of the first dehydratase;
  • glycine and aldehyde organic matter as substrates, and use the enzyme produced by overexpression of the third recombinant microorganism containing a D-acetalase gene, a racemase gene, and a second dehydratase gene as a catalyst to perform an enzymatic reaction; glycine And aldehyde organic matter is converted into ⁇ -hydroxy- ⁇ amino acid under the joint catalysis of D-acetalase and racemase, and ⁇ -hydroxy- ⁇ amino acid generates keto acid under the catalysis of the second dehydratase.
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 or several;
  • it is ilvA or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is shown in SEQ ID NO.3 or SEQ ID NO.4.
  • the D-acetalase gene is selected from one or more of A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CH LRE; More preferably, the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7; the racemase gene is selected from one or more of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, MJ8_44540 ; Preferably ILE2E_LENBU; More preferably, the nucleotide sequence of the ILE2E_LENBU gene
  • the method includes constructing the first recombinant microorganism, the second recombinant microorganism or the third recombinant microorganism through genetic engineering methods, and the genetic engineering methods include plasmid expression or genome integration.
  • the plasmid vector used when constructing using plasmid expression is one or both of pZAlac and pZElac.
  • the culture method of the recombinant microorganism is as follows: inoculating the recombinant microorganism into a 2-xyT medium containing ampicillin, kanamycin and chloramphenicol, and cultivating the recombinant microorganism at 20 to 60 Cultivate for 3 to 6 hours at °C, add IPTG to a final concentration of 0.3mM, continue culturing for 15 to 30 hours, then centrifuge, and discard the supernatant culture fluid.
  • the reaction temperature is 20-90°C
  • the pH of the reaction buffer is 6.5-8.5.
  • the recombinant microorganism includes one or more of recombinant Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces.
  • the recombinant microorganism is selected from the group consisting of recombinant Escherichia coli, Bacillus subtilis, nicotine-degrading bacteria (Pseudomonas sp.), Bacillus megaterium, and Bacillus amyloliquefaciens ), one or more of Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris.
  • a third aspect provides a recombinant microorganism for preparing keto acids, the recombinant microorganism being a first recombinant microorganism containing L-aldolase and a first dehydratase gene and a second recombinant microorganism containing a dehydrogenase; or, the recombinant microorganism is The microorganism is a third recombinant microorganism containing a D-acetylase gene, a racemase gene, a second dehydratase gene, and a second recombinant microorganism containing a dehydrogenase gene. group of microorganisms;
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 Or several; preferably ilvA or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is shown in SEQ ID NO.3 or SEQ ID NO.4; the dehydrogenas
  • the D-acetalase gene is selected from one or more of A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CHLRE ;More
  • the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7;
  • the racemase gene is selected from one or more of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, and MJ8_44540;
  • it is ILE2E_LENBU; more preferably, the nucleotide sequence of the ILE
  • the first recombinant microorganism also contains an enamine/imine deaminase gene; preferably it is ridA; more preferably, the nucleotide sequence of the ridA gene is as shown in SEQ ID NO: 5.
  • the fourth aspect provides a recombinant microorganism for preparing keto acids.
  • the recombinant microorganism is the first recombinant microorganism containing L-aldolase and the first dehydratase gene; or the recombinant microorganism is the first recombinant microorganism containing the D-aldolase gene.
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 Or several; preferably ilvA or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is as shown in SEQ ID NO.3 or SEQ ID NO.4;
  • the D-acetalase gene is selected from A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, One or more of guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CHLRE; more preferably, the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7; the elimination The gyrase gene is selected from one or more of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, MJ8_44540; preferably ILE2E_LENBU; more preferably, the nucleotide sequence of the ILE2E_LENBU;
  • ketoacid includes but is not limited to ketoacid with the following general formula:
  • R can be (CH 3 ) 2 CH 2 -, (CH 3 )C-, CH3-, wait.
  • the recombinant microorganism is selected from recombinant Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), nicotine-degrading bacteria (Pseudomonas sp.) Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens ( One or more of Bacillus amyloliquefaciens), Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • nicotine-degrading bacteria Pseudomonas sp.
  • Bacillus megaterium Bacillus megaterium
  • Bacillus amyloliquefaciens One or more of Bacillus amyloliquefaciens
  • Corynebacterium glutamicum Saccharomyces cerevisia
  • the fifth aspect provides a recombinant DNA or biological material for preparing keto acids
  • the recombinant DNA or biological material contains L-aldolase and first dehydratase genes and contains a dehydrogenase; or, the recombinant DNA or biological material
  • the material contains D-acetalase gene, racemase gene, second dehydratase gene and dehydrogenase;
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 or several; preferably ilvA Or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is shown in SEQ ID NO.3 or SEQ ID NO.4; the dehydrogenas
  • the D-acetalase gene is selected from one or more of A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CHLRE ;More
  • the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7;
  • the racemase gene is selected from one or more of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, and MJ8_44540;
  • it is ILE2E_LENBU; more preferably, the nucleotide sequence of the ILE
  • the recombinant DNA or biological material also contains an enamine/imine deaminase gene; preferably it is ridA; more preferably, the nucleotide sequence of the ridA gene is as shown in SEQ ID NO: 5;
  • the biological material is an expression cassette, transposon, plasmid vector, phage vector or viral vector.
  • a sixth aspect provides recombinant DNA or biological materials for preparing keto acids, the recombinant DNA or biological materials containing L-aldolase and first dehydratase genes; or the recombinant DNA or biological materials containing D- Acetalase gene, racemase gene, second dehydratase gene;
  • the L-aldolase gene is selected from one or more of ltaE, ltaE_Pp, psald, dhaa, CC_3093, fbaA, itaA, glyA or URA1; preferably ltaE_Pp or ltaE; more preferably, ltaE_Pp or
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the first dehydratase gene is selected from one of ilvA, tdcB, TDH, CHA1, TD2, A8H32_14290, Saut_1089, and C0627_08730 Or several; preferably ilvA or A8H32_14290; more preferably, the nucleotide sequence of the ilvA or A8H32_14290 gene is as shown in SEQ ID NO.3 or SEQ ID NO.4;
  • the D-acetalase gene is selected from one or more of A0A1C9ZZ39_CHLRE, tasS, dna, cghG, folB, guaB, dus, dhaa, bhcC, NCTC12151_01614, A4G23_03658, OJAG_33340, GGC03_18995; preferably A0A1C9ZZ39_CHLRE ;More
  • the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO.7; the racemase gene is selected from one of ILE2E_LENBU, agiA, puuE, PS659_05479, HRbin10_02390, CVS47_02795, HRbin08_01795, MJ8_44540 or Several; preferably ILE2E_LENBU; more preferably, the nucleotide sequence of the ILE2E_
  • the biological material is an expression cassette, transposon, plasmid vector, phage vector or viral vector.
  • ketoacid includes but is not limited to ketoacid with the following general formula:
  • R can be (CH 3 ) 2 CH 2 -, (CH 3 )C-, CH3-, wait.
  • the seventh aspect provides the use of the recombinant microorganism for preparing keto acids described in the third or fourth aspect, the recombinant DNA or biological material for preparing keto acids described in the fifth or sixth aspect in the preparation of keto acids.
  • the eighth aspect provides the use of the recombinant microorganism for preparing keto acids described in the third or fourth aspect, the recombinant DNA or biological material for preparing keto acids described in the fifth or sixth aspect in the preparation of amino acids or amino acid derivatives. application.
  • a ninth aspect provides a method for preparing diol organic matter.
  • the ⁇ -hydroxy- ⁇ amino acid obtained by the method of the first aspect generates diol organic matter under the action of deaminase, decarboxylase and reductase.
  • the method for preparing ketoacids of the present invention provides a novel multi-enzyme catalytic system. Glycine and different types of aldehydes produce ketoacids in the multi-enzyme catalytic system to perform enzymatic catalytic reactions.
  • the number of enzymes used is much less than that of natural enzymes.
  • the synthetic route has low production cost. It builds an artificial metabolism platform for keto acids and can produce a variety of important keto acids, such as phenylpyruvic acid, 4-methyl 2-oxopentanoic acid, pyruvate, and 2-oxobutyric acid. etc., with simple operation, high yield, high substrate utilization and low pollution.
  • the method for preparing ketoacids of the present invention is applied in the preparation process of amino acids and amino acid derivatives, and can achieve high-yield and high-yield preparation of various amino acids and amino acid derivatives, and realize industrial mass production.
  • the present invention can also be applied to the preparation of diol organic compounds.
  • Figure 1 is the process curve for the production of phenylpyruvic acid from benzaldehyde and glycine in the R2 system.
  • Figure 2 is the process curve for the production of 4-methyl-2-oxopentanoic acid from isobutyraldehyde and glycine in the R3 and R4 systems.
  • Figure 3 is the process curve for the production of 2-oxobutyric acid from acetaldehyde and glycine in the R5 system.
  • gene synthesis means produced using recombinant DNA technology or obtained using synthetic DNA or amino acid sequence technology available and well known in the art.
  • Coding refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for the synthesis of other polymers and macromolecules used in biological processes. Molecules have either a defined sequence of nucleotides (ie, rRNA, tRNA, and mRNA) or a defined sequence of amino acids and biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produce a protein in a cell or other biological system.
  • Both the coding strand where the nucleotide sequence is equivalent to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcribing a gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.
  • endogenous refers to any substance from or produced within an organism, cell, tissue or system.
  • exogenous refers to any substance introduced from or produced outside an organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter. Translated.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • the phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may in some versions include intron(s).
  • the term "vector” is a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell.
  • the transferred nucleic acid is typically ligated, eg, inserted, into a vector nucleic acid molecule.
  • the vector may contain sequences that direct autonomous replication in the cell or may contain sequences sufficient to allow integration into the host cell DNA.
  • Many vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacterial phages, and animal viruses.
  • the term “vector” includes autonomously replicating plasmids or viruses.
  • the L-acetalase encoding gene includes the ltaE gene, the fbaA gene of Bacillus subtilis, the psald gene of Pseudomonas, etc.
  • the dehydratase gene includes the ilvA gene of Escherichia coli, the thadh gene of Pseudomonas, The A8H32_14290_14160 gene of Burkholderia, the thrc gene of Bacillus subtilis, etc.
  • A8H32_14290 represents the dehydratase A8H32_14290_14160 derived from Burkholderia sp.
  • the gene can be wild-type or mutant.
  • the A8H32_14290 gene can be wild-type A8H32_14290 or mutant A8H32_14290m.
  • Mutant A8H32_14290m refers to the A8H32_14290 mutant gene that uses PLP as a coenzyme.
  • This mutation Type A8H32_14290 is different from wild-type A8H32_14290, which uses PLP as a coenzyme.
  • Mutant type A8H32_14290 carries mutations in S246A and E291A.
  • the L-acetalase (ltaE), dehydratase (ilvA, tdcB), enamine/imine deaminase (ridA) are from the E. coli genome.
  • the L-acetalase (ltaE) can be from Pseudomonas putida, and in the present invention, ltaE-Pp represents the L-acetalase from Pseudomonas putida.
  • the dehydratase (A8H32_14290) can be from Burkholderia sp.
  • the above genes were obtained through PCR reaction using DNA primers.
  • the vector used in the present invention contains any of the polynucleotide molecules mentioned above, and the vector also contains commonly used elements, including but not limited to the origin of replication (ORI), antibiotic resistance genes, One or more of multiple cloning sites (MCS), promoters, enhancers, primer binding sites, and selectable markers.
  • ORI origin of replication
  • MCS multiple cloning sites
  • promoters enhancers
  • primer binding sites and selectable markers.
  • the vector is a plasmid vector, and the plasmid is one or both of pZAlac and pZElac.
  • ltaE_Pp-R AGGTCGACATAGTTAATTTCTCCTACTAGTTCAGCCACCAATGATCGTGCGGATATCCGC;
  • ilvA-R CTTTCGTTTTATTTGATGCCTCTAGACTAACCCGCCAAAAAGAACCTGAACGCCG;
  • pdh-F CATCCGCATTTAAGCTAGCAGGAGAAATTAACTATGAGCCTGGTGGAAAAAACCAGCAT;
  • A0A1C9ZZ39_CHLRE-F GAATTCATTAAAGAGGAGAAAGGTACCATGCGCGCACTGGTTAGCAAAGCACG;
  • A0A1C9ZZ39_CHLRE-R GCCCATAGTTAATTTCTCCTgctagcTCACTGGCCAGGACCACGACCACGAATC;
  • ILE2E_LENBU-F CCAGTGAGCTAGCAGGAGAAATTAACTATGGGCAAACTGGACAAAGCGAGCAAAC;
  • ILE2E_LENBU-R GACATAGTTAATTTCTCCTAAGCTTTCACCAGCCAATTTTACCGGTGTCTTTCGGA;
  • A8H32_14290-F GGTGGCTGAACTAGTAGGAGAAATTAACTATGTCGACCTCACCCCACCGCCCCGCTCAT;
  • A8H32_14290-R ATAGTTTTGCTCATAGTTAATTTCTCCTGCTAGCTCACGGCCACGACATGCGATGCAGCCGAGC;
  • A8H32_14290’-R CGTTTTATTTGATGCTCTAGATCACGGCCACGACATGCGATGCAGCCGAG
  • ridA-F CATGTCGTGGCCGTGAGCTAGCAGGAGAAATTAACTATGAGCAAAACTATCGCGACGGAAAATGC;
  • ridA-R GCCTTTCGTTTTATTTGATGCCTCTAGATTAGCGACGAACAGCGATCGCTTCGATC;
  • xylB-F TCATTAAAGAGGAGAAAGGTACCATGGCGGTATTTGCCAGTGACTCTTTTG;
  • xylB-R CATAGTTAATTTCTCCTGCTAGCTTAAATGCGGATGATGGTCGTCTTTG;
  • the corresponding gene fragments were obtained through PCR, digested with restriction enzymes, and connected to plasmid pZElac through ligase to obtain recombinant expression plasmid vectors pZE-ltaE_Pp-A8H32_14290, pZE-ltaE_Pp-A8H32_14290-ridA, and pZE-ltaE- ilvA, pZE-ltaE-ilvA---ridA, pZA-xylB-pdh, pZE-A0A1C9ZZ39_CHLRE-ILE2E_LENBU-A8H32_14290'.
  • the nucleotide sequence of the ltaE_Pp gene is shown in SEQ ID NO: 1.
  • the nucleotide sequence of the ltaE gene is shown in SEQ ID NO: 2.
  • the nucleotide sequence of the ilvA gene is shown in SEQ ID NO: 3.
  • the nucleotide sequence of the A8H32_14290 gene is shown in SEQ ID NO: 4.
  • the nucleotide sequence of the ridA gene is shown in SEQ ID NO: 5.
  • the nucleotide sequence of the xylB gene is shown in SEQ ID NO: 6.
  • the nucleotide sequence of the A0A1C9ZZ39_CHLRE gene is shown in SEQ ID NO: 7.
  • the nucleotide sequence of the ILE2E_LENBU gene is shown in SEQ ID NO: 8.
  • the nucleotide sequence of the pdh gene used in Example 4 is shown in SEQ ID NO: 9.
  • glycine and benzaldehyde are used as raw materials, and the product is phenylpyruvic acid.
  • the recombinant expression plasmid vectors pZE-ltaE_Pp-A8H32_14290 and pZE-ltaE_Pp-A8H32_14290-ridA were transformed into the expression strain Escherichia coli BL21 respectively to obtain recombinant E. coli, labeled R1 and R2.
  • glycine and isobutyraldehyde are used as raw materials, and the product is 4-methyl-2-oxopentanoic acid.
  • the recombinant expression plasmid vectors pZE-ltaE-ilvA and pZE-ltaE-ilvA--ridA were transformed into the expression strain Escherichia coli BL21 respectively to obtain recombinant E. coli, labeled R3 and R4. Plug the recombinant E. coli into 2-xyT culture medium containing ampicillin and chloramphenicol, culture it in a 50mlenmeyer flask (10mL liquid volume) at 30°C for 3-6 hours, rotate at 240rpm, add IPTG until the end The concentration is 0.3mM, continue culturing for 20 hours, and then centrifuge at 4°C for 5 minutes at 8000rpm. Pour off the supernatant culture liquid to obtain bacterial liquid, and keep it in ice bath for later use.
  • the system contains 50mM Tris-HCl buffer (pH7.5), 20g/L glycine and 2.6g/L isobutyraldehyde, shake at 30°C, 240rpm
  • the reaction was carried out with shaking at high temperature for 24 hours to obtain a conversion solution containing phenylpyruvic acid.
  • samples were taken every 2 hours of catalysis to detect the production of oxopentanoic acid; the obtained conversion solution was diluted 20 times with deionized water, centrifuged at 12500 rpm for 10 to 15 minutes, and the supernatant was diluted and analyzed by high performance liquid chromatography. The concentration of the product in the catalytic system.
  • system R3 produced 1.9g/L of 4-methyl-2-oxopentanoic acid
  • system R4 produced 3.1g/L of 4-methyl-2-oxopentanoic acid.
  • glycine and acetaldehyde are used as raw materials, and the product is 2-oxobutyric acid.
  • the recombinant expression plasmid vector pZE-ltaE-ilvA was transformed into the expression strain Escherichia coli BL21 to obtain recombinant E. coli, labeled R5.
  • the system contains 50mM Tris-HCl buffer with a pH of 8.0, add glycine at an amount of 20g/L, and add 15ml acetaldehyde, and shake at 30°C and 240rpm.
  • glycine and benzyl alcohol are used as raw materials, and the products are phenylpyruvic acid and phenylalanine.
  • the recombinant expression plasmid vectors pZE-ltaE_Pp-A8H32_14290 and pZA-xylB-pdh were simultaneously electrotransduced into the expression strain Escherichia coli BL21 to obtain recombinant E. coli, labeled R6.
  • the pdh gene encodes an enzyme that reduces phenylpyruvic acid to phenylalanine.
  • the substrate ketoacid is further converted into an amino acid).
  • glycine and benzaldehyde are used as raw materials, and the product is phenylpyruvic acid (D-acetalase)
  • the recombinant expression plasmid vector pZE-A0A1C9ZZ39_CHLRE-ILE2E_LENBU-A8H32_14290' was transformed into the expression strain Escherichia coli BL21 to obtain recombinant E. coli, labeled R7.
  • the recombinant Escherichia coli was inserted into 2-xyT culture medium containing ampicillin and chloramphenicol, and placed in a 50 ml Erlenmeyer flask at 30°C. Liquid volume: 10 mL) and culture for 3-6 hours at a rotation speed of 240 rpm. Add IPTG to a final concentration of 0.3 mM and continue culturing for 20 hours for protein expression. Centrifuge for 5 minutes at 4°C at 8000 rpm. Pour off the supernatant culture medium to obtain bacterial liquid. Keep in ice bath for later use.

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Abstract

本发明涉及基因工程技术领域，具体涉及一种制备酮酸的方法，其以甘氨酸和醇类有机物为底物进行酶催化反应，酶催化反应过程中，醇类有机物转化为醛类有机物，甘氨酸和醛类有机物转化成β-羟基-α氨基酸，β-羟基-α氨基酸再转化为酮酸。本发明还公开了酮酸的制备方法在制备氨基酸中的应用。本发明所用酶的数量大大少于天然合成途径，生产成本低，其搭建了酮酸的人工代谢平台，能够产生多种中药的酮酸，如苯丙酮酸、4-甲基2-氧戊酸、丙酮酸、2-氧代丁酸等，操作简单，产率高，底物利用率高，污染低。

Description

一种制备酮酸的方法及该方法在制备氨基酸或氨基酸衍生物中的应用
相关申请的交叉引用
本发明要求于2022年4月11日提交中国专利局、申请号为202210371436.6、发明名称为“一种制备酮酸的方法及该方法在制备氨基酸或氨基酸衍生物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物工程技术领域，具体涉及一种制备酮酸的方法及该方法在制备氨基酸中的应用。
背景技术
氨基酸是生物功能大分子蛋白质的基本组成单位，在人和动物的生命活动以及营养健康方面发挥着至关重要的作用。氨基酸是含有碱性氨基(-NH2)和酸性羧基(-COOH)的有机化合物，且氨基和羧基连接在同一个碳原子上，其化学通式是RCHNH2COOH。根据对人体需要程度，可分为必需氨基酸和非必需氨基酸。除了生物合成的天然氨基酸外，还包括人工合成的非天然氨基酸。氨基酸类产品在医疗/保健，食品(调味品)，饲料添加剂，化妆品，化学合成等多领域有着广泛的用途，已然成为全球性一大产业。
目前世界上氨基酸的主要生产方法包括发酵法，化学合成法，水解法以及化学合成-酶法。发酵法是利用微生物自身所具有的合成各种氨基酸能力，通过菌株诱变等处理，选育出各种缺陷型及抗性的变异菌株，以解除代谢通路中的反馈与阻遏，以实现某种氨基酸过量生产为目的的一种生产方法。发酵法生产氨基酸不可避免的缺点是难以控制各种非目标氨基酸的产生与积累，生产周期长，提纯难度大，成本高。化学合成法是借助于各种有机物之间复杂的反应生产氨基酸的一种方法。虽然化学合成法可以生产各种天然氨基酸和非天然氨基酸，但所得氨基酸大多为光学消旋体(含有D和L两种旋光异构体)，大大制约了其工业价值。同时，化学合成法也存在反应复杂，步骤多，污染环境的问题。水解法是以富含蛋白质的物质如毛发等为原料，通过酸、碱或蛋白质水解酶进行水解，再经过 分离纯化获得各种氨基酸的工艺过程。水解法原料较为丰富，投产相对容易，但产量低，成本较高且对环境的污染比较大。
目前，国内除谷氨酸、赖氨酸等少量氨基酸外，大部分氨基酸依然无法实现产业化生产，至今仍为国外企业垄断。而大多数氨基酸均可通过酮酸经一步转氨酶合成，因此，酮酸的生产和制备极为重要。目前来说，酮酸的制备存在的问题是使用有毒的催化剂，并且产量和转化率低的现象。公开号为US08153839B2的美国专利公开了通过乙炔化合物水合合成酮酸或氨基酸的方法，其使用金属盐和过渡金属络合物的水合允许在温和条件下由乙炔-羧酸合成酮酸(包括酮酸和酮酸衍生物)，而不使用极其有害的汞催化剂，并且可以在同一容器中通过随后的还原胺化由合成的酮酸(包括酮酸和酮酸衍生物)容易地合成氨基酸(包括氨基酸和氨基酸衍生物)，然而其操作复杂，生产过程难以控制，且产量和转化率较低，难以实现工业化大批量生产。
因此，本发明旨在从酮酸的制备着手突破技术创新，全面提高多种天然氨基酸或非天然氨基酸及衍生物产量，提高产品市场竞争力。
发明内容
本发明所解决的技术问题之一在于提供一种底物利用率高、污染底、产量高、生产成本低的一种制备酮酸的方法。
本发明所解决的技术问题之二在于提供一种底物利用率高、污染底、产量高、生产成本低的一种制备二醇类有机物的方法。
本发明所解决的技术问题之三在于能够产生多种重要的酮酸：苯丙酮酸、4-甲基2-氧戊酸、丙酮酸、2-氧代丁酸等，并且该途径可应用于其它酮酸及氨基酸的生产中。
本发明所解决的技术问题采用以下技术方案来实现：
第一方面，提供一种制备酮酸的方法，以甘氨酸和醇类有机物为底物进行酶催化反应，酶催化反应过程中，醇类有机物转化为醛类有机物，甘氨酸和醛类有机物转化成β-羟基-α氨基酸，β-羟基-α氨基酸再转化为酮酸。
优选地，制备得到的酮酸包括但不限于具有如下通式的酮酸：
其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
进一步地，以甘氨酸和醇类有机物为底物，以含有L-醛缩酶和第一脱水酶基因的第一重组微生物及含有脱氢酶的第二重组微生物过表达产生的酶为催化剂进行酶催化反应；醇类有机物在脱氢酶的作用下转化为醛类有机物，甘氨酸和醛类有机物在L-醛缩酶的单独催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第一脱水酶的催化下生成酮酸；
或以含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物及含有所述脱氢酶的第二重组微生物过表达产生的酶为催化剂进行酶催化反应；醇类有机物在脱氢酶的作用下转化为醛类有机物，甘氨酸和醛类有机物在D-缩醛酶、消旋酶的共同催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第二脱水酶的催化下生成酮酸。
进一步地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因包括adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示。
进一步地，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID  NO.3所示。
进一步地，第一重组微生物还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示。
进一步地，所述醇类有机物选自苯甲醇、4-咪唑甲醇、2甲硫基乙醇、吲哚-3-甲醇、2-羟乙基-甲基次磷酸、对羟基苯甲醇、3，4-二羟基苯甲醇、对甲基苯甲醇、苯乙醇、特戊醇、异丁醇、乙醇中的一种或几种。
第二方面，提供一种制备酮酸的方法，以甘氨酸及醛类有机物为底物进行酶催化反应，酶催化反应过程中，甘氨酸和醛类有机物转化成β-羟基-α氨基酸，β-羟基-α氨基酸再转化为酮酸。
优选地，制备得到的酮酸包括但不限于具有如下通式的酮酸：
其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
进一步地，以甘氨酸及醛类有机物为底物，以含有L-醛缩酶和第一脱水酶基因的第一重组微生物过表达产生的酶为催化剂进行酶催化反应；甘氨酸和醛类有机物在L-醛缩酶的单独催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第一脱水酶的催化下生成酮酸；
或以甘氨酸及醛类有机物为底物，以所述含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物过表达产生的酶为催化剂进行酶催化反应；甘氨酸和醛类有机物在D-缩醛酶、消旋酶的共同催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第二脱水酶的催化下生成酮酸。
进一步地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种； 优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示。
进一步地，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
进一步地，包括通过基因工程方法构建所述第一重组微生物、第二重组微生物或第三重组微生物，所述基因工程方法包括质粒表达或基因组整合。
进一步地，采用质粒表达方式构建时采用的质粒载体为pZAlac、pZElac中的一种或两种。
进一步地，构建的重组微生物培养后进行酶催化反应，重组微生物的培养方法为：将重组微生物接种至含有氨苄青霉素、卡那霉素和氯霉素的2-xyT培养基中，在20～60℃的条件下培养3～6h，加入IPTG至终浓度0.3mM，继续培养15～30h后离心，倒去上清培养液即可。
进一步地，酶催化反应过程中，反应温度为20-90℃，反应缓冲液的PH为6.5-8.5。
进一步地，重组微生物包括重组的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
进一步地，重组微生物选自重组的大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、尼古丁降解菌(Pseudomonas sp.)巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
第三方面，提供制备酮酸的重组微生物，所述重组微生物为含有L-醛缩酶和第一脱水酶基因的第一重组微生物及含有脱氢酶的第二重组微生物；或者，所述重组微生物为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物及含有脱氢酶的第二重 组微生物；
优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因选自adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示；
或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示；
进一步优选地，所述第一重组微生物还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示。
第四方面，提供制备酮酸的重组微生物，所述重组微生物为含有L-醛缩酶和第一脱水酶基因的第一重组微生物；或者，所述重组微生物为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物；
优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、 guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
进一步地，所述酮酸包括但不限于具有如下通式的酮酸：
其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
进一步地，所述重组微生物选自重组的大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、尼古丁降解菌(Pseudomonas sp.)巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
第五方面，提供用于制备酮酸的重组DNA或生物材料，所述重组DNA或生物材料含有L-醛缩酶和第一脱水酶基因及含有脱氢酶；或者，所述重组DNA或生物材料含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因及含有脱氢酶；
优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA 或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因选自adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示；
或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示；
进一步优选地，所述重组DNA或生物材料还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示；
优选地，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
第六方面，提供用于制备酮酸的重组DNA或生物材料，所述重组DNA或生物材料含有L-醛缩酶和第一脱水酶基因；或者，所述重组DNA或生物材料为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因；
优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或 几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
优选地，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
进一步地，所述酮酸包括但不限于具有如下通式的酮酸：
其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
第七方面，提供第三方面或第四方面所述的制备酮酸的重组微生物、第五方面或第六方面所述的制备酮酸的重组DNA或生物材料在制备酮酸中的应用。
第八方面，提供第三方面或第四方面所述的制备酮酸的重组微生物、第五方面或第六方面所述的制备酮酸的重组DNA或生物材料在制备氨基酸或氨基酸衍生物中的应用。
第九方面，提供一种制备二醇类有机物的方法，第一方面的方法获得的β-羟基-α氨基酸在脱氨酶、脱羧酶及还原酶的作用下生成二醇类有机物。
有益效果：
本发明所述的酮酸制备的方法，其通过提供新型的多酶催化体系，甘氨酸和不同种类的醛生产酮酸在多酶催化体系的作用进行酶催化反应，所用酶的数量大大少于天然合成途径，生产成本低，其搭建了酮酸的人工代谢平台，能够产生多种重要的酮酸，如苯丙酮酸、4-甲基2-氧戊酸、丙酮酸、2-氧代丁酸等，操作简单，产率高，底物利用率高，污染低。
本发明所述的酮酸制备的方法，应用于氨基酸及氨基酸衍生物的制备过程中，可实现多种氨基酸及氨基酸衍生物的高产量和高产率制备，实现工业化大批量生产。
本发明还可应用于二醇类有机物的制备。
附图说明
图1为R2体系苯甲醛和甘氨酸生产苯丙酮酸的过程曲线。
图2为R3、R4体系异丁醛和甘氨酸生产4-甲基-2-氧戊酸的过程曲线。
图3为R5体系乙醛和甘氨酸生产2-氧代丁酸的过程曲线。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例进一步阐述本发明。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
也应理解本文使用的术语仅是为了描述具体实施方式的目的，并不意欲是限制性的。
本文中使用冠词“一”和“所述”来指代冠词的语法宾语中的一个或多于一个。
替代方案(例如，“或”)的使用应当被理解为意指替代方案中的一个、两个或其任何组合。术语“和/或”应当被理解为意指替代方案中的一个或两个。
如本文所用，术语“基因合成”，指利用重组DNA技术产生或利用本领域可用和公知的合成DNA或氨基酸序列技术获得。
“编码”指的是多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异性序列用作模板合成在生物学过程中的其他多聚体和大分子的固有性质，所述多聚体和大分子具有核苷酸(即，rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性质。因此，如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学系统中产生蛋白质，则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中的编码链，和用作转录基因或cDNA的模板的非编码链两者，都可被称为编码那个基因或cDNA的蛋白质或其他产物。
如本文所用，术语“内源的”指的是来自有机体、细胞、组织或系统的或在有机体、细胞、组织或系统内产生的任何物质。
如本文所用，术语“外源的”指的是任何从有机体、细胞、组织或系统引入的或在有机体、细胞、组织或系统外产生的物质。
如本文所用，术语“表达”被定义为由它的启动子驱动的特定核苷酸序列的转录和/或翻 译。
除非另有规定，“编码氨基酸序列的多核苷酸序列”包括为彼此简并版本并编码相同的氨基酸序列的所有的核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子，其程度为编码该蛋白质的核苷酸序列可在某些版本中包含内含子(一个或多个)。
如本文所用的，术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。转移的核酸通常连接到例如插入到载体核酸分子中。载体可以包含引导细胞中的自主复制的序列或可以包含足以允许整合到宿主细胞DNA中的序列。很多载体在本领域中是已知的，包含但不限于质粒、噬菌粒、人工染色体、细菌噬菌体以及动物病毒。因此，术语“载体”包括自主复制的质粒或病毒。
在一些实施例中，L-缩醛酶编码基因包括ltaE基因、枯草芽孢杆菌的fbaA基因、假单胞菌的psald基因等、脱水酶基因包括大肠杆菌ilvA基因、假单胞杆菌的thadh基因、伯克霍尔德菌的A8H32_14290_14160基因、枯草芽孢杆菌的thrc基因等。
以A8H32_14290表示来自伯克霍尔德菌的脱水酶A8H32_14290_14160。
所述基因可以是野生型或突变型，例如在一些实施例中，所述A8H32_14290基因可以为野生型的A8H32_14290或突变型A8H32_14290m，突变型A8H32_14290m是指使用PLP作为辅酶的A8H32_14290突变型基因，该突变型A8H32_14290与使用PLP为辅酶的野生型A8H32_14290不同，突变型A8H32_14290携带S246A、E291A的变异。
在一些实施例中，所述L-缩醛酶(ltaE)、脱水酶(ilvA，tdcB)，烯胺/亚胺脱氨酶(ridA)来自大肠杆菌基因组。
在一些实施例中，所述L-缩醛酶(ltaE)可以来自恶臭假单胞菌，在本发明中以ltaE-Pp表示来自恶臭假单胞杆菌的L-缩醛酶。
在一些实施例中，所述脱水酶(A8H32_14290)可以来自伯克霍尔德菌。
上述基因利用DNA引物通过PCR反应获得。
本发明用到的载体中，包含前述任一项的多核苷酸分子，所述载体中还包含常用元件，包括但不限于复制起始位点(Origin of replication，ORI)、抗生素抗性基因、多克隆位点(multiple cloning site，MCS)、启动子、增强子、引物结合位点、选择标记中的一种或多种。
在一些实施例中，载体为质粒载体，质粒为pZAlac、pZElac中的一种或两种。
根据NCBI公布的恶臭假单胞菌(Pseudomonas putida)的基因组序列设计引物：
ltaE_Pp-F：GAATTCATTAAAGAGGAGAAAGGTACCATGACAGACAAGAGCCAAC AATTCGCCAGCG；
ltaE_Pp-R：AGGTCGACATAGTTAATTTCTCCTACTAGTTCAGCCACCAATGATCGTGCGGATATCCGC；
根据NCBI公布的大肠杆菌MG1655的基因组序列设计引物：
ltaE-F：TTAAAGAGGAGAAAGGTACCATGATTGATTTACGCAGTGATACCG；
ltaE-R：CAGCCATAGTTAATTTCTCCTACTAGTTCAGCCACCAATGATCGTGCGGATATCC；
ilvA-F：GCGCGTTAAACTAGTAGGAGAAATTAACTATGGCTGACTCGCAACCCCTGTCC)；
ilvA-R：CTTTCGTTTTATTTGATGCCTCTAGACTAACCCGCCAAAAAGAACCTGAACGCCG；
根据NCBI公布的芽孢杆菌(Bacillus badius)的基因组序列设计引物：
pdh-F：CATCCGCATTTAAGCTAGCAGGAGAAATTAACTATGAGCCTGGTGGAAAAAACCAGCAT；
pdh-R：ATTTGATGCCTCTAGAGCTAGCTTAATTACGAATATCCCATTTCGGTTTAAC；
根据NCBI公布的莱茵衣藻(Chlamydomonas reinhardtii)的基因组序列设计引物：
A0A1C9ZZ39_CHLRE-F：GAATTCATTAAAGAGGAGAAAGGTACCATGCGCGCACTGGTTAGCAAAGCACG；
A0A1C9ZZ39_CHLRE-R：GCCCATAGTTAATTTCTCCTgctagcTCACTGGCCAGGACCACGACCACGAATC；
根据NCBI公布的布氏乳杆菌(Lactobacillus buchneri)的基因组序列设计引物：
ILE2E_LENBU-F：CCAGTGAGCTAGCAGGAGAAATTAACTATGGGCAAACTGGACAAAGCGAGCAAAC；
ILE2E_LENBU-R：GACATAGTTAATTTCTCCTAAGCTTTCACCAGCCAATTTTACCGGTGTCTTTCGGA；
根据NCBI公布的伯克霍尔德菌(Burkholderia thailandensis E264)的基因组序列设计引物有两组：
A8H32_14290-F：GGTGGCTGAACTAGTAGGAGAAATTAACTATGTCGACCTCACCCCACCGCCCCGCTCAT；
A8H32_14290-R：ATAGTTTTGCTCATAGTTAATTTCTCCTGCTAGCTCACGGCCACGACATGCGATGCAGCCGAGC；
A8H32_14290’-F：TGGCTGGTAAAAGCTTAGGAGAAATTAACTATGTCGACCTCACCCCACCGCCCCG
A8H32_14290’-R：CGTTTTATTTGATGCCTCTAGATCACGGCCACGACATGCGATGCAGCCGAG
根据NCBI公布的大肠杆菌MG1655的基因组序列设计引物：
ridA-F：CATGTCGTGGCCGTGAGCTAGCAGGAGAAATTAACTATGAGCAAAACTATCGCGACGGAAAATGC；
ridA-R：GCCTTTCGTTTTATTTGATGCCTCTAGATTAGCGACGAACAGCGATCGCTTCGATC；
根据NCBI公布的恶臭假单胞菌(Pseudomonas putida)的基因组序列设计引物：
xylB-F：TCATTAAAGAGGAGAAAGGTACCATGGCGGTATTTGCCAGTGACTCTTTTG；
xylB-R：CATAGTTAATTTCTCCTGCTAGCTTAAATGCGGATGATGGTCGTCTTTG；
通过PCR获取对应的基因片段，通过限制性内切酶消化后，通过连接酶，连接在质粒pZElac上，得到重组表达质粒载体pZE-ltaE_Pp-A8H32_14290、pZE-ltaE_Pp-A8H32_14290-ridA、pZE-ltaE-ilvA、pZE-ltaE-ilvA--ridA、pZA-xylB-pdh、pZE-A0A1C9ZZ39_CHLRE-ILE2E_LENBU-A8H32_14290’。
其中：
ltaE_Pp基因的核苷酸序列如SEQ ID NO：1所示。
ltaE基因的核苷酸序列如SEQ ID NO：2所示。
ilvA基因的核苷酸序列如SEQ ID NO：3所示。
A8H32_14290基因的核苷酸序列如SEQ ID NO：4所示。
ridA基因的核苷酸序列如SEQ ID NO：5所示。
xylB基因的核苷酸序列如SEQ ID NO：6所示。
A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO：7所示。
ILE2E_LENBU基因的核苷酸序列如SEQ ID NO：8所示。
实施例4用到的pdh基因的核苷酸序列如SEQ ID NO：9所示。
实施例1
本实施例以甘氨酸和苯甲醛为原料，产物为苯丙酮酸。
将重组表达质粒载体pZE-ltaE_Pp-A8H32_14290，pZE-ltaE_Pp-A8H32_14290-ridA分别转化至表达菌株大肠杆菌Escherichia coli BL21中，得到重组大肠杆菌，标记为R1，R2。将重组大肠杆菌接入含有氨苄青霉素和氯霉素的2-xyT培养基中，在30℃下于50ml三角瓶中(装液量10mL)培养3-6个小时，转速240rpm，加入IPTG至终浓度0.3mM，继续培养20h，以进行蛋白质表达。在4℃条件下离心5分钟，转速8000rpm，倒去上清培养液，得到菌液，冰浴待用。
在一个2ml体系中，加入上述菌液，体系中含有50mM的pH为7.5的Tris-HCl缓冲液，以20g/L的比例加入甘氨酸(该比例为甘氨酸与体系的比例)，并加入4.6g/L苯甲醛，在30℃摇床、240rpm的条件下振荡反应24个小时，得到含苯丙酮酸的转化液。其中，每催化2小时取样检测苯丙酮酸的生成；将所得的转化液用去离子水稀释20倍，12500转/分离心10～15分钟，吸取上清稀释进行高效液相色谱分析，测定催化体系中底物和产物的浓度。经测定，催化反应24h之后，R1体系产生苯丙酮酸3.6g/L，催化反应12h后，R2体系产生苯丙酮酸5g/L。R2体系中，增加基因ridA能加快从中间体烯胺到终产物的速度，进而让反应更快，并提高产物浓度及产量(如图1所示)。
实施例2
本实施例以甘氨酸和异丁醛为原料，产物为4-甲基-2-氧戊酸。
将重组表达质粒载体pZE-ltaE-ilvA、pZE-ltaE-ilvA--ridA，分别转化至表达菌株大肠杆菌Escherichia coli BL21中，得到重组大肠杆菌，标记为R3，R4。将重组大肠杆菌接入含有氨苄青霉素和氯霉素的2-xyT培养基中，在30℃下于50ml三角瓶中(装液量10mL)培养3-6个小时，转速240rpm，加入IPTG至终浓度0.3mM，继续培养20个小时，然后在4℃离心5分钟，转速8000rpm，倒去上清培养液，得到菌液，冰浴待用。
在一个2ml体系中，加入上述菌液，体系中含有50mM Tris-HCl缓冲液(pH7.5)，20g/L的甘氨酸和2.6g/L的异丁醛，在30℃摇床，240rpm的条件下振荡反应24个小时，得到含苯丙酮酸的转化液。其中，每催化2小时取样检测氧代戊酸的生成；将所得的转化液用去离子水稀释20倍，12500转/分离心10～15分钟，吸取上清稀释进行高效液相色谱分析，测定催化体系中产物的浓度。经测定，催化反应26h，体系R3产生4-甲基-2-氧戊酸1.9g/L，体系R4产生4-甲基-2-氧戊酸3.1g/L。增加基因ridA能加快从中间体烯胺到终产物的速度，进而让反应更快，并提高产物浓度及产量(如图2所示)。
实施例3
本实施例以甘氨酸和乙醛为原料，产物为2-氧代丁酸。
将重组表达质粒载体pZE-ltaE-ilvA转化至表达菌株大肠杆菌Escherichia coli BL21中，得到重组大肠杆菌，标记为R5。将重组大肠杆菌接入含有氨苄青霉素和氯霉素的2-xyT培养基中，在30℃下于50ml三角瓶中(装液量10mL)培养3-6个小时，转速240rpm，加入IPTG至终浓度0.3mM继续培养20个小时。在4℃条件下离心5分钟，转速8000rpm，倒去上清培养液，得到菌液，冰浴待用。
在一个2ml体系中，加入上述菌液，体系中含有50mM的pH为8.0的Tris-HCl缓冲液，以20g/L的量加入甘氨酸，并加入15ml乙醛，在30℃、240rpm的条件下摇床发酵，振荡反应3个小时，得到含2-氧代丁酸的转化液，将所得的转化液用去离子水稀释10倍，12500转/分离心10～15分钟，吸取上清稀释进行高效液相色谱分析，测定产物的浓度。经测定，催化反应18h后，共产生2-氧代丁酸1.4g/L(如图3所示)。
实施例4
本实施例以甘氨酸和苯甲醇为原料，产物为苯丙酮酸和苯丙氨酸。
将重组表达质粒载体pZE-ltaE_Pp-A8H32_14290、pZA-xylB-pdh同时电转至表达菌株大肠杆菌Escherichia coli BL21中，得到重组大肠杆菌，标记为R6。(其中pdh基因编码把苯丙酮酸还原为苯丙氨酸的酶，该实施例中，底物酮酸进一步转化为氨基酸)。将菌种接入含有氨苄青霉素、卡那霉素和氯霉素的2-xyT培养基中，在30℃下于50ml三角瓶中(装液量10mL)培养3-6个小时，转速240rpm，加入IPTG至终浓度0.3mM，继续培养20个小时。在4℃离心5分钟，转速8000rpm，倒去上清培养液，得到菌液，冰浴待用。
在一个2ml体系中，加入上述菌液，使之含有50mM的pH为7.5的Tris-HCl缓冲液，以20g/L的比例加入甘氨酸并以4.6g/L的比例加入苯甲醇，在30℃、240rpm的条件下摇床发酵，振荡反应24h，得到含苯丙氨酸的转化液。催化反应24h之后，将所得的转化液用去离子水稀释20倍，12500转/分离心10～15分钟，吸取上清稀释进行高效液相色谱分析，测定催化体系中底物和产物的浓度。经测定，R6体系产生苯丙酮酸2.3g/L，苯丙氨酸1.2g/L。
实施例5
本实施例以甘氨酸和苯甲醛为原料，产物为苯丙酮酸(D-缩醛酶)
将重组表达质粒载体pZE-A0A1C9ZZ39_CHLRE-ILE2E_LENBU-A8H32_14290’转化至表达菌株大肠杆菌Escherichia coli BL21中，得到重组大肠杆菌，标记为R7。将重组大肠杆菌接入含有氨苄青霉素和氯霉素的2-xyT培养基中，在30℃下于50ml三角瓶中(装 液量10mL)培养3-6个小时，转速240rpm，加入IPTG至终浓度0.3mM，继续培养20h，以进行蛋白质表达。在4℃条件下离心5分钟，转速8000rpm，倒去上清培养液，得到菌液，冰浴待用。
在一个2ml体系中，加入上述菌液，体系中含有50mM的pH为7.5的Tris-HCl缓冲液，以20g/L的比例加入甘氨酸(该比例为甘氨酸与体系的比例)，并加入4.6g/L苯甲醛，在30℃摇床、240rpm的条件下振荡反应24个小时，得到含苯丙酮酸的转化液。其中，每催化2小时取样检测苯丙酮酸的生成；将所得的转化液用去离子水稀释20倍，12500转/分离心10～15分钟，吸取上清稀释进行高效液相色谱分析，测定催化体系中底物和产物的浓度。经测定，催化反应24h之后，R7体系产生苯丙酮酸2.6g/L。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (28)

  1. 一种制备酮酸的方法，其特征在于，以甘氨酸和醇类有机物为底物进行酶催化反应，酶催化反应过程中，醇类有机物转化为醛类有机物，甘氨酸和醛类有机物转化成β-羟基-α氨基酸，β-羟基-α氨基酸再转化为酮酸。
  2. 根据权利要求1所述的方法，其特征在于，制备得到的酮酸包括但不限于具有如下通式的酮酸：
    其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
  3. 根据权利要求1或2所述的制备酮酸的方法，其特征在于，以甘氨酸和醇类有机物为底物，以含有L-醛缩酶和第一脱水酶基因的第一重组微生物及含有脱氢酶的第二重组微生物过表达产生的酶为催化剂进行酶催化反应；醇类有机物在脱氢酶的作用下转化为醛类有机物，甘氨酸和醛类有机物在L-醛缩酶的单独催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第一脱水酶的催化下生成酮酸；
    或以含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物及含有所述脱氢酶的第二重组微生物过表达产生的酶为催化剂进行酶催化反应；醇类有机物在脱氢酶的作用下转化为醛类有机物，甘氨酸和醛类有机物在D-缩醛酶、消旋酶的共同催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第二脱水酶的催化下生成酮酸。
  4. 根据权利要求3所述的制备酮酸的方法，其特征在于，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、 A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因包括adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示。
  5. 根据权利要求3所述的制备酮酸的方法，其特征在于，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
  6. 根据权利要求1-5任一项所述的制备酮酸的方法，其特征在于，第一重组微生物还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示。
  7. 根据权利要求1-6任一项所述的制备酮酸的方法，其特征在于，所述醇类有机物选自苯甲醇、4-咪唑甲醇、2甲硫基乙醇、吲哚-3-甲醇、2-羟乙基-甲基次磷酸、对羟基苯甲醇、3，4-二羟基苯甲醇、对甲基苯甲醇、苯乙醇、特戊醇、异丁醇、乙醇中的一种或几种。
  8. 一种制备酮酸的方法，其特征在于，以甘氨酸及醛类有机物为底物进行酶催化反应，酶催化反应过程中，甘氨酸和醛类有机物转化成β-羟基-α氨基酸，β-羟基-α氨基酸再转化为酮酸。
  9. 根据权利要求8所述的方法，其特征在于，制备得到的酮酸包括但不限于具有如下通式的酮酸：
    其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
  10. 根据权利要求8或9所述的方法，其特征在于，以甘氨酸及醛类有机物为底物，以含有L-醛缩酶和第一脱水酶基因的第一重组微生物过表达产生的酶为催化剂进行酶催化反应；甘氨酸和醛类有机物在L-醛缩酶的单独催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第一脱水酶的催化下生成酮酸；
    或以甘氨酸及醛类有机物为底物，以所述含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物过表达产生的酶为催化剂进行酶催化反应；甘氨酸和醛类有机物在D-缩醛酶、消旋酶的共同催化下转化成β-羟基-α氨基酸，β-羟基-α氨基酸在第二脱水酶的催化下生成酮酸。
  11. 根据权利要求10所述的方法，其特征在于，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；
    所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示。
  12. 根据权利要求10所述的方法，其特征在于，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
  13. 根据权利要求1-12所述的方法，其特征在于，包括通过基因工程方法构建所述第一重组微生物、第二重组微生物或第三重组微生物，所述基因工程方法包括质粒表达或基因组整合。
  14. 根据权利要求13所述的方法，其特征在于，采用质粒表达方式构建时采用的质粒载体为pZAlac、pZElac中的一种或两种。
  15. 根据权利要求14所述的方法，其特征在于，构建的重组微生物培养后进行酶催化反应，重组微生物的培养方法为：将重组微生物接种至含有氨苄青霉素、卡那霉素和氯霉素的2-xyT培养基中，在20～60℃的条件下培养3～6h，加入IPTG至终浓度0.3mM，继续培养15～30h后离心，倒去上清培养液即可。
  16. 根据权利要求15所述的方法，其特征在于，酶催化反应过程中，反应温度为20-90℃，反应缓冲液的PH为6.5-8.5。
  17. 根据权利要求13-16任一项所述酮酸的制备方法，其特征在于，重组微生物包括重组的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
  18. 根据权利要求17所述制备酮酸的方法，其特征在于，重组微生物选自重组的大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、尼古丁降解菌(Pseudomonas sp.)巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
  19. 制备酮酸的重组微生物，所述重组微生物为含有L-醛缩酶和第一脱水酶基因的第一重组微生物及含有脱氢酶的第二重组微生物；或者，所述重组微生物为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物及含有脱氢酶的第二重组微生物；
    优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因选自adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示；
    或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、 guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示；
    进一步优选地，所述第一重组微生物还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示。
  20. 制备酮酸的重组微生物，所述重组微生物为含有L-醛缩酶和第一脱水酶基因的第一重组微生物；或者，所述重组微生物为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因的第三重组微生物；
    优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
    或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
  21. 根据权利要求19或20所述的重组微生物，其特征在于，所述酮酸包括但不限于具有如下通式的酮酸：
    其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
  22. 权利要求19-21任一项所述的重组微生物，其特征在于，所述重组微生物选自重组的大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、尼古丁降解菌(Pseudomonas sp.)巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
  23. 用于制备酮酸的重组DNA或生物材料，其特征在于，所述重组DNA或生物材料含有L-醛缩酶和第一脱水酶基因及含有脱氢酶；或者，所述重组DNA或生物材料含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因及含有脱氢酶；
    优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；所述脱氢酶基因选自adhE、adh、ADH7、xylB、adhA、xylW、ped、leuB、BADH、aldh、ACIAD1578、qbdA中的一种或几种；优选为xylB；更优选地，xylB基因的核苷酸序列如SEQ ID NO.6所示；
    或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、 PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示；
    进一步优选地，所述重组DNA或生物材料还含有烯胺/亚胺脱氨酶基因；优选为ridA；更优选地，ridA基因的核苷酸序列如SEQ ID NO：5所示；
    优选地，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
  24. 用于制备酮酸的重组DNA或生物材料，其特征在于，所述重组DNA或生物材料含有L-醛缩酶和第一脱水酶基因；或者，所述重组DNA或生物材料为含有D-缩醛酶基因、消旋酶基因、第二脱水酶基因；
    优选地，所述L-醛缩酶基因选自ltaE、ltaE_Pp、psald、dhaa、CC_3093、fbaA、itaA、glyA或URA1中的一种或几种；优选为ltaE_Pp或ltaE；更优选地，ltaE_Pp或ltaE基因的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；所述第一脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；优选为ilvA或A8H32_14290；更优选地，ilvA或A8H32_14290基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
    或者，所述D-缩醛酶基因选自A0A1C9ZZ39_CHLRE、tasS、dna、cghG、folB、guaB、dus、dhaa、bhcC、NCTC12151_01614、A4G23_03658、OJAG_33340、GGC03_18995中的一种或几种；优选为A0A1C9ZZ39_CHLRE；更优选地，A0A1C9ZZ39_CHLRE基因的核苷酸序列如SEQ ID NO.7所示；所述消旋酶基因选自ILE2E_LENBU、agiA、puuE、PS659_05479、HRbin10_02390、CVS47_02795、HRbin08_01795、MJ8_44540中的一种或几种；优选为ILE2E_LENBU；更优选地，ILE2E_LENBU基因的核苷酸序列如SEQ ID NO.8所示；所述第二脱水酶基因选自ilvA、tdcB、TDH、CHA1、TD2、A8H32_14290、Saut_1089、C0627_08730中的一种或几种；更优选地，ilvA基因的核苷酸序列如SEQ ID NO.3所示。
    优选地，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体或病毒载体。
  25. 根据权利要求23或24所述的重组DNA或生物材料，其特征在于，所述酮酸包括但不限于具有如下通式的酮酸：
    其中R的结构式可以为(CH3)2CH2-、(CH3)C-、CH3-、等。
  26. 权利要求19-22任一项所述的制备酮酸的重组微生物、权利要求23-25任一项所述的制备酮酸的重组DNA或生物材料在制备酮酸中的应用。
  27. 权利要求19-22任一项所述的制备酮酸的重组微生物、权利要求23-25任一项所述的制备酮酸的重组DNA或生物材料在制备氨基酸或氨基酸衍生物中的应用。
  28. 一种制备二醇类有机物的方法，其特征在于，权利要求1-19任一项方法获得的β-羟基-α氨基酸在脱氨酶、脱羧酶及还原酶的作用下生成二醇类有机物。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008109924A (ja) * 2006-08-30 2008-05-15 Ajinomoto Co Inc ４−ヒドロキシ−ｌ−イソロイシン又はその塩の製造法
CN102245779A (zh) * 2008-12-11 2011-11-16 生物结构实验室公司 大宗化学品的生物合成
US8153839B2 (en) 2005-09-14 2012-04-10 Japan Science And Technology Agency Method for synthesis of keto acids or amino acids by hydration of acetylene compound
CN107586797A (zh) * 2017-09-25 2018-01-16 长兴制药股份有限公司 一锅酶法制备左旋多巴的方法
US20190177713A1 (en) * 2016-06-30 2019-06-13 Ardra Bio Inc. Methods and microorganisms for producing flavors and fragrance chemicals

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010187658A (ja) * 2008-08-25 2010-09-02 Kochi Univ Ｄ−フェニルセリンデアミナーゼ及びその利用
CA2933470C (en) * 2013-12-12 2023-10-03 Dow Global Technologies Llc Processes to prepare elongated 2-ketoacids and c6-c10 compounds therefrom via genetic modifications to microbial metabolic pathways
CN104372035B (zh) * 2014-10-17 2017-10-31 湖南宝利士生物技术有限公司 合成高纯2‑酮酸盐的方法
CN104673814B (zh) * 2015-02-27 2018-02-02 台州职业技术学院 一种来自于阴沟肠杆菌的l‑苏氨酸醛缩酶及其应用
CN104774881B (zh) * 2015-04-10 2018-06-19 湖南福来格生物技术有限公司 一种生物催化生产L-α-氨基丁酸的方法
CN111406104B (zh) * 2017-11-29 2023-08-08 上海凯赛生物技术股份有限公司 减少为了生产氨基酸或氨基酸衍生产物的亚胺/烯胺的积累
CN108841844B (zh) * 2018-06-26 2022-09-06 江南大学 一种高效生产苯丙酮酸的方法
JP6894650B2 (ja) * 2018-10-30 2021-06-30 ＧｒｅｅｎＥａｒｔｈＩｎｓｔｉｔｕｔｅ株式会社 有機化合物の製造方法およびコリネ型細菌
CN113481188B (zh) * 2018-11-06 2023-03-24 王喆明 苏氨酸醛缩酶的突变体及其在制备取代苯丝氨酸衍生物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8153839B2 (en) 2005-09-14 2012-04-10 Japan Science And Technology Agency Method for synthesis of keto acids or amino acids by hydration of acetylene compound
JP2008109924A (ja) * 2006-08-30 2008-05-15 Ajinomoto Co Inc ４−ヒドロキシ−ｌ−イソロイシン又はその塩の製造法
CN102245779A (zh) * 2008-12-11 2011-11-16 生物结构实验室公司 大宗化学品的生物合成
US20190177713A1 (en) * 2016-06-30 2019-06-13 Ardra Bio Inc. Methods and microorganisms for producing flavors and fragrance chemicals
CN107586797A (zh) * 2017-09-25 2018-01-16 长兴制药股份有限公司 一锅酶法制备左旋多巴的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Clinical Biochemistry", 31 May 1999, TIANJIN SCIENCE AND TECHNOLOGY PRESS, CN, ISBN: 9787530824733, article TANG, XINZHI ET AL.: "Other Bioconvertase Systems and their Catalytic Reactions", pages: 837 - 840, XP009549413 *
See also references of EP4509610A4

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