WO2023197968A1 - 一种异丙醇胺的制备方法 - Google Patents

一种异丙醇胺的制备方法 Download PDF

Info

Publication number
WO2023197968A1
WO2023197968A1 PCT/CN2023/087079 CN2023087079W WO2023197968A1 WO 2023197968 A1 WO2023197968 A1 WO 2023197968A1 CN 2023087079 W CN2023087079 W CN 2023087079W WO 2023197968 A1 WO2023197968 A1 WO 2023197968A1
Authority
WO
WIPO (PCT)
Prior art keywords
isopropanolamine
tdh
gre2p
gene
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/087079
Other languages
English (en)
French (fr)
Inventor
曹晨凯
张科春
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mint Biotechnologies Co Ltd
Original Assignee
Mint Biotechnologies Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mint Biotechnologies Co Ltd filed Critical Mint Biotechnologies Co Ltd
Priority to EP23787612.3A priority Critical patent/EP4516918A4/en
Priority to US18/855,814 priority patent/US20250290106A1/en
Publication of WO2023197968A1 publication Critical patent/WO2023197968A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01103—L-Threonine 3-dehydrogenase (1.1.1.103)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the technical fields of genetic engineering and fermentation engineering, and in particular to a preparation method of isopropanolamine.
  • Isopropanolamine is an important basic chemical raw material. It is widely used in metal protective agents, acid gas absorbers, cement grinding aids and other fields. It can also be used as fiber industry refining agent, antistatic agent, dyeing agent and fiber wetting agent. , it can also be used to synthesize detergents, and be used in the preparation of cosmetic lubricants, antioxidants, plasticizers, emulsifiers and solvents for cutting oils, etc.
  • the current methods for producing isopropanolamine include propylene oxide method, calcium cyanamide method and supercritical fluid method.
  • the propylene oxide method is to mix propylene oxide and ammonia, perform a ring-opening reaction through preheating to generate a mixture, and then undergo dehydrogenation, dehydration, vacuum distillation, and rectification to obtain isopropanolamine. During this process, Depending on the feeding ratio of propylene oxide and ammonia, diisopropanolamine and triisopropanolamine can also be generated.
  • the calcium cyanamide method uses propylene oxide and calcium cyanamide to form 2-amino-3-(2-hydroxypropyl)-5methyl-1,3-dioxazolidine and calcium carbonate; oxazole Alkanes generate diisopropanolamine in the presence of potassium hydroxide or potassium carbonate.
  • the supercritical fluid method uses ammonia and propylene oxide as raw materials and water as a catalyst. In the supercritical state, it undergoes processes such as flash evaporation, two-step absorption deamination, continuous distillation under reduced pressure, and falling film evaporation to continuously generate isopropyl alcohol. amine.
  • the above synthesis methods face problems such as complex steps, unenvironmental-friendly raw materials and intermediate products, the use of highly toxic cyanide, harsh reaction conditions, complex by-products, low conversion rates, and low yields, and they need to be improved urgently.
  • the technical problem solved by the present invention is to provide a preparation method of isopropanolamine that is more suitable for industrial production, more environmentally friendly, and has higher yield.
  • the invention provides a method for preparing isopropanolamine, which is characterized in that threonine is converted into isopropanolamine under the action of oxidase and reductase, and the isopropanolamine is selected from 1-amino-(R)- 2-propanol and/or 1-amino-(S)-2-propanol; isopropanolamine is preferably 1-amino-(S)-2-propanol.
  • the invention also provides a preparation method of isopropanolamine, which is characterized in that threonine is converted into L-2-amino-3-oxobutyric acid under the action of oxidase, and L-2-amino-3-oxobutyric acid is Butyric acid spontaneously decarboxylates to aminoacetone, which reacts with the action of reductase It is converted into isopropanolamine, which includes 1-amino-(R)-2-propanol or 1-amino-(S)-2-propanol.
  • transformation is accomplished in vivo in bacteria or fungi.
  • threonine is used as a substrate, and a recombinant microorganism containing an oxidase encoding gene and a reductase encoding gene is added for fermentation culture. During the fermentation process, the recombinant microorganism is overexpressed to produce the oxidase and reductase.
  • the oxidase encoding gene includes any one or more of tdh, yiaY, adhB, preferably tdh; more preferably, the nucleotide sequence of the tdh gene is as shown in SEQ ID NO: 1.
  • the reductase encoding gene includes any one or more of gre2p, egsA, SU7, and VIN7, preferably gre2p; more preferably, the nucleotide sequence of the gre2p gene is such as SEQ ID NO: 2 shown.
  • the preparation method further includes constructing the recombinant microorganism by genetic engineering methods, which preferably include plasmid expression or genome integration.
  • the recombinant microorganism is constructed by a plasmid expression method.
  • the construction method is: obtaining the oxidase encoding gene and the reductase encoding gene through PCR amplification, and jointly connecting the obtained genes to a plasmid vector (for example, containing IPTG inducible
  • a plasmid vector for example, containing IPTG inducible
  • the plasmid vector of the promoter preferably the pZElac vector containing the IPTG inducible promoter
  • competent cells such as E. coli dh5a competent cells
  • the recombinant vector is pZE-tdh-gre2p; preferably, the construction method of pZE-tdh-gre2p is: amplify the tdh gene and gre2p gene through PCR, and jointly connect the tdh gene and gre2p gene to a protein containing IPTG-induced type promoter on the pZElac vector and transformed into competent cells. After sequencing, the plasmid pZE-tdh-gre2p is obtained; the exemplary construction method of pZE-tdh-gre2p is: using the genomes of Escherichia coli MG1655 and Saccharomyces cerevisiae S288C as templates respectively.
  • the tdh gene and gre2p gene were amplified by PCR.
  • the tdh gene and gre2p gene were jointly connected to the pZElac vector containing the IPTG inducible promoter and transformed into E. coli dh5a competent cells. After sequencing, the plasmid pZE-tdh was obtained -gre2p.
  • the microorganism is selected from one or more of Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces.
  • the microorganism is selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum ( Corynebacterium glutamicum), Saccharomyces cerevisiae, Candida utilis or Pichia pastoris.
  • the fermentation temperature ranges from 20 to 90°C.
  • the culture medium used includes raw materials in the following proportions: M9 salt 11-13g/L, magnesium sulfate 1-5g/L, calcium chloride 0.1-0.5g/L, and thiamine 0.01 ⁇ 0.05g/L, excipients 10 ⁇ 100g/L, yeast powder 3 ⁇ 8g/L, IPTG 1 ⁇ 3mM/L, ampicillin 30 ⁇ 60 ⁇ g/mL.
  • the excipient is D-glucose.
  • the present invention also provides a recombinant microorganism for preparing isopropanolamine, the recombinant microorganism contains an oxidase encoding gene and a reductase encoding gene;
  • the oxidase encoding gene includes any one or more of tdh, yiaY, and adhB, preferably tdh; more preferably, the nucleotide sequence of the tdh gene is as shown in SEQ ID NO: 1;
  • the reductase encoding gene includes any one or more of gre2p, egsA, SU7, and VIN7, preferably gre2p; more preferably, the nucleotide sequence of the gre2p gene is as shown in SEQ ID NO: 2 ;
  • the microorganism is selected from one or more of Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; more preferably, the microorganism is selected from Escherichia coli, Bacillus subtilis ), Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris ( Pichia pastoris) one or more.
  • the present invention also provides recombinant DNA or biological materials for preparing isopropanolamine, the recombinant microorganisms or biological materials contain oxidase encoding genes and reductase encoding genes;
  • the oxidase encoding gene includes any one or more of tdh, yiaY, and adhB, preferably tdh; more preferably, the nucleotide sequence of the tdh gene is as shown in SEQ ID NO: 1;
  • the reductase encoding gene includes any one or more of gre2p, egsA, SU7, and VIN7, preferably gre2p; more preferably, the nucleotide sequence of the gre2p gene is as shown in SEQ ID NO: 2 ;
  • the biological material is an expression cassette, transposon, plasmid vector, phage vector or viral vector.
  • the present invention also provides the use of the above-mentioned recombinant microorganism, the above-mentioned recombinant DNA or biological materials in the preparation of isopropanolamine.
  • the preparation method of isopropanolamine according to the present invention uses enzyme to oxidize threonine to L-2-amino-3-oxobutyric acid, and obtain L-2-amino-3-oxobutyric acid. Spontaneous decarboxylation of butyric acid yields aminoacetone, which is then reduced to isopropanolamine using reductase.
  • the invention uses low-cost raw materials, has simple preparation procedures, mild operating means, can avoid the use of highly toxic cyanide, is an environmentally friendly process, has high conversion rate and yield, and is suitable for industrial processing and production.
  • the invention can produce two different configurations of isopropanolamine respectively in a mild environment. Has good production promotion and application Use value.
  • 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.
  • 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, which To the extent that the nucleotide sequence encoding the protein may in some versions contain 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.
  • DNA polymerase Phanta Max Super-Fidelity DNA Polymerase and ligase-independent single fragment rapid cloning kit used in examples of the present invention II One Step was purchased from Nanjing Novozan Biotechnology Co., Ltd.
  • LB medium ingredients tryptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, and add 1.5% agar powder to the solid medium.
  • the antibiotic concentration is: ampicillin 50 ⁇ g/mL.
  • Detection method of isopropanolamine Use HPLC-RID equipped with Agilent chromatography column (Agilent InfinityLab Poroshell 120 Columns) to quantitatively detect isopropanolamine.
  • Example 1 The recombinant vector used in Example 1 and Example 2 was constructed as follows;
  • the tdh gene fragment was amplified by PCR using the genome of E. coli MG1655 as the template, and the gre2p gene fragment was amplified by PCR using the genome of Saccharomyces cerevisiae S288C as the template, and they were ligated using the ligase-independent single fragment rapid cloning kit. into the vector pZElac containing the IPTG inducible promoter, then transformed into BW25113 competent cells, spread on kanamycin sulfate-resistant plates and cultured overnight, select positive clones for sequencing verification, and name the correct recombinant vector is pZE-tdh-gre2p.
  • the tdh gene fragment and gldA gene fragment were amplified by PCR using the genome of E. coli MG1655 as a template, and ligated into the vector pZElac containing an IPTG-inducible promoter through a ligase-independent single fragment rapid cloning kit, and then Transform into BW25113 competent cells, coat kanamycin sulfate-resistant plates and culture overnight, select positive clones for sequencing verification, and the correct recombinant vector is named pZE-tdh-gldA.
  • nucleotide sequence of the tdh gene is shown in SEQ ID NO:1.
  • the nucleotide sequence of the gre2p gene is shown in SEQ ID NO:2.
  • the nucleotide sequence of gldA gene is shown in SEQ ID NO:3.
  • the recombinant vector pZE-tdh-gre2p was transferred into E. coli BW25113 to obtain the recombinant bacteria.
  • Single colonies of the above recombinant bacteria were inoculated into 2 mL of LB liquid medium containing 50 ⁇ g/mL ampicillin, and cultured overnight at 37°C and 220 rpm (about 14 hours ).
  • the initial OD of 0.05 transfer a 100mL Erlenmeyer flask containing 10mL of fermentation medium, and add threonine as a substrate at a ratio of 8.57g/L to the fermentation medium (this ratio is the ratio of threonine to fermentation medium) ), culture at 30°C, 220rpm.
  • the fermentation medium used is as follows:
  • This example is a comparative example.
  • the recombinant vector pZE-tdh-gldA was transformed into E. coli BW25113 to obtain recombinant
  • To form bacteria single colonies of the above recombinant bacteria were inoculated into 2 mL of LB liquid medium containing 50 ⁇ g/mL ampicillin, and cultured at 37°C and 220 rpm overnight (about 14 hours).
  • Example 1 The test results of Example 1 and Example 2 are shown in Table 1. It can be seen that in Example 1, the concentration of isopropanolamine in the fermentation broth after 12 hours of fermentation is 0.43g/L, and the conversion rate is 22.6%. After 24 hours of fermentation, the concentration of isopropanolamine in the fermentation broth is as high as 1.48g/L, and the conversion rate is 43.8 %, the isopropanolamine obtained in this example is all S-configuration isopropanolamine. In other embodiments, the configuration of the isopropanolamine product can be controlled through the selection of enzyme genes. . In Example 2, during the fermentation process, threonine was converted due to the presence of oxidase. However, although gldA is also a gene encoding a reductase, it cannot catalyze the production of isopropanolamine.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明涉及基因工程及发酵工程技术领域，具体公开了一种异丙醇胺的制备方法，其特征在于，苏氨酸在氧化酶的作用下转化为L-2-氨基-3氧代丁酸，L-2-氨基-3氧代丁酸自发脱羧得到氨基丙酮，氨基丙酮在还原酶的作用下转化为异丙醇胺，异丙醇胺包括1-氨基-(R)-2-丙醇或1-氨基-(S)-2-丙醇。

Description

一种异丙醇胺的制备方法
本申请要求于2022年4月11日提交中国专利局、申请号为202210371284.X、发明名称为“一种异丙醇胺的制备方法”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
本发明涉及基因工程及发酵工程技术领域，具体涉及一种异丙醇胺的制备方法。
背景技术
异丙醇胺是重要的基础性化工原料，广泛应用于金属保护剂、酸性气体吸收剂、水泥助磨剂等领域，还可以用作纤维工业精炼剂、抗静电剂、染色剂、纤维湿润剂，还可合成洗涤剂，及用于化妆品润滑油，切削油的抗氧剂、增塑剂、乳化剂和溶剂的制备等。
目前生产异丙醇胺的方法有环氧丙烷法、氰氨化钙法和超临界流体法。环氧丙烷法是将环氧丙烷与氨混合后，经预热进行开环反应，生成混合物，然后经脱氢、脱水、减压蒸馏、精馏而得到异丙醇胺，在此过程中，根据环氧丙烷和氨的投料比，还可生成二异丙醇胺和三异丙醇胺。氰氨化钙法是利用环氧丙烷与氰氨化钙作用，生成2-氨基-3-(2-羟丙基)-5甲基-1,3-二噁唑烷和碳酸钙；噁唑烷在氢氧化钾或碳酸钾存在下生成二异丙醇胺。超临界流体法是以氨和环氧丙烷为原料，水为催化剂，在超临界状态下经过闪蒸、吸收二步脱氨和减压连续精馏、降膜蒸发等工艺，连续生成异丙醇胺。
以上合成方法面临着步骤复杂，原料及中间产物不环保，涉及到剧毒氰化物的使用，且反应条件苛刻，副产物复杂，转化率低下、产量不高等问题，亟待改进。
发明内容
本发明所解决的技术问题在于提供一种更适合工业化生产且更环保，产量更高的异丙醇胺的制备方法。
本发明提供一种异丙醇胺的制备方法，其特征在于，苏氨酸在氧化酶、还原酶的作用下转化为异丙醇胺，异丙醇胺选自1-氨基-(R)-2-丙醇和/或1-氨基-(S)-2-丙醇；异丙醇胺优选为1-氨基-(S)-2-丙醇。
本发明还提供一种异丙醇胺的制备方法，其特征在于，苏氨酸在氧化酶的作用下转化为L-2-氨基-3氧代丁酸，L-2-氨基-3氧代丁酸自发脱羧得到氨基丙酮，氨基丙酮在还原酶的作 用下转化为异丙醇胺，异丙醇胺包括1-氨基-(R)-2-丙醇或1-氨基-(S)-2-丙醇。
在一些实施方案中，转化在细菌或真菌体内完成。
在一些实施方案中，以苏氨酸为底物，加入含有氧化酶编码基因及还原酶编码基因的重组微生物进行发酵培养，发酵过程中，重组微生物过表达产生所述氧化酶及还原酶。
在一些实施方案中，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示。
在一些实施方案中，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述制备方法还包括通过基因工程方法构建所述重组微生物，基因工程方法优选地包括质粒表达或基因组整合。
在一些实施方案中，重组微生物通过质粒表达的方法进行构建，构建方法为：通过PCR扩增获得氧化酶编码基因及还原酶编码基因，将获得的基因共同连接至质粒载体(例如含有IPTG诱导型启动子的质粒载体，优选为含有IPTG诱导型启动子的pZElac载体)上、并转化至感受态细胞(例如大肠杆菌E.coli dh5a感受态细胞)中，测序后获得重组载体；将重组载体转化至微生物中，即得到重组微生物。
在一些实施方案中，重组载体为pZE-tdh-gre2p；优选地，pZE-tdh-gre2p构建方法为：通过PCR扩增得tdh基因和gre2p基因，将tdh基因和gre2p基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至感受态细胞中，测序后得到质粒pZE-tdh-gre2p；pZE-tdh-gre2p构建方法示例性为：以大肠杆菌MG1655及酿酒酵母S288C的基因组为模板分别通过PCR扩增得tdh基因和gre2p基因，将tdh基因和gre2p基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中，测序后得到质粒pZE-tdh-gre2p。
在一些实施方案中，微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
在一些实施方案中，微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、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。
在一些实施方案中，发酵过程中，发酵温度为20～90℃。
在一些实施方案中，发酵过程中，采用的培养基包括如下比例的原料：M9盐11～13g/L，硫酸镁1～5g/L，氯化钙0.1～0.5g/L、硫胺素0.01～0.05g/L、辅料10～100g/L、酵母粉3～8g/L、IPTG 1～3mM/L、氨苄霉素30～60μg/mL。
在一些实施方案中，辅料为D-葡萄糖。
本发明还提供制备异丙醇胺的重组微生物，所述重组微生物含有氧化酶编码基因和还原酶编码基因；
优选地，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示；
和/或，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2所示；
优选地，微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种；更优选地，微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、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。
本发明还提供用于制备异丙醇胺的重组DNA或生物材料，所述重组微生物或生物材料含有氧化酶编码基因和还原酶编码基因；
优选地，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示；
和/或，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2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。
本发明还提供上述重组微生物、上述重组DNA或生物材料在制备异丙醇胺中的应用。
有益效果：本发明所述的一种异丙醇胺的制备方法，其利用酶将苏氨酸氧化为L-2-氨基-3-氧代丁酸，得到的L-2-氨基-3氧代丁酸自发脱羧得到氨基丙酮，然后利用还原酶将氨基丙酮还原为异丙醇胺。本发明采用成本低廉的原料，制备工序简单，操作手段温和，能避免剧毒氰化物的使用，过程环保，转化率及产率高，适合工业化加工生产。
本发明能在温和的环境下分别生产两种不同构型的异丙醇胺。具有很好的生产推广和应 用价值。
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。
在本公开中，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。
本发明实例所用的DNA聚合酶Phanta Max Super-Fidelity DNA Polymerase、非连接酶依赖型单片段快速克隆试剂盒II One Step购自南京诺唯赞生物科技股份有限公司。
LB培养基成分：胰蛋白胨10g/L，酵母粉5g/L,氯化钠10g/L，固体培养基中添加1.5％的琼脂粉。
抗生素浓度为：氨苄霉素50μg/mL。
异丙醇胺的检测方法：使用配备安捷伦色谱柱(Agilent InfinityLab Poroshell 120 Columns)的HPLC-RID定量检测异丙醇胺。
实施例1和实施例2用到的重组载体构建如下；
根据NCBI公布的大肠杆菌MG1655和酿酒酵母S288C的基因组序列分别设计引物：
以大肠杆菌MG1655的基因组为模板通过PCR扩增得tdh基因片段，以酿酒酵母S288C的基因组为模板通过PCR扩增得gre2p基因片段，并通过非连接酶依赖型单片段快速克隆试剂盒将其连接到含有IPTG诱导型启动子的载体pZElac上，然后转化至BW25113感受态细胞，涂布硫酸卡那霉素抗性平板过夜培养，挑阳性克隆进行测序验证，正确的重组载体命名 为pZE-tdh-gre2p。
以大肠杆菌MG1655的基因组为模板通过PCR扩增得tdh基因片段及gldA基因片段，并通过非连接酶依赖型单片段快速克隆试剂盒将其连接到含有IPTG诱导型启动子的载体pZElac上，然后转化至BW25113感受态细胞，涂布硫酸卡那霉素抗性平板过夜培养，挑阳性克隆进行测序验证，正确的重组载体命名为pZE-tdh-gldA。
其中，tdh基因的核苷酸序列如SEQ ID NO:1所示。
gre2p基因的核苷酸序列如SEQ ID NO:2所示。
gldA基因的核苷酸序列如SEQ ID NO:3所示。
实施例1
将重组载体pZE-tdh-gre2p转入大肠杆菌BW25113中，得到重组菌，将上述重组菌单菌落分别接种2mL含有50μg/mL氨苄霉素的LB液体培养基，37℃，220rpm过夜培养(约14h)。按照初始OD为0.05转接装有10mL发酵培养基的100mL三角瓶，往发酵培养基中按8.57g/L的比例加入苏氨酸作为底物(该比例为苏氨酸与发酵培养基的比值)，30℃，220rpm培养。每个发酵瓶中加入0.5g CaCO3调节发酵液的pH。培养12h及24h后收集发酵液，检测苏氨酸和异丙醇胺的浓度(参见表1)。苏氨酸在tdh编码的氧化酶的作用下转化为L-2-氨基-3氧代丁酸，L-2-氨基-3氧代丁酸自发脱羧得到氨基丙酮，氨基丙酮在gre2p编码的还原酶的作用下转化为异丙醇胺。
采用的发酵培养基如下：
实施例2
本实施例为对照实施例。将重组载体pZE-tdh-gldA转入大肠杆菌BW25113中，得到重 组菌，将上述重组菌单菌落分别接种2mL含有50μg/mL氨苄霉素的LB液体培养基，37℃，220rpm过夜培养(约14h)。按照初始OD为0.05转接装有10mL发酵培养基的100mL三角瓶，往发酵培养基中按8.57g/L的比例加入苏氨酸作为底物(该比例为苏氨酸与发酵培养基的比值)，32℃，220rpm培养。每个发酵瓶中加入0.5g CaCO3调节发酵液的pH。培养12h及24h后收集发酵液，检测苏氨酸和异丙醇胺的浓度(参见表1)。
实施例1及实施例2的检测结果如表1所示。可知，实施例1中发酵12h后发酵液中异丙醇胺浓度为0.43g/L，转化率为22.6％，发酵24h后发酵液中的异丙醇胺高达1.48g/L，转化率为43.8％，本实施例中得到的异丙醇胺均为S-构型的异丙醇胺，在其它的一些实施例中，可通过酶基因的选择实现对异丙醇胺产物的构型的控制。而实施例2在发酵过程中，由于氧化酶的存在，苏氨酸有所转化，然而gldA虽然也为还原酶编码基因，却并不能催化产生异丙醇胺。
表1检测结果表
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。
序列信息：



Claims (15)

  1. 一种异丙醇胺的制备方法，其特征在于，苏氨酸在氧化酶、还原酶的作用下转化为异丙醇胺，异丙醇胺选自1-氨基-(R)-2-丙醇和/或1-氨基-(S)-2-丙醇；异丙醇胺优选为1-氨基-(S)-2-丙醇。
  2. 一种异丙醇胺的制备方法，其特征在于，苏氨酸在氧化酶的作用下转化为L-2-氨基-3氧代丁酸，L-2-氨基-3氧代丁酸自发脱羧得到氨基丙酮，氨基丙酮在还原酶的作用下转化为异丙醇胺，异丙醇胺包括1-氨基-(R)-2-丙醇或1-氨基-(S)-2-丙醇。
  3. 根据权利要求1或2所述的异丙醇胺的制备方法，其特征在于，转化在细菌或真菌体内完成。
  4. 根据权利要求1-3任一项所述的异丙醇胺的制备方法，其特征在于，以苏氨酸为底物，加入含有氧化酶编码基因及还原酶编码基因的重组微生物进行发酵培养，发酵过程中，重组微生物过表达产生所述氧化酶及还原酶。
  5. 根据权利要求1-4任一项所述的异丙醇胺的制备方法，其特征在于，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示；
    和/或，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2所示。
  6. 根据权利要求4或5所述的异丙醇胺的制备方法，其特征在于，所述制备方法还包括通过基因工程方法构建所述重组微生物，基因工程方法优选地包括质粒表达或基因组整合。
  7. 根据权利要求6所述的异丙醇胺的制备方法，其特征在于，重组微生物通过质粒表达的方法进行构建，构建方法为：通过PCR扩增获得氧化酶编码基因及还原酶编码基因，将获得的基因共同连接至质粒载体(例如含有IPTG诱导型启动子的质粒载体，优选为含有IPTG诱导型启动子的pZElac载体)上、并转化至感受态细胞(例如大肠杆菌E.coli dh5a感受态细胞)中，测序后获得重组载体；将重组载体转化至微生物中，即得到重组微生物。
  8. 根据权利要求7所述的异丙醇胺的制备方法，其特征在于，重组载体为pZE-tdh-gre2p；优选地，pZE-tdh-gre2p构建方法为：通过PCR扩增得tdh基因和gre2p基因，将tdh基因和gre2p基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至感受态细胞中，测序后得到质粒pZE-tdh-gre2p；pZE-tdh-gre2p构建方法示例性为：以大肠杆菌MG1655及酿酒酵 母S288C的基因组为模板分别通过PCR扩增得tdh基因和gre2p基因，将tdh基因和gre2p基因共同连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中，测序后得到质粒pZE-tdh-gre2p。
  9. 根据权利要求7所述的异丙醇胺的制备方法，其特征在于，微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
  10. 根据权利要求7所述的异丙醇胺的制备方法，其特征在于，微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、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。
  11. 根据权利要求4-10任一项所述的异丙醇胺的制备方法，其特征在于，发酵过程中，发酵温度为20～90℃。
  12. 根据权利要求4-10任一项所述的异丙醇胺的制备方法，其特征在于，发酵过程中，采用的培养基包括如下比例的原料：M9盐11～13g/L，硫酸镁1～5g/L，氯化钙0.1～0.5g/L、硫胺素0.01～0.05g/L、辅料10～100g/L、酵母粉3～8g/L、IPTG 1～3mM/L、氨苄霉素30～60μg/mL。
  13. 制备异丙醇胺的重组微生物，所述重组微生物含有氧化酶编码基因和还原酶编码基因；
    优选地，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示；
    和/或，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2所示；
    优选地，微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种；更优选地，微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、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。
  14. 用于制备异丙醇胺的重组DNA或生物材料，其特征在于，所述重组微生物或生物材料含有氧化酶编码基因和还原酶编码基因；
    优选地，氧化酶编码基因包括tdh、yiaY、adhB中的任一种或几种，优选为tdh；更优选地，tdh基因的核苷酸序列如SEQ ID NO:1所示；
    和/或，所述还原酶编码基因包括gre2p、egsA、SU7、VIN7中的任一种或几种，优选为gre2p；更优选地，gre2p基因的核苷酸序列如SEQ ID NO:2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。
  15. 权利要求13所述重组微生物、权利要求14所述的重组DNA或生物材料在制备异丙醇胺中的应用。
PCT/CN2023/087079 2022-04-11 2023-04-07 一种异丙醇胺的制备方法 Ceased WO2023197968A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP23787612.3A EP4516918A4 (en) 2022-04-11 2023-04-07 PROCESS FOR THE PREPARATION OF ISOPROPANOLAMINE
US18/855,814 US20250290106A1 (en) 2022-04-11 2023-04-07 Method for preparing isopropanolamine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210371284.X 2022-04-11
CN202210371284.XA CN116376993B (zh) 2022-04-11 2022-04-11 一种异丙醇胺的制备方法

Publications (1)

Publication Number Publication Date
WO2023197968A1 true WO2023197968A1 (zh) 2023-10-19

Family

ID=86971726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/087079 Ceased WO2023197968A1 (zh) 2022-04-11 2023-04-07 一种异丙醇胺的制备方法

Country Status (4)

Country Link
US (1) US20250290106A1 (zh)
EP (1) EP4516918A4 (zh)
CN (1) CN116376993B (zh)
WO (1) WO2023197968A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117756650A (zh) * 2023-12-19 2024-03-26 万华化学集团股份有限公司 一种单异丙醇胺的制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1610745A (zh) * 2001-03-02 2005-04-27 第一精密化学股份有限公司 氨基酮不对称还原酶及其核酸
US20050222430A1 (en) * 2004-03-30 2005-10-06 Keisuke Yaegashi Process for preparing amines
CN107162920A (zh) * 2016-03-08 2017-09-15 沈阳金久奇科技有限公司 一种r-1-氨基-2-丙醇的制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101502634B1 (ko) * 2007-02-08 2015-03-16 코덱시스, 인코포레이티드 케토 환원 효소 및 이의 용도
CN105218385A (zh) * 2014-07-04 2016-01-06 沈阳斯特朗外加剂有限公司 一种异丙醇胺的制备方法
CN106906248A (zh) * 2017-03-28 2017-06-30 清华大学 一种利用重组微生物发酵生产1,3‑丙二醇的方法
CN112076780B (zh) * 2020-09-22 2021-04-30 山东达民化工股份有限公司 一种异丙醇胺的催化制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1610745A (zh) * 2001-03-02 2005-04-27 第一精密化学股份有限公司 氨基酮不对称还原酶及其核酸
US20050222430A1 (en) * 2004-03-30 2005-10-06 Keisuke Yaegashi Process for preparing amines
CN107162920A (zh) * 2016-03-08 2017-09-15 沈阳金久奇科技有限公司 一种r-1-氨基-2-丙醇的制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAMPBELL, R. L. ET AL.: "Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K12", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 53, no. 2, 17 July 1973 (1973-07-17), XP024841171, DOI: 10.1016/0006-291X(73)90680-3 *
See also references of EP4516918A4
TURNER JM: "Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in escherichia coli", BIOCHEMICAL JOURNAL, BIOCHEMICAL SOCIETY, LONDON, GB, vol. 99, no. 2, 1 May 1966 (1966-05-01), GB , pages 427 - 433, XP009549645, ISSN: 0006-2936, DOI: 10.1042/bj0990427 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117756650A (zh) * 2023-12-19 2024-03-26 万华化学集团股份有限公司 一种单异丙醇胺的制备方法

Also Published As

Publication number Publication date
CN116376993A (zh) 2023-07-04
EP4516918A4 (en) 2025-05-28
US20250290106A1 (en) 2025-09-18
EP4516918A1 (en) 2025-03-05
CN116376993B (zh) 2023-11-28

Similar Documents

Publication Publication Date Title
JP6848067B2 (ja) 新規なイソプロピルリンゴ酸シンターゼ変異体及びそれを用いたｌ−ロイシンの生産方法
US20220186271A1 (en) Xylose-Induced Genetically Engineered Bacteria Used for Producing Ectoine and Use Thereof
JP2017523787A (ja) フィードバック抵抗性アセトヒドロキシ酸シンターゼ変異体及びそれを用いたｌ−バリンの生産方法
JP2004535163A (ja) Ｒｎａポリメラーゼから誘導されたポリペプチド及びそれらの使用
KR101483012B1 (ko) 재조합 대장균을 이용하여 3-히드록시프로피온산을 고수율로 생산하는 방법
CN109777763A (zh) 一株用于l-茶氨酸生产的基因工程菌及其构建与应用
JP7753355B2 (ja) 改善特性を有する遺伝子改変メチロバチルス細菌
CN117757651A (zh) 优化苏氨酸脱水酶或脱氨酶表达提高l-异亮氨酸产量的方法
CN116376993B (zh) 一种异丙醇胺的制备方法
CN104673734B (zh) 用于生产β-丙氨酸的工程菌及生产β-丙氨酸的方法
CN108913724A (zh) 一种以丙二酸盐为原料合成3-羟基丙酸的制备方法及其相应重组细胞和应用
CN119955704B (zh) 一种用于发酵生产d-泛醇的重组菌及其应用
CN108546697B (zh) 酶法制备beta丙氨酸
CN115404224A (zh) 产l-苏氨酸基因工程菌及其构建方法与应用
CN108220317B (zh) 一种重组表达质粒及其制备方法、用途
EP4361268A1 (en) Mdh gene-based polynucleotide having promoter activity and use thereof
WO2023186037A1 (zh) 一种利用苏氨酸制备甘氨酸、乙酰辅酶a及乙酰辅酶a衍生物的方法
JP2024536957A (ja) Ｄ－プシコースの転化合成に安定的に繰り返し使用可能なバチルス・リケニフォルミス細胞
CN110904102A (zh) 一种用于重组蛋白质表达的启动子
CN118909905B (zh) 一种优化的l-异亮氨酸生产菌株构建方法及其应用
CN116376989B (zh) 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
CN106282083B (zh) 一种利用葡萄糖合成d-乳酸的重组菌及其构建方法与应用
CN119193529B (zh) 一种t7 rna聚合酶突变体及其制备与应用
EP4509612A1 (en) Method for preparing s-lactoylglutathione
CN118638759B (zh) 一种烟酰胺磷酸核糖转移酶突变体及其应用

Legal Events

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

Ref document number: 23787612

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023787612

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023787612

Country of ref document: EP

Effective date: 20241111

WWP Wipo information: published in national office

Ref document number: 18855814

Country of ref document: US

WWW Wipo information: withdrawn in national office

Ref document number: 2023787612

Country of ref document: EP