WO2022127887A1 - 利用生物多酶偶联法制备l-草铵膦的方法 - Google Patents

利用生物多酶偶联法制备l-草铵膦的方法 Download PDF

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WO2022127887A1
WO2022127887A1 PCT/CN2021/139010 CN2021139010W WO2022127887A1 WO 2022127887 A1 WO2022127887 A1 WO 2022127887A1 CN 2021139010 W CN2021139010 W CN 2021139010W WO 2022127887 A1 WO2022127887 A1 WO 2022127887A1
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glufosinate
transaminase
ammonium
reaction
amino
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French (fr)
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王华磊
魏东芝
吴承骏
刘清海
罗中华
张长雷
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永农生物科学有限公司
华东理工大学
宁夏永农生物科学有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)

Definitions

  • the present application relates to the field of biotechnology, and in particular to a method for preparing L-glufosinate-ammonium using a biological multi-enzyme coupling method.
  • Glufosinate-ammonium also known as bialaphos, glufosinate, the trade names include Baoshida, Besutun, etc., the English name is phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) ) phosphono]butyric acid), is the world's second largest genetically modified crop tolerant herbicide, developed and produced by Hearst Company.
  • Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthase inhibitor, a non-selective (killing) contact herbicide.
  • glufosinate-ammonium There are two optical isomers of glufosinate-ammonium, namely L-glufosinate-ammonium and D-glufosinate-ammonium, but only the L-type has herbicidal activity, and it is easily decomposed in the soil and is less toxic to humans and animals. Weeding has a wide spectrum and little damage to the environment.
  • glufosinate-ammonium sold on the market is generally a racemic mixture. If glufosinate-ammonium products can be used in the form of pure optical isomers of L-configuration, the usage amount of glufosinate-ammonium can be significantly reduced, which is of great significance for improving atom economy, reducing use cost and reducing environmental pressure.
  • the chemical method includes chemical separation method and chemical synthesis method.
  • the chemical resolution method is to separate the racemic D,L-glufosinate or its derivatives synthesized by the external chemical method by chiral resolution reagents, so as to obtain optically pure L-glufosinate.
  • this process has disadvantages such as the need to use a chiral resolution reagent, the need for racemization of D-glufosinate-ammonium, and the need for multiple resolutions, making it difficult for large-scale industrial production.
  • Chemical synthesis methods can be used to synthesize L-glufosinate from natural chiral amino acids or asymmetric methods. However, the amino protection and deprotection process of this process is complicated and involves many steps, which are mostly found in laboratory studies.
  • the chemical synthesis method can also adopt the asymmetric catalytic hydrogenation method to synthesize L-glufosinate-ammonium.
  • the catalyst used in this route is expensive, the reaction route is long, and the industrialization cost is high.
  • biocatalysis Compared with chemical methods, which have the disadvantages of high energy consumption, heavy pollution, and high cost, biocatalysis has the advantages of mild reaction conditions, high stereoselectivity, and high yield, and is an important trend for industrial preparation of L-glufosinate-ammonium.
  • L-glufosinate-ammonium Most of the methods for preparing L-glufosinate-ammonium currently use 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) as a substrate, and then asymmetrically synthesize L-glufosinate through an ammoniation reaction.
  • PPO 2-oxo-4-(hydroxymethylphosphinyl)butyric acid
  • CN1349561A describes the use of screened aspartate aminotransferase (Asp-TA) to synthesize L-glufosinate from PPO by transamination with L-aspartate as amino donor.
  • Patent CN105603015B describes a method for producing L-glufosinate using aminotransferase with L-alanine as amino donor.
  • This process can completely convert 100 mM PPO into L-Glufosinate-ammonium, but in order to inhibit the reverse reaction, the dosage of alanine is too high, and 300 mM L-alanine still exists in the system after the reaction, which causes difficulties for subsequent separation and purification , while the reaction temperature is as high as 50 ° C, the energy consumption is high, which is not conducive to production.
  • PPO under the mediation of amino acid dehydrogenase, using inorganic ammonia as a donor, PPO is reductively aminated to prepare L-fine glufosinate-ammonium, such as CN106978453A. Inorganic ammonia is used in the reaction process, and the conversion rate can reach 99%. Above, however, this process requires an additional coenzyme regeneration system, and the produced by-products increase the difficulty of separation and purification.
  • the application provides a method for preparing L-glufosinate-ammonium using a biological multi-enzyme coupling method, which uses D,L-glufosinate as raw material, and obtains L-glufosinate through a multi-enzyme catalytic system.
  • the catalytic system includes: (R)-transaminase and (S)-transaminase.
  • the method has high raw material conversion rate, simple separation and purification process, high product yield, low production cost and easy industrialization.
  • the present application provides a method of producing L-glufosinate-ammonium, the method comprising: in the presence of (R)-transaminase, (S)-transaminase, amino acceptor and amino donor, Convert D,L-glufosinate to L-glufosinate.
  • the method of the present application only needs to add racemic glufosinate-ammonium, a trace amount of amino acceptor (such as pyruvic acid) (to initiate the first step reaction), and amino donor (such as L-aspartic acid) (for start the second step reaction), and the biocatalysts (R)-transaminase and (S)-transaminase.
  • the method of the present application involves a two-step reaction.
  • (R)-transaminase mediates the deamination of D-glufosinate in racemic glufosinate to generate the intermediate keto acid 2-carbonyl-4 -(Hydroxymethylphosphono)butyric acid (PPO), while L-glufosinate does not participate in the reaction and is completely retained;
  • PPO is reductively aminated by (S)-transaminase in time to generate L-glufosinate Ammonium phosphine, generated by-products such as oxaloacetate, spontaneously degrades to amino acceptors, complementing the deamination reaction in the first step.
  • the content of amino acceptor is always maintained at a low concentration, which reduces the degree of inhibition of the reaction, and due to the spontaneous degradation of by-products, the reaction equilibrium is always moved to the product side, breaking the limit of thermodynamic equilibrium to achieve complete transform.
  • the method of the present application can realize the one-pot and one-step deracemization of racemic glufosinate-ammonium only through the coupling of two stereoselective complementary transaminases, without introducing a cofactor in situ regeneration system to remove the amino acceptor, only The recycling of amino acceptors is realized through spontaneous degradation of by-products, which greatly reduces the complexity of the reaction system.
  • Transaminase (Amine Transaminase, ATA, EC 2.6.1.X) belongs to the class of transferases, which catalyze the transfer of an amino group on an amino donor (amino acid or simple amine) to a prochiral acceptor ketone to obtain a chiral amine
  • a class of enzymes with by-products ketones or ⁇ -keto acids, the reactions catalyzed are reversible.
  • Transaminases can be further divided into ⁇ -transaminases and omega-transaminases (EC 2.6.1.1), depending on the amino acceptor at which the amino group is transferred.
  • the ⁇ -transaminase reaction process can be divided into two steps.
  • the first reaction is to transfer the amino group on the amino donor to the carbonyl of PLP under the action of ⁇ -transaminase, thereby forming pyridoxamine 5-phosphate (PMP) and pyridoxamine 5-phosphate (PMP).
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • the ketone corresponding to the amino donor; the second step of the reaction also transfers the amino group on the PMP to the amino acceptor under the action of ⁇ -transaminase, and the PMP is converted into PLP to realize the cycle.
  • Stereoselectivity of transaminases can be determined in racemic resolution of racemic mixtures of substrates containing chiral centers.
  • ⁇ -aminotransferases can be divided into "(R)-aminotransferases” ((R)-amine transaminase) and (S)-aminotransferases ((S)-amine transaminase).
  • (R)-transaminase may be an enzyme that preferentially induces a transamination reaction of D-glufosinate from racemic D,L-glufosinate in the presence of a ketone substrate such as pyruvate .
  • the (R)-transaminase can be any enzyme known in the art having (R)-transaminase activity.
  • the (R)-transaminase is any one selected from the group consisting of: APH1 (eg, NCBI SEQ ID NO: WP_015938787.1), HEA-2 (eg, NCBI SEQ ID NO: ABX05998.1), TSP -1 (eg NCBI serial number is WP_013128145.1), DEP-2 (eg NCBI serial number is WP_013615256.1) and MPH (eg NCBI serial number is WP_013863226.1).
  • the (R)-transaminase is APH1.
  • the (R)-transaminase is from Pseudarthrobacter chlorophenolicus, eg, APH1 from Pseudarthrobacter chlorophenolicus.
  • the sequence of the (R)-transaminase has an NCBI accession number of WP_015938787.1.
  • the amino acid sequence of the (R)-transaminase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the (R)-transaminase is at least 60%, 70%, 80%, 90%, 91%, 91% of the nucleotide sequence shown in SEQ ID No. 2 , 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the amino acid sequence of the (R)-transaminase is SEQ ID No. 1.
  • the nucleotide sequence of the (R)-transaminase is SEQ ID No. 2.
  • (S)-transaminase is an enzyme that preferentially induces a transamination reaction of L-glufosinate from racemic D,L-glufosinate in the presence of a ketone substrate such as pyruvate.
  • the (S)-transaminase described herein can be any enzyme known in the art having (S)-transaminase activity.
  • the (S)-transaminase is any one selected from the group consisting of LMG42 (i.e. EN5) (e.g. NCBI serial number PZN34824.1) and ATA-0303 (e.g. NCBI serial number WP_012204645. 1).
  • the (S)-transaminase is from Corynebacterium vitaeruminis, such as EN5 from Corynebacterium vitaeruminis. In some embodiments, the (S)-transaminase is from Corynebacterium vitaeruminis DSM 20294. In some embodiments, the amino acid sequence of the (S)-transaminase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the (S)-transaminase is at least 60%, 70%, 80%, 90%, 91%, 91% of the nucleotide sequence shown in SEQ ID No. 4 , 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the amino acid sequence of the (S)-transaminase is SEQ ID No.3.
  • the nucleotide sequence of the (S)-transaminase is SEQ ID No. 4.
  • the enzyme preparation forms of the (R)-transaminase and (S)-transaminase can be purified enzymes; partially purified enzymes; cell-free extracts or crude cell extracts; liquid, powder or fixed forms; Processed cells, whole cells or whole fermentation broth, lyophilized cells or any other suitable form.
  • the (R)-transaminase and (S)-transaminase forms are each independently selected from: partially purified enzymes; cell-free extracts or crude cell extracts; liquid, powder, or fixed forms; containing Enzyme permeabilized cells, whole cells or whole fermentation broth, lyophilized cells, or any combination thereof.
  • the (R)-transaminase and (S)-transaminase used in the methods of the present application are co-expressed from a single recombinant microorganism.
  • the methods of the present application may comprise: converting D,L-glufosinate to L-grass in the presence of a recombinant microorganism co-expressing (R)-transaminase and (S)-transaminase, an amino acceptor and an amino donor Ammonium phosphine.
  • the recombinant microorganism can be constructed using any method known in the art.
  • the recombinant microorganism can be constructed as follows: constructing a recombinant vector containing the (R)-transaminase and (S)-transaminase genes, transforming the recombinant vector into the microorganism, inducing and culturing the obtained recombinant microorganism, and isolating and culturing it.
  • a recombinant microorganism containing (R)-transaminase and (S)-transaminase genes is obtained.
  • the amount of the recombinant microorganism added is 5g/L-200g/L reaction solution; more preferably, 30g/L-100g/L reaction solution; most preferably , is 30g/L reaction solution.
  • the added amount of the recombinant microorganism is 1-50 g dry cell/L reaction solution.
  • the recombinant microorganism can be any engineered bacteria suitable for enzyme expression.
  • the recombinant microorganism belongs to one of the following genera: Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida Genus (Candida), Hansenula (Hansenula), Humicola (Humicola), Issatchenkia (Issatchenkia), Trichosporon (Trichosporon), Brettanomyces (Brettanomyces), Pachysolen), Yarrowia or Escherichia.
  • the recombinant microorganism is selected from the group consisting of: Saccharomyces cerevisiae, Yarrowia lipolitica, Candida krusei, Issatchenkia orientalis ) or Escherichia coli. In some more preferred embodiments, the recombinant microorganism is E. coli.
  • the amino acceptor is pyruvate.
  • the amino donor is L-aspartic acid or L-alanine. In some preferred embodiments, the amino donor is L-aspartic acid.
  • the method is carried out in the presence of a reaction buffer, preferably the reaction buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7-10, preferably 8-9.
  • a reaction buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7-10, preferably 8-9.
  • a better reaction efficiency can be obtained when the reaction is carried out in this disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • the molar ratio of amino acceptor to D,L-glufosinate is 1:500-1:5, eg, 1:500-1:10; more preferably, 1:50-1:10 .
  • the method is performed in the presence of the coenzyme pyridoxal phosphate.
  • the molar ratio of pyridoxal phosphate to substrate is from 1:10 to 1:200.
  • pyridoxal phosphate is added in an amount of 0.1-2 mM on a molar basis; more preferably 1 mM.
  • the method is performed at a temperature of 20-70°C for 6-96 hours; more preferably, at a temperature of 30-50°C, such as 30-45°C, for a time of 48-72 hours, most preferably Yes, the time is 48h.
  • the method is performed at pH 6-10, such as pH 7-9 or pH 8-9.
  • the methods described herein can be carried out in one or more reaction vessels. Preferably, the methods described herein are carried out in one reaction vessel (ie "one-pot, one-step process").
  • the yield of the methods of the present application can be measured by any method known in the art.
  • the content of both configurations in the glufosinate product obtained can be measured by chiral HPLC.
  • the glufosinate product is obtained in an enantiomeric excess (e.e.) of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%.
  • the present invention can directly use D,L-glufosinate-ammonium as a substrate for splitting, without expensive splitting reagents, without synthesizing glufosinate derivatives, and without the need to separate and recycle D-glufosinate-ammonium. Racemization, re-resolution and other steps.
  • This method overcomes the defect of chemical synthesis of L-glufosinate precursor 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and is a green, environmentally friendly, low-carbon process route, Suitable for large-scale industrial production applications.
  • SEQ ID NO: 1 is the amino acid sequence annotated as (R)-transaminase (APH1) derived from Pseudarthrobacter chlorophenolicus.
  • SEQ ID NO: 2 is the nucleotide sequence annotated as (R)-transaminase (APH1) derived from Pseudarthrobacter chlorophenolicus.
  • SEQ ID NO:3 is the amino acid sequence annotated as (S)-transaminase (EN5) derived from Corynebacterium vitaeruminis DSM 20294.
  • SEQ ID NO: 4 is the nucleotide sequence annotated as (S)-transaminase (EN5) derived from Corynebacterium vitaeruminis DSM 20294.
  • Fig. 1 is the reaction formula for producing L-glufosinate-ammonium by the multi-enzyme system splitting method adopted in some embodiments of the method of the application.
  • Figure 2 exemplarily shows the APH1-EN5 construct.
  • FIG. 3 exemplarily shows the reaction process of one-pot and one-step deracemization of single bacteria and multi-enzymes to prepare L-glufosinate-ammonium.
  • genome extraction kit plasmid extraction kit, DNA purification and recovery kit
  • One-step cloning kit was obtained from Vazyme Co., Ltd. MultiS One Step Cloning Kit
  • E.coli DH5 ⁇ , E.coli BL21(DE3), plasmid pET-28a(+), pCDFduet-1 vector, etc. were purchased from Shanghai Xuguan Biotechnology Development Co., Ltd.
  • DNA marker, low molecular weight standard protein , protein glue, etc. were purchased from Beijing GenStar Co., Ltd.
  • catalase was purchased from Ningxia Xiasheng Industrial Group Co., Ltd., the commodity number is CAT-400. Refer to the product manual for the usage of the above reagents.
  • the reagents used in the catalytic process 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO for short), D,L-glufosinate-ammonium were purchased from Yongnong Bioscience Co., Ltd.; other commonly used reagents were purchased from Sinopharm Group Chemical Reagent Co., Ltd.
  • HPLC high performance liquid chromatography
  • the content of two configurations of glufosinate-ammonium was checked by chiral HPLC analysis method.
  • the chiral HPLC analysis method was: chromatographic column/OA-5000L; mobile phase/0.5% copper sulfate pentahydrate; detection wavelength/254nm; flow rate/0.7mL /min; column temperature/35°C.
  • E.coli BL21(DE3)/pCDFduet-1-APH1, E.coli BL21(DE3)/pCDFduet-1-EN5, E.coli BL21(DE3)/pCDFduet-1-APH1-EN5 were streaked on a plate After activation, single colonies were inoculated into 10 mL of LB liquid medium containing 50 ⁇ g/mL kanamycin, and incubated at 37°C with shaking for 10 h.
  • APH1 The sequence derived from Pseudarthrobacter chlorophenolicus annotated as (R)-transaminase (APH1) (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) was synthesized after the whole gene was inserted.
  • the plasmid pET-28a(+) was expressed to give pET28a-APH1.
  • pET28a-APH1 was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • sequence (amino acid sequence shown in SEQ ID NO.3, nucleotide sequence shown in SEQ ID NO.4) derived from Corynebacterium vitaeruminis DSM 20294 annotated as (S)-transaminase (EN5) was subjected to full gene synthesis, Insert into expression plasmid pET-28a(+) to obtain pET28a-EN5. After sequencing and verification, pET28a-EN3 was transferred into the expression host E. coli BL21 (DE3) for subsequent recombinase expression.
  • Example 3 Construction of co-expression strains containing (R)-transaminase and (S)-transaminase systems:
  • the APH1 gene used in Example 2 was connected to the multi-cloning site vector pCDFduet-1 by a one-step cloning kit, the restriction sites were HindIII and XhoI, and the one-step cloning primers were C1-F and C1-R (Table 1) , the plasmid pCDFduet-1-APH1 was constructed.
  • the EN5 used in Example 2 was connected to the second cloning site of the multi-cloning site vector pCDFduet-1 by a one-step cloning kit, and the restriction sites were NdeI and XhoI, the one-step cloning primers were C2-F and C2-R, the plasmid pCDFduet-1-APH1-EN5 was constructed and the co-expression strain E.coli BL21(DE3)/pCDFduet-1-APH1-EN5 was constructed.
  • the APH1-EN5 construct is shown in Figure 2.
  • the co-expression strain E.coli BL21(DE3)/pCDFduet-1-APH1-EN5 capable of expressing (R)-transaminase and (S)-transaminase systems was constructed and cultured according to the method of Example 3, and the bacterial cells were collected by centrifugation.
  • the 30ml reaction system contains 500mM D, L-PPT, 10mM pyruvate, 600mM L-aspartic acid, 100mM phosphate buffer, the pH of the reaction system is adjusted to 8.0 with ammonia, and the co-expression strain E.coli BL21 ( DE3)/pCDFduet-1-APH1-EN5 30g/L stem cells.
  • the reaction conditions were as follows: temperature at 30° C. and rotation speed at 250 rpm. Samples (100 ⁇ l) were taken at regular intervals, 900 ⁇ l of deionized water was added, and the reaction was terminated by heating. The conversion of D-PPT and the generation of L-PPT were detected by HPLC, and the reaction progress curve was shown in Figure 3.

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Abstract

本申请涉及一种利用生物多酶偶联法制备L-草铵膦的方法，其包括：在(R)-转氨酶、(S)-转氨酶、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。本申请的方法能够实现高浓度D,L-草铵膦的高效拆分来制备L-草铵膦。

Description

利用生物多酶偶联法制备L-草铵膦的方法
相关中请的交叉引用
本申请要求于2020年12月17日提交中国专利局的申请号为202011496575.9、名称为“利用生物多酶偶联法制备L-草铵膦的方法”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
本申请涉及生物技术领域，特别是涉及一种利用生物多酶偶联法制备L-草铵膦的方法。
背景技术
草铵膦(又名双丙氨膦、草丁膦，商品名包括保试达、百速顿等，英文名为phosphinothricin(简称PPT)，化学名为2-氨基-4-[羟基(甲基)膦酰基]丁酸)，是世界第二大转基因作物耐受除草剂，由赫斯特公司开发生产。草铵膦属膦酸类除草剂，是谷氨酰胺合成酶抑制剂，非选择性(灭生性)触杀型除草剂。目前，世界三大除草剂分别为百草枯，草甘膦，草铵膦。在市场使用方面，草甘膦独占鳌头，但是由于其长期使用，使得大量杂草产生抗性，而草甘膦也趋于失效；百草枯由于其剧毒性，已被列入《鹿特丹公约》，全球越来越多国家禁用或限用，中国农业部已发布公告说明，百草枯在2014年7月1日停止生产，2016年7月1日禁止使用；而目前草铵膦产量虽小，却具有优异的除草性能和较小的药害副作用，因此，在未来一段时间内拥有巨大的市场潜力。
草铵膦有两种光学异构体，分别为L-草铵膦和D-草铵膦，但只有L-型具有除草活性，且在土壤中易分解，对人类和动物的毒性较小，除草谱广，对环境的破坏力小。
目前，市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用，可显著降低草铵膦的使用量，这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
现有制备手性纯L-草铵膦的方法主要两种：化学法和生物法。
其中化学法包括化学拆分法和化学合成法。化学拆分法是通过手性拆分试剂拆分外化学法合成的消旋D,L-草铵膦或其衍生物，从而制得光学纯的L-草铵膦。然而该工艺存在需要使用手性拆分试剂、D-草铵膦需要消旋再利用，需要多次拆分等缺点，难以规模工业化生产。化学合成法可以以天然手性氨基酸或者不对称法合成L-草铵膦。但是该过程氨基保护与脱保护过程复杂，步骤较多，多见于实验室研究中。化学合成法还可以采用不对称催化加氢法合成L-草铵膦。但该路线所使用的催化剂价格昂贵，反应路线较长，工业化成本较高。
相比化学法能耗高、污染重、成本高等劣势，生物催化法具有反应条件温和、立体选择性高、收率高等优点，是工业化制备L-草铵膦的重要趋势。
制备L-草铵膦的方法目前大多是2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)为底物，经氨化反应，不对称合成L-草铵膦。其中CN1349561A描述了利用筛选的天冬氨酸转氨酶(Asp-TA)，以L-天冬氨酸作为氨基供体，通过转氨作用将PPO合成L-草铵膦。该工艺使用与PPO大约等摩尔量的氨基供体，生成的草酰乙酸在水中水解成丙酮酸，可以经酶促反应去除，不存在可逆反应。然而此工艺中底物PPO转化率仅有52％，效率较低，并且反应条件苛刻(80℃)。专利CN105603015B描述了一种以L-丙氨酸为氨基供体的氨基转移酶生产L-草铵膦的方法。此工艺能够将100mM的PPO完全转化成L-草铵膦，但是为了抑制逆反应，丙氨酸投量过高，反应结束仍有300mM的L-丙氨酸存在体系中，为后续分离纯化造成困难，同时反应温度高达50℃，能耗较高，不利于生产。另外还有的方法在氨基酸脱氢酶的介导下，以无机氨为供体，将PPO还原胺化制备L-精草铵膦，例如CN106978453A，反应过程采用无机氨，转化率能够达到99％以上，然而该工艺需要额外添加辅酶再生系统，产生的副产物提高了分离纯化的难度。
上述工艺都以PPO为原料，但PPO的成本很高，从而导致这些工艺生产L-草铵膦的成本很高，难以实现工业化。因此，国际专利WO2017151573A1描述了一种以D，L-草铵膦为原料生产L-精草铵膦的方法，该工艺采用D-氨基酸 氧化酶氧化D-草铵膦获得PPO，再由转氨酶经转氨反应将PPO转化为L-草铵膦。尽管该工艺实现了草铵膦消旋体的动态动力学拆分，但是该工艺存在明显缺陷：一是底物投量难以提升(仅有300mM D,L-草铵膦)；二是转氨酶介导的PPO到L-草铵膦的反应，由于受到可逆反应影响，仅能实现90％的转化率；三是由于胺供体是L-谷氨酸，反应后仍有大量剩余，产物分离纯化困难。
因此迫切需要开发一种能够高效实现动态拆分高浓度D,L-草铵膦来制备L-草铵膦的方法。
发明内容
本申请提供了一种利用生物多酶偶联法制备L-草铵膦的方法，其以D,L-草铵膦为原料，经多酶催化体系获得L-草铵膦，所述多酶催化体系包括：(R)-转氨酶和(S)-转氨酶。该方法原料转化率高、分离精制过程简单、产品收率高、生产成本低，易于工业化。
在一些实施方式中，本申请提供了一种生产L-草铵膦的方法，所述方法包括：在(R)-转氨酶、(S)-转氨酶、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。
本申请方法在初始时仅需加入外消旋草铵膦、微量氨基受体(例如丙酮酸)(用以启动第一步反应)，氨基供体(例如L-天冬氨酸)(用以启动第二步反应)，以及生物催化剂(R)-转氨酶和(S)-转氨酶。本申请的方法涉及两步反应，在第一步反应中，(R)-转氨酶介导外消旋草铵膦中的D-草铵膦的脱氨反应生成中间体酮酸2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)，而L-草铵膦不参与反应而完全保留；在第二步反应中，PPO及时被(S)-转氨酶还原胺化，生成L-草铵膦，生成的副产物例如草酰乙酸自发降解为氨基受体，回补第一步的脱氨化反应。整个反应过程中，氨基受体的含量始终维持在低浓度，从而降低了对反应的抑制程度，而且由于副产物的自发降解，推动反应平衡始终移向产物一侧，打破热力学平衡的限制实现完全转化。本申请方法仅通过两种立体选择性互补的转氨酶的偶联，即可以实现一锅一步的外消旋草铵膦的去消旋化，无需引入辅因子原位再生系统除去氨基受体，仅通过副产物的自发降解实现氨基受体的循环利用，大大降低了反应体系的复杂性。
转氨酶(Amine Transaminase,ATA,EC 2.6.1.X)属于转移酶类，是催化1个氨基供体(氨基酸或简单的胺)上的氨基转移到前手性的受体酮，得到手性胺和副产物酮或者α-酮酸的一类酶，其催化的反应是可逆的。根据氨基被转移到不同位置的氨基受体上，转氨酶又可以被分为α-转氨酶和ω-转氨酶(EC 2.6.1.1)。ω-转氨酶反应过程可分为两步，第一步反应是在ω-转氨酶的作用下将氨基供体上的氨基转移到PLP的羰基上，从而形成5-磷酸吡哆胺(PMP)和与氨基供体对应的酮；第二步反应同样在ω-转氨酶的作用下将PMP上的氨基转移到氨基受体上，PMP又转变为PLP实现循环。转氨酶的立体选择性可在包含手性中心的底物外消旋混合物的外消旋拆分中测定。ω-转氨酶根据立体选择性可分为“(R)-转氨酶”((R)-amine transaminase)和(S)-转氨酶((S)-amine transaminase)。
根据本申请，(R)-转氨酶可以是在存在酮底物(例如丙酮酸)的情况下，优先从外消旋D,L-草铵膦中诱导D-草铵膦的转氨反应的酶。(R)-转氨酶可以为本领域已知的任何具有(R)-转氨酶活性的酶。在一些实施方式中，所述(R)-转氨酶为选自以下中的任一种：APH1(例如NCBI序列号为WP_015938787.1)、HEA-2(例如NCBI序列号为ABX05998.1)、TSP-1(例如NCBI序列号为WP_013128145.1)、DEP-2(例如NCBI序列号为WP_013615256.1)和MPH(例如NCBI序列号为WP_013863226.1)。在一些实施方式中，所述(R)-转氨酶为APH1。在一些实施方式中，所述(R)-转氨酶来自Pseudarthrobacter chlorophenolicus，例如来自Pseudarthrobacter chlorophenolicus的APH1。在一些实施方式中，(R)-转氨酶的序列的NCBI登录号为WP_015938787.1。在一些实施方式中，所述(R)-转氨酶的氨基酸序列与SEQ ID No.1所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的核苷酸序列与SEQ ID No.2所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的氨基酸序列为SEQ ID No.1。在一些实施方式中，所述(R)-转氨酶的核苷酸序列为SEQ ID No.2。
根据本发明，(S)-转氨酶是在存在酮底物比如丙酮酸的情况下，优先从外消旋D,L-草铵膦诱导L-草铵膦的转氨反应的酶。本申请所述的(S)-转氨酶可以为本领域已知的任何具有(S)-转氨酶活性的酶。在一些实施方式中，所述(S)-转 氨酶为选自以下中的任一种：LMG42(即EN5)(例如NCBI序列号为PZN34824.1)和ATA-0303(例如NCBI序列号为WP_012204645.1)。在一些实施方式中，所述(S)-转氨酶来自Corynebacterium vitaeruminis，例如来自Corynebacterium vitaeruminis的EN5。在一些实施方式中，所述(S)-转氨酶来自Corynebacterium vitaeruminis DSM 20294。在一些实施方式中，所述(S)-转氨酶的氨基酸序列与SEQ ID No.3所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的核苷酸序列与SEQ ID No.4所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的氨基酸序列为SEQ ID No.3。在一些实施方式中，所述(S)-转氨酶的核苷酸序列为SEQ ID No.4。
所述(R)-转氨酶和(S)-转氨酶的酶制剂形式可以为纯化的酶；部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其他任何合适形式。因此，在一些实施方式中，(R)-转氨酶和(S)-转氨酶的形式各自独立地选自：部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其任何组合。
在一些优选的实施方式中，本申请方法中使用的(R)-转氨酶和(S)-转氨酶由单一重组微生物共表达。因此，本申请方法可以包括：在共表达(R)-转氨酶和(S)-转氨酶的重组微生物、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。可以利用本领域已知的任何方法构建所述重组微生物。例如，所述重组微生物可以如下构建：构建含所述(R)-转氨酶和(S)-转氨酶基因的重组载体，将所述重组载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有(R)-转氨酶和(S)-转氨酶基因的重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述重组微生物的添加量为5g/L-200g/L反应液；更优选地，30g/L-100g/L反应液；最优选地，为30g/L反应液。优选地，以干菌体重量计，所述重组微生物的添加量为1-50g干菌体/L反应液。
所述重组微生物可以是任何适用于酶表达的工程菌。在一些实施方式中，所述重组微生物属于以下属中的一种：酵母属(Saccharomyces)、曲霉属 (Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)或埃希氏杆菌属(Escherichia)。在一些优选的实施方式中，所述重组微生物选自：酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)或大肠杆菌(Escherichia coli)。在一些更优选的实施方式中，所述重组微生物是大肠杆菌。
在一些实施方式中，所述氨基受体为丙酮酸。
在一些实施方式中，所述氨基供体为L-天冬氨酸或L-丙氨酸。在一些优选的实施方式中，所述氨基供体为L-天冬氨酸。
在一些实施方式中，所述方法在反应缓冲液的存在下进行，优选地，所述反应缓冲液是pH为7-10，优选8-9的磷酸氢二钠-磷酸二氢钠缓冲液。在这种磷酸氢二钠-磷酸二氢钠缓冲液中进行反应时可以获得更优的反应效率。
在一些实施方式中，氨基受体和D,L-草铵膦的摩尔比为1:500-1:5，例如1:500-1:10；更优选地，为1:50-1:10。
在一些实施方式中，所述方法在辅酶磷酸吡哆醛的存在下进行。在一些实施方式中，磷酸吡哆醛与底物的摩尔比为1:10-1:200。在一些实施方式中，以摩尔浓度计，磷酸吡哆醛的添加量为0.1-2mM；更优选为1mM。
在一些实施方式中，所述方法进行的温度为20-70℃，时间为6-96小时；更优选，温度为30-50℃，例如30-45℃，时间为48-72小时，最优选的，时间为48h。
在一些实施方式中，所述方法在pH 6-10，例如pH 7-9或pH 8-9的条件下进行。
本申请所述的方法可以在一个或更多个反应容器中进行。优选地，本申请所述的方法在一个反应容器中进行(即“一锅一步法”)。
本申请方法的产率可以通过本领域已知的任何方法测量。例如，可以通过手性HPLC来测量所获得的草铵膦产物中两个构型含量。在一些实施方式中， 获得的草铵膦产物的对映体过量(e.e.)至少为80％、85％、90％、95％、96％、97％、98％、99％或99.9％。
本申请方法具有以下有益效果：
(1)D-草铵膦被氧化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，L-草铵膦因不参与反应而被完全保留；产物2-羰基-4-[羟基(甲基)膦酰基]丁酸又可以被转氨酶继续催化还原为L-草铵膦，进而实现D,L-草铵膦的原位去消旋化。而传统的氧化方法则需要将D-草铵膦和L-草铵膦都转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，造成了原料的浪费。
(2)本发明能够直接以D,L-草铵膦为底物进行拆分，无需昂贵的拆分试剂，也无需合成草铵膦衍生物，更无需对D-草铵膦进行分离、再消旋、再拆分等步骤。
(3)本方法克服了化学法合成L-草铵膦前体2-羰基-4-[羟基(甲基)膦酰基]丁酸的缺陷，是一种绿色，环保，低碳的工艺路线，适合大规模工业化生产应用。
对序列表的描述
SEQ ID NO:1是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的氨基酸序列。
SEQ ID NO:2是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的核苷酸序列。
SEQ ID NO:3是来源于Corynebacterium vitaeruminis DSM 20294的注释为(S)-转氨酶(EN5)的氨基酸序列。
SEQ ID NO:4是来源于Corynebacterium vitaeruminis DSM 20294的注释为(S)-转氨酶(EN5)的核苷酸序列。
附图说明
图1为本申请方法的一些实施例中采用的多酶体系拆分法生产L-草铵膦的反应式。
图2示例性显示了APH1-EN5构建体。
图3示例性显示了单菌多酶一锅一步去消旋化制备L-草铵膦的反应进程。
具体实施方式
实施例
材料和方法
基因工程所用材料和试剂：基因组提取试剂盒、质粒提取试剂盒、DNA纯化回收试剂盒购自康宁生命科学(吴江)有限公司；一步克隆试剂盒是诺唯赞有限公司(Vazyme)的
Figure PCTCN2021139010-appb-000001
MultiS One Step Cloning Kit；E.coli DH5α、E.coli BL21(DE3)、质粒pET-28a(+)、pCDFduet-1载体等购自上海旭冠生物科技发展有限公司；DNA marker、低分子量标准蛋白、蛋白胶等购自北京GenStar有限公司；过氧化氢酶购自宁夏夏盛实业集团有限公司，商品编号为CAT-400。以上试剂使用方法参考商品说明书。
序列合成，序列测序工作由杭州擎科梓熙生物技术有限公司完成。
催化工艺所用试剂2-羰基-4-[羟基(甲基)膦酰基]丁酸(简称PPO)，D,L-草铵膦购自永农生物科学有限公司；其他常用试剂购自国药集团化学试剂有限公司。
通过高效液相色谱(HPLC)检测反应的进行，并对PPO进行分析。HPLC分析方法为：色谱柱/PBr；柱温/30℃；流速/0.8mL/min；检测波长/210nm；流动相：50mM(NH4)2HPO4，加入12％的乙腈。
通过手性HPLC分析方法检查草铵膦的两个构型含量，手性HPLC分析方法为：色谱柱/OA-5000L；流动相/0.5％五水硫酸铜；检测波长/254nm；流速/0.7mL/min；柱温/35℃。
实施例1：工程菌菌体的培养
将工程菌E.coli BL21(DE3)/pCDFduet-1-APH1、E.coli BL21(DE3)/pCDFduet-1-EN5、E.coli BL21(DE3)/pCDFduet-1-APH1-EN5经平皿划线活化后，挑单菌落接种至含有50μg/mL卡那霉素的10mL LB液体培养基中，37℃震荡培养10h。按2％的接种量转接至50mL同样含有50μg/mL卡那霉素的LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入终浓度为0.5mM的IPTG，28℃下震荡培养12h。培养结束后，将培养液8000rpm离心10min，弃上清，收集菌体，放到-80℃超低温冰箱中保存，待用。
实施例2：酶序列合成和菌株构建
将来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的序列(氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.2所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-APH1。测序验证无误后将pET28a-APH1转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Corynebacterium vitaeruminis DSM 20294注释为(S)-转氨酶(EN5)的序列(氨基酸序列为SEQ ID NO.3所示，核苷酸序列为SEQ ID NO.4所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-EN5。测序验证无误后将pET28a-EN3转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
实施例3：含(R)-转氨酶和(S)-转氨酶系统的共表达菌株的构建：
将实施例2中使用的APH1基因通过一步克隆试剂盒连接到多克隆位点载体pCDFduet-1上，酶切位点为HindIII和XhoI，一步克隆引物为C1-F和C1-R(表1)，构建得到质粒pCDFduet-1-APH1。再在pCDFduet-1-APH1质粒的基础上，通过一步克隆试剂盒将实施例2中使用的EN5连接到多克隆位点载体pCDFduet-1第二个克隆位点上，酶切位点为NdeI和XhoI，一步克隆引物为C2-F和C2-R，构建得到质粒pCDFduet-1-APH1-EN5，构建得到共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5。APH1-EN5构建体如图2所示。
表1：克隆引物序列
引物 序列
C1-F CCCAAGCTTAAGGAGATATACATATGACCTCTCCCGCTTCCGT
C1-R CCGCTCGAGCTATTGGATTCCGGCGTAAAGC
C2-F CCCAAGCTTATGACGCCACAGGGCACGTTCTTCCTCCCGGGGCC
C2-R CCGCTCGAG TTATCAGATGACTTCCCCTATCACGGCGAGGAGGGCGT
实施例4：单菌多酶一锅一步去消旋化制备L-草铵膦
按照实施例3的方法构建和培养能够表达(R)-转氨酶和(S)-转氨酶系统的共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5，离心收集菌体细胞。
30ml的反应体系包含500mM D,L-PPT，10mM丙酮酸，600mM L-天冬氨酸，100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5 30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测D-PPT的转化情况和L-PPT的生成情况，反应进程曲线如图3所示。
结果显示，反应48小时，液相检测D-PPT为0mM，D-PPT转化率为99.9％，PPO为2mM，L-PPT为488mM，产品草铵膦的e.e.值为99.9％。

Claims (13)

  1. 一种制备L-草铵膦的方法，其包括：在(R)-转氨酶、(S)-转氨酶、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。
  2. 权利要求1所述的方法，其中所述方法在同一反应器中进行。
  3. 根据前述权利要求中任一项所述的方法，其中所述氨基受体为丙酮酸。
  4. 根据前述权利要求中任一项所述的方法，其中所述氨基供体为L-天冬氨酸或L-丙氨酸。
  5. 根据前述权利要求中任一项所述的方法，其中所述(R)-转氨酶和所述(S)-转氨酶的形式各自独立地选自：部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其任何组合。
  6. 根据前述权利要求中任一项所述的方法，其中所述方法包括：在共表达(R)-转氨酶和(S)-转氨酶的重组微生物、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。
  7. 根据前述权利要求中任一项所述的方法，其中所述重组微生物选自：酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)或大肠杆菌(Escherichia coli)。
  8. 根据权利要求4-7中任一项所述的方法，其中，以干菌体重量计，所述重组微生物的添加量为1-50g干菌体/L反应液。
  9. 根据权利要求4-7中任一项所述的方法，其中，以湿菌体重量计，所述重组微生物的添加量为5-200g湿菌体/L反应液。
  10. 根据前述权利要求中任一项所述的方法，其在反应缓冲液的存在下进行，优选地，所述反应缓冲液是pH为7-10的磷酸氢二钠-磷酸二氢钠缓冲液。
  11. 根据前述权利要求中任一项所述的方法，其中氨基受体和D,L-草铵膦的摩尔比为1:500-1:5。
  12. 根据前述权利要求中任一项所述的方法，所述方法在30-45℃的温度下进行6-48小时。
  13. 根据前述权利要求中任一项所述的方法，其中所述方法在辅酶磷酸吡哆醛的存在下进行，优选地磷酸吡哆醛与底物的摩尔比为1:10-1:200。
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