CN112852652A

CN112852652A - Recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, construction and application

Info

Publication number: CN112852652A
Application number: CN202110054935.8A
Authority: CN
Inventors: 史劲松; 徐国强; 许正宏; 李会; 龚劲松
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2021-05-28

Abstract

The invention discloses a recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, and construction and application thereof. At present, the conversion rate of the substrate CDCA reaches 80%.

Description

Recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, construction and application

Technical Field

The invention belongs to the technical field of biosynthesis, and particularly relates to a recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, and construction and application thereof.

Background

Ursodeoxycholic acid (UDCA) is the main effective component contained in the rare Chinese medicine bear bile, and it and its Corresponding Diastereoisomer Chenodeoxycholic Acid (CDCA) are clinically used for treating various cholelithiasis and various acute and chronic liver diseases, and have good effect. The extraction of UDCA from artificially cultured bear gall has low yield and limited source, and is out of the animal protection, so that the artificial synthesis of UDCA has important significance. The synthesis method of UDCA mainly comprises a method combining total chemical synthesis and chemical enzyme method, wherein the starting material is animal-derived Cholic Acid (CA) or deoxycholic acid (such as CDCA).

Ursodeoxycholic acid (UDCA) is an effective component of the traditional Chinese medicine, has very wide clinical application and excellent medicinal value, has very good curative effect in treating gallstone, promoting liver transplantation, bile reflux gastritis, alcoholic liver, biliary cirrhosis and hepatitis induced by medicines, and has large market dosage. At present, two methods of bile taking from live bears and artificial synthesis are mainly used for preparing UDCA, wherein the natural source is that live bears take bile or bear bile, the live bears are protected by an animal protection method, and the extraction source is limited, so that the source of natural bear bile tends to be reduced. The artificial synthesis is to synthesize UDCA by using Chenodeoxycholic acid (CDCA) extracted from cow and goose bile which can be obtained in large quantity through a chemical method, and the 7-OH is subjected to configuration inversion through an oxidation-reduction method, but the synthetic method has a series of problems of complex reaction process, low selectivity, harsh reaction conditions, high energy consumption, high pollution and the like, particularly needs toxic and dangerous reagents in protection and deprotection processes, and seriously limits the industrial application of the chemical method. The existing UDCA produced by a chemical method accounts for about 30 percent of the market share, has low preparation purity of about 80 percent, and still can not meet the requirements of the market on the dosage and quality of the UDCA.

UDCA biosynthesis by CDCA is highly efficient and relatively environmentally friendly compared to chemical epimerization. The microbial transformation or the bio-enzyme catalysis is mainly developed around 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH), and the bioconversion of CDCA to UDCA is realized by using 7 alpha-HSDH and 7 beta-HSDH producing slime clostridium, inharmonic clostridium, clostridium pasteurianum and xanthomonas maltophilia, and the bio-enzyme catalysis reaction process of UDCA is shown in figure 1.

However, high concentrations of CDCA inhibit the accumulation of cellular biomass, presenting difficulties in product recovery and purification. In addition, previous studies have shown that the production of intermediates increases and UDCA decreases with the increase of culture time, and thus industrial production cannot be achieved.

Hirano and Masuda describe NADP + -dependent 7 β -HSDH from Coprinus aerogenes ATCC 25986 (apple Environ Microbiol,1982,43(5): 1057-. The genome sequencing of ATCC 25986 was completed in 2007, and Rolf D.Schmid and Germany cell pharmaceutical company expressed the 7 beta-HSDH gene in Escherichia coli in 2011, identified its enzymological properties and used for reducing 7, 12-diketone-LCA or 7-KLCA to obtain 12-keto-UDCA or UDCA (Appl Microbiol Biotechnol,2011,90: 127-. On the basis of the sequence, German cell pharmaceutical company continuously optimizes to obtain mutants with improved activity and removed substrate inhibition (CN201080062617, CN201180067680), and the high conversion rate and high specificity of the enzyme 7 beta-HSDH generated by recombination enable the enzyme method large-scale production of UDCA. In addition, the 7 beta-HSDH gene of the Ruminococcus angularis is cloned and efficiently expressed from ATCC35915 by the university of eastern China, and the enzyme synthesis test of UDCA proves that the enzyme also has high conversion rate and high specificity on a substrate 7-KLCA similar to the 7 beta-HSDH from Coprinus aerogenes.

In summary, only reports on the expression of 7 α -HSDH and 7 β -HSDH in Escherichia coli, but whether the same expression can be achieved by the enzyme in other strains, are not clear, and further research and study by researchers are needed.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a recombinant yeast strain for efficiently converting ursodeoxycholic acid and application thereof, wherein yeast cells are used as chassis cells to realize heterologous expression of 7 alpha-HSDH and 7 beta-HSDH, so that the recombinant yeast strain for efficiently converting the ursodeoxycholic acid is obtained.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:

the invention also aims to provide a recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, in particular to an engineering bacterium obtained by transferring a recombinant expression plasmid containing 7 alpha-HSDH and 7 beta-HSDH genes by taking a yeast strain S.

Furthermore, the nucleotide sequence of the 7 alpha-HSDH gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the 7 beta-HSDH gene is shown as SEQ ID NO. 2.

Further, the name of the recombinant yeast strain is S.cerevisiae CEN.PK2-1C 7 alpha-HSDH ≠ 7 beta-HSDH ≠ ═ 7.

The invention also aims to provide a construction method of the recombinant yeast strain for efficiently converting ursodeoxycholic acid, which takes a saccharomyces cerevisiae chassis cell strain S.cerevisiae CEN.PK2-1C as a host, entrusts a company to synthesize a whole gene sequence according to a nucleotide sequence of a 7 alpha-HSDH gene and a nucleotide sequence of a 7 beta-HSDH gene, further inserts the 7 alpha-HSDH and the 7 beta-HSDH gene into shuttle vectors pY15TEF1 and pYX212 to obtain recombinant expression plasmids pY15TEF1-7 alpha-HSDH and pYX212-7 beta-HSDH, and then converts the recombinant expression plasmids into the mutant yeast strain S.cerevisiae CEN.PK2-1C.

The invention also aims to provide the application of the recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid in the production of the ursodeoxycholic acid.

Another object of the present invention is to provide ursodeoxycholic acid obtained by the above-mentioned application.

Has the advantages that: compared with the prior art, the recombinant yeast strain for efficiently converting ursodeoxycholic acid provided by the invention has the following advantages:

the invention takes Saccharomyces cerevisiae strain S.cerevisiae CEN.PK2-1C as a chassis cell, heterogeneously expresses 7 alpha-HSDH and 7 beta-HSDH coding genes of clostridium, and aims to realize biosynthesis of UDCA by taking CDCA as a substrate. At present, the conversion rate of the substrate CDCA reaches 80%.

Drawings

FIG. 1 is a schematic diagram of the process of the bio-enzyme catalyzed reaction of UDCA.

FIG. 2 is an LC-MS map of UDCA. (a) Liquid chromatography of UDCA standard; (b) liquid chromatogram of the recombinant yeast strain fermentation liquid; (c) mass spectrometry spectra of UDCA standards; (d) and (3) mass spectrum of the recombinant yeast strain fermentation liquor.

FIG. 3 shows the conversion of substrate CDCA as a function of time.

Detailed Description

The invention heterologously expresses 7 alpha-HSDH and 7 beta-HSDH encoding genes from clostridium in saccharomyces cerevisiae S.cerevisiae CEN.PK2-1C, thereby improving the substrate conversion efficiency.

The invention is further described with reference to the following figures and examples.

Examples

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

Example 1: construction of transformed chenodeoxycholic acid engineering yeast

pY15TEF1 and pYX212 are used as expression plasmids to construct pY15TEF1-7 alpha-HSDH and pYX212-7 beta-HSDH over-expression vectors, and the specific method comprises the following steps: the gene products 7 α -HSDH and 7 β -HSDH having restriction sites were synthesized by Shanghai Bioengineering services Co., Ltd, after which the above gene products 7 α -HSDH and 7 β -HSDH and plasmids pY15TEF1, pYX212 were double-digested with XbaI and BamHI, EcoRI and BamHI (primer sequences are shown in SEQ. NO.03-SEQ. NO.06 of Table 1) at the same time, ligated with T4 DNA ligase, 5. mu.L of the ligated product was transformed into 50. mu.L of competent cells of s.cerevisiae strain, LA plates were coated, the transformants obtained were verified for correctness by colony PCR and digestion, double-digested with XbaI and BamHI, EcoRI and BamHI, and the correct transformants were verified by sequencing to obtain recombinant plasmids pY15TEF1-7 α -HSDH and pYX212-7 β -HSDH.

The plasmid is transformed into a saccharomyces cerevisiae strain S.cerevisiae CEN.PK2-1C by electric shock, transformants are selected on a resistant plate, colony PCR verification is carried out on 7 alpha-HSDH-F, 7 alpha-HSDH-R, 7 beta-HSDH-F and 7 beta-HSDH-R (table 1) by using primers, and a recombinant strain S.cerevisiae CEN.PK2-1C 7 alpha-HSDH × [ 7 ] beta-HSDH × [ [) is successfully constructed.

The s.cerevisiae strain used in this example was obtained from eurocarf, cen.pk2-1C, see in particular: http:// web. uni-frankfurt. de/fb 15/mikro/eurocarf/data/cen. html.

TABLE 1 primer sequences

Numbering	Primer and method for producing the same	Sequence (5 '-3')
			SEQ.NO.03	7α-HSDH-F	GCtctagaATGAAAAGATTAGAAGGAAA(XbaI)
SEQ.NO.04	7α-HSDH-R	CGCggatccTTATCTTGGACAGTATTCT(BamHI)
			SEQ.NO.05	7β-HSDH-F	CGgattcATGAATTTTAGAGAAAAATA(EcoRI)
SEQ.NO.06	7β-HSDH-R	CGCggatccTTATGCCATATGATCAAACA(BamHI)

Example 2: fermentation for converting chenodeoxycholic acid engineering yeast

The culture conditions of the saccharomyces cerevisiae are as follows: taking the strain from a strain storage tube at the temperature of-80 ℃, activating on a YPD (transformant containing nutritional marker plasmid in a corresponding SD defect culture medium) plate, and culturing for 3d at the temperature of 30 ℃; the activated single colonies were picked and inoculated into 3mL tubes containing YPD (transformants containing the nutritional marker plasmid in the corresponding SD-deficient medium) medium, and cultured overnight at 220rpm at 30 ℃ until saturation.

Fermentation culture conditions: activated single colonies were picked from the plates and inoculated into seed medium and shake-cultured at 30 ℃ and 220rpm for 24h to saturation. The starting OD was transferred to a shake flask of the fermentation medium at 0.2 and incubated at 30 ℃ and 220 rpm. The cells were cultured to stationary phase (48h), harvested by centrifugation, resuspended in 0.1M PBS, 2% CDCA added, and reacted in 30 ℃ thermostatic water bath for 4 h.

Seed medium (g/L): glucose 20, Yeast Nitrogen Source 1.7, (NH)₄)₂

SO

₄5, 10 xAA 100mL/L (lacking 5 amino acids amino acid mixture), 100 xUra 10mL/L,100 xHis 10mL/L,100 xLeu 10mL/L,100 xArg 10mL/L,100 xTrp 10mL/L, pH 6.8-7.0 per 250mL triangle bottle in liquid 25mL, 121 ℃ sterilization for 20 min.

Fermentation medium (g/L): glucose 40, yeast nitrogen source 3.4, (NH)₄)₂

SO

₄5, 10 xAA 100mL/L (lacking 5 amino acids of the amino acid mixture), 100 xUra 10mL/L,100 xHis 10mL/L,100 xLeu 10mL/L,100 xArg 10mL/L,100 xTrp 10mL/L, pH 6.8-7.0, 500mL of each triangle bottle 50mL, 121 ℃ sterilization for 20 min.

Wherein, the amino acid mixed solution is 10 xAA: 5.9g of an amino acid mixture containing 1.5g of valine, 0.2g of arginine, 0.3g of lysine, 0.5g of phenylalanine, 2g of threonine, 0.4g of tryptophan, 0.3g of tyrosine, 0.2g of methionine, 0.3g of isoleucine and 0.2g of adenine sulfate was added with deionized water to a volume of 1L. 100 × Ura: dissolving 2g Uracine in deionized water to constant volume of 1L; 100 × His: dissolving 2g Histidine in deionized water, and fixing the volume to 1L; 100 × Leu: dissolving 10g of Leucine in deionized water, and fixing the volume to 1L; 100 × Arg: dissolving 10g of Arginine in deionized water, and keeping the volume to 1L; 100 × Trp: 10g of Tryptophane is dissolved in deionized water, and the volume is increased to 1L.

Example 3: product validation

The fermentation product of example 2 was assayed using liquid chromatography and mass spectrometry, the assay method being:

and (3) standard substance and preparation: 100mg of UDCA (98%) and CDCA (98%) standard substances are precisely weighed in a 10mL volumetric flask, dissolved in methanol by ultrasonic waves and diluted to a scale mark, filtered by a 0.22-micron organic filter membrane, and then diluted 2500 times by using methanol to prepare 4mg/L for later use. The sample is dissolved by methanol in an ultrasonic mode, diluted to a scale and filtered by a 0.22 mu m organic filter membrane for later use.

LC-MS, the chromatographic column is a 5cm Hypersil gold C18 column, the column temperature is 30 ℃, C is acetonitrile, D is 0.1% formic acid water, a gradient elution mode (Table 2) is adopted, and the mass spectrum adopts a negative ion mode full sweep of 200-500.

TABLE 2 gradient elution conditions

	Retention time (min)	Flow rate (mL/min)	C(％)	D(％)
					1	0.000	0.300	40.0	60.0
2	0.000	0.300	40.0	60.0
					3	1.000	0.300	40.0	60.0
4	4.000	0.300	99.0	1.0
					5	5.000	0.300	99.0	1.0
6	5.100	0.300	40.0	60.0
					7	8.000	0.300	40.0	60.0

The results of the profile determination are shown in FIG. 2, wherein (a) is a liquid chromatogram of the UDCA standard and (b) is a liquid chromatogram of the fermentation broth of the recombinant yeast strain; (c) is the mass spectrum of the UDCA standard product, and (d) is the mass spectrum of the fermentation liquor of the recombinant yeast strain. Indicating successful fermentation to yield UDCA.

Example 4: determination of conversion

The method for measuring the surplus of substrate CDCA and the yield of UDCA comprises the following steps:

standard curve of standard: a100 mg to 10mL volumetric flask was prepared by precisely weighing UDCA (98%) and CDCA (98%) standards, dissolving them in methanol with ultrasound, diluting them to a predetermined scale, filtering them with a 0.22 μm organic filter, diluting them 2500 times with methanol to 4mg/L for further use, further diluting them to 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L, and 3.0mg/L, and preparing a standard curve by an external standard method according to the LC chromatographic conditions in example 3.

Testing the substrate and product, wherein the reaction system comprises 0.1M PBS buffer solution with pH8.0, and 2% coenzyme NADP⁺And the reaction temperature is 38 ℃, recombinant yeast cells are added, the residue of the substrate CDCA and the yield of the UDCA are respectively measured after the reaction is carried out for 3h, 6h, 9h, 12h, 15h and 18h by sampling, and researches show that after the reaction is carried out for 12h, the amount of the substrate CDCA is greatly reduced, a large amount of UDCA is correspondingly synthesized, the conversion rate of the CDCA is as high as 80 percent (shown in figure 3), and the efficient conversion of the CDCA is fully demonstrated.

In conclusion, the saccharomyces cerevisiae strain S.cerevisiae CEN.PK2-1C is taken as a chassis cell, and the 7 alpha-HSDH and 7 beta-HSDH encoding genes from clostridium are heterologously expressed, compared with the method for heterologously expressing 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH) by taking escherichia coli as the chassis cell which is commonly used at present, the saccharomyces cerevisiae strain S.cerevisiae CEN.PK2-1C is taken as a host, and the 7 alpha-HSDH and 7 beta-HSDH from clostridium are heterologously expressed, so that the product has higher safety, the potential safety problem of toxin production by the escherichia coli is solved, meanwhile, the yeast cannot be impregnated by phage, the industrial production is facilitated, and an unexpected technical effect is obtained.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

<110> university of south of the Yangtze river

<120> recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, construction and application

<141> 2021-01-15

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atgaaaagat tagaaggaaa agtcgcaata gtaacatcat ctactagagg aataggacgt 60

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aacttaactg ctggagatac agatgaattc tttagaatat taaaagataa cgttcaaagt 360

gtatacttac cagcaaaagc tgctatacca catatggaaa aagtaggcgg tggaagcata 420

gttaatatct caactatagg atcagttgtt ccagatatat caagaatagc ttactgtgta 480

tcaaaatccg ctataaactc tttaactcaa aacatagcat tacaatatgc aagaaagaat 540

atcagatgta atgcagtatt acctggttta ataggaacta gagcagcact cgaaaatatg 600

actgatgaat ttagagactc attcttagga catgttcctt taaatagagt aggaagacca 660

gaagatatag caaatgcagt tttatactat gcctctgatg attcaggtta tgtaacagga 720

atgattcatg aagttgcagg aggttttgca ttaggaactc ctcaatattc agaatactgt 780

ccaagataa 789

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atgaatttta gagaaaaata tggacaatgg ggaattgttt taggggcaac agaaggaatt 60

ggtaaagcta gtgcttttga attagctaaa agagggatgg atgttatttt agttggaaga 120

agaaaagaag cattagaaga gttagctaag gcaatacatg aagaaacagg aaaagaaatc 180

agagtattac cacaagattt atctgaatat gatgctgcag aaagattaat agaagcaact 240

aaagatttag atatgggagt cattgagtat gttgcatgtc tacatgcaat gggacaatat 300

aataaagttg actacgctaa atatgaacaa atgtatagag ttaatataag aacattctca 360

aaattattac atcactatat aggtgaattc aaagaaagag atagaggtgc attcataaca 420

ataggatctt tatcaggatg gacatcatta ccattctgtg cagaatatgc agcagaaaaa 480

gcttatatga tgacagtaac agaaggagtt gcttacgaat gtgcaaatac taatgttgac 540

gtaatgcttt tatcagcggg ttcaacaatc acacctactt ggttaaaaaa taaaccatca 600

gatcctaagg cggttgcagc agcaatgtat ccagaagatg ttataaaaga tggatttgaa 660

caattaggaa agaaatttac ttatttagct ggagagttaa atagagaaaa aatgaaggaa 720

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gctctagaat gaaaagatta gaaggaaa 28

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cgcggatcct tatcttggac agtattct 28

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cggattcatg aattttagag aaaaata 27

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Claims

1. A recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid is characterized in that: the engineering bacteria are obtained by transferring recombinant expression plasmids containing 7 alpha-HSDH and 7 beta-HSDH genes by taking a yeast strain S.cerevisiae CEN.PK2-1C as an original strain.

2. The recombinant yeast strain for synthesizing ursodeoxycholic acid by efficiently converting chenodeoxycholic acid according to claim 1, which is characterized in that: the nucleotide sequence of the 7 alpha-HSDH gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the 7 beta-HSDH gene is shown as SEQ ID NO. 2.

3. The recombinant yeast strain for synthesizing ursodeoxycholic acid by efficiently converting chenodeoxycholic acid according to claim 1, which is characterized in that: the name of the recombinant yeast strain is S.cerevisiae CEN.PK2-1C 7 alpha-HSDH ≠ 7 beta-HSDH ═ ℃ @.

4. The method for constructing the recombinant yeast strain for synthesizing ursodeoxycholic acid by efficiently converting chenodeoxycholic acid according to any one of claims 1 to 4, which is characterized in that: comprises the following steps:

(1) constructing a recombinant expression plasmid containing 7 alpha-HSDH and 7 beta-HSDH genes: inserting the 7 alpha-HSDH and 7 beta-HSDH genes into shuttle vectors pY15TEF1 and pYX212 to obtain recombinant expression plasmids pY15TEF1-7 alpha-HSDH and pYX212-7 beta-HSDH;

(2) transforming the recombinant expression plasmid into the yeast strain S.cerevisiae CEN.PK2-1C.

5. The method for constructing the recombinant yeast strain for synthesizing ursodeoxycholic acid by efficiently converting chenodeoxycholic acid according to claim 4, which is characterized in that: the culture medium used by the recombinant yeast strain is an YNB culture medium.

6. The use of the recombinant yeast strain for synthesizing ursodeoxycholic acid by efficiently converting chenodeoxycholic acid according to any one of claims 1 to 3 in the production of ursodeoxycholic acid.

7. Ursodeoxycholic acid obtainable by the use according to claim 6.