KR20200087220A - Method for producing taurorusodeoxycholic acid by bioconversion and its application - Google Patents

Abstract

This application discloses methods and applications for the production of taurorusodeoxycholic acid by bioconversion, including gene codon optimization, engineering bacterial construction, engineering bacterial embryos, substrate conversion and product production; Taurorusodeoxycholic acid is produced by fermenting a direct conversion substrate using engineering bacteria, and the substrate is taurorusodeoxycholic acid. The substrate concentration may be as high as 250 g/L, the reaction time is short, the conversion rate to the substrate may be as high as 98% or more, and the obtained product purity is 99% or more; In the reaction system, NAD ⁺ is recirculated, greatly reducing the amount of the protective enzyme NAD ⁺ , the cost of the enzyme catalytic reaction is lowered, and it is advantageous for industrial expansion; The hydroxysteroid dehydrogenase and the protective enzyme regeneration enzyme are linked together through a soft polypeptide sequence to form a fusion protein polymer, so that the binding distance between the substrate and the coenzyme is closer, more advantageous for the progress of the conversion reaction, and industrialization In production, it reduces the recovery of fermentation, simplifies the process, and saves time and raw material costs.

Description

Method for producing taurorusodeoxycholic acid by bioconversion and its application

This application was filed with the Chinese Patent Office on November 29, 2018, the application number is 201811446689.5, and the title of the invention is the priority of Chinese patent application, which is "Method and method for manufacturing taurorusodeoxycholic acid by biological conversion". Claims, all of which are incorporated herein by reference.

Technology field

The present application relates to the field of biotechnology, and relates to a method for catalyzing the bioconversion of taurochenodeoxycholic acid with high efficiency after manipulation of a bioenzyme using genetic engineering means, in particular to tau by bioconversion. It relates to the application of a method for producing laurusodeoxycholic acid and a method for producing taurorusodeoxycholic acid by bioconversion.

Background

)”인 타우로우루소데옥시콜산 캡슐이 중국에서 판매하도록 허락되었다. 이는 주요하게 콜레스테롤 결석을 용해시키는데 사용된다. 우루소데옥시콜산은 친수성의 담즙산으로서, 돌 용해율이 제한적이고, 안정송이 좋으며, 부작용이 비교적 적어, 이미 널리 임상에 적용되고 있다. 타우로우루소데옥시콜산은 우루소데옥시콜산과 타우린의 공액체이고, 우루소데옥시콜산에 비하여 친수성이 더욱 강하고, 돌 용해 속도가 더욱 빠르며, 안전성이 더욱 좋다.Taurorusodeoxycholic acid has a chemical name of 3α,7β-dihydroxycholanacyl-N-taurine, and has functions such as relieving cramps, preventing competition, dissolving anti-inflammatory and gallstones. Taurorusodeoxycholic acid is mainly present in black bear bile, and is a representative active ingredient in bile. In 2007, the product name was “Taurolite(

)”, taurorusodeoxycholic acid capsule, was allowed to be sold in China. It is primarily used to dissolve cholesterol stones. Urusodeoxycholic acid is a hydrophilic bile acid, has a limited stone dissolution rate, has good stable transport, and has relatively few side effects, and is already widely applied in clinical practice. Taurorusodeoxycholic acid is a conjugate of urusodeoxycholic acid and taurine, has a stronger hydrophilicity, faster stone dissolution rate, and better safety than urusodeoxycholic acid.

Initially, taurorusodeoxycholic acid was extracted from “artificially drawn” black bear bile, so its source was limited, yield was low, the difference between batches was large, and it was not human to animals. Later, it was gradually replaced by artificial synthetic methods. Artificial chemical synthetic methods are mainly divided into three types, firstly, by forming active intermediates such as mixed acid anhydrides and active thioesters, sodium taurate. To proceed with the reaction; The second is to form amides under the action of condensing agents; The third is to obtain a target product by forming a sulfuride and oxidizing it again through a cystamine-based material. These methods have low selectivity, use large amounts of organic reagents, and pollute the environment.

To address the shortcomings of bear bile “artificial withdrawal” extraction and artificial chemical synthesis methods, we gradually developed a method for producing taurorusodeoxycholic acid using bioconversion. Taurokenodeoxycholic acid is widely present in bile of livestock such as chickens, ducks, and crabs, and is a 7-digit hydroxy phase epimer from taurorusodeoxycholic acid. Chinese Patent No. CN102994604A discloses a method for converting taurochenodeoxycholic acid to taurorusodeoxycholic acid through two-step catalysis of 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase. . In this method, the substrate concentration is relatively low (1 g/L), the substrate conversion is not complete, a large amount of expensive coenzyme must be used, and the reaction intermediate taurine 7-ketonlitochoric acid is difficult to remove as a by-product. Chinese invention patent CN107287272A discloses a method for producing taurorusodeoxycholic acid. This constitutes an expression vector or both co-expression vectors containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, respectively, adding substrate to the medium and fermenting it, and simultaneously converting, Taurokenodeoxycholic acid is converted to taurorusodeoxycholic acid. However, this method has a low substrate concentration, a low conversion rate, a high content of the reaction intermediate taurine 7-ketone ritolinic acid, a long conversion cycle, and is not easy to proceed with industrial production.

Technical challenges

The embodiments of the present application provide a method for manufacturing taurorusodeoxycholic acid by bioconversion and its application, so that the substrate concentration present when preparing taurorusodeoxycholic acid using conventional bioconversion technology is low One object is to solve the problem that the conversion rate is low, the reaction intermediate taurine 7-ketoneritocholic acid content is high, the conversion cycle is long, and it is not easy to proceed with industrial production.

Technical solutions

The technical method of the present application for solving the above technical problem is as follows.

In a first aspect, there is provided a method for producing taurorusodeoxycholic acid by bioconversion, comprising a genetic codon optimization step, an engineering bacterial construction step, an engineering bacterial culture step, a substrate conversion and product production step; At this time, the engineering bacteria directly ferment the conversion substrate to produce taurorusodeoxycholic acid; The substrate is taurochenodeoxycholic acid; The engineering bacteria are selected from the family of engineering bacteria capable of expressing 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase.

In a second aspect, an application during the production of ursodeoxycholic acid of the method is provided.

The beneficial effects of the method for producing taurorusodeoxycholic acid by bioconversion provided in the Examples of the present application are as follows.

(1) When the bioconversion method provided in the present application is used, the substrate concentration to be converted is high, the substrate concentration may be high as 250 g/L, the reaction time is short, and the conversion rate to the substrate is 98% or more. May be high, and the obtained product purity is 99% or more;

(2) When bioengineering bacteria using the specific expression of E. coli are used, the efficiency of converting the reaction intermediate taurine 7-ketone ritolinic acid to taurorusodeoxycholic acid in the bioconversion process is high, and almost by-products in the final product This is not included;

(3) Using 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase and 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, NAD+ in the reaction system is circulated and regenerated. Greatly lowering the use amount, lowering the cost of the enzyme catalytic reaction, and advantageous for industrial expansion;

(4) The hydroxysteroid dehydrogenase and the protective enzyme regeneration enzyme are linked together through a soft polypeptide sequence to form a fusion protein polymer, so that the binding distance between the substrate and the coenzyme is closer, and it is more advantageous for the progress of the conversion reaction. To reduce the number of fermentation, simplify the process, and save time and raw material costs in industrialized production;

(5) The present application uses a whole cell of 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase and 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase to produce taurorusodeoxycholic acid. The conversion can be carried out to avoid large steps in industrial costs, such as cell disruption, cell fluid purification, and enzyme-friendly purification, thereby reducing a large amount of cost, and also the process is simple and controllable.

If the beneficial effect of the application of the method for producing taurorusodeoxycholic acid by bioconversion provided in the Examples of the present invention, in the taururusodeoxycholic acid conversion solution prepared by bioconversion, alkali decomposition proceeds to urusode Preparing oxycholic acid, avoiding the use of large amounts of organic solvents when preparing ursodeoxycholic acid by chemical methods; In addition, the method has a short reaction time, the reaction is gentle and controllable, and the operation is simple.

Drawing description
In order to further clarify the technical solutions in the embodiments of the present application, a brief description is given of the drawings to be used in the following embodiments or the demonstration technology description, and the drawings in the following description are only some embodiments of the present application. Other technical drawings can be obtained by these drawings without requiring creative efforts.
1 is a principle diagram of manufacturing taurorusodeoxycholic acid using the bioconversion method provided in the Examples of the present application.
Figure 2 is a diagram of the hydroxysteroid dehydrogenase and coenzyme regeneration enzyme provided in an embodiment of the present application catalyzes the fusion expression of the substrate.
3 is an HPLC spectrum of taururusodeoxycholic acid prepared and obtained in Example 9 provided in the present application.

Method of implementation of the invention

In order to better understand the objects, technical solutions, and advantages of the present application, detailed description of the present application will be made with reference to the following examples and the accompanying drawings. It should be understood that the specific embodiments described herein are merely interpretation of the present application and are not intended to limit the present application.

The terms "first" and "second" are for convenience of explanation only, and do not imply relative importance or quantity of technical features indicating. Unless otherwise specified, "majority" means two or more.

Since the weight of the related components mentioned in the specification of the embodiments of the present application can only refer to the specific content of each component, and can also indicate the proportional relationship between the weights of the components, the components related to the specification of the embodiments of the present application Anything that expands or contracts the content of the product in proportion falls within the scope disclosed by the embodiment specification of the present invention. Specifically, the weight in the specification of the present application example may be a mass unit known in the chemical field such as μg, mg, g, kg.

In order to describe the technical method of the present application, it will be described in detail with reference to specific drawings and examples below.

In a first aspect, in some embodiments of the present application, there is provided a method for producing taurorusodeoxycholic acid by bioconversion, the method comprising a gene codon optimization step, engineering bacterial construction step, engineering bacterial culture step, substrate conversion And a product preparation step; At this time, the engineering bacteria directly ferment the conversion substrate to produce taurorusodeoxycholic acid; The substrate is taurochenodeoxycholic acid, and the engineering bacteria are capable of expressing 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. It is selected from the family of engineering bacteria.

The beneficial effects of the method for producing taurorusodeoxycholic acid by bioconversion provided in the Examples of the present application are as follows.

(1) When the bioconversion method provided in the present application is used, the substrate concentration to be converted is high, the substrate concentration may be high as 250 g/L, the reaction time is short, and the conversion rate to the substrate is 98% or more. May be high, and the obtained product purity is 99% or more;

(2) When bioengineering bacteria using the specific expression of E. coli are used, the efficiency of converting the reaction intermediate taurine 7-ketone ritolinic acid to taurorusodeoxycholic acid in the bioconversion process is high, and almost by-products in the final product This is not included;

(3) Using 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase and 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, NAD+ in the reaction system is circulated and regenerated. Greatly lowering the use amount, lowering the cost of the enzyme catalytic reaction, and advantageous for industrial expansion;

(4) The hydroxysteroid dehydrogenase and the protective enzyme regeneration enzyme are linked together through a soft polypeptide sequence to form a fusion protein polymer, so that the binding distance between the substrate and the coenzyme is closer, and it is more advantageous for the progress of the conversion reaction. To reduce the number of fermentation, simplify the process, and save time and raw material costs in industrialized production;

(5) The present application uses a whole cell of 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase and 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase to produce taurorusodeoxycholic acid. The conversion can be carried out to avoid large steps in industrial costs, such as cell disruption, cell fluid purification, and enzyme-friendly purification, thereby reducing a large amount of cost, and also the process is simple and controllable.

As shown in Fig. 1, the principle of the method for producing taurorusodeoxycholic acid by bioconversion provided in the present application is 7α-hydroxy after manipulation of genetic engineering means using taurorusodeoxycholic acid as a substrate. Through the use of the steroid dehydrogenase, it is converted to taurine 7-ketonlitocholic acid, and co-expression or fusion expression of lactic acid dehydrogenase in the presence of sodium pyruvate recirculates the coenzyme NAD+. ; Subsequently, 7β-hydroxysteroid dehydrogenase converts taurine 7-ketonolitocholic acid to taurusoledeoxycholic acid, and co-expression or fusion expression of glucose dehydrogenase NAD+ in the presence of glucose Circulate and recycle. Among the above methods, the fusion-expressing protein forms a fusion protein polymer by linking hydroxysteroid dehydrogenase and a protective enzyme regeneration enzyme together through a soft polypeptide sequence, so that the binding distance between the substrate and the coenzyme is closer and the conversion reaction proceeds. By making it more advantageous, it is possible to realize the production of high yield and high purity taurorusodeoxycholic acid.

The engineering bacteria are selected from the family of engineering bacteria capable of expressing 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, 7α- The four enzymes, hydroxysteroid dehydrogenase, lactate dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, are not necessarily completely expressed among the same engineering bacteria, but two or two. Expression of one or several of 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase through a series of engineering bacteria formed by the above engineering bacteria And finally, it expresses the expression of four enzymes: 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. Also under most circumstances, 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase through an engineering bacterial family formed by two or more engineering bacteria. The kinase implements the expression of four enzymes. It goes without saying that many of the engineering bacteria in the engineering bacteria family may be the same type of engineering bacteria. In some embodiments, engineering bacteria are capable of expressing 7α-hydroxysteroid dehydrogenase and engineering bacteria capable of expressing lactic acid dehydrogenase, and 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. Engineering bacteria. The construction method of engineering bacteria in the engineering bacteria family is not limited thereto, and can express 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. As long as you have, you will understand what is of course. In addition, in some of the above embodiments, the engineering bacteria expressing the enzyme may be E. coli.

In some embodiments, the 7α-hydroxysteroid dehydrogenase and the lactic acid dehydrogenase expression enzyme are 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase single expression enzyme, 7α-hydroxysteroid Dehydrogenase and lactate dehydrogenase co-expression enzymes, 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase pair 4 fusion enzyme, 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase Consisting of a pair tetramer fusion enzyme and a lactic acid dehydrogenase co-expression enzyme, a 7α-hydroxysteroid dehydrogenase and a lactate dehydrogenase co-expressing enzyme It is selected from the military.

In some embodiments, the 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase expression enzymes are 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase single expression enzymes, 7β-hydroxysteroid dehydrate Rosenase and glucose dehydrogenase co-expression enzymes, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair tetramer fusion enzyme, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair A group consisting of a tetrameric fusion enzyme and glucose dehydrogenase co-expression enzyme, a 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair tetrameric fusion enzyme and a 7β-hydroxysteroid dehydrogenase co-expression enzyme It is selected from.

In some embodiments, the engineering bacteria are 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase single expression enzymes, or 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase co-expression enzymes, or 7α -Hydroxysteroid dehydrogenase and lactate dehydrogenase pair tetramer fusion enzyme, or 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase pair tetramer fusion enzyme and lactate dehydrogenase co-expression enzyme , Or an engineering bacterium capable of expressing a 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase pair tetramer fusion enzyme and a 7α-hydroxysteroid dehydrogenase co-expressing enzyme; 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase single expression enzyme, or 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase co-expression enzyme, or 7β-hydroxysteroid dehydrogenase and Glucose dehydrogenase 4-mer fusion enzyme, or 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase 4-mer fusion enzyme and glucose dehydrogenase co-expression enzyme, or 7β-hydroxysteroid dehydrogenase And glucose dehydrogenase 4 fusion enzyme and 7β-hydroxysteroid dehydrogenase co-expressing enzyme.

In an embodiment of the present application, the DNA sequence encoding 7α-hydroxysteroid dehydrogenase is SEQ ID NO:1, the DNA sequence encoding lactic acid dehydrogenase is SEQ ID NO:3, and 7β-hydr The DNA sequence encoding hydroxysteroid dehydrogenase is SEQ ID NO:5, and the DNA sequence encoding glucose dehydrogenase is SEQ ID NO:7.

In some examples, the DNA sequence encoding 7β-hydroxysteroid dehydrogenase is mutated at the A78 and V116 sites.

In an embodiment of the present application, the protein sequence of the 7α-hydroxysteroid dehydrogenase is SEQ ID NO:2, the protein sequence of the lactic acid dehydrogenase is SEQ ID NO:4, and the 7β-hydroxy The protein sequence of the steroid dehydrogenase is SEQ ID NO:6, and the protein sequence of the glucose dehydrogenase is SEQ ID NO:8.

In an embodiment of the present application, when producing taurorusodeoxycholic acid by bioconversion using the above principle, engineering bacteria have 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid Genes expressing dehydrogenase and glucose dehydrogenase should be constructed, and gene codon optimization should be performed.

In some embodiments, the method of optimizing the gene codon is a step of optimizing E. coli expression codon for a gene sequence, adding an affinity tag, and also performing full-length gene synthesis, wherein the 7α-hydroxysteroid dehydro The gene encoding the genease is 7α-HSDH, the gene LDH encoding the lactic acid dehydrogenase, the gene encoding the 7β-hydroxysteroid dehydrogenase is 7β-HSDH, and the glucose dehydrogenase is encoded. The gene to be expressed is designated as GDH.

After optimizing the gene codon, engineering bacteria containing the optimized codon gene are constructed, and the constructed expression vector is transformed into soluble cells of E. coli BL21 (DE3) to obtain engineering bacteria, and culture is also performed. .

In some embodiments, the method of engineering bacterial construction comprises:

The gene expression vector of the 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase obtained by constructing and constructing the gene expression vector was obtained from E. coli BL21. Transforming the soluble cells to obtain engineering bacteria.

In some embodiments, the gene encoding 7α-hydroxysteroid dehydrogenase is designated 7α-HSDH, the gene encoding lactic acid dehydrogenase is designated LDH, and 7β-hydroxysteroid dehydrogenase The gene encoding is expressed as 7β-HSDH, the gene encoding glucose dehydrogenase is designated as GDH, and the method for constructing the gene expression vector is:

7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, and each single gene expression vector pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, pETDuet- Obtaining 1-GDH; or

Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in pETDuet-1 vector, respectively, to obtain dual gene expression vectors pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH, respectively; or

7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in pETDuet-1 vector, respectively, and each single gene fusion protein Obtaining expression vectors pETDuet-1-(LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH); or

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Genes were constructed in the pETDuet-1 vector, respectively, so that a single gene fusion protein and a dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β) -Obtaining HSDH)/GDH; or

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and a single gene fusion protein and a hydroxysteroid dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/7α -HSDH, pETDuet-1- (GDH-Linker-7β-HSDH) / acquiring 7β-HSDH; to include.

It will derived from: (CDQ67_02445 UniProt), wherein the LDH is a human (Human) In some embodiments, the 7α-HSDH is Campylobacter high Te sustaining day devices (Campylobacter hyointestinalis): will derived from (UniProt P00338), the 7β-HSDH is choline Cellar Aero Pacifico Enschede (Collinsella aerofaciens) ATCC 25986: will derived from (UniProt A4ECA9), the GDH is Bacillus subtilis (Bacillus subtilis) (strain 168) is derived from (UniProt P12310) .

In some embodiments, the DNA sequence encoding the 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene is SEQ ID NO:9, and the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase The DNA sequence encoding a single gene gene is SEQ ID NO:11.

In some embodiments, the protein sequence encoded by the 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene is SEQ ID NO:10, and the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrate The protein sequence encoded by the single gene is a SEQ ID NO:12.

After completing the construction of the engineering bacteria, culture is performed on this, so that it is fermented and expressed in large quantities. In some embodiments, the engineering bacterial culture includes a small amount of engineering bacteria fermentation and expression steps and a large amount of engineering bacteria fermentation and expression steps. Among them, the method of fermentation and expression of a small amount of engineering bacteria, the engineering bacterial fungus was applied to an ampicillin-resistant LB plate, a single clone was selected, and inoculated into 5 mL of LB medium containing ampicillin, and at 37°C, 220 rpm. Cultivation with, and when the OD value is 0.8-1.2, 1mM IPTG is added for 2 h to induce, and the expression level is examined by SDS-PAGE to select a clone with a high expression level and to preserve the species; Inoculate 20 μL of the bacterial species into 200 mL of ampicillin-resistant LB medium and incubate overnight, and when the OD value is 2.5-4.0, take 2 mL culture and inoculate into ampicillin-resistant medium and culture, when the OD value is 1, And adding IPTG to induce expression overnight and collecting the cells. For the expression and method of mass fermentation of engineering bacteria, engineering bacteria are selected and inoculated into 1 L Erlenmeyer flasks of ampicillin resistant LB medium, cultured at 37° C. at 220 rpm overnight, 20 mL each when the OD600 value is 2.5-4.0 The culture was taken and inoculated into a 3 L Erlenmeyer flask containing 10 1 L ampicillin resistant media, and cultured at 37° C. at 140 rpm overnight; 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, incubated at 37° C. for 8 hours while passing gas and passing gas, and then added to the fermentation tank with an IPTG solution with a final concentration of 0.1 mM. To proceed with induction, to terminate fermentation after 10-12h induction, to drain the liquid, collect the cells by centrifugation, and also store at 4°C, take a small amount of cells and suspend them in 100 mM phosphate buffer again, and crush with ultrasonic waves It includes the step of obtaining a coenzyme solution.

After the engineering bacteria have been cultured and subjected to mass fermentation, activity measurement can be performed on the enzymes expressed by the engineering bacteria.

In this example, the method for measuring the enzyme activity of 7α-hydroxysteroid dehydrogenase is 2.97 mL of 100 mM phosphate buffer (pH 8.0) in one 3 mL reaction system using taurusodeoxycholic acid as a substrate. A final concentration of 0.5 mM tauurorusodeoxycholic acid, 10 μL of dilute enzyme solution, and a final concentration of 0.5 mM NADP+ was added, reacted at pH 8.0 and 25° C. for 1 min, and measured the increase in absorbance at 340 nm. to be.

In the example of the present application, the method for measuring the enzyme activity of lactic acid dehydrogenase is 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 100 mM sodium pyruvate in one 3 mL reaction system using sodium pyruvate as a substrate. , 50 μL of dilute enzyme solution, NADH having a final concentration of 0.2 mM was added, reacted at pH 8.0 and 25° C. for 1 min, and the decrease in absorbance at 340 nm was measured.

In the example of the present application, the method for measuring the enzyme activity of 7β-hydroxysteroid dehydrogenase is 2.97 mL of 100 mM phosphate buffer (pH 8.0) in one 3 mL reaction system using taurokenodeoxycholic acid as a substrate. , Taurokenodeoxycholic acid having a final concentration of 0.5 mM, 10 μL of dilute enzyme solution, and adding NADP+ having a final concentration of 0.5 mM, reacting for 1 min at pH 8.0 and 25° C., and measuring the increase in absorbance at 340 nm. .

In the embodiment of the present application, the method for measuring the enzyme activity of glucose dehydrogenase, using glucose as a substrate, 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 1.5 M glucose in one 3 mL reaction system, To add 50 μL of diluted enzyme solution, NADP+ having a final concentration of 2 mM, react for 2 min at pH 8.0 and 25° C., and measure the increase in absorbance at 340 nm.

In the examples of the present application, after engineering bacteria are cultivated until they can stably express the target enzyme, the substrate is directly converted using engineering bacteria to prepare taurorusodeoxycholic acid. The substrate is taurochenodeoxycholic acid. In some examples, the concentration of the substrate is 20 g/L-250 g/L. When the low-concentration reaction, the substrate has a large reaction volume and a large amount of coenzyme. By optimizing the amount of 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, the substrate concentration is improved to 250 g/L, and the reaction system Reduced volume and use of coenzymes. When the substrate concentration continues to increase and exceeds 250 g/L, the substrate dissolving performance deteriorates, and the conversion of the substrate is insufficient.

In some embodiments, using the taurochenodeoxycholic acid as a substrate, the method for directly converting the substrate to engineering bacteria includes:

Taurochenodeoxycholic acid was dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD ⁺ was added, 5-60 g/L of sodium pyruvate was added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydro Purified or partially purified E. coli cells expressing kinase, or cell lysates thereof, or cell resuspensions thereof are added, supplemented with 20-100 mM glycine buffer to the final volume, pH with sodium hydroxide Adjusted to 6.5-8.5, and reacted at 25° C. for 6-18 h; Purified or partially purified E. coli cells, or cell lysates thereof, which are added to 1.8-100 g/L of glucose and express 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase And adding the cell resuspension solution, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25°C for 6-18h.

In the examples of the present application, after the substrate conversion is completed, the product taurorusodeoxycholic acid in the engineering bacteria is extracted. In some embodiments, in the manufacturing method of the taurorusodeoxycholic acid, the converted reaction solution is rotary evaporated into a paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and centrifugation or filtering is performed. The precipitate is removed, and when the supernatant is dried, taurorusodeoxycholic acid is obtained. The crude product of taurorusodeoxycholic acid is dissolved using acetonitrile, filtered through a 0.22um filtration membrane to remove insoluble matters, and the column supply solution. To form; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is carried out in a methanol-water fluidized bed of different concentrations, and the collected eluent is poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; It is then placed in a vacuum drying oven and dried, using high performance liquid chromatography to measure the purity of taurusodeoxycholic acid in the sample.

In some embodiments, a method for producing taurorusodeoxycholic acid by bioconversion includes the following steps.

(1) Gene codon optimization

As a step of optimizing E. coli expression codons for gene sequences, adding affinity tags, and also performing full-length gene synthesis, genes encoding 7α-hydroxysteroid dehydrogenase are 7α-HSDH, respectively, and lactic acid The gene encoding dehydrogenase is LDH, the gene encoding 7β-hydroxysteroid dehydrogenase is 7β-HSDH, and the gene encoding glucose dehydrogenase is designated GDH, respectively;

(2) Engineering bacteria composition

The gene expression vector of the 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase obtained by constructing and constructing the gene expression vector was obtained from E. coli BL21. Transforming soluble cells to obtain engineering bacteria;

(3) Engineering bacteria culture

As a step of fermenting and expressing a small amount of engineering bacteria: engineering bacterial fungus is applied to an ampicillin resistant LB plate, a single clone is selected, inoculated in 5 mL of LB medium containing ampicillin, and cultured at 37°C at 220 rpm. When the OD value is 0.8-1.2, 1 mM IPTG is added to induce it for 2 h, and the expression level is examined by SDS-PAGE to select a clone with a high expression level and to preserve the species; Inoculate 20 μL of the bacterial species into 200 mL of ampicillin-resistant LB medium and incubate overnight, and when the OD value is 2.5-4.0, take 2 mL culture and inoculate into ampicillin-resistant medium and culture, when the OD value is 1, Adding IPTG to induce expression overnight and collecting cells;

As a step of mass fermentation and expression of engineering bacteria: engineering bacteria are selected and inoculated into 1 L Erlenmeyer flasks of ampicillin resistant LB medium, and cultured overnight at 37° C. at 220 rpm, with an OD600 value of 2.5-4.0, Each 20 mL culture was taken and inoculated into a 3 L Erlenmeyer flask containing 10 1 L ampicillin resistant media, and cultured at 37° C. at 140 rpm overnight; 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, incubated at 37° C. for 8 hours while passing gas and passing gas, and then added to the fermentation tank with an IPTG solution with a final concentration of 0.1 mM. To proceed with induction, to terminate fermentation after 10-12h induction, to drain the liquid, collect the cells by centrifugation, and also store at 4°C, take a small amount of cells and suspend them in 100 mM phosphate buffer again, and crush with ultrasonic waves To obtain a coenzyme solution;

(4) substrate conversion

Taurokenodeoxycholic acid solution was dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD ⁺ was added, 5-60 g/L of sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase and lactic acid dehydrate were added. Purified or partially purified E. coli cells expressing a logase, or a cell lysate thereof, or a cell resuspension solution thereof are added, and supplemented with 20-100 mM glycine buffer until the final volume, with sodium hydroxide adjusting the pH to 6.5-8.5 and reacting at 25° C. for 6-18 h; And 1.8-100 g/L of glucose, and expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, or a purified or partially purified E. coli cell, or a cell lysate thereof, or Adding the cell resuspension solution, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25° C. for 6-18 h;

(5) Product preparation

In step (4), the completed reaction solution is rotary evaporated into a paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and the resulting mixture is centrifuged or filtered to remove precipitation, and the supernatant is removed. Drying to obtain taurorusodeoxycholic acid, dissolving the taurorusodeoxycholic acid crude product using acetonitrile, filtering through a 0.22um filter membrane to remove insoluble substances to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is carried out with different concentrations of methanol-water fluidized bed, the eluent is collected and poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; It is then placed in a vacuum drying oven and dried, using a high performance liquid chromatography to measure the purity of taurorusodeoxycholic acid in the sample.

In some embodiments, after step (2), the method of constructing the gene expression vector is:

7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, and each single gene expression vector pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, pETDuet- Obtaining 1-GDH; or

Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in pETDuet-1 vector, respectively, to obtain dual gene expression vectors pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH, respectively; or

7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in pETDuet-1 vector, respectively, and each single gene fusion protein Obtaining expression vectors pETDuet-1-(LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH); or

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Genes were constructed in the pETDuet-1 vector, respectively, so that a single gene fusion protein and a dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β) -Obtaining HSDH)/GDH; or

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and a single gene fusion protein and a hydroxysteroid dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/7α It is a step of acquiring -HSDH, pETDuet-1-(GDH-Linker-7β-HSDH)/7β-HSDH.

In an embodiment of the present application, the DNA sequence encoding the 7α-hydroxysteroid dehydrogenase is SEQ ID NO:1, and the DNA sequence encoding the lactic acid dehydrogenase is SEQ ID NO:3, the The DNA sequence encoding 7β-hydroxysteroid dehydrogenase is SEQ ID NO:5, and the DNA sequence encoding the glucose dehydrogenase is SEQ ID NO:7.

In an embodiment of the present application, the 7α-hydroxysteroid dehydrogenase, the lactic acid dehydrogenase, the 7β-hydroxysteroid dehydrogenase and the glucose dehydrogenase are independently liquid enzymes or immobilization enzymes. Selected from the above, the 7α-hydroxysteroid dehydrogenase, the lactic acid dehydrogenase, the 7β-hydroxysteroid dehydrogenase and the glucose dehydrogenase are independently whole cells, unpurified enzymes Or purified enzyme.

In some examples, specific steps of the bioconversion method are as follows.

(1) Gene codon optimization

As a step of optimizing the codon expression codon for the gene sequence, adding an affinity tag, and also performing full-length gene synthesis, the gene encoding 7α-hydroxysteroid dehydrogenase is 7α-HSDH, lactic acid dehydrate The gene encoding the logase is LDH, the gene encoding 7β-hydroxysteroid dehydrogenase is 7β-HSDH, and the gene encoding glucose dehydrogenase is designated GDH, respectively;

(2) Single gene expression vector construction

7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, to obtain pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, and pETDuet-1-GDH To do;

(3) Double gene expression vector construction

Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in the pETDuet-1 vector, respectively, to obtain pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH;

(4) Single gene fusion protein expression vector construction

The 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene and the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in the pETDuet-1 vector, respectively, and pETDuet-1-( Obtaining LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH);

(5) Construction of a single gene fusion protein and a dehydrogenase co-expression vector

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Constructing genes in pETDuet-1 vectors, respectively, to obtain pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β-HSDH)/GDH;

(6) Single gene fusion protein and hydroxysteroid dehydrogenase co-expression vector construction

7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and pETDuet-1-(LDH-Linker-7α-HSDH)/7α-HSDH, pETDuet-1-(GDH-Linker-7β-HSDH) Obtaining /7β-HSDH;

(7) Engineering bacteria composition

(2)-(6) transforming all of the expression vectors constructed in steps into soluble cells of E. coli BL21 (DE3) to obtain engineering bacteria;

(8) Small amount fermentation and expression of engineering bacteria

The engineering bacterial fungus was applied to an ampicillin-resistant LB plate, a single clone was selected, inoculated into 5 mL of LB medium containing ampicillin, and cultured at 37°C at 220 rpm, and the OD value was 0.8-1.2 days. At this time, 1 mM IPTG was added to induce it for 2 h, and the expression level was examined by SDS-PAGE to select a clone with a high expression level and to preserve the species; Inoculate 20 μL of the bacterial species into 200 mL of ampicillin-resistant LB medium and incubate overnight, and when the OD value is 2.5-4.0, take 2 mL culture and inoculate into ampicillin-resistant medium and culture, when the OD value is 1, Adding IPTG to induce expression overnight and collecting cells;

(9) Mass fermentation and expression of engineering bacteria

Engineering bacteria were selected and inoculated into 1 L Erlenmeyer flasks of ampicillin-resistant LB medium, cultured overnight at 37°C at 220 rpm, and when the OD600 value was 2.5-4.0, each 20 mL culture was taken and 10 1 L ampicillin resistance Inoculated into a 3 L Erlenmeyer flask containing medium, and cultured at 37° C. at 140 rpm overnight. 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, incubated at 37° C. for 8 hours while passing gas and passing gas, and then added to the fermentation tank with an IPTG solution with a final concentration of 0.1 mM. To proceed with induction, and after induction for 10-12 h, fermentation is terminated, liquid is discharged, and the cells are collected by centrifugation and preserved at 4°C. A small amount of cells is again suspended in 100 mM phosphate buffer and ultrasonically Crushing to obtain a coenzyme solution;

(10) Enzyme activity measurement

As a method for measuring the enzyme activity of 7α-hydroxysteroid dehydrogenase: tauurusodeoxycholic acid as a substrate, 2.97 mL of 100 mM phosphate buffer (pH 8.0) in a 3 mL reaction system, final concentration of 0.5 mM Phosphorous phosphorus deoxycholic acid, dilute enzyme solution of 10 μL, NADP+ having a final concentration of 0.5 mM was added, reacted at pH 8.0 and 25° C. for 1 min, and measured the increase in absorbance at 340 nm;

As a method for measuring the enzyme activity of lactic acid dehydrogenase: using sodium pyruvate as a substrate, 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 100 mM sodium pyruvate, 50 μL of diluted enzyme solution in a 3 mL reaction system, final Adding NADH having a concentration of 0.2 mM, reacting with pH 8.0 at 25° C. for 1 min, and measuring a decrease in absorbance at 340 nm;

As a method for measuring the enzyme activity of 7β-hydroxysteroid dehydrogenase: using taurochenodeoxycholic acid as a substrate, 2.97 mL of 100 mM phosphate buffer (pH8.0) in a 3 mL reaction system, the final concentration being 0.5 mM Adding taurochenodeoxycholic acid, 10 μL of diluted enzyme solution, NADP+ having a final concentration of 0.5 mM, reacting at pH 8.0 and 25° C. for 1 min, and measuring an increase in absorbance at 340 nm;

As a method for measuring the enzyme activity of glucose dehydrogenase: glucose as a substrate, 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 1.5M glucose, 50 μL of dilute enzyme solution, final concentration in one 3 mL reaction system Adding NADP+ of 2 mM, reacting with pH 8.0 at 25° C. for 2 min, measuring an increase in absorbance at 340 nm;

(11) Conversion of taurokenodeoxycholic acid to taurorusodeoxycholic acid

Taurokenodeoxycholic acid solution was dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD+ was added, 5-60 g/L of sodium pyruvate was added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydro Purified or partially purified E. coli cells expressing kinase, or cell lysate thereof, or cell resuspension thereof are added, supplemented with 20-100 mM glycine buffer to the final volume, pH with sodium hydroxide Adjusted to 6.5-8.5, and reacted at 25° C. for 6-18 h; Purified or partially purified E. coli cells, or cell lysates thereof, which are added to 1.8-100 g/L of glucose and express 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase Adding the cell resuspension, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25° C. for 6-18 h;

(12) Preparation of taurorusodeoxycholic acid

In step (11), the completed reaction solution is rotary evaporated in a paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and the precipitate is removed by centrifugation or filtering, and when the supernatant is dried, Taulow To obtain the rusodeoxycholic acid, and dissolve the crude taurorusodeoxycholic acid product using acetonitrile, filter with a 0.22um filter membrane to remove the insoluble material to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is carried out in a methanol-water fluidized bed of different concentrations, and the collected eluent is poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; After that, it is put in a vacuum drying oven and dried, and it is a step of measuring the purity of taurorusodeoxycholic acid in a sample using high performance liquid chromatography.

In a second aspect, the examples of the present application provide the use of the method in the production of ursodeoxycholic acid.

If the beneficial effect of the application of the method for producing taurorusodeoxycholic acid by bioconversion provided in the Examples of the present invention, in the taururusodeoxycholic acid conversion solution prepared by bioconversion, alkali decomposition proceeds to urusode Preparing oxycholic acid, avoiding the use of large amounts of organic solvents when preparing ursodeoxycholic acid by chemical methods; In addition, the method has a short reaction time, the reaction is gentle and controllable, and the operation is simple.

In some embodiments, the process for preparing urusodeoxycholic acid includes the following steps, and the pH is adjusted to 8-11 by adding sodium hydroxide in the conversion-produced taurorusodeoxycholic acid solution, 80 It is a step of reacting for 18-24h by raising the temperature to -100℃, lowering the temperature to 10-15℃ and adjusting the pH to 3-5 by adding hydrochloric acid to precipitate urusodeoxycholic acid. In the conversion solution of taurorusodeoxycholic acid prepared by the bioconversion method, sodium hydroxide is added to adjust the pH to 8-11 to proceed with alkaline decomposition, and then neutralized by adding hydrochloric acid to precipitate urusodeoxycholic acid. Thus, avoiding the use of large amounts of organic solvents when preparing ursodeoxycholic acid by chemical methods; The reaction time is short, the reaction is gentle and controllable, and the operation is simple.

The concentration of the substrate in the course of the bioconversion reaction is 20-250 g/L.

All of the above enzymes may be liquid enzymes or solid enzymes, and may also be whole cells, unpurified enzymes or purified enzymes.

Below, with reference to specific embodiments to proceed with the description

In the following specific examples, the method of constructing the recombinant plasmid is specifically as follows.

One. Construction of a single gene expression vector

a) Preparation of the recombinant plasmid pETDuet-1-7α-HSDH containing the 7α-hydroxysteroid dehydrogenase gene

Campylobacter-high dehydrogenase kinase gene 7α- hydroxy steroid dehydrogenase derived from te day sustaining devices (Campylobacter hyointestinalis) (DNA sequence: SEQ ID NO: 1, the encoded protein sequence: SEQ ID NO: 2) a pair of primers ( Expansion by PCR using SEQ ID NO:13)5'-CGGGATCCATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:14)5'-CGGAATTCTTATTTAAAGGTGGTGCCA-3', enzyme digestion using BamH I and EcoR I Proceed, digest template using Dpn I enzyme. The pETDuet-1 vector is enzymatically digested using BamH I and EcoR I. The vector is linked to the 7α-hydroxysteroid dehydrogenase gene fragment using a linking enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

b) Preparation of recombinant plasmid pETDuet-1-LDH containing lactic acid dehydrogenase gene

Primer pair (SEQ ID NO:15)5´-GGAATTCCATATGGGCAGCAGCCATCATCA-3´ with the lactic acid dehydrogenase gene (DNA sequence: SEQ ID NO:3, encoded protein sequence: SEQ ID NO:4) originated from Human And (SEQ ID NO:16)5'-TCCCTCGAGTTAAAACTGCAGTTCTTTCT-3' is used to expand through PCR, digestion is performed using Nde I and Ava I, and a template is digested using Dpn I enzyme . The pETDuet-1 plasmid is enzymatically cleaved using Nde I and Ava I, and the ligation dehydrogenase gene fragment and vector are linked using a ligation enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

c) Preparation of the recombinant plasmid pETDuet-1-7β-HSDH containing the 7β-hydroxysteroid dehydrogenase gene

Colin Cellar Aero Pacifico Enschede (Collinsella aerofaciens) myuton (A78C, V116C) a dehydrogenase kinase gene 7β- hydroxy steroid dehydrogenase derived from ATCC 25986 (DNA sequence: SEQ ID NO: 5, the encoded protein sequence: SEQ ID NO: 6 ) Using a primer pair (SEQ ID NO:17)5'-CGGGATCCATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:18)5'-CGGAATTCTTAGTCACGGTAGAAAGAAC-3' to proceed with expansion through PCR, BamH I and EcoR I Enzyme digestion is performed using Dpn I enzyme to digest the template. The pETDuet-1 vector is enzymatically digested using BamH I and EcoR I. The linking enzyme is used to link the vector with the 7β-hydroxysteroid dehydrogenase gene fragment. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

d) Preparation of recombinant plasmid pETDuet-1-GDH containing glucose dehydrogenase gene

Bacillus subtilis (Bacillus subtilis) dehydrogenase kinase gene by glucose dehydrogenase derived from (strain 168) (DNA sequence: SEQ ID NO: 7, the encoded protein sequence: SEQ ID NO: 8) a pair of primers (SEQ ID NO: 15 ) 5'-GGAATTCCATATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:19) and 5'-TCCCTCGAGTTAACCACGACCGGCCTGAAAGCT-3' to proceed through expansion through PCR, using Nde I and Ava I to perform enzyme digestion, and Dpn I Digest the template using enzymes. The pETDuet-1 plasmid is enzymatically cleaved using Nde I and Ava I, and the ligation dehydrogenase fragment and vector are linked using a ligation enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

2. Construction of double gene co-expression vector

a) Preparation of the recombinant plasmid pETDuet-1-7α-HSDH/LDH containing the 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase genes

The lactic acid dehydrogenase gene originating from Human is PCR through PCR using primer pair (SEQ ID NO:15)5'-GGAATTCCATATGATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:16)5'-TCCCTCGAGTTAAAACTGCAGTTCTTTCT-3' Proceed with expansion, enzyme digestion with Nde I and Ava I, and digest template with Dpn I enzyme. The sequencing-accurate pETDuet-1-7α-HSDH plasmid is enzymatically cleaved using Nde I and Ava I, and a ligation dehydrogenase gene fragment is linked to the vector using a ligation enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

b) Preparation of recombinant plasmid pETDuet-1-7β-HSDH/GDH containing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase genes

Bacillus subtilis (Bacillus subtilis) (strain 168) Grape aldehyde dehydrogenase derived from a dehydratase gene primer pair (SEQ ID NO: 15) 5'-and 3'-GGAATTCCATATGATGGGCAGCAGCCATCATCA (SEQ ID NO: 19) 5' -TCCCTCGAGTTAACCACGACCGGCCTGAAAGCT- Use 3´ to expand through PCR, digest the enzyme using Nde I and Ava I, and digest the template using the Dpn I enzyme. The sequencing-accurate pETDuet-1-7β-HSDH plasmid is enzymatically cleaved using Nde I and Ava I, and a ligation dehydrogenase fragment and vector are linked using a ligation enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

3. Construction of single gene expression fusion protein vector

a) Preparation of 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene recombinant plasmid pETDuet-1-(LDH-Linker-7α-HSDH)

7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene (DNA sequence: SEQ ID NO:9, encoded protein sequence: SEQ ID NO:10) primer pair (SEQ ID NO:15) 5' -GGAATTCCATATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:14) and 5'-CGGAATTCTTATTTAAAGGTGGTGCCA-3' are used for expansion through PCR, enzyme digestion is performed using BamH I and EcoR I, and Dpn I enzyme is used. To digest the template. The pETDuet-1 vector is enzymatically digested using BamH I and EcoR I. The linking enzyme is used to link the vector with the 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene gene fragment. The ligation product is converted to DH5α and applied to the ampicillin resistant LB plate for screening. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

b) Preparation of 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene recombinant plasmid pETDuet-1-(GDH-Linker-7β-HSDH)

7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene (DNA sequence: SEQ ID NO:11, encoded protein sequence: SEQ ID NO:12) primer pair (SEQ ID NO:15) 5' -GGAATTCCATATGATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:18) and 5'-GAATTCTTAGTCACGGTAGAAAGAAC-3' are expanded through PCR, enzyme digestion is performed using BamH I and EcoR I, and Dpn I enzyme is used. To digest the template. The pETDuet-1 vector is enzymatically digested using BamH I and EcoR I. The linking enzyme is used to link the vector with the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene gene fragment. The ligation product is converted to DH5α and applied to the ampicillin resistant LB plate for screening. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

4. Construction of a single gene fusion protein and a dehydrogenase co-expression vector

a) Preparation of recombinant plasmid pETDuet-1-(LDH-Linker-7α-HSDH)/LDH containing co-expression of 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase and lactic acid dehydrogenase genes

Human lactic acid dehydrogenase gene derived from human primer pair (SEQ ID NO:15)5'-GGAATTCCATATGGGCAGCAGCCATCATCA-3' and (SEQ ID NO:16)5'-TCCCTCGAGTTAAAACTGCAGTTCTTTCT-3' to expand through PCR Then, the enzyme is digested using Nde I and Ava I, and the template is digested using the Dpn I enzyme. The pETDuet-1-(LDH-Linker-7α-HSDH) plasmid is enzymatically cleaved using Nde I and Ava I, and a ligation dehydrogenase gene fragment is linked to the vector using a ligation enzyme. The ligation product is converted to DH5α and applied to the ampicillin resistant LB plate for screening. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

b) Preparation of recombinant plasmid pETDuet-1-(GDH-Linker-7β-HSDH)/GDH containing co-expression of 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase and glucose dehydrogenase genes

Primer pair of grape dehydrogenase gene derived from Bacillus subtilis (Strain 168) (SEQ ID NO:15) 5´-GGAATTCCATATGGGCAGCAGCCATCATCA-3′ and (SEQ ID NO:19)5´-TCCCTCGAGTTAACCACGACCGGCCTGAAAGCT- Use 3´ to expand through PCR, digest the enzyme using Nde I and Ava I, and digest the template using the Dpn I enzyme. The pETDuet-1-(GDH-Linker-7β-HSDH) plasmid is digested with Nde I and Ava I, and a linking enzyme is used to link the glucose dehydrogenase fragment with the vector. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

5. Construction of single gene fusion protein and hydroxysteroid dehydrogenase co-expression vector

a) Fusion of 7α-hydroxysteroid dehydrogenase 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase gene Recombinant plasmid pETDuet-1-(LDH-Linker-7α-HSDH)/7α- Preparation of HSDH

Primer pair (SEQ ID NO:15) 5´-GGAATTCCATATGGGCAGCAGCCATCATCA-3′ with 7α-hydroxysteroid dehydrogenase gene derived from Campylobacter hyointestinalis (SEQ ID NO:20) 5'-TCCCTCGAGTTATTTAAAGGTGGTGCCA-3' is used to expand through PCR, enzyme digestion is performed using Nde I and Ava I, and a template is digested using Dpn I enzyme. The pETDuet-1-(LDH-Linker-7α-HSDH) plasmid is enzymatically cleaved using Nde I and Ava I, and the linking enzyme is used to link the vector with the 7α-hydroxysteroid dehydrogenase gene fragment. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

b) Fusion of 7β-hydroxysteroid dehydrogenase 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase gene Recombinant plasmid pETDuet-1-(GDH-Linker-7β-HSDH)/7β- Preparation of HSDH

Primer pair (SEQ ID NO:15)5´-GGAATTCCATATGGGCAGCAGCCATCATCA-3′ with mutone (A78C, V116C) of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens ATCC 25986 NO:21)5'-TCCCTCGAGTTAGTCACGGTAGAAAGAAC-3' is used to expand through PCR, enzyme digestion is performed using Nde I and Ava I, and a template is digested using Dpn I enzyme. The pETDuet-1-(GDH-Linker-7β-HSDH) plasmid was enzymatically cleaved using Nde I and Ava I, and the vector was linked to the 7β-hydroxysteroid dehydrogenase fragment using a linking enzyme. The ligation product is converted to DH5α and screened by applying to ampicillin resistant LB plates. A single clone was selected and inoculated in 5 mL LB to incubate overnight. The cells were collected, the plasmid was extracted using the TIANGEN plasmid extraction kit, and sequencing was performed. Sequencing preserves the correct plasmid.

Example 1 Fermentation and expression of E. coli containing recombinant plasmid in Erlenmeyer flask

20 μL of E. coli BL21 (DE3) strain containing the recombinant plasmid was taken, inoculated into 200 mL of ampicillin-resistant LB medium, and cultured at 37° C. for one night at 220 rpm until the OD600 value was 2.5-4.0. A 20 mL culture is taken and inoculated into 1 L of ampicillin-resistant culture medium, cultured at 37° C. for 140 hours at 140 rpm, and when the OD600 value is 1, 0.5 mM IPTG is added to induce expression overnight. The cells are collected by centrifugation. A small amount of the cells was taken, suspended again in 100 mM phosphate buffer, and crushed with ultrasound to obtain a coenzyme solution. Enzyme activity is measured according to the method in the technical method.

Example 2 Fermentation and expression of E. coli containing recombinant plasmid in fermentation tank

20 μL of E. coli BL21 (DE3) strain containing the recombinant plasmid was taken, inoculated into 200 mL of ampicillin-resistant LB medium, and cultured at 37° C. for one night at 220 rpm until the OD600 value was 2.5-4.0. The 20 mL culture was taken and inoculated into 1 L of ampicillin resistant culture medium, and cultured at 37° C. at 140 rpm overnight. 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, and cultured at 37° C. for 8 hours while passing gas. In the E. coli high-density fermentation medium, 18 g/L potassium dihydrogen phosphate dodecahydrate, 6.8 g/L potassium dihydrogen phosphate, 0.7 g/L anhydrous sodium sulfate, 0.48 g/L magnesium sulfate, 2.25 g/L Contains glycine, 2.5 g/L yeast powder, and 5 g/L peptone. After incubating for 8 hours while passing the gas, the induction proceeds by adding an IPTG solution having a final concentration of 0.1 mM to the fermentation tank. It is also stored at 4°C. A small amount of the cells was taken, suspended again in 100 mM phosphate buffer, and crushed with ultrasound to obtain a coenzyme solution. Enzyme activity is measured according to the method in the technical method.

Example 3 Conversion of taurorusodeoxycholic acid using a single gene expression protein in a 1L reaction system

250 g of taurochenodeoxycholic acid was dissolved in 700 mL of 100 mM glycine buffer, 0.25 mM NAD ⁺ was added, 60 g/L sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase (approximately 5 g of pure enzyme) ) And purified or partially purified E. coli cells expressing lactic acid dehydrogenase (including approximately 2 g of pure enzyme), or a cell lysate thereof, or a cell resuspension thereof, and 100 mM glycine up to 1 L The buffer is added supplementally and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. Purified or partially purified to which 100 g/L glucose is added and expresses 7β-hydroxysteroid dehydrogenase (including approximately 5 g of pure enzyme) and glucose dehydrogenase (including approximately 2 g of pure enzyme). E. coli cells, or a cell lysate thereof, or a cell resuspension solution are added, and the pH is adjusted to 7.5 with 5M NaOH. It is reacted at 25°C for 6-18h. The substrate conversion rate is 98% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

Example 4 Taurorusodeoxycholic acid conversion using double gene co-expression protein in 1L reaction system

250 g of taurochenodeoxycholic acid is dissolved in 700 mL of 100 mM glycine buffer, 0.25 mM NAD ⁺ is added, 60 g/L sodium pyruvate is added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogena A purified or partially purified E. coli cell, or a cell lysate thereof, or a cell resuspension solution, which co-expresses an azease (enzyme content total approximately 10 g) is added, supplemented with 100 mM glycine buffer to 1 L, , Adjust pH to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. Purified or partially purified E. coli cells, wherein 100 g/L glucose is added and co-express 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase (enzyme content total approximately 10 g), or Cell lysate or cell resuspension is added, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. The substrate conversion rate is 98% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

Example 5 Taurorusodeoxycholic acid conversion using single gene expression fusion protein in 1L reaction system

250 g of taurochenodeoxycholic acid was dissolved in 700 mL of 100 mM glycine buffer, 0.25 mM NAD ⁺ was added, 60 g/L sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase fused lactic acid dehydrogena Purified or partially purified E. coli cells expressing an azease (enzyme content approximately 5 g), or cell lysate thereof, or cell resuspension thereof are added, supplemented with 100 mM glycine buffer up to 1 L, and 5 M of The pH is adjusted to 7.5 with NaOH. At 25° C., react for 6-18 h. Purified or partially purified E. coli cells, or cell lysis thereof, wherein 100 g/L glucose is added and expresses 7β-hydroxysteroid dehydrogenase fused glucose dehydrogenase (enzyme content approximately 5 g) The solution or cell resuspension is added, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. The substrate conversion rate is 99.7% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

Example 6 Conversion of taurorusodeoxycholic acid using a single gene fusion protein and a dehydrogenase co-expression protein in a 1L reaction system

250 g of taurochenodeoxycholic acid was dissolved in 700 mL of 100 mM glycine buffer, 0.20 mM NAD ⁺ was added, 60 g/L sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase fused lactic acid dehydrogena A purified or partially purified E. coli cell, or a cell lysate thereof, or a cell resuspension solution thereof, which co-expresses an azeostat and lactic acid dehydrogenase (enzyme content total approximately 7 g) is added, and 100 mM up to 1 L Glycine buffer is added supplementally, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. Purified or partially purified which adds 100 g/L glucose and co-expresses 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase and glucose dehydrogenase (enzyme content total approximately 7 g) E. coli cells, or cell lysate thereof, or cell resuspension solution is added, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. The substrate conversion rate is 98.5% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

Example 7 Conversion of taurorusodeoxycholic acid using single gene fusion protein and hydroxysteroid dehydrogenase co-expression protein in 1L reaction system

250 g of taurochenodeoxycholic acid was dissolved in 700 mL of 100 mM glycine buffer, 0.50 mM NAD ⁺ was added, 60 g/L sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase fusion 7α-hydroxy Purified or partially purified E. coli cells, or cell lysates thereof, or cell resuspensions, which co-express steroid dehydrogenase and lactic acid dehydrogenase (enzyme content total approximately 7 g), , Supplemented with 100 mM glycine buffer until 1 L, and adjusted the pH to 7.5 with 5 M NaOH. At 25° C., react for 6-18 h. Purified by adding 100 g/L glucose and co-expressing 7β-hydroxysteroid dehydrogenase fused glucose dehydrogenase and 7β-hydroxysteroid dehydrogenase (enzyme content total approximately 7g) Alternatively, a partially purified E. coli cell, or a cell lysate thereof, or a cell resuspension solution is added, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. The substrate conversion rate is 99.5% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

Example 8 Converting taururusodeoxycholic acid to a single gene expression fusion protein in a 100L reaction system

25 Kg of taurochenodeoxycholic acid was dissolved in 70 L of 100 mM glycine buffer, 0.25 mM NAD ⁺ was added, 60 g/L sodium pyruvate was added, and 7α-hydroxysteroid dehydrogenase fused lactic acid dehydrogena Purified or partially purified E. coli cells expressing an azease (enzyme content approximately 500 g), or cell lysate thereof, or cell resuspension thereof are added, supplemented with 100 mM glycine buffer up to 100 L, and 5 M of The pH is adjusted to 7.5 with NaOH. At 25° C., react for 6-18 h. Purified or partially purified E. coli cells, or cell lysis thereof, wherein 100 g/L glucose is added and expresses 7β-hydroxysteroid dehydrogenase fused glucose dehydrogenase (enzyme content approximately 500 g) The solution or cell resuspension is added, and the pH is adjusted to 7.5 with 5M NaOH. At 25° C., react for 6-18 h. The substrate conversion rate is 99.5% or more, the finished product content is 96.8% or more, and the yield is 85% or more.

In Example 3-8, the amount of 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase, the amount of 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, the amount of NAD ⁺ and the substrate conversion were It is as shown in Table 1 below.

7α-HSDH+LDH enzyme consumption (g/L) 7β-HSDH+GDH enzyme usage (g/L) NAD ⁺ addition amount (mM) Substrate conversion rate Example 3 7 7 0.25 98% Example 4 10 10 0.25 98% Example 5 5 5 0.25 99.7% Example 6 7 7 0.20 98.5% Example 7 7 7 0.50 99.5% Example 8 5 5 0.25 99.5%

Example 9 Preparation of taurorusodeoxycholic acid

The converted reaction solution is evaporated by rotary evaporation in a paste form, and 10 times by volume of anhydrous ethanol or 95% ethanol is added, and precipitation is removed by centrifugation or filtering. The supernatant is dried to obtain taurorusodeoxycholic acid. After the reaction, the crude product of taurorusodeoxycholic acid was dissolved using acetonitrile, filtered through a 0.22 um filter membrane to remove insoluble matter, to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column (column specification is 15*255 mm) loaded with a C18 silica gel filler using preparative HPLC; After the elution proceeds by injecting the fluidized bed A prepared with 30% methanol-aqueous solution into a stainless column, the elution rate is 240 mL/h, the elution time is 175 minutes, and the eluent 1 is collected; Again, the fluidized bed gradient was linearly improved to 50% fluidized bed B (80% methanol-aqueous solution) within 50 minutes to proceed with elution, and eluent 2 was collected by eluting for 75 minutes while maintaining 50% fluidized bed B; Eluting to improve the fluidized bed gradient linearly to 100% fluidized bed B within 40 minutes, and eluting for 70 minutes while maintaining 100% fluidized bed B to collect eluent 3; The collected eluent is poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; Thereafter, the mixture was dried in a vacuum drying oven, and the purity of taurusodeoxycholic acid in the sample was measured using high performance liquid chromatography. Among them, taurusodeoxycholic acid content in eluent 1 was 5.77%, and the recovery rate. Silver is 8.2%, the content of taurorusodeoxycholic acid in eluent 2 is 99.3%, the recovery rate is 81.5%, the content of taurorusodeoxycholic acid in eluent 3 is 14.9%, and the recovery rate is 10.3%. The HPLC spectrum of taurorusodeoxycholic acid prepared and obtained in Example 9 is as shown in FIG. 3.

Example 10 Preparation of urusodeoxycholic acid

Sodium hydroxide was added to the converted reaction solution to adjust the pH to 10, heated to 100°C for 24h reaction, the temperature was reduced to 10°C and hydrochloric acid was added to adjust the pH to 4, urusodeoxycholic acid To precipitate. Then, a crude product of urusodeoxycholic acid is obtained.

In the above, the present invention has been described and described with respect to selectable examples. Speaking of technical personnel in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the scope of the basic ideology and principles of this application should all fall within the scope of the claims of this application.

SEQUENCE LISTING <110> NOVABAND BIOMEDICAL TECHNOLOGY INC. <120> METHOD FOR PREPARING TAUROURSODEOXYCHOLIC ACID BY BIOTRANSFORMATION AND APPLICATION THEREOF <130> PKR204577-1-APC-2020-0328 <150> CN 201811446689.5 <151> November 29, 2018 <160> 21 <170> SIPOSequenceListing 1.0 <210> 1 <211> 840 <212> DNA <213> Campylobacter hyointestinalis <400> 1 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catgagttgc 60 tataacgatg agtttaaagg taaaaccctg gttattagcg gtggcacccg cggtattggt 120 cgcgccattg ttctggaatt tgccaatgca ggtgcaaata ttgcattcac ttttaatagc 180 aacaaggaaa tggccgaaga acaggcccgt gaactggaaa ataagtttgg tattaaggcc 240 cgcgcctatg cactgaatat tctggaaccg gaaagttata aagaactgtt tctgcagatt 300 gatgaagatt ttgatcgtgt ggatttcttt attagcaatg ccattattag cggccgtgcc 360 gttgcaggcg gttataccaa attcatgaaa ctgaaaccgc gcggtattaa taatattttt 420 accgccaccg tgaatgcatt tgtttgcggt acacaggaag ccgccaaacg tatggaaaaa 480 gttggtggtg gtagcgttat tagtctgagc agtaccggca atctggtgta tattgaacat 540 tatagcggcc atggcaccgc caaagcagca gtggaagcca tggcccgcta tgcagcaacc 600 gaactgggcg ataaaaatat tcgtgttaat gttgttagcg gcggtccgat tgaaaccgat 660 gcactgcgtg cctttaccaa ttatgaagaa gtgcgcgatg caaccgcagc cctgagcccg 720 ctgggtcgta tgggccagcc gaccgatctg gccggcgcat gtctgtttct gtgtagcagc 780 aaagccagtt gggtgaccgg tcataccttt attattgatg gtggcaccac ctttaaataa 840 <210> 2 <211> 279 <212> PRT <213> Campylobacter hyointestinalis <400> 2 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Ser Cys Tyr Asn Asp Glu Phe Lys Gly Lys Thr Leu Val Ile 20 25 30 Ser Gly Gly Thr Arg Gly Ile Gly Arg Ala Ile Val Leu Glu Phe Ala 35 40 45 Asn Ala Gly Ala Asn Ile Ala Phe Thr Phe Asn Ser Asn Lys Glu Met 50 55 60 Ala Glu Glu Gln Ala Arg Glu Leu Glu Asn Lys Phe Gly Ile Lys Ala 65 70 75 80 Arg Ala Tyr Ala Leu Asn Ile Leu Glu Pro Glu Ser Tyr Lys Glu Leu 85 90 95 Phe Leu Gln Ile Asp Glu Asp Phe Asp Arg Val Asp Phe Phe Ile Ser 100 105 110 Asn Ala Ile Ile Ser Gly Arg Ala Val Ala Gly Gly Tyr Thr Lys Phe 115 120 125 Met Lys Leu Lys Pro Arg Gly Ile Asn Asn Ile Phe Thr Ala Thr Val 130 135 140 Asn Ala Phe Val Cys Gly Thr Gln Glu Ala Ala Lys Arg Met Glu Lys 145 150 155 160 Val Gly Gly Gly Ser Val Ile Ser Leu Ser Ser Thr Gly Asn Leu Val 165 170 175 Tyr Ile Glu His Tyr Ser Gly His Gly Thr Ala Lys Ala Ala Val Glu 180 185 190 Ala Met Ala Arg Tyr Ala Ala Thr Glu Leu Gly Asp Lys Asn Ile Arg 195 200 205 Val Asn Val Val Ser Gly Gly Pro Ile Glu Thr Asp Ala Leu Arg Ala 210 215 220 Phe Thr Asn Tyr Glu Glu Val Arg Asp Ala Thr Ala Ala Leu Ser Pro 225 230 235 240 Leu Gly Arg Met Gly Gln Pro Thr Asp Leu Ala Gly Ala Cys Leu Phe 245 250 255 Leu Cys Ser Ser Lys Ala Ser Trp Val Thr Gly His Thr Phe Ile Ile 260 265 270 Asp Gly Gly Thr Thr Phe Lys 275 <210> 3 <211> 1050 <212> DNA <213> human <400> 3 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catggcaacc 60 ctgaaagatc agctgatcta taatctgctg aaagaagaac agaccccgca gaataagatt 120 accgtggtgg gcgtgggtgc agtgggcatg gcctgcgcaa ttagcattct gatgaaagat 180 ctggcagatg aactggcact ggtggatgtg attgaagata aactgaaagg cgaaatgatg 240 gatctgcagc atggtagtct gtttctgcgt accccgaaaa ttgtgagtgg taaagattat 300 aatgtgaccg caaatagcaa actggttatt attaccgcag gcgcccgtca gcaggaaggt 360 gaaagtcgtc tgaatctggt gcagcgtaat gtgaatattt ttaaattcat catccctaac 420 gtggttaaat atagcccgaa ttgcaaactg ctgattgtga gcaatccggt ggatattctg 480 acctatgttg cctggaaaat tagtggtttt ccgaaaaatc gtgttattgg cagcggctgt 540 aatctggata gtgcacgttt tcgctatctg atgggcgaac gtctgggcgt tcatccgctg 600 agttgccacg gttgggtgct gggtgaacat ggtgacagca gcgttccggt ttggagcggc 660 atgaatgtgg ccggtgttag tctgaaaacc ctgcatccgg atctgggtac agataaagat 720 aaagaacagt ggaaagaagt tcataaacag gtggttgaaa gtgcatacga agtgattaag 780 ctgaaaggct ataccagctg ggcaattggt ctgagcgttg cagatctggc agaaagtatt 840 atgaaaaatc tgcgccgtgt gcatccggtt agcaccatga ttaagggcct gtatggtatt 900 aaggatgatg tgtttctgag cgtgccgtgc attctgggcc agaatggtat tagtgatctg 960 gttaaagtta ccctgaccag tgaagaagaa gcacgtctga aaaaatctgc agataccctg 1020 tggggcattc agaaagaact gcagttttaa 1050 <210> 4 <211> 349 <212> PRT <213> human <400> 4 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu 20 25 30 Glu Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val 35 40 45 Gly Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu 50 55 60 Leu Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met 65 70 75 80 Asp Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser 85 90 95 Gly Lys Asp Tyr Asn Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr 100 105 110 Ala Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln 115 120 125 Arg Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr 130 135 140 Ser Pro Asn Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu 145 150 155 160 Thr Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile 165 170 175 Gly Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly 180 185 190 Glu Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly 195 200 205 Glu His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Met Asn Val Ala 210 215 220 Gly Val Ser Leu Lys Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp 225 230 235 240 Lys Glu Gln Trp Lys Glu Val His Lys Gln Val Val Glu Ser Ala Tyr 245 250 255 Glu Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser 260 265 270 Val Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His 275 280 285 Pro Val Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Asp Val 290 295 300 Phe Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Leu 305 310 315 320 Val Lys Val Thr Leu Thr Ser Glu Glu Glu Ala Arg Leu Lys Lys Ser 325 330 335 Ala Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe 340 345 <210> 5 <211> 843 <212> DNA <213> Collinsella aerofaciens <400> 5 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catgaacctg 60 cgtgaaaaat acggtgaatg gggtctgatc ctgggtgcta ccgaaggtgt tggtaaagct 120 ttctgcgaaa aaatcgctgc tggtggtatg aacgttgtta tggttggtcg tcgtgaagaa 180 aaactgaacg ttctggctgg tgaaatccgt gaaacctacg gtgttgaaac caaagttgtt 240 cgtgctgact tctctcagcc gggtgctgct gaaaccgttt tctgcgctac cgaaggtctg 300 gacatgggtt tcatgtctta cgttgcttgc ctgcactctt tcggtaaaat ccaggacacc 360 ccgtgggaaa aacacgaagc tatgatcaac gttaactgcg ttaccttcct gaaatgcttc 420 caccactaca tgcgtatctt cgctgctcag gaccgtggtg ctgttatcaa cgtttcttct 480 atgaccggta tctcttcttc tccgtggaac ggtcagtacg gtgctggtaa agctttcatc 540 ctgaaaatga ccgaagctgt tgcttgcgaa tgcgaaggta ccggtgttga cgttgaagtt 600 atcaccctgg gtaccaccct gaccccgtct ctgctgtcta acctgccggg tggtccgcag 660 ggtgaagctg ttatgaaaat cgctctgacc ccggaagaat gcgttgacga agctttcgaa 720 aaactgggta aagaactgtc tgttatcgct ggtcagcgta acaaagactc tgttcacgac 780 tggaaagcta accacaccga agacgaatac atccgttaca tgggttcttt ctaccgtgac 840 taa 843 <210> 6 <211> 280 <212> PRT <213> Collinsella aerofaciens <400> 6 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Leu Ile Leu Gly 20 25 30 Ala Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ala Gly 35 40 45 Gly Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Asn Val 50 55 60 Leu Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu Thr Lys Val Val 65 70 75 80 Arg Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr Val Phe Cys Ala 85 90 95 Thr Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Leu His 100 105 110 Ser Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met 115 120 125 Ile Asn Val Asn Cys Val Thr Phe Leu Lys Cys Phe His His Tyr Met 130 135 140 Arg Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser 145 150 155 160 Met Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly 165 170 175 Lys Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Cys Glu 180 185 190 Gly Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr 195 200 205 Pro Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val 210 215 220 Met Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp Glu Ala Phe Glu 225 230 235 240 Lys Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln Arg Asn Lys Asp 245 250 255 Ser Val His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Tyr Ile Arg 260 265 270 Tyr Met Gly Ser Phe Tyr Arg Asp 275 280 <210> 7 <211> 837 <212> DNA <213> Bacillus subtilis <400> 7 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catgtacccg 60 gatctgaaag gcaaagttgt tgcaattacc ggtgccgcca gtggcctggg caaagccatg 120 gcaattcgtt ttggcaaaga acaggccaaa gtggttatta attattatag caacaagcag 180 gacccgaatg aagttaaaga agaagtgatt aaggccggtg gtgaagcagt tgttgttcag 240 ggtgacgtga ccaaagaaga agatgttaaa aatatcgtgc agaccgccat taaggaattt 300 ggcaccctgg atattatgat taataatgcc ggcctggaaa atccggttcc gagccatgaa 360 atgccgctga aagattggga taaagttatt ggcaccaatc tgaccggcgc atttctgggt 420 agtcgtgaag ccattaagta ttttgtggaa aatgatatta agggcaacgt tattaacatg 480 agcagcgtgc atgaagtgat tccgtggccg ctgtttgttc attatgcagc aagcaaaggt 540 ggtattaagc tgatgaccga aaccctggca ctggaatatg caccgaaagg tattcgcgtg 600 aataatattg gcccgggtgc cattaatacc ccgattaatg cagaaaaatt cgccgatccg 660 aaacagaaag cagatgtgga aagtatgatt ccgatgggct atattggtga accggaagaa 720 attgccgcag tggcagcatg gctggcaagc aaagaagcaa gctatgttac cggtattacc 780 ctgtttgcag atggcggtat gacccagtat ccgagctttc aggccggtcg tggttaa 837 <210> 8 <211> 278 <212> PRT <213> Bacillus subtilis <400> 8 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala 20 25 30 Ala Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln 35 40 45 Ala Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu 50 55 60 Val Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln 65 70 75 80 Gly Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala 85 90 95 Ile Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu 100 105 110 Glu Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys 115 120 125 Val Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala 130 135 140 Ile Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met 145 150 155 160 Ser Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala 165 170 175 Ala Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu 180 185 190 Tyr Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile 195 200 205 Asn Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala 210 215 220 Asp Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu 225 230 235 240 Ile Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val 245 250 255 Thr Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser 260 265 270 Phe Gln Ala Gly Arg Gly 275 <210> 9 <211> 1881 <212> DNA <213> Artificial Sequence <400> 9 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catggcaacc 60 ctgaaagatc agctgatcta taatctgctg aaagaagaac agaccccgca gaataagatt 120 accgtggtgg gcgtgggtgc agtgggcatg gcctgcgcaa ttagcattct gatgaaagat 180 ctggcagatg aactggcact ggtggatgtg attgaagata aactgaaagg cgaaatgatg 240 gatctgcagc atggtagtct gtttctgcgt accccgaaaa ttgtgagtgg taaagattat 300 aatgtgaccg caaatagcaa actggttatt attaccgcag gcgcccgtca gcaggaaggt 360 gaaagtcgtc tgaatctggt gcagcgtaat gtgaatattt ttaaattcat catccctaac 420 gtggttaaat atagcccgaa ttgcaaactg ctgattgtga gcaatccggt ggatattctg 480 acctatgttg cctggaaaat tagtggtttt ccgaaaaatc gtgttattgg cagcggctgt 540 aatctggata gtgcacgttt tcgctatctg atgggcgaac gtctgggcgt tcatccgctg 600 agttgccacg gttgggtgct gggtgaacat ggtgacagca gcgttccggt ttggagcggc 660 atgaatgtgg ccggtgttag tctgaaaacc ctgcatccgg atctgggtac agataaagat 720 aaagaacagt ggaaagaagt tcataaacag gtggttgaaa gtgcatacga agtgattaag 780 ctgaaaggct ataccagctg ggcaattggt ctgagcgttg cagatctggc agaaagtatt 840 atgaaaaatc tgcgccgtgt gcatccggtt agcaccatga ttaagggcct gtatggtatt 900 aaggatgatg tgtttctgag cgtgccgtgc attctgggcc agaatggtat tagtgatctg 960 gttaaagtta ccctgaccag tgaagaagaa gcacgtctga aaaaatctgc agataccctg 1020 tggggcattc agaaagaact gcagtttggc ggcggcggca gcggcggcgg cggcagcggc 1080 ggcggcggca gcatgagttg ctataacgat gagtttaaag gtaaaaccct ggttattagc 1140 ggtggcaccc gcggtattgg tcgcgccatt gttctggaat ttgccaatgc aggtgcaaat 1200 attgcattca cttttaatag caacaaggaa atggccgaag aacaggcccg tgaactggaa 1260 aataagtttg gtattaaggc ccgcgcctat gcactgaata ttctggaacc ggaaagttat 1320 aaagaactgt ttctgcagat tgatgaagat tttgatcgtg tggatttctt tattagcaat 1380 gccattatta gcggccgtgc cgttgcaggc ggttatacca aattcatgaa actgaaaccg 1440 cgcggtatta ataatatttt taccgccacc gtgaatgcat ttgtttgcgg tacacaggaa 1500 gccgccaaac gtatggaaaa agttggtggt ggtagcgtta ttagtctgag cagtaccggc 1560 aatctggtgt atattgaaca ttatagcggc catggcaccg ccaaagcagc agtggaagcc 1620 atggcccgct atgcagcaac cgaactgggc gataaaaata ttcgtgttaa tgttgttagc 1680 ggcggtccga ttgaaaccga tgcactgcgt gcctttacca attatgaaga agtgcgcgat 1740 gcaaccgcag ccctgagccc gctgggtcgt atgggccagc cgaccgatct ggccggcgca 1800 tgtctgtttc tgtgtagcag caaagccagt tgggtgaccg gtcatacctt tattattgat 1860 ggtggcacca cctttaaata a 1881 <210> 10 <211> 626 <212> PRT <213> Artificial Sequence <400> 10 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu 20 25 30 Glu Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val 35 40 45 Gly Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu 50 55 60 Leu Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met 65 70 75 80 Asp Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser 85 90 95 Gly Lys Asp Tyr Asn Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr 100 105 110 Ala Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln 115 120 125 Arg Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr 130 135 140 Ser Pro Asn Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu 145 150 155 160 Thr Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile 165 170 175 Gly Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly 180 185 190 Glu Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly 195 200 205 Glu His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Met Asn Val Ala 210 215 220 Gly Val Ser Leu Lys Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp 225 230 235 240 Lys Glu Gln Trp Lys Glu Val His Lys Gln Val Val Glu Ser Ala Tyr 245 250 255 Glu Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser 260 265 270 Val Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His 275 280 285 Pro Val Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Asp Val 290 295 300 Phe Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Leu 305 310 315 320 Val Lys Val Thr Leu Thr Ser Glu Glu Glu Ala Arg Leu Lys Lys Ser 325 330 335 Ala Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe Gly Gly Gly 340 345 350 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Cys Tyr 355 360 365 Asn Asp Glu Phe Lys Gly Lys Thr Leu Val Ile Ser Gly Gly Thr Arg 370 375 380 Gly Ile Gly Arg Ala Ile Val Leu Glu Phe Ala Asn Ala Gly Ala Asn 385 390 395 400 Ile Ala Phe Thr Phe Asn Ser Asn Lys Glu Met Ala Glu Glu Gln Ala 405 410 415 Arg Glu Leu Glu Asn Lys Phe Gly Ile Lys Ala Arg Ala Tyr Ala Leu 420 425 430 Asn Ile Leu Glu Pro Glu Ser Tyr Lys Glu Leu Phe Leu Gln Ile Asp 435 440 445 Glu Asp Phe Asp Arg Val Asp Phe Phe Ile Ser Asn Ala Ile Ile Ser 450 455 460 Gly Arg Ala Val Ala Gly Gly Tyr Thr Lys Phe Met Lys Leu Lys Pro 465 470 475 480 Arg Gly Ile Asn Asn Ile Phe Thr Ala Thr Val Asn Ala Phe Val Cys 485 490 495 Gly Thr Gln Glu Ala Ala Lys Arg Met Glu Lys Val Gly Gly Gly Ser 500 505 510 Val Ile Ser Leu Ser Ser Thr Gly Asn Leu Val Tyr Ile Glu His Tyr 515 520 525 Ser Gly His Gly Thr Ala Lys Ala Ala Val Glu Ala Met Ala Arg Tyr 530 535 540 Ala Ala Thr Glu Leu Gly Asp Lys Asn Ile Arg Val Asn Val Val Ser 545 550 555 560 Gly Gly Pro Ile Glu Thr Asp Ala Leu Arg Ala Phe Thr Asn Tyr Glu 565 570 575 Glu Val Arg Asp Ala Thr Ala Ala Leu Ser Pro Leu Gly Arg Met Gly 580 585 590 Gln Pro Thr Asp Leu Ala Gly Ala Cys Leu Phe Leu Cys Ser Ser Lys 595 600 605 Ala Ser Trp Val Thr Gly His Thr Phe Ile Ile Asp Gly Gly Thr Thr 610 615 620 Phe Lys 625 <210> 11 <211> 1671 <212> DNA <213> Artificial Sequence <400> 11 atgggcagca gccatcatca tcatcatcac gaaaacctgt attttcaggg catgtacccg 60 gatctgaaag gcaaagttgt tgcaattacc ggtgccgcca gtggcctggg caaagccatg 120 gcaattcgtt ttggcaaaga acaggccaaa gtggttatta attattatag caacaagcag 180 gacccgaatg aagttaaaga agaagtgatt aaggccggtg gtgaagcagt tgttgttcag 240 ggtgacgtga ccaaagaaga agatgttaaa aatatcgtgc agaccgccat taaggaattt 300 ggcaccctgg atattatgat taataatgcc ggcctggaaa atccggttcc gagccatgaa 360 atgccgctga aagattggga taaagttatt ggcaccaatc tgaccggcgc atttctgggt 420 agtcgtgaag ccattaagta ttttgtggaa aatgatatta agggcaacgt tattaacatg 480 agcagcgtgc atgaagtgat tccgtggccg ctgtttgttc attatgcagc aagcaaaggt 540 ggtattaagc tgatgaccga aaccctggca ctggaatatg caccgaaagg tattcgcgtg 600 aataatattg gcccgggtgc cattaatacc ccgattaatg cagaaaaatt cgccgatccg 660 aaacagaaag cagatgtgga aagtatgatt ccgatgggct atattggtga accggaagaa 720 attgccgcag tggcagcatg gctggcaagc aaagaagcaa gctatgttac cggtattacc 780 ctgtttgcag atggcggtat gacccagtat ccgagctttc aggccggtcg tggtggcggc 840 ggcggcagcg gcggcggcgg cagcggcggc ggcggcagca tgaacctgcg tgaaaaatac 900 ggtgaatggg gtctgatcct gggtgctacc gaaggtgttg gtaaagcttt ctgcgaaaaa 960 atcgctgctg gtggtatgaa cgttgttatg gttggtcgtc gtgaagaaaa actgaacgtt 1020 ctggctggtg aaatccgtga aacctacggt gttgaaacca aagttgttcg tgctgacttc 1080 tctcagccgg gtgctgctga aaccgttttc tgcgctaccg aaggtctgga catgggtttc 1140 atgtcttacg ttgcttgcct gcactctttc ggtaaaatcc aggacacccc gtgggaaaaa 1200 cacgaagcta tgatcaacgt taactgcgtt accttcctga aatgcttcca ccactacatg 1260 cgtatcttcg ctgctcagga ccgtggtgct gttatcaacg tttcttctat gaccggtatc 1320 tcttcttctc cgtggaacgg tcagtacggt gctggtaaag ctttcatcct gaaaatgacc 1380 gaagctgttg cttgcgaatg cgaaggtacc ggtgttgacg ttgaagttat caccctgggt 1440 accaccctga ccccgtctct gctgtctaac ctgccgggtg gtccgcaggg tgaagctgtt 1500 atgaaaatcg ctctgacccc ggaagaatgc gttgacgaag ctttcgaaaa actgggtaaa 1560 gaactgtctg ttatcgctgg tcagcgtaac aaagactctg ttcacgactg gaaagctaac 1620 cacaccgaag acgaatacat ccgttacatg ggttctttct accgtgacta a 1671 <210> 12 <211> 556 <212> PRT <213> Artificial Sequence <400> 12 Met Gly Ser Ser His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 15 Gly Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala 20 25 30 Ala Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln 35 40 45 Ala Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu 50 55 60 Val Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln 65 70 75 80 Gly Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala 85 90 95 Ile Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu 100 105 110 Glu Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys 115 120 125 Val Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala 130 135 140 Ile Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met 145 150 155 160 Ser Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala 165 170 175 Ala Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu 180 185 190 Tyr Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile 195 200 205 Asn Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala 210 215 220 Asp Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu 225 230 235 240 Ile Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val 245 250 255 Thr Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser 260 265 270 Phe Gln Ala Gly Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 275 280 285 Gly Gly Gly Gly Ser Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly 290 295 300 Leu Ile Leu Gly Ala Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys 305 310 315 320 Ile Ala Ala Gly Gly Met Asn Val Val Met Val Gly Arg Arg Glu Glu 325 330 335 Lys Leu Asn Val Leu Ala Gly Glu Ile Arg Glu Thr Tyr Gly Val Glu 340 345 350 Thr Lys Val Val Arg Ala Asp Phe Ser Gln Pro Gly Ala Ala Glu Thr 355 360 365 Val Phe Cys Ala Thr Glu Gly Leu Asp Met Gly Phe Met Ser Tyr Val 370 375 380 Ala Cys Leu His Ser Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys 385 390 395 400 His Glu Ala Met Ile Asn Val Asn Cys Val Thr Phe Leu Lys Cys Phe 405 410 415 His His Tyr Met Arg Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile 420 425 430 Asn Val Ser Ser Met Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln 435 440 445 Tyr Gly Ala Gly Lys Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala 450 455 460 Cys Glu Cys Glu Gly Thr Gly Val Asp Val Glu Val Ile Thr Leu Gly 465 470 475 480 Thr Thr Leu Thr Pro Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln 485 490 495 Gly Glu Ala Val Met Lys Ile Ala Leu Thr Pro Glu Glu Cys Val Asp 500 505 510 Glu Ala Phe Glu Lys Leu Gly Lys Glu Leu Ser Val Ile Ala Gly Gln 515 520 525 Arg Asn Lys Asp Ser Val His Asp Trp Lys Ala Asn His Thr Glu Asp 530 535 540 Glu Tyr Ile Arg Tyr Met Gly Ser Phe Tyr Arg Asp 545 550 555 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <400> 13 cgggatccat gggcagcagc catcatca 28 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <400> 14 cggaattctt atttaaaggt ggtgcca 27 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <400> 15 ggaattccat atgggcagca gccatcatca 30 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <400> 16 tccctcgagt taaaactgca gttctttct 29 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <400> 17 cgggatccat gggcagcagc catcatca 28 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <400> 18 cggaattctt agtcacggta gaaagaac 28 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <400> 19 tccctcgagt taaccacgac cggcctgaaa gct 33 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <400> 20 tccctcgagt tatttaaagg tggtgcca 28 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <400> 21 tccctcgagt tagtcacggt agaaagaac 29

Claims (20)

A method for producing taurorusodeoxycholic acid by bioconversion, comprising a genetic codon optimization step, engineering bacterial construction step, engineering bacterial culture step, substrate conversion and product production step;
At this time, the engineering bacteria directly ferment the conversion substrate to produce taurorusodeoxycholic acid;
The substrate is taurochenodeoxycholic acid;
The engineering bacteria are selected from the family of engineering bacteria capable of expressing 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. Method of producing taurorusodeoxycholic acid by bioconversion.
According to claim 1, wherein the 7α-hydroxysteroid dehydrogenase and the lactic acid dehydrogenase expression enzyme is 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase single expression enzyme, 7α-hydroxy Steroid dehydrogenase and lactate dehydrogenase co-expression enzymes, 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase pair tetramer fusion enzyme, 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase Azeomeric pair tetramer fusion enzyme and lactic acid dehydrogenase co-expression enzyme, 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogenase pair tetramer fusion enzyme and 7α-hydroxysteroid dehydrogenase co-expression enzyme Is selected from the group consisting of;
The 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase expression enzymes are 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase single expression enzymes, 7β-hydroxysteroid dehydrogenase and glucose Dehydrogenase co-expression enzyme, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair tetramer fusion enzyme, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair tetramer fusion enzyme Characterized by a group selected from the group consisting of glucose dehydrogenase co-expression enzyme, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase pair tetramer fusion enzyme and 7β-hydroxysteroid dehydrogenase co-expression enzyme Method for producing taurorusodeoxycholic acid by bioconversion.
According to claim 1, DNA sequence of 7α-hydroxysteroid dehydrogenase is SEQ ID NO:1, DNA sequence of lactic acid dehydrogenase is SEQ ID NO:3, 7β-hydroxysteroid dehydrogenase A method of producing taurusodeoxycholic acid by bioconversion, characterized in that the DNA sequence of the aze is SEQ ID NO:5 and the DNA sequence of glucose dehydrogenase is SEQ ID NO:7.
The method of claim 3, wherein the DNA sequence encoding the 7β-hydroxysteroid dehydrogenase is mutated at the A78 and V116 sites, and the method for producing taurorusodeoxycholic acid by bioconversion.
The protein sequence of claim 3 or 4, wherein the protein sequence of the 7α-hydroxysteroid dehydrogenase is SEQ ID NO:2, the protein sequence of the lactic acid dehydrogenase is SEQ ID NO:4, and the 7β -The protein sequence of hydroxysteroid dehydrogenase is SEQ ID NO:6, and the protein sequence of the glucose dehydrogenase is SEQ ID NO:8. Method of manufacture.
The method of claim 1, wherein the engineering bacteria construction step,
Gene expression vector construct, gene expression vector of 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, respectively, is soluble in E. coli BL21 A method for producing taurorusodeoxycholic acid by biotransformation, comprising transforming cells to obtain engineering bacteria.
The method of claim 6, wherein the gene expressing 7α-hydroxysteroid dehydrogenase is indicated by 7α-HSDH, the gene expressing lactic acid dehydrogenase is indicated by LDH, and 7β-hydroxysteroid dehydrogenase. The gene expressing the azease is designated as 7β-HSDH, and the gene expressing the glucose dehydrogenase is designated as GDH; Constructing the gene expression vector,
7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, so that a single gene expression vector pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, pETDuet- Obtaining 1-GDH; or
Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in pETDuet-1 vector, respectively, to obtain dual gene expression vectors pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH, respectively; or
7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in pETDuet-1 vector, respectively, and each single gene fusion protein Obtaining expression vectors pETDuet-1-(LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH); or
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Genes were constructed in the pETDuet-1 vector, respectively, so that a single gene fusion protein and a dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β) -Obtaining HSDH)/GDH; or
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and a single gene fusion protein and a hydroxysteroid dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/7α -Obtaining HSDH, pETDuet-1-(GDH-Linker-7β-HSDH)/7β-HSDH; A method for producing taurorusodeoxycholic acid by bioconversion comprising the steps of:
The method of claim 7, wherein the 7α-HSDH is Campylobacter high Te sustaining day devices (Campylobacter hyointestinalis): will derived from (UniProt CDQ67_02445), wherein the LDH is a human (Human): will derived from (UniProt P00338) the 7β-HSDH is choline Cellar Aero Pacifico Enschede (Collinsella aerofaciens) ATCC 25986: will derived from (UniProt A4ECA9), the GDH is Bacillus subtilis (Bacillus subtilis) (strain 168) derived from (UniProt P12310) A method for producing taurorusodeoxycholic acid by bioconversion.
The DNA sequence of claim 7, wherein the 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene is SEQ ID NO:9, and the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrate. A method for producing taurorusodeoxycholic acid by bioconversion, characterized in that the DNA sequence encoding the single gene is a SEQ ID NO:11.
According to claim 7, The 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase protein sequence encoded by a single gene is SEQ ID NO:10, the 7β-hydroxysteroid dehydrogenase fusion glucose A method for producing taurorusodeoxycholic acid by bioconversion, characterized in that the protein sequence encoded by a dehydrogenase single gene is SEQ ID NO:12.
The method of claim 1, wherein the concentration of the substrate is 20 g/L-250 g/L, characterized in that the method for producing taurorusodeoxycholic acid by bioconversion.
The method of claim 11, wherein the step of substrate conversion,
Taurochenodeoxycholic acid was dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD ⁺ was added, 5-60 g/L of sodium pyruvate was added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydro Purified or partially purified E. coli cells expressing kinase, or cell lysate thereof, or cell resuspension thereof are added, supplemented with 20-100 mM glycine buffer to the final volume, pH with sodium hydroxide Adjusted to 6.5-8.5, and reacted at 25° C. for 6-18 h; And 1.8-100 g/L of glucose, and expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, or a purified or partially purified E. coli cell, or a cell lysate thereof, or Characterized in that it comprises; adding the cell resuspension, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25° C., 6-18 h; to produce taurorusodeoxycholic acid by bioconversion. Way.
The method of claim 12, wherein the manufacturing step of the taurorusodeoxycholic acid, rotary evaporation of the converted reaction solution in paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and the resulting mixture is mixed. The precipitate is removed by centrifugation or filtering, and the supernatant is dried to obtain taurorusodeoxycholic acid, the crude product of taurorusodeoxycholic acid is dissolved using acetonitrile, and filtered with a 0.22um filter membrane to dissolve insoluble substances. Removed to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is carried out with different concentrations of methanol-water fluidized bed, the eluent is collected and poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; It is then placed in a vacuum drying oven and dried, using a high performance liquid chromatography to measure the purity of taururusodeoxycholic acid in the sample, characterized in that it comprises the step of bioconversion to produce taururusodeoxycholic acid How to.
The method of claim 1, wherein the method comprises the following steps:
(1) Gene codon optimization
As a step of optimizing E. coli expression codon for the gene sequence, adding an affinity tag, and also performing full-length gene synthesis, wherein the gene encoding 7α-hydroxysteroid dehydrogenase is 7α-HSDH, lactic acid The gene encoding dehydrogenase is LDH, the gene encoding 7β-hydroxysteroid dehydrogenase is 7β-HSDH, and the gene encoding glucose dehydrogenase is designated GDH, respectively;
(2) Engineering bacteria composition
The gene expression vector of the 7α-hydroxysteroid dehydrogenase, lactic acid dehydrogenase, 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase obtained by constructing and constructing the gene expression vector was obtained from E. coli BL21. Transforming soluble cells to obtain engineering bacteria;
(3) Engineering bacteria culture
As a step of fermenting and expressing a small amount of engineering bacteria: engineering bacterial fungus is applied to an ampicillin resistant LB plate, a single clone is selected, inoculated in 5 mL of LB medium containing ampicillin, and cultured at 37°C at 220 rpm. When the OD value is 0.8-1.2, 1 mM IPTG is added for 2 h to induce, and the expression level is examined by SDS-PAGE to select a clone with a high expression level and to preserve the species; Inoculate 20 μL of the bacterial species into 200 mL of ampicillin-resistant LB medium and incubate overnight, and when the OD value is 2.5-4.0, take 2 mL culture and inoculate into ampicillin-resistant medium and culture, when the OD value is 1, Including expression by adding IPTG to induce expression during the night and collecting the cells;
As a step of mass fermentation and expression of engineering bacteria: engineering bacteria are selected and inoculated into 1 L Erlenmeyer flasks of ampicillin resistant LB medium, and cultured overnight at 37° C. at 220 rpm, with an OD600 value of 2.5-4.0 Each 20 mL culture was taken and inoculated into a 3 L Erlenmeyer flask containing 10 1 L ampicillin resistant media, and cultured at 37° C. at 140 rpm overnight; 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, incubated at 37° C. for 8 hours while passing gas, and gas was passed, and then IPTG solution with a final concentration of 0.1 mM was added to the fermentation tank. To proceed with induction, to terminate fermentation after 10-12h induction, to drain the liquid, collect the cells by centrifugation, and also store at 4°C, take a small amount of cells and suspend them in 100 mM phosphate buffer again, and crush with ultrasonic waves To obtain a coenzyme solution; including;
(4) substrate conversion
Taurochenodeoxycholic acid was dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD ⁺ was added, 5-60 g/L of sodium pyruvate was added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydro Purified or partially purified E. coli cells expressing kinase, or cell lysates thereof, or cell resuspensions thereof are added, supplemented with 20-100 mM glycine buffer to the final volume, pH with sodium hydroxide Adjusted to 6.5-8.5, and reacted at 25° C. for 6-18 h; And 1.8-100 g/L of glucose, and expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, or a purified or partially purified E. coli cell, or a cell lysate thereof, or Adding the cell resuspension solution, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25° C. for 6-18 h;
(5) Product preparation
Conversion in step (4) The completed reaction solution is rotary evaporated in a paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and the precipitate is removed by centrifugation or filtering, and the supernatant is dried to dry the taurow. To obtain the rusodeoxycholic acid, and dissolve the crude taurorusodeoxycholic acid product using acetonitrile, filter with a 0.22um filter membrane to remove the insoluble material to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is performed with methanol-water fluidized beds of different concentrations, the eluent is collected, poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; Then, put in a vacuum drying oven and dry, and measuring the purity of taurorusodeoxycholic acid in the sample using high performance liquid chromatography.
15. The method of claim 14, wherein the step of constructing the gene expression vector comprises the following steps: A method for producing taurorusodeoxycholic acid by bioconversion:
7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, so that a single gene expression vector pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, pETDuet- Obtaining 1-GDH; or
Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in pETDuet-1 vector, respectively, to obtain dual gene expression vectors pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH, respectively; or
7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in pETDuet-1 vector, respectively, and each single gene fusion protein Obtaining expression vectors pETDuet-1-(LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH); or
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Genes were constructed in the pETDuet-1 vector, respectively, so that a single gene fusion protein and a dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β) -Obtaining HSDH)/GDH; or
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and a single gene fusion protein and a hydroxysteroid dehydrogenase co-expression vector pETDuet-1-(LDH-Linker-7α-HSDH)/7α -Acquiring HSDH, pETDuet-1-(GDH-Linker-7β-HSDH)/7β-HSDH.
The method of claim 1, wherein the DNA sequence encoding the 7α-hydroxysteroid dehydrogenase is SEQ ID NO:1, the DNA sequence encoding the lactic acid dehydrogenase is SEQ ID NO:3, and the 7β -DNA sequence encoding hydroxysteroid dehydrogenase is SEQ ID NO:5, DNA sequence encoding the glucose dehydrogenase is SEQ ID NO:7, Tauurorusode as a bioconversion. Method for producing oxycholic acid.
The method of claim 1, wherein the 7α-hydroxysteroid dehydrogenase, the lactic acid dehydrogenase, the 7β-hydroxysteroid dehydrogenase and the glucose dehydrogenase are independently from a liquid enzyme or an immobilized enzyme. Selected, the 7α-hydroxysteroid dehydrogenase, the lactic acid dehydrogenase, the 7β-hydroxysteroid dehydrogenase and the glucose dehydrogenase are independently whole cells, unpurified enzymes, or A method for producing taurorusodeoxycholic acid by bioconversion, characterized in that it is selected from purified enzymes.
The method according to claim 1, wherein the bioconversion method specifically comprises the following steps: A method for producing taurorusodeoxycholic acid by bioconversion:
(1) Gene codon optimization
As a step of optimizing the codon expression codon for the gene sequence, adding an affinity tag, and also performing full-length gene synthesis, the gene encoding 7α-hydroxysteroid dehydrogenase is 7α-HSDH, lactic acid dehydrate The gene encoding the logase is LDH, the gene encoding 7β-hydroxysteroid dehydrogenase is 7β-HSDH, and the gene encoding glucose dehydrogenase is designated GDH, respectively;
(2) Single gene expression vector construction
7α-HSDH, LDH, 7β-HSDH and GDH were constructed in the pETDuet-1 vector, respectively, to obtain pETDuet-1-7α-HSDH, pETDuet-1-LDH, pETDuet-1-7β-HSDH, and pETDuet-1-GDH To do;
(3) Double gene expression vector construction
Constructing 7α-HSDH and LDH, 7β-HSDH and GDH in the pETDuet-1 vector, respectively, to obtain pETDuet-1-7α-HSDH/LDH, pETDuet-1-7β-HSDH/GDH;
(4) Single gene fusion protein expression vector construction
The 7α-hydroxysteroid dehydrogenase fusion lactic acid dehydrogenase single gene and the 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene were constructed in the pETDuet-1 vector, respectively, and pETDuet-1-( Obtaining LDH-Linker-7α-HSDH), pETDuet-1-(GDH-Linker-7β-HSDH);
(5) Construction of a single gene fusion protein and a dehydrogenase co-expression vector
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and lactic acid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and glucose dehydrogenase single gene Constructing genes in pETDuet-1 vectors, respectively, to obtain pETDuet-1-(LDH-Linker-7α-HSDH)/LDH, pETDuet-1-(GDH-Linker-7β-HSDH)/GDH;
(6) Single gene fusion protein and hydroxysteroid dehydrogenase co-expression vector construction
7α-hydroxysteroid dehydrogenase fusion lactate dehydrogenase single gene and 7α-hydroxysteroid dehydrogenase single gene, 7β-hydroxysteroid dehydrogenase fusion glucose dehydrogenase single gene and 7β- A single hydroxysteroid dehydrogenase gene was constructed in the pETDuet-1 vector, and pETDuet-1-(LDH-Linker-7α-HSDH)/7α-HSDH, pETDuet-1-(GDH-Linker-7β-HSDH) Obtaining /7β-HSDH;
(7) Engineering bacteria composition
(2)-(6) transforming all of the expression vectors constructed in steps into soluble cells of E. coli BL21 (DE3) to obtain engineering bacteria;
(8) Small amount fermentation and expression of engineering bacteria
The engineering bacterial fungus was applied to an ampicillin-resistant LB plate, a single clone was selected, inoculated into 5 mL of LB medium containing ampicillin, and cultured at 37°C at 220 rpm, and the OD value was 0.8-1.2 days. At this time, 1 mM IPTG was added to induce it for 2 h, and the expression level was examined by SDS-PAGE to select a clone with a high expression level and to preserve the species; Inoculate 20 μL of the bacterial species into 200 mL of ampicillin-resistant LB medium and incubate overnight, and when the OD value is 2.5-4.0, take 2 mL culture and inoculate into ampicillin-resistant medium and culture, when the OD value is 1, Adding IPTG to induce expression overnight and collecting cells;
(9) Mass fermentation and expression of engineering bacteria
Engineering bacteria were selected and inoculated into 1 L Erlenmeyer flasks of ampicillin-resistant LB medium, cultured overnight at 37°C at 220 rpm, and when the OD600 value was 2.5-4.0, each 20 mL culture was taken and 10 1 L ampicillin resistance Inoculated into a 3 L Erlenmeyer flask containing medium, and cultured at 37° C. at 140 rpm overnight. 10 L of seed solution was inoculated into a fermentation tank containing 200 L of E. coli high-density fermentation medium, incubated at 37° C. for 8 hours while passing gas, and gas was passed, and then IPTG solution with a final concentration of 0.1 mM was added to the fermentation tank. To proceed with induction, and after induction for 10-12 h, fermentation is terminated, liquid is discharged, and the cells are collected by centrifugation and preserved at 4°C. A small amount of cells is again suspended in 100 mM phosphate buffer and ultrasonically Crushing to obtain a coenzyme solution;
(10) Enzyme activity measurement
As a method for measuring the enzyme activity of 7α-hydroxysteroid dehydrogenase: tauurusodeoxycholic acid as a substrate, 2.97 mL of 100 mM phosphate buffer (pH 8.0) in a 3 mL reaction system, final concentration of 0.5 mM Phosphorous phosphorus deoxycholic acid, dilute enzyme solution of 10 μL, NADP+ having a final concentration of 0.5 mM was added, reacted at pH 8.0 and 25° C. for 1 min, and measured the increase in absorbance at 340 nm;
As a method for measuring the enzyme activity of lactic acid dehydrogenase: using sodium pyruvate as a substrate, 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 100 mM sodium pyruvate, 50 μL of diluted enzyme solution in a 3 mL reaction system, final Adding NADH having a concentration of 0.2 mM, reacting with pH 8.0 at 25° C. for 1 min, and measuring a decrease in absorbance at 340 nm;
As a method for measuring the enzyme activity of 7β-hydroxysteroid dehydrogenase: using taurochenodeoxycholic acid as a substrate, 2.97 mL of 100 mM phosphate buffer (pH8.0) in a 3 mL reaction system, the final concentration being 0.5 mM Adding taurochenodeoxycholic acid, 10 μL of diluted enzyme solution, NADP+ having a final concentration of 0.5 mM, reacting at pH 8.0 and 25° C. for 1 min, and measuring an increase in absorbance at 340 nm;
As a method for measuring the enzyme activity of glucose dehydrogenase: glucose as a substrate, 2.7 mL of 100 mM phosphate buffer (pH8.0), 0.2 mL of 1.5M glucose, 50 μL of dilute enzyme solution, final concentration in one 3 mL reaction system Adding NADP+ of 2 mM, reacting with pH 8.0 at 25° C. for 2 min, and measuring an increase in absorbance at 340 nm;
(11) Conversion of taurokenodeoxycholic acid to taurorusodeoxycholic acid
Taurokenodeoxycholic acid is dissolved in 20-100 mM glycine buffer, 0.01-0.8 mM NAD+ is added, 5-60 g/L sodium pyruvate is added, 7α-hydroxysteroid dehydrogenase and lactic acid dehydrogena Purified or partially purified E. coli cells, or cell lysates thereof, which express the azease, are added, or a cell resuspension thereof, supplemented with 20-100 mM glycine buffer until the final volume, and the pH is adjusted with sodium hydroxide. Adjusted to 6.5-8.5 and reacted at 25° C. for 6-18 h; And 1.8-100 g/L of glucose, and expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, or a purified or partially purified E. coli cell, or a cell lysate thereof, or Adding a resuspension of the cells, adjusting the pH to 6.5-8.5 with sodium hydroxide, and reacting at 25° C. for 6-18 h;
(12) Preparation of taurorusodeoxycholic acid
In step (11), the completed reaction solution is rotary evaporated in a paste form, 2-10 times anhydrous ethanol or 95% ethanol is added, and the precipitate is removed by centrifugation or filtering, and when the supernatant is dried, Taulow To obtain the rusodeoxycholic acid, and dissolve the crude taurorusodeoxycholic acid product using acetonitrile, filter with a 0.22um filter membrane to remove the insoluble material to form a column feed solution; Injecting the column feed solution into a high pressure stainless steel column loaded with a silica gel chromatography filler using a preparative high performance liquid manufacturing apparatus; Then, step-wise elution is carried out in a methanol-water fluidized bed of different concentrations, and the collected eluent is poured into a rotary evaporator, rotary evaporated until thickened, and methanol is recovered; Then, put in a vacuum drying oven and dry, and using high performance liquid chromatography to measure the purity of taururusodeoxycholic acid in the sample.
Use of the method according to any one of claims 1 to 18 in the production of ursodeoxycholic acid.
The method of claim 19, wherein the process of preparing urusodeoxycholic acid comprises the following steps: Use: To adjust the pH to 8-11 by adding sodium hydroxide in a conversion-produced taurorusodeoxycholic acid solution. , Reacted for 18-24h by raising the temperature to 80-100°C, lowering the temperature to 10-15°C and adjusting the pH to 3-5 by adding hydrochloric acid to precipitate urusodeoxycholic acid.

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