阅读说明：本技术 一种泛解酸、泛酸、泛醇及其盐的制备方法 (Preparation method of pantoic acid, pantothenic acid, panthenol and salt thereof ) 是由 张科春 王吉龙 于 2021-06-28 设计创作，主要内容包括：本申请公开了一种泛解酸、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-醛基丁酸经微生物发酵得到。该方法对于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。本申请还进一步公开了一种D-泛酸和D-泛醇的制备方法。(The application discloses a preparation method of pantoic acid, pantothenic acid, panthenol and salts thereof, which is obtained by fermenting a substrate 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid by microorganisms. According to the method, 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid can be directly fermented without in vitro reduction of aldehyde group, and 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is reduced into 2, 4-dihydroxy-3, 3-dimethyl-butyric acid by using in vivo reductase. The application further discloses a process for the preparation of D-pantothenic acid and D-panthenol.)

1. A process for the preparation of pantoic acid, pantothenic acid, panthenol or salts thereof, comprising the step of subjecting a substrate to microbial fermentation, wherein the substrate comprises 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid.

2. The method according to claim 1, wherein the microorganism is selected from bacteria or fungi; optionally, the microorganism is selected from wild or genetically engineered escherichia coli, bacillus, corynebacterium, yeast, or streptomyces; optionally, the microorganism is selected from wild or genetically engineered Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida utilis (Candida utilis), or Pichia pastoris (Pichia pastoris); optionally, the microorganism is selected from wild or genetically engineered escherichia coli; optionally, the microorganism is selected from recombinant e.coli containing an exogenous gene expressing a reductase, wherein the reductase comprises YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5, or AdH 6.

3. The production method according to claim 1 or 2, wherein the enzyme for fermenting the 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid is a reductase capable of converting aldehydes into alcohols;

optionally, the reductase includes, but is not limited to, wild or genetically engineered YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5, or AdH 6;

alternatively, the transformation is accomplished in vivo in bacteria or fungi using the reductase.

4. The production method according to any one of claims 1 to 3, wherein the fermentation temperature is 20 to 90 ℃; optionally, the fermentation temperature is about 30 ℃ to 65 ℃; optionally, the fermentation temperature is about 30-35 ℃.

5. The production method according to any one of claims 1 to 4, wherein the 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid is produced by condensation of glyoxylic acid with isobutyraldehyde; optionally, the substrate further comprises beta-alanine;

optionally, the fermentation is adjusted for pH with calcium carbonate, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, or potassium hydroxide, optionally, the salt is selected from calcium pantothenate, potassium pantothenate, sodium pantothenate, or ammonium pantothenate.

6. A process for producing pantothenic acid, characterized by reacting pantoic acid obtained by the process of any one of claims 1 to 5 with β -alanine; alternatively, the pantothenic acid is D-pantothenic acid.

7. A process for the production of pantothenic acid or a salt thereof, comprising subjecting a substrate to a microbial fermentative conversion step, wherein the substrate comprises 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid and β -alanine; alternatively, the pantothenic acid is D-pantothenic acid; optionally, the fermentation is adjusted for pH with calcium carbonate, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, or potassium hydroxide, optionally, the salt is selected from calcium pantothenate, potassium pantothenate, sodium pantothenate, or ammonium pantothenate.

8. The method according to claim 7, wherein the microorganism is selected from bacteria or fungi; optionally, the microorganism is selected from wild or genetically engineered escherichia coli, bacillus, corynebacterium, yeast, or streptomyces; optionally, the microorganism is selected from wild or genetically engineered Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida utilis (Candida utilis), or Pichia pastoris (Pichia pastoris); optionally, the microorganism is selected from wild or genetically engineered escherichia coli; optionally, the microorganism is selected from recombinant e.coli containing an exogenous gene expressing a reductase, wherein the reductase comprises YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5 or AdH 6.

9. The method according to claim 7, wherein the enzyme for fermenting 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid is a reductase capable of converting aldehydes into alcohols;

optionally, the reductase includes, but is not limited to, wild or genetically engineered YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5, or AdH 6;

alternatively, the transformation is accomplished in vivo in bacteria or fungi using the reductase.

10. The production method according to any one of claims 7 to 9, wherein the fermentation temperature is 20 to 90 ℃; optionally, the fermentation temperature is about 30 ℃ to 65 ℃; optionally, the fermentation temperature is about 30-35 ℃.

11. The production method according to any one of claims 7 to 10, wherein the 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid is produced by condensation of glyoxylic acid and isobutyraldehyde.

12. A process for producing panthenol, which comprises chemically reacting pantoic acid obtained by the process according to any one of claims 1 to 5 with β -alaninol; optionally, the panthenol is D-panthenol.

13. A method for producing panthenol, comprising subjecting a substrate to a microbial fermentation conversion step, wherein the substrate comprises 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid and β -alaninol; optionally, the panthenol is D-panthenol.

14. The method of claim 13, wherein the microorganism is selected from bacteria or fungi; optionally, the microorganism is selected from wild or genetically engineered escherichia coli, bacillus, corynebacterium, yeast, or streptomyces; optionally, the microorganism is selected from wild or genetically engineered Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida utilis (Candida utilis), or Pichia pastoris (Pichia pastoris); optionally, the microorganism is selected from wild or genetically engineered escherichia coli; optionally, the microorganism is selected from recombinant e.coli containing an exogenous gene expressing a reductase, wherein the reductase comprises YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5 or AdH 6.

Technical Field

The application relates to the technical field of biochemical synthesis, in particular to a preparation method of pantoic acid, pantothenic acid, panthenol and salts thereof.

Background

Pantothenic acid is also known as vitamin B5. Pantoic acid is an important intermediate for producing pantothenic acid and panthenol. Calcium D-pantothenate is the most commercially important product form of D-pantothenic acid 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. In cells, pantothenic acid is primarily used for the biosynthesis of coenzyme A (CoA) and Acyl Carrier Protein (ACP). These coenzymes are important for all cells, and they are involved in over 100 different intermediate reactions in cellular metabolism. Panthenol is one of pantothenic acid derivatives, and D-panthenol is mainly used for preparing hair care products and cosmetics for topical application, and can be used for preventing and treating small wrinkles, inflammation, solarization, and erosion, preventing alopecia, and promoting hair growth.

The existing method for synthesizing the D-calcium pantothenate comprises the steps of reacting beta-aminopropionic acid, alpha-hydroxy-beta, beta' -dimethyl-gamma-butyrolactone (pantoic acid lactone) and calcium metal to generate DL-calcium pantothenate, and then splitting to obtain the D-calcium pantothenate. The pantoic acid lactone is produced mainly by the isobutyraldehyde-formaldehyde-hydrocyanic acid method (U.S. Pat. No. 4,4020103,1977; JP.47-35314,1973; JP.49-41361,1974) and the isobutyraldehyde-aldehyde acetic acid method (JP.55-62080,1980). Isobutyraldehyde-formaldehyde-hydrocyanic acid method, said method comprises making isobutyraldehyde and formaldehyde into alpha, alpha-dimethyl-beta-hydroxypropionaldehyde by hydroxymethylation, then producing cyanohydrin reaction with hydrocyanic acid to produce alpha, gamma-dihydroxy-beta, beta-dimethylbutyraldehyde, obtaining alpha, gamma-dihydroxy-beta, beta-dimethylbutyrate (pantoic acid) by acid hydrolysis, then obtaining DL-pantolactone by dehydration lactonization. The so-called isobutyraldehyde-hydroxyacetonitrile process is actually an improvement over this process, in that instead of hydrocyanic acid, hydroxyacetonitrile is used, which is obtained by the addition of formaldehyde and hydrocyanic acid. The so-called isobutyraldehyde-hydroxyacetonitrile process is actually an improvement over this process, in that instead of hydrocyanic acid, hydroxyacetonitrile is used, which is obtained by the addition of formaldehyde and hydrocyanic acid. The method has the main problems that a plurality of steps of hydroxymethylation, cyanohydrin, hydrolysis, lactonization and the like are required, virulent hydrocyanic acid is used in the reaction, the technical requirement is high, great troubles are brought to management and use, and the cost for taking related safety measures is increased. The isobutyraldehyde-glyoxylic acid process has two distinct processes: the condensation-disproportionation process (JP 55-62080, 1980) comprises aldol condensation of isobutyraldehyde and glyoxylic acid, disproportionation of the aldol condensation with another molecule of glyoxylic acid, and dehydration cyclization to produce pantoic acid lactone; the condensation-hydrogenation process (EP0171046) is carried out by catalytically hydrogenating the aldol condensation product at a high pressure of 250bar to prepare DL-pantolactone. The former has poor economy and environmental protection without competitiveness because of adopting twice the amount of glyoxylic acid and generating a large amount of wastewater; the latter requires high pressures of 250bar, which results in high equipment costs, maintenance costs and poor safety, and greatly limits the use thereof.

To overcome the above-mentioned disadvantages and to improve the production of pantothenic acid, D-pantothenate and the like, processes for microbial synthesis have been the subject of research and have also been developed in recent years. Pure microbial synthesis methods have not been used for industrial production because of low yields (EP 2163629). The current industrial processes are all chemical precursors, followed by enzymatic resolution to give chiral D-pantothenic acid.

Disclosure of Invention

The present invention proposes a new process for the preparation of pantoic acid, pantothenic acid, panthenol and salts thereof, which solves at least partly the above mentioned technical problems.

One of the objects of the present invention is to provide a process for the preparation of pantoic acid, pantothenic acid or panthenol, which comprises converting 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid into pantoic acid, pantothenic acid or panthenol by bacterial or yeast fermentation.

Another object of the present invention is to provide a process for producing D-pantothenic acid, which comprises: 1) 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is converted into pantoic acid by bacterial or yeast fermentation; 2) reacting said pantoic acid with beta-alanine to produce D-pantothenic acid.

The invention also aims to provide a preparation method of D-panthenol, which comprises the following steps: 1) 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is converted into pantoic acid by bacterial or yeast fermentation; 2) reacting said pantoic acid with β -alaninol to obtain D-panthenol.

According to the preparation method provided by the invention, the aldehyde group of the 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid can be directly fermented without in vitro reduction, and the 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is reduced into the 2, 4-dihydroxy-3, 3-dimethyl-butyric acid by using in vivo reductase. The method utilizes low-cost raw materials, avoids toxic cyanide and avoids a high-cost chiral resolution step.

Detailed Description

In the present disclosure, unless defined otherwise, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Meanwhile, for better understanding of the present disclosure, definitions and explanations of related terms are provided below.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein and unless otherwise specified, the term "pantoic acid" is 2, 4-dihydroxy-3, 3-dimethylbutyric acid. The term "pantothenic acid" means 2, 4-dihydroxy-3, 3-, dimethylbutyryl-3-alanine. The term "panthenol" refers to 2, 4-dihydroxy-N- (3-hydroxypropyl) -3, 3-dimethylbutanamide.

The term "amplification" means that the intracellular activity of one or more enzymes in a microorganism which are encoded by suitable DNA is increased, for example by increasing the gene copy number, using strong promoters or using genes which encode suitable enzymes with high activity and optionally combining these measures.

The term "wild" refers to an object that can be found in nature.

Unless specifically stated otherwise, the terms "first," "second," and the like, do not denote any order or importance, but rather the terms first, second, and the like are used to distinguish one object from another.

As used herein and unless otherwise specified, the term "about" refers to a measurable value such as an amount, time period, or the like, and is meant to encompass a variation of ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from a given value, so long as such variation is suitable for practicing the disclosed methods.

As described above, an object of the present invention is to provide a process for producing 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 a method for preparing pantoic acid, pantothenic acid, panthenol or salts thereof, by adding 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid to a medium as a fermentation substrate, and fermenting the fermentation substrate by bacteria or fungi.

According to some embodiments of the invention, wherein the fermentation substrate does not contain beta-alanine, more pantoate and less pantothenate are obtained by bacterial or fungal fermentation.

According to some embodiments of the present invention, wherein 2-hydroxy-3, 3-dimethyl-4-aldehyde butyrate and beta-alanine are added into the culture medium at the same time as fermentation substrate for fermentation, pantoic acid and pantothenic acid can be obtained at the same time, and the amount of produced pantoic acid is increased obviously.

The reaction mechanism for preparing pantoic acid and pantothenic acid according to the invention is as follows:

wherein, after the production of pantoic acid, the subsequent production of pantothenic acid can be realized by a chemical method or a biological fermentation method which is already reported; the replacement of beta-alanine with beta-alaninol enables the production of panthenol, which can likewise be achieved by publicly reported chemical or biological fermentation methods. According to some embodiments of the invention, wherein the bacteria or fungi include, but are not limited to, wild or genetically engineered Escherichia coli, Bacillus, yeast, Corynebacterium or Streptomyces. For example Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida utilis (Candida utilis) or Pichia pastoris (Pichia pastoris).

According to some embodiments of the invention, wherein the microorganism is selected from the group consisting of wild or genetically engineered escherichia coli; optionally, the microorganism is selected from recombinant e.coli containing an exogenous gene expressing a reductase, wherein the reductase comprises YqhD, YqhE, DkgA, AdhE, yihU, AdH1, AdH2, AdH5, or AdH 6.

According to some embodiments of the invention, wherein the enzyme that ferments the 2-hydroxy-3, 3-dimethyl-4-aldehyde butyrate is a reductase capable of converting aldehydes to alcohols. The reductase includes, but is not limited to, wild or genetically engineered YqhD, YqhE, AdhE, DkgA, Adh1, Adh2, Adh5, Adh6, or yihU.

According to some embodiments of the invention, wherein the fermentative conversion of 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid to pantoic acid, pantothenic acid or panthenol is performed in the bacteria or fungi using the reductase.

According to some embodiments of the invention, wherein said converting of 2-hydroxy-3, 3-dimethyl-4-formylbutyric acid into pantoic acid, pantothenic acid or panthenol by fermentation is performed at 20-40 ℃ plus a carbon source. The carbon source includes, but is not limited to, glucose, starch, and the like. When fermentation is carried out using thermophilic bacteria or thermophilic enzymes, the fermentation temperature may be up to 90 deg.C, and the fermentation temperature may be about 20 deg.C, about 30 deg.C, about 35 deg.C, about 65 deg.C, about 90 deg.C.

During fermentation, a pH regulator can be added according to the fermentation requirement. Calcium carbonate, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate or potassium hydroxide are adopted to adjust the pH, and the fermentation product pantothenic acid and the pH adjusting agent generate corresponding salt to obtain calcium pantothenate, potassium pantothenate, sodium pantothenate or ammonium pantothenate.

According to some embodiments of the invention, the 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is prepared by condensation of glyoxylic acid and isobutyraldehyde. The condensation reaction can be achieved by publicly reported preparation methods.

In another aspect, the invention provides a process for the preparation of pantothenic acid and its salts by reacting pantoic acid obtained from the fermentation with beta-alanine.

The invention provides a preparation method of D-pantothenic acid, which comprises the following steps: 1) 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is converted into pantoic acid by bacterial or yeast fermentation; 2) reacting said pantoic acid with beta-alanine to produce D-pantothenic acid.

In another aspect, the present invention provides a process for the preparation of panthenol, wherein pantoic acid obtained by fermentation is reacted with β -alaninol.

The starting reagents added or the equipment or procedures used in the examples below are those routinely determined by one of ordinary skill in the art.

Examples

Materials and methods

DNA Polymerase Phanta Max Super-Fidelity DNA Polymerase, ligase independent single-fragment rapid cloning kit used in the embodiment of the inventionII One Step Cloning Kit and agarose gel electrophoresis recovery Kit were purchased from Nanjing Novophilin Biotechnology GmbH.

Spanish Agarose (Biowest Agarose) was used for DNA electrophoresis.

Tryptone, yeast powder, D- (+) -glucose, potassium dihydrogen phosphate, sodium chloride, calcium chloride, ammonium chloride, D-calcium pantothenate, beta-alanine, IPTG, thiamine, and kanamycin sulfate were all purchased from Shanghai Producers.

Calcium carbonate, sodium hydroxide and magnesium sulfate were purchased from national medicine.

Glyoxylic acid and isobutyraldehyde were purchased from alatin.

Agar powder used for preparing the solid medium was purchased from shanghai.

Plasmid construction sequencing validation was done with the Kingchi.

LB medium composition: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride, and 1.5% of agar powder is added into a solid culture medium.

The antibiotic concentrations were: kanamycin sulfate 50. mu.g/mL.

The fermentation substrate 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is prepared by condensation of glyoxylic acid and isobutyraldehyde by the conventional method.

Fermentation medium components:

detection methods for pantoate and pantothenate: pantothenate was quantitatively detected using HPLC-RID equipped with a Berkele Column (1250140Aminex HPX-87H Column 300X 7.8 mm).

Example 1 construction of E.coli reductase expression vector

Primers were designed based on the genomic sequence of E.coli MG1655 published by NCBI:

yqhE_F(gaccgaattcattaaagaggagaaaggtaccatggctaatccaaccgttattaagctacag)/yqhE_R(ctttcgttttatttgatgcctctagagctagcttagccgccgaactggtcaggatcgggaccgag)

and

yihU_F(gaccgaattcattaaagaggagaaaggtaccatggcagcaatcgcgtttatcggtttagg)/yihU_R(ctttcgttttatttgatgcctctagagctagcttacatttttactttggcagtcatcccggcactg)

the genes of yqhE and yihU are respectively obtained by PCR amplification by taking MG1655 genome as a template, and are connected to a pZA vector containing IPTG inducible promoter through a single-chip quick cloning kit of non-ligase dependent type, BW25113 competent cells are transformed, kanamycin sulfate resistant plates are coated for overnight culture, positive clones are selected for sequencing verification, and correct recombinant vectors are respectively named as pZA-yqhE and pZA-yihU.

Example 2 fermentation of recombinant E.coli to produce pantoate and pantothenate

The single colonies of the recombinant bacteria were inoculated into 2mL of LB liquid medium containing 50. mu.g/mL of kanamycin sulfate, and cultured overnight (about 14 hours) at 37 ℃ and 220 rpm. A100 mL Erlenmeyer flask containing 5mL of the fermentation medium was transferred at an initial OD of 0.05, and cultured at 30 ℃ and 220 rpm. 0.5g CaCO was added to each fermentation flask₃Adjusting the pH of the fermentation broth. After 24h of incubation the fermentation broth was collected and the concentrations of pantoate and pantothenate were determined (see table below). YqhE and YIhU protein promoting substratesReducing 2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid into pantoic acid. When the fermentation broth is supplemented with another substrate, beta-alanine, the final product pantothenic acid can be produced. However, the fermentation results show that there is a rate-limiting step in the metabolic pathway for converting pantoate to pantothenate, and that in order to increase pantothenate production, further increases in the expression of enzymes required for downstream metabolic pathways are desired. CaCO was used in this example₃Adjusting the pH value of the fermentation liquor, and generating calcium pantothenate from the fermentation product pantothenic acid in the fermentation liquor. If NaOH, KOH or ammonia water is added to adjust the pH value in the fermentation liquor, sodium salt, potassium salt and ammonium salt of pantothenic acid are correspondingly generated in the fermentation liquor.