US-12624335-B2 - Production method of recombinant Escherichia coli and high-purity ursodeoxycholic acid

Abstract

The present invention belongs to the field of bioengineering technologies, and in particular, to a production method of recombinant Escherichia coli ( E. coli ) and high-purity ursodeoxycholic acid (UDCA). The present invention constructs novel double-enzyme co-expression gene engineered bacteria, that is, recombinant E. coli . The bacteria simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). The bacteria is applicable to the production of high-purity UDCA. The yield of a target product is increased through the joint expression and application of 7β-HSDH and GDH. The production method of high-purity UDCA in the present invention is simple, generates a small amount of impurities in a production process, is a green process that satisfies environmental protection requirements, and has significant industrial application value.

Inventors

• Tian Zhang
• Tong XUE
• Lihui ZHU

Assignees

• Tian Zhang
• Tong XUE
• Lihui ZHU

Dates

Publication Date

20260512

Application Date

20221211

Priority Date

20220311

Claims (1)

1. 1 . A recombinant Escherichia coli ( E. coli ), wherein the recombinant E. coli simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH), wherein the recombinant E. coli is constructed by using following steps: (1) performing a codon optimization on genes of the 7β-HSDH and the GDH, and performing a complete gene synthesis, respectively; (2) first connecting the synthesized gene of the 7β-HSDH to a vector pETDuet1, to obtain a recombinant plasmid pETDuet1-7β-HSDH, and then connecting the gene of the GDH to the plasmid pETDuet1-7β-HSDH, to obtain a recombinant plasmid pETDuet1-GDH-7β-HSDH co-expressing the 7β-HSDH and the GDH; (3) transferring the pETDuet1-GDH-7β-HSDH into an E. coli BL21(DE3) competent cell, to obtain E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH carrying the recombinant plasmid; and (4) culturing the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH, to obtain the recombinant E. coli; wherein in step (1), after the codon optimization, the 7β-HSDH comprises a sequence of SEQ ID NO:1 and the GDH comprises a sequence of SEQ ID NO: 2; wherein in step (4), the culture of E. coli comprises the following steps: inoculating the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH into a lysogeny broth (LB) solid plate culture medium for activation and culture, then picking a single colony from the LB solid plate culture medium, inoculating the single colony into an Erlenmeyer flask with an LB liquid culture medium containing 100 mg/L of ampicillin (Amp + ), and performing shaking culture; transferring the culture from inoculating the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH into a tuberculosis (TB) liquid culture medium containing 100 mg/L of ampicillin (Amp + ), performing shaking culture for a period of time, then adding 0.4 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG), and continuing with the culture; and after induced expression ends, centrifugally collecting cells, to obtain the recombinant E. coli as engineered bacteria; wherein the LB solid plate culture medium comprises the following components: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride, and 15.0 g/L of agar; the LB liquid culture medium comprises the following components: 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride; and the TB liquid culture medium comprises the following components: 12 g/L of tryptone, 24 g/L of yeast extract, 5 g/L of glycerol, 9.4 g/L of dipotassium phosphate, and 2.2 g/L of monopotassium phosphate; and wherein the recombinant E. coli is adapted for producing ursodeoxycholic acid (UDCA) using 7-oxo-lithocholic acid with a conversion rate of greater than 99%.

Description

This application claims priority to Chinese Patent Application No. CN 202210285129.6, filed on Mar. 11, 2022, which is incorporated by reference for all purposes as if fully set forth herein. A Sequence Listing XML file named “10025_0099.xml” created on Feb. 22, 2026, and having a size of 5,215 bytes, is filed concurrently with the specification. The sequence listing contained in the XML file is part of the specification and is herein incorporated by reference in its entirety. TECHNICAL FIELD The present invention belongs to the field of bioengineering technologies, and in particular, to a production method of recombinant Escherichia coli (E. coli) and high-purity ursodeoxycholic acid (UDCA). BACKGROUND As a traditional Chinese medicine active ingredient, UDCA (3a,7β-dihydroxy-5β-cholan-24-oic acid) has found very wide clinical application and is of excellent pharmaceutical value. UDCA produces an excellent therapeutic effect in treating gallstones, facilitated liver transplantation, bile reflux gastritis, alcoholic liver, biliary cirrhosis, and drug-induced hepatitis, leading to high market demand. At present, UDCA is mainly prepared from a natural source and through synthesis. The natural source is bear gallbladders or bile extracted from live bears that are protected by animal protection laws, and an extraction source is limited, causing a gradual decline in the source of natural bear gallbladders. In the synthesis, UDCA is chemically synthesized using chenodeoxycholic acid (CDCA) extracted from readily available cow or goose bile, and 7-OH undergoes a conformational change by using an oxidation-reduction method. However, there are a series of problems such as a complex reaction process, low selectivity, stringent reaction conditions, high energy consumption, and serious contamination. Especially, toxic and hazardous reagents are required during protection and deprotection, which severely limits the industrial application of chemical methods. At present, UDCA produced using chemical methods accounts for a market share of approximately 30% and has a relatively low purity of approximately 80%, which is far from satisfying the requirements for the usage and quality of UDCA in the market. Compared with chemical epimerization, the biological synthesis of UDCA is efficient and relatively environmentally friendly. Microbial transformation or biological enzyme catalysis mainly involves the expansion of 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). Clostridium limosum, Clostridium absonum, Clostridium pasteurianum, and Xanthomonas maltophilia that produce 7α-HSDH and 7β-HSDH are used to implement the biological transformation from CDCA into UDCA. However, high-concentration CDCA inhibits the accumulation of cell biomass, making it difficult to recycle and purify products. In addition, previous researches show that as the culture time elapses, the yield of intermediates increases, and UDCA decreases, making it impossible to implement industrial production. In recent years, the joint application of 7α-HSDH and 7β-HSDH to generate UDCA with CDCA as a substrate by using a two-step method becomes a research hotspot. At present, a comprehensive transformation rate that has been researched can exceed 90%. However, a reaction amount is only at a milliliter level and is still far from industrial production. The major limitative bottlenecks are as follows: 1) A key enzyme 7β-HSDH in the two-step method with CDCA as a substrate is extremely unstable. The enzyme is active enough, but quickly becomes inactive in a catalysis reaction system.2) The reaction system in the two-step method can carry an excessively low substrate amount (with a volume ratio of 1%), and the system is unstable.3) Two key enzymes in the two-step method face the problem of large-scale sources in industrial application.4) The costs of separating and purifying enzyme protein are excessively high. SUMMARY To resolve the foregoing problems in the prior art, the present invention provides a production method of high-purity UDCA, constructs engineered bacteria co-expressing multiple enzymes, and implements efficient production of UDCA. The present invention provides recombinant engineered bacteria that can produce UDCA at low costs. In addition, the present invention is intended to resolve the technical problems of strain construction and application. To achieve the foregoing objectives, the following technical solutions are adopted in the present invention: A first objective of the present invention is to provide recombinant E. coli that can produce UDCA at low costs. The recombinant E. coli may simultaneously express two enzymes, which are respectively 7β-HSDH and glucose dehydrogenase (GDH). The recombinant E. coli has been deposited in the China Center for Type Culture Collection on Dec. 27, 2021. The deposition number thereof is CCTCC NO: M20211644. Preferably, the 7β-HSDH comes from clostridium absonum, and the