WO2022001939A1 - 一种泛解酸、泛酸、泛醇及其盐的制备方法 - Google Patents

一种泛解酸、泛酸、泛醇及其盐的制备方法 Download PDF

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WO2022001939A1
WO2022001939A1 PCT/CN2021/102698 CN2021102698W WO2022001939A1 WO 2022001939 A1 WO2022001939 A1 WO 2022001939A1 CN 2021102698 W CN2021102698 W CN 2021102698W WO 2022001939 A1 WO2022001939 A1 WO 2022001939A1
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escherichia coli
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张科春
王吉龙
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • 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)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • 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)
    • C12Y101/01021—Aldehyde reductase (1.1.1.21), i.e. aldose-reductase

Definitions

  • the present application relates to the technical field of biochemical synthesis, in particular to a preparation method of pantoic acid, pantothenic acid, panthenol and their salts.
  • Pantothenic acid is also known as vitamin B5.
  • Pantothenic acid is an important intermediate in the production of pantothenic acid and panthenol.
  • Calcium D-pantothenate is the most commercially important product form of D-pantothenate and has been widely used in the pharmaceutical, feed and food industries.
  • D-pantothenic acid or vitamin B5 is a member of the vitamin B complex, which is naturally required by mammals.
  • pantothenic acid is mainly used in the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). These coenzymes are important to all cells, and they are involved in more than 100 different intermediate reactions in cellular metabolism.
  • Panthenol is one of the derivatives of pantothenic acid.
  • D-panthenol is mainly used to formulate hair care products and topical cosmetics. It can prevent small wrinkles, inflammation, sun exposure, erosion, prevent hair loss and promote hair growth.
  • the current synthesis method of calcium D-pantothenate is to react ⁇ -aminopropionic acid, ⁇ -hydroxy- ⁇ , ⁇ '-dimethyl- ⁇ -butyrolactone (pantolactone) and metal calcium to generate DL-pantothenic acid Calcium, and then split to obtain D-calcium pantothenate.
  • pantolactone mainly adopts isobutyraldehyde-formaldehyde-hydrocyanic acid method (US.4020103,1977; JP.47-35314,1973; JP.49-41361,1974) and isobutyraldehyde-aldehyde acetic acid method (JP. 55-62080, 1980).
  • Isobutyraldehyde-formaldehyde-hydrocyanic acid method in this method, isobutyraldehyde and formaldehyde are methylolated to obtain ⁇ , ⁇ -dimethyl- ⁇ -hydroxypropionaldehyde, which is then reacted with hydrocyanic acid to generate cyanohydrin ⁇ , ⁇ -Dihydroxy- ⁇ , ⁇ -dimethylbutyronitrile, which is acid hydrolyzed to obtain ⁇ , ⁇ -dihydroxy- ⁇ , ⁇ -dimethylbutyric acid (pantoic acid), which is then dehydrated to lactonization That is, DL-pantolactone is obtained.
  • the so-called isobutyraldehyde-hydroxyacetonitrile method is actually an improvement of this method, instead of directly using hydrocyanic acid, but using hydroxyacetonitrile instead, but hydroxyacetonitrile is still obtained by addition of formaldehyde and hydrocyanic acid.
  • the so-called isobutyraldehyde-hydroxyacetonitrile method is actually an improvement of this method, instead of directly using hydrocyanic acid, but using hydroxyacetonitrile instead, but hydroxyacetonitrile is still obtained by addition of formaldehyde and hydrocyanic acid.
  • the main problem of this method is that it needs to go through a multi-step reaction such as methylolation, cyanoalcoholation, hydrolysis, and lactonization.
  • the former is not competitive due to the use of twice the amount of glyoxylic acid and the production of a large amount of waste water, which is not economical and environmentally friendly; the latter requires high pressure of 250 bar, resulting in high equipment costs, maintenance costs and poor safety. limit its application.
  • microbial synthesis methods have been the subject of research and have been developed in recent years. Pure microbial synthesis methods have not been used for industrial production due to low yields (EP2163629). The current industrial methods are all chemically generated precursors, and then enzymatically resolved to obtain chiral D-pantothenic acid.
  • the present invention provides a new preparation method of pantoic acid, pantothenic acid, panthenol and their salts, which can at least partially solve the above-mentioned technical problems.
  • One of the objects of the present invention is to provide a preparation method of pantoic acid, pantothenic acid or panthenol, which converts 2-hydroxy-3,3-dimethyl-4-aldol butyric acid into pantoic acid by fermentation with bacteria or yeast , pantothenic acid or panthenol.
  • the second object of the present invention is to provide a preparation method of D-pantothenic acid, comprising: 1) converting 2-hydroxy-3,3-dimethyl-4-aldol butyric acid into pantothenic acid through bacterial or yeast fermentation; 2) The pantothenic acid is reacted with ⁇ -alanine to obtain D-pantothenic acid.
  • the third object of the present invention is to provide a method for preparing D-panthenol, comprising: 1) converting 2-hydroxy-3,3-dimethyl-4-aldol butyric acid into pantoic acid through bacterial or yeast fermentation 2) The pantothenic acid is reacted with ⁇ -alaninol to obtain D-panthenol.
  • the aldehyde group can be directly fermented without in vitro reduction, and the 2-hydroxy- 3,3-Dimethyl-4-aldobutyric acid is reduced to 2,4-dihydroxy-3,3-dimethyl-butyric acid.
  • This method utilizes low-cost raw materials, and avoids toxic cyanide and expensive chiral resolution steps.
  • pantoic acid is 2,4-dihydroxy-3,3-dimethylbutyric acid.
  • pantothenic acid refers to 2,4-dihydroxy-3,3-,dimethylbutyryl-3-alanine.
  • panthenol refers to 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutanamide.
  • amplification refers to the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism, for example, by increasing gene copy number, using strong promoters, or using genes encoding appropriate enzymes with high activity, and selectively It is improved by combining these methods.
  • wild refers to objects that can be found in nature.
  • first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one object from another.
  • the term "about" when referring to a measurable value such as an amount, a period of time, etc., is meant to include a ⁇ 10% variation from a given value, more preferably ⁇ 5%, even more preferably ⁇ 1%, and even more preferably ⁇ 0.1% variation, so long as such variation is suitable for carrying out the disclosed method.
  • one of the objects of the present invention is to provide a preparation method of pantoic acid, pantothenic acid, panthenol and salts thereof, which is safe and environmentally friendly and has a high yield.
  • Some embodiments of the present invention disclose the preparation methods of pantoic acid, pantothenic acid, panthenol and salts thereof, and 2-hydroxy-3,3-dimethyl-4-aldolbutyric acid is added to the culture medium as a fermentation substrate It is obtained by fermenting the fermentation substrate by bacteria or fungi.
  • pantothenic acid and less pantothenic acid are obtained through bacterial or fungal fermentation.
  • pantothenic acid wherein 2-hydroxy-3,3-dimethyl-4-aldolbutyric acid and ⁇ -alanine are simultaneously added to the culture medium as fermentation substrates for fermentation, the Pantothenic acid and pantothenic acid, the amount of pantothenic acid generated will be significantly increased.
  • the present invention prepares the reaction mechanism of pantothenic acid and pantothenic acid as follows:
  • pantothenic acid after pantothenic acid is generated, the subsequent generation of pantothenic acid can be achieved by a publicly reported chemical method or a biological fermentation method; ⁇ -alanine can be replaced by ⁇ -alaninol to generate panthenol, and the publicly reported chemical method can also be used. method or biological fermentation method.
  • the bacteria or fungi include but are not limited to wild or genetically engineered Escherichia coli, Bacillus, yeast, Corynebacterium or Streptomyces.
  • Escherichia coli for example Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae), Candida utilis or Pichia pastoris.
  • the microorganism is selected from wild or genetically engineered Escherichia coli; alternatively, the microorganism is selected from recombinant Escherichia coli, and the recombinant Escherichia coli contains an exogenous reductase-expressing enzyme A gene, wherein the reductase comprises YqhD, YqhE, DkgA, AdhE, yihU, AdH1, ADH2, ADH5 or ADH6.
  • the enzyme that ferments the 2-hydroxy-3,3-dimethyl-4-aldolbutyric acid is a reductase capable of converting aldehydes to alcohols.
  • the reductases include, but are not limited to, wild or genetically engineered YqhD, YqhE, AdhE, DkgA, Adhl, Adh2, Adh5, ADH6, or yihU.
  • pantothenic acid or panthenol utilizes the reductase enzyme done in the bacteria or fungi.
  • pantothenic acid or panthenol is carried out at 20- 40 °C, plus carbon source for fermentation.
  • the carbon source includes, but is not limited to, glucose, starch, and the like.
  • the fermentation temperature can reach 90°C, and the fermentation temperature can be about 20°C, about 30°C, about 35°C, about 65°C, and about 90°C.
  • pH regulators can be added according to fermentation needs.
  • Calcium carbonate, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate or potassium hydroxide are used to adjust pH, and the fermentation product pantothenic acid and pH adjuster generate corresponding salts to obtain calcium pantothenate, potassium pantothenate, sodium pantothenate or Ammonium pantothenate.
  • the 2-hydroxy-3,3-dimethyl-4-aldol butyric acid is prepared by condensation of glyoxylic acid and isobutyraldehyde.
  • the condensation reaction can be achieved by publicly reported preparation methods.
  • the present invention provides a preparation method of pantothenic acid and its salts, which is obtained by reacting the pantoic acid obtained by fermentation with ⁇ -alanine.
  • the invention provides a preparation method of D-pantothenic acid, comprising: 1) converting 2-hydroxy-3,3-dimethyl-4-aldehyde butyric acid into pantothenic acid through bacterial or yeast fermentation; 2) converting The pantothenic acid reacts with ⁇ -alanine to obtain D-pantothenic acid.
  • the present invention provides a method for preparing panthenol, which is obtained by reacting pantothenic acid obtained by fermentation with ⁇ -alaninol.
  • DNA polymerase Phanta Max Super-Fidelity DNA Polymerase, ligase-independent single-fragment rapid cloning kit used in the examples of the present invention One Step Cloning Kit and agarose gel electrophoresis recovery kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.
  • Glyoxylic acid and isobutyraldehyde were purchased from Aladdin.
  • the agar powder used to prepare the solid medium was purchased from Shanghai Shengsi.
  • LB medium components tryptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, and 1.5% agar powder added to the solid medium.
  • the antibiotic concentration was: kanamycin sulfate 50 ⁇ g/mL.
  • the fermentation substrate 2-hydroxy-3,3-dimethyl-4-aldobutyric acid is obtained by condensation of glyoxylic acid and isobutyraldehyde by the conventional method.
  • Detection method of pantothenic acid and pantothenic acid quantitative detection of pantothenic acid by HPLC-RID equipped with a Biohler column (1250140Aminex HPX-87H Column 300 x 7.8mm).
  • yihU_F (gaccgaattcattaaagaggagaaaggtaccatggcagcaatcgcgtttatcggtttagg)/yihU_R(ctttcgttttatttgatgcctctagagctagcttacatttttactttggcagtcatcccggcactg)
  • the yqhE and yihU genes were obtained by PCR amplification using the MG1655 genome as a template, and ligated to the pZA vector containing the IPTG-inducible promoter by a ligase-independent single-fragment rapid cloning kit to transform BW25113 competent cells. Plates coated with kanamycin sulfate resistance were cultured overnight, and positive clones were picked for sequencing verification. The correct recombinant vectors were named pZA-yqhE and pZA-yihU, respectively.
  • the single colonies of the recombinant bacteria were inoculated into 2 mL of LB liquid medium containing 50 ⁇ g/mL kanamycin sulfate, and cultured at 37° C. and 220 rpm overnight (about 14 h). According to the initial OD of 0.05, a 100 mL Erlenmeyer flask containing 5 mL of fermentation medium was transferred, and cultured at 30°C and 220 rpm. Each flask was added 0.5g CaCO 3 fermentation broth is adjusted pH. The fermentation broth was collected after 24 h of incubation, and the concentrations of pantothenic acid and pantothenic acid were determined (see table below).
  • YqhE and YihU proteins promote the reduction of the substrate 2-hydroxy-3,3-dimethyl-4-aldolbutyrate to pantoate.
  • beta-alanine is added to the fermentation broth, the final product pantothenic acid can be produced.
  • the fermentation results show that there is a rate-limiting step in the metabolic pathway that converts pantoate into pantothenic acid.
  • CaCO 3 was used to adjust the pH in the fermentation broth, and the fermentation product pantothenic acid produced calcium pantothenate in the fermentation broth. If NaOH, KOH or ammonia water is added to adjust the pH of the fermentation broth, the sodium, potassium and ammonium salts of pantothenic acid will be generated in the fermentation broth accordingly.

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Abstract

一种泛解酸、泛酸、泛醇及其盐的制备方法，通过将底物2-羟基-3,3-二甲基-4-醛基丁酸经细菌或酵母发酵得到。所述微生物选自细菌或真菌；所述微生物选自野生或基因工程改造过的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌。一种泛酸的制备方法，将上述方法得到的泛解酸与β-丙氨酸进行化学反应得到。一种泛醇的制备方法，将上述方法得到的泛解酸与β-丙氨醇进行化学反应得到。

Description

一种泛解酸、泛酸、泛醇及其盐的制备方法
本申请要求享有在先申请的申请日为2020年6月28日，申请号为202010595583.2，名称为“一种泛解酸、泛酸及其盐、泛醇的制备方法及其应用”的中国发明专利申请的优先权。
技术领域
本申请涉及生化合成技术领域，尤其涉及一种泛解酸、泛酸、泛醇及其盐的制备方法。
背景技术
泛酸是也称维生素B5。泛解酸是生产泛酸、泛醇的一种重要的中间产物。D-泛酸钙是D-泛酸在商业上最为重要的产品形式，已广泛应用于医药，饲料和食品行业。D-泛酸或维生素B5是维生素B复合物的成员，其是哺乳动物天然所需的。细胞中，泛酸主要用于辅酶A(CoA)和酰基载体蛋白(ACP)的生物合成。这些辅酶对于所有细胞来说都是重要的，它们参与细胞代谢中超过100种不同的中间反应。泛醇是泛酸衍生物之一，D-泛醇主要用于配制护发制品和局部使用的化妆品，可以防治小皱纹、炎症、日晒、糜烂，防止脱发，促进生发。
目前D-泛酸钙的合成方法是将β-氨基丙酸、α-羟基-β,β’-二甲基-γ-丁内酯(泛解酸内酯)和金属钙反应，生成DL-泛酸钙，再进行拆分得D-泛酸钙。泛解酸内酯的制备主要采用异丁醛-甲醛-氢氰酸法(US.4020103，1977；JP.47-35314，1973；JP.49-41361，1974)和异丁醛-醛乙酸法(JP.55-62080，1980)。异丁醛-甲醛-氢氰酸法，该方法以异丁醛和甲醛经羟甲基化制得α,α-二甲基-β-羟基丙醛，再与氢氰酸发生氰醇化反应生成α,γ-二羟基-β,β-二甲基丁腈，经酸水解得到α,γ-二羟基-β,β-二甲基丁酸(泛解酸)，然后将其脱水内酯化即得DL-泛内酯。所谓的异丁醛-羟乙腈法实际是该方法的改进，不直接用氢氰酸，而用羟乙腈代替，但羟乙腈仍是有甲醛和氢氰酸加成而得。所谓的异丁醛-羟乙腈法实际是该方法的改进，不直接用氢氰酸，而用羟乙腈代替，但羟乙腈仍是有甲醛和氢氰酸加成而得。这种方法存在的主要问题是需要经过羟甲基化、氰醇化、水解、内酯化等多步反应，反应中要使用剧毒的氢氰酸，对技术要求高，给管理、使用带来很大的麻烦，同时也增加了采取有关安全措施所需的费用。异丁醛-乙醛酸法有两种不同工艺：缩合-歧化工艺(JP.55-62080，1980)由异丁醛和乙醛酸进行羟醛缩合，再与另一分子乙醛酸歧化、脱水环化生成泛解酸内酯；缩合-加氢工艺(EP0171046)则是将羟醛缩合产物在250bar的高压下进行催化加氢制备DL-泛内酯。前者由于采用两倍量的乙醛酸和产生大量废水，经济性和环保性差而没有竞争力；后者由于在需250bar的高压，而造成使设备成本、维护费用高和安全性差，极大程度上限制了其应用。
为克服上述缺陷，以及为了改进对泛酸、D-泛酸盐等的生产，近年来微生物合成方法已成为研究主题，也已得到了发展。纯微生物合成方法因为产率低，一直没有用于工业生产(EP2163629)。现在工业方法都是用化学法生成前体，然后酶拆分得到手性D-泛酸。
发明内容
本发明提出一种新的泛解酸、泛酸、泛醇及其盐的制备方法，能至少部分地解决上述技术问题。
本发明目的之一是提供一种泛解酸、泛酸或泛醇的制备方法，将2-羟基-3,3-二甲基-4-醛基丁酸经细菌或酵母发酵转化成泛解酸、泛酸或者泛醇。
本发明目的之二是提供一种D-泛酸的制备方法，包括：1)将2-羟基-3,3-二甲基-4-醛基丁酸经细菌或酵母发酵转化成泛解酸；2)将所述泛解酸与β-丙氨酸反应得到D-泛酸。
本发明目的之三是提供一种D-泛醇的制备方法，包括：1)将2-羟基-3,3-二甲基-4-醛基丁酸经细菌或酵母发酵转化成泛解酸；2)将所述泛解酸与β-丙氨醇反应得到D-泛醇。
本发明提供的上述制备方法，对于2-羟基-3,3-二甲基-4-醛基丁酸，醛基无需经过体外还原，可直接进行发酵，利用生物体内还原酶将2-羟基-3,3-二甲基-4-醛基丁酸还原为2,4-二羟基-3,3-二甲基-丁酸。此方法利用成本低的原料，又避免了有毒的氰化物，和成本高昂的手性拆分步骤。
具体实施方式
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。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。
如本文使用的和除非另作说明，术语“泛解酸”是2,4-二羟基-3,3-二甲基丁酸。术语“泛酸”是指2,4-二羟基-3,3-，二甲基丁酰-3-丙氨酸。术语“泛醇”是指2,4-二羟基-N-(3-羟丙基)-3,3-二甲基丁酰胺。
术语“扩增”是指微生物中由适当DNA编码的一种或多种酶的细胞内活性例如通过增加基因拷贝数、使用强启动子或使用编码具有高活性的适当酶的基因以及选择性地结合这些方法而得到提高。
术语“野生”指在自然界中可以找到的对象。
除非具体陈述，术语“第一”、“第二”不表示任何次序或重要性，而是使用术语第一、第二等来区分一个对象与另一对象。
如本文使用的和除非另作说明，术语“大约”指的是可测量的值诸如量、时间期间等时，表示包括从给定值±10％的变化，更优选±5％，甚至更优选±1％，和还要更优选±0.1％的变化，只要这种变化适于实施公开的方法。
如上所述，本发明的目的之一在于提供一种泛解酸、泛酸、泛醇及其盐的制备方法，安全环保，产率较高。
本发明的一些实施方式公开了泛解酸、泛酸、泛醇及其盐的制备方法，将2-羟基-3,3- 二甲基-4-醛基丁酸加入到培养基中作为发酵底物，通过细菌或真菌将发酵底物进行发酵得到。
根据本发明的一些实施方式，其中，当发酵底物不含有β-丙氨酸时，经细菌或真菌发酵得到泛解酸较多，而泛酸较少。
根据本发明的一些实施方式，其中，将2-羟基-3,3-二甲基-4-醛基丁酸和β-丙氨酸同时加入到培养基中作为发酵底物发酵，可同时得到泛解酸和泛酸，生成的泛酸量将明显增多。
本发明制备泛解酸和泛酸的反应机理如下：
Figure PCTCN2021102698-appb-000001
其中，在生成泛解酸后，后续生成泛酸可用已公开报道的化学方法或生物发酵方法实现；将β-丙氨酸替换为β-丙氨醇能够生成泛醇，同样可用已公开报道的化学方法或生物发酵方法实现。根据本发明的一些实施方式，其中，细菌或真菌包括但不限于野生或者基因工程改造过的大肠杆菌、芽孢杆菌、酵母、棒状杆菌或链霉菌。例如大肠埃希氏菌(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)。
根据本发明的一些实施方式，其中，所述微生物选自野生或基因工程改造过的大肠杆菌；可选地，所述微生物选自重组大肠杆菌，所述重组大肠杆菌含有表达还原酶的外源基因，其中所述还原酶包括YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6。
根据本发明的一些实施方式，其中，将所述2-羟基-3,3-二甲基-4-醛基丁酸发酵的酶是能把醛类转化成醇类的还原酶。所述还原酶包括但不限于野生或者基因工程改造过的YqhD、YqhE、AdhE、DkgA、Adh1、Adh2、Adh5、ADH6或yihU。
根据本发明的一些实施方式，其中，将2-羟基-3,3-二甲基-4-醛基丁酸发酵转化成泛解酸、泛酸或者泛醇的所述转化是利用所述还原酶在所述细菌或真菌体内完成。
根据本发明的一些实施方式，其中，将2-羟基-3,3-二甲基-4-醛基丁酸发酵转化成泛解酸、泛酸或者泛醇的所述转化的条件是在20-40℃，加上碳源进行发酵。所述碳源包括但不限于葡萄糖，淀粉等。当采用耐高温菌或耐高温酶进行发酵时，发酵温度可达90℃，发酵温度可以为大约20℃，大约30℃，大约35℃，大约65℃，大约90℃。
发酵时可根据发酵需求加入pH调节剂。采用碳酸钙、氢氧化钙、碳酸钠、碳酸氢钠、氢氧化钠、碳酸钾或氢氧化钾调节pH，发酵产物泛酸和pH调节剂生成对应的盐，得到泛酸钙、泛酸钾、泛酸钠或泛酸铵。
根据本发明的一些实施方式，其中，所述2-羟基-3,3-二甲基-4-醛基丁酸是经乙醛酸与异 丁醛缩合制得的。缩合反应可用已公开报道的制备方法实现。
另一方面，本发明提供了一种泛酸及其盐的制备方法，将发酵得到的泛解酸与β-丙氨酸反应得到。
本发明提供了一种D-泛酸的制备方法，包括：1)将2-羟基-3,3-二甲基-4-醛基丁酸经细菌或酵母发酵转化成泛解酸；2)将所述泛解酸与β-丙氨酸反应得到D-泛酸。
另一方面，本发明提供了一种泛醇的制备方法，将发酵得到的泛解酸与β-丙氨醇反应得到。
以下实施例中加入的原料试剂或使用的仪器或操作步骤均是本领域普通技术人员可常规确定的内容。
实施例
材料与方法
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
Figure PCTCN2021102698-appb-000002
One Step Cloning Kit和琼脂糖凝胶电泳回收试剂盒均购自南京诺唯赞生物科技股份有限公司。
DNA电泳使用西班牙琼脂糖(Biowest Agarose)。
胰蛋白胨、酵母粉、D-(+)-葡萄糖、磷酸二氢钾、氯化钠、氯化钙、氯化铵、D-泛酸钙、β-丙氨酸、IPTG、硫胺素和硫酸卡那霉素均购自上海生工。
碳酸钙、氢氧化钠和硫酸镁购自国药。
乙醛酸和异丁醛购自阿拉丁。
配制固体培养基使用的琼脂粉购自上海盛思。
质粒构建测序验证由金唯智完成。
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：硫酸卡那霉素50μg/mL。
发酵底物2-羟基-3,3-二甲基-4-醛基丁酸是经乙醛酸与异丁醛以现有常规方法缩合制得。
发酵培养基组分：
Figure PCTCN2021102698-appb-000003
Figure PCTCN2021102698-appb-000004
泛解酸和泛酸的检测方法：使用配备伯乐色谱柱(1250140Aminex HPX-87H Column 300 x 7.8mm)的HPLC-RID定量检测泛酸。
实施例1大肠杆菌还原酶表达载体构建
根据NCBI公布的大肠杆菌MG1655的基因组序列分别设计引物：
yqhE_F(gaccgaattcattaaagaggagaaaggtaccatggctaatccaaccgttattaagctacag)/yqhE_R(ctttcgttttatttgatgcctctagagctagcttagccgccgaactggtcaggatcgggaccgag)
和
yihU_F(gaccgaattcattaaagaggagaaaggtaccatggcagcaatcgcgtttatcggtttagg)/yihU_R(ctttcgttttatttgatgcctctagagctagcttacatttttactttggcagtcatcccggcactg)
以MG1655基因组为模板通过PCR扩增分别得yqhE和yihU基因，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pZA载体上，转化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，正确的重组载体分别命名为pZA-yqhE和pZA-yihU。
实施例2重组大肠杆菌发酵生产泛解酸和泛酸
将上述重组菌单菌落分别接种2mL含有50μg/mL硫酸卡那霉素的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5mL发酵培养基的100mL三角瓶，30℃，220rpm培养。每个发酵瓶中加入0.5g CaCO 3调节发酵液的pH。培养24h后收集发酵液，检测泛解酸和泛酸的浓度(参见下面表格)。YqhE和YihU蛋白促进底物2-羟基-3,3-二甲基-4-醛基丁酸还原成泛解酸。当发酵液中添加另一种底物β-丙氨酸后，可以生产终产物泛酸。但该发酵结果显示将泛解酸转化成泛酸的代谢途径存在限速步骤，为提高泛酸的产量，需进一步提高下游代谢途径所需要的酶的表达。本实施例中采用CaCO 3调节发酵液中的pH，发酵产物泛酸在发酵液中生成泛酸钙。如果加入NaOH、KOH或氨水调节发酵液中的pH，发酵液中相应地将生成泛酸的钠盐、钾盐、铵盐。
Figure PCTCN2021102698-appb-000005

Claims (14)

  1. 一种泛解酸、泛酸、泛醇或者其盐的制备方法，其特征在于，包括将底物进行微生物发酵步骤，其中，所述底物包括2-羟基-3,3-二甲基-4-醛基丁酸。
  2. 根据权利要求1所述的制备方法，其中，所述微生物选自细菌或真菌；可选地，所述微生物选自野生或基因工程改造过的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌；可选地，所述微生物选自野生或基因工程改造过的大肠埃希氏菌(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)；可选地，所述微生物选自野生或基因工程改造过的大肠杆菌；可选地，所述微生物选自重组大肠杆菌，所述重组大肠杆菌含有表达还原酶的外源基因，其中所述还原酶包括YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6。
  3. 根据权利要求1或2所述的制备方法，其中，将所述2-羟基-3,3-二甲基-4-醛基丁酸进行发酵的酶是能把醛类转化成醇类的还原酶；
    可选地，所述还原酶包括但不限于野生或者基因工程改造过的YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6；
    可选地，所述转化是利用所述还原酶在细菌或真菌体内完成。
  4. 根据权利要求1-3任一项所述的制备方法，其中，所述发酵温度为20-90℃；可选地，所述发酵温度为大约30℃-65℃；可选地，所述发酵温度为大约30-35℃。
  5. 根据权利要求1-4任一项所述的制备方法，其中，所述2-羟基-3,3-二甲基-4-醛基丁酸是经乙醛酸与异丁醛缩合制得的；可选地，所述底物还包括β-丙氨酸；
    可选地，发酵采用碳酸钙、氢氧化钙、碳酸钠、碳酸氢钠、氢氧化钠、碳酸钾或氢氧化钾调节pH，可选地，所述盐选自泛酸钙、泛酸钾、泛酸钠或泛酸铵。
  6. 一种泛酸的制备方法，其特征在于，将权利要求1-5任一项制备方法得到的泛解酸与β-丙氨酸进行化学反应得到；可选地，所述泛酸为D-泛酸。
  7. 一种泛酸或其盐的制备方法，其特征在于，包括将底物进行微生物发酵转化步骤，其中，所述底物包括2-羟基-3,3-二甲基-4-醛基丁酸和β-丙氨酸；可选地，所述泛酸为D-泛酸；可选地，发酵采用碳酸钙、氢氧化钙、碳酸钠、碳酸氢钠、氢氧化钠、碳酸钾或氢氧化钾调节pH，可选地，所述盐选自泛酸钙、泛酸钾、泛酸钠或泛酸铵。
  8. 根据权利要求7所述的制备方法，其中，所述微生物选自细菌或真菌；可选地，所述微生物选自野生或基因工程改造过的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌；可选地，所述微生物选自野生或基因工程改造过的大肠埃希氏菌(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)；可选地，所述微 生物选自野生或基因工程改造过的大肠杆菌；可选地，所述微生物选自重组大肠杆菌，所述重组大肠杆菌含有表达还原酶的外源基因，其中还原酶包括YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6。
  9. 根据权利要求7所述的制备方法，其中，将所述2-羟基-3,3-二甲基-4-醛基丁酸进行发酵的酶是能把醛类转化成醇类的还原酶；
    可选地，所述还原酶包括但不限于野生或者基因工程改造过的YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6；
    可选地，所述转化是利用所述还原酶在细菌或真菌体内完成。
  10. 根据权利要求7-9任一项所述的制备方法，其中，所述发酵温度为20-90℃；可选地，所述发酵温度为大约30℃-65℃；可选地，所述发酵温度为大约30-35℃。
  11. 根据权利要求7-10任一项所述的制备方法，其中，所述2-羟基-3,3-二甲基-4-醛基丁酸是经乙醛酸与异丁醛缩合制得的。
  12. 一种泛醇的制备方法，其特征在于，将权利要求1-5任一项制备方法得到的泛解酸与β-丙氨醇进行化学反应得到；可选地，所述泛醇为D-泛醇。
  13. 一种泛醇的制备方法，其特征在于，包括将底物进行微生物发酵转化步骤，其中，所述底物包括2-羟基-3,3-二甲基-4-醛基丁酸和β-丙氨醇；可选地，所述泛醇为D-泛醇。
  14. 根据权利要求13所述的制备方法，其中，所述微生物选自细菌或真菌；可选地，所述微生物选自野生或基因工程改造过的大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌；可选地，所述微生物选自野生或基因工程改造过的大肠埃希氏菌(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)；可选地，所述微生物选自野生或基因工程改造过的大肠杆菌；可选地，所述微生物选自重组大肠杆菌，所述重组大肠杆菌含有表达还原酶的外源基因，其中还原酶包括YqhD、YqhE、DkgA、AdhE、yihU、AdH1、ADH2、ADH5或ADH6。
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US12509706B2 (en) 2025-12-30
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