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CN-122012425-A - Steroid 11 alpha-hydroxylase and use thereof

CN122012425ACN 122012425 ACN122012425 ACN 122012425ACN-122012425-A

Abstract

The invention provides steroid 11 alpha-hydroxylase and application thereof. The co-expression vector provided by the invention can simultaneously express the steroid 11 alpha-hydroxylase and the cytochrome P450 reductase in escherichia coli or mycobacterium, and the genetic engineering strain containing the co-expression vector can be suitable for 11 alpha-hydroxyl conversion of various steroid substrates, has strong specificity, has the highest conversion rate of 90.39%, and has the advantages of simple, efficient and controllable production technology and important industrial application value.

Inventors

  • CHANG ZUNXUE
  • SUI JING
  • ZHANG DAI
  • Meng Xinda

Assignees

  • 沈阳博泰生物制药有限公司

Dates

Publication Date
20260512
Application Date
20260408

Claims (20)

  1. 1. A steroid 11 alpha-hydroxylase, characterized in that the amino acid sequence thereof consists of the amino acid sequence shown in SEQ ID NO. 1.
  2. 2. A gene encoding the steroid 11 a-hydroxylase of claim 1.
  3. 3. The gene encoding a steroid 11 a-hydroxylase according to claim 2, characterized in that its nucleotide sequence consists of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1.
  4. 4. A co-expression vector having steroid 11 a-hydroxylase activity, characterized in that it comprises a gene encoding steroid 11 a-hydroxylase and a gene encoding cytochrome P450 reductase according to claim 2 or 3.
  5. 5. The co-expression vector of claim 4, wherein the amino acid sequence of the cytochrome P450 reductase consists of the amino acid sequence shown in SEQ ID No. 3.
  6. 6. The co-expression vector according to claim 4, wherein the nucleotide sequence of the gene encoding cytochrome P450 reductase consists of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 3.
  7. 7. The co-expression vector according to claim 4, wherein the gene encoding steroid 11. Alpha. -hydroxylase and the gene encoding cytochrome P450 reductase are integrated on the chromosome of the host bacterium or on an episomal plasmid located outside the chromosome of the host bacterium.
  8. 8. The co-expression vector according to claim 4, wherein the gene encoding steroid 11. Alpha. -hydroxylase and the gene encoding cytochrome P450 reductase are controlled by the same or different promoters.
  9. 9. The co-expression vector of claim 8, wherein the promoter is a eukaryotic promoter or a prokaryotic promoter.
  10. 10. The co-expression vector of claim 4, wherein the initial vector of the co-expression vector is a eukaryotic expression vector or a prokaryotic expression vector.
  11. 11. The co-expression vector according to claim 4, wherein the nucleotide sequences of the gene encoding steroid 11. Alpha. -hydroxylase and the gene encoding cytochrome P450 reductase comprise nucleotide sequences encoding ribosome binding sites, respectively, upstream thereof.
  12. 12. Genetically engineered strain for the preparation of 11 a-hydroxysteroids, characterized in that it expresses a steroid 11 a-hydroxylase according to claim 1, comprises a gene encoding a steroid 11 a-hydroxylase according to claim 2 or 3 and/or comprises a co-expression vector according to any of claims 4-11.
  13. 13. The genetically engineered strain of claim 12, wherein the initial strain of the genetically engineered strain is selected from the group consisting of strains of streptomyces, bacillus, pseudomonas, corynebacterium, arthrobacter, rhodococcus, mycobacterium, escherichia, and saccharomyces.
  14. 14. The genetically engineered strain of claim 13, wherein the original strain of the genetically engineered strain is escherichia coli or mycobacterium.
  15. 15. A process for preparing 11 a-hydroxysteroids, characterized in that it comprises converting a steroid into an 11 a-hydroxysteroid using a steroid 11 a-hydroxylase according to claim 1, a gene encoding a steroid 11 a-hydroxylase according to claim 2 or 3, a co-expression vector according to any one of claims 4-11 and/or a genetically engineered strain according to any one of claims 12-14.
  16. 16. The method according to claim 15, wherein the transformation is achieved by using the genetically engineered strain according to any one of claims 12-14 in a growing or resting state in a reaction solution containing a steroid.
  17. 17. The method of claim 15 or 16, wherein the steroid is a steroid drug intermediate and the 11 a-hydroxy steroid is an 11 a-hydroxylation product of the steroid drug intermediate converted by the steroid 11 a-hydroxylase.
  18. 18. The method according to claim 15 or 16, wherein the steroid is selected from one or more of androstenedione, bisnoralcohol, progesterone and 17 a-hydroxy progesterone, and the 11 a-hydroxylation product of the steroid is selected from one or more of 11 a-hydroxy androstenedione, 11 a-hydroxy bisnoralcohol, 11 a-hydroxy progesterone and 11a, 17 a-bishydroxy progesterone.
  19. 19. Use of a steroid 11 a-hydroxylase according to claim 1, a gene encoding a steroid 11 a-hydroxylase according to claim 2 or 3, a co-expression vector according to any of claims 4-11 and/or a genetically engineered strain according to any of claims 12-14 for the preparation of 11 a-hydroxysteroids by transformation of a steroid.
  20. 20. The use according to claim 19, wherein the steroid is selected from one or more of androstenedione, bisnoralcohol, progesterone and 17 a-hydroxy progesterone, and the 11 a-hydroxylation product of the steroid is selected from one or more of 11 a-hydroxy androstenedione, 11 a-hydroxy bisnoralcohol, 11 a-hydroxy progesterone and 11 a, 17 a-dihydroxy progesterone.

Description

Steroid 11 alpha-hydroxylase and use thereof Technical Field The invention belongs to the technical field of bioconversion, and particularly relates to steroid 11 alpha-hydroxylase and application thereof. Background Steroid drugs are the second most important drugs which are second to antibiotics in clinical application, have various therapeutic actions such as anti-inflammatory, immunosuppressive, antithrombotic, antitumor, anticoagulant and antishock, and occupy a huge share in the modern pharmaceutical industry. Currently, the main modes for preparing steroid drugs include traditional chemical synthesis methods and microbial transformation methods. Among them, the microbial transformation method is receiving increasingly wide attention by virtue of the remarkable advantages of specific and efficient reaction, low cost, mild condition, safety, environmental protection and the like, and the attention is also increasing. The hydroxyl group is an essential group for various steroid drugs to exert their physiological activities, and the 11 alpha-hydroxylated steroid compound is an important corticosteroid drug intermediate, and has an irreplaceable effect in drug synthesis. As early as 1952, murray and Peterson initially utilized Rhizopus arrhizus (Rhizopus arrhizus) and Rhizopus nigrum (Rhizopus nigricans) to transform progesterone, successfully producing 11 alpha-OH progesterone. The achievement not only skillfully solves the hydroxylation problem in the production process of the corticoids, but also obviously reduces the production cost of the subsequent corticoids, and opens up a new way for the production of steroid medicines. However, the technological development of 11 a-hydroxylated steroid drug intermediates prepared by microbial conversion has remained a relatively primitive stage for many years. At present, the whole cell transformation mode of the original fungi or the hydroxylase over-expressed fungi is mainly relied on, and the mode has a plurality of defects. For example, longer conversion cycles result in lower production efficiency, and more byproducts make it difficult to achieve the desired level of product purity. These factors have greatly limited the wide application of this technology in industrial production. The identification and isolation of individual hydroxylases from fungal cells and their functional expression in a suitable heterologous host is an important research direction in which fungal steroid hydroxylases are fully exploited. However, E.coli (ESCHERICHIA COLI), which is the preferred bacterial host for heterologous protein expression, has the advantages of clear genetic background, easy genetic manipulation, rapid propagation, easy achievement of high density and low cost, but does not have a typical intracellular organelle membrane, thus making it difficult for fungal hydroxylases requiring membrane anchoring to fold, localize and form active proteins correctly in E.coli. In addition, there are problems such as lack of compatible electron transfer systems in E.coli, which further cause difficulty in smooth hydroxylation reaction, thereby seriously affecting the activity of 11. Alpha. -hydroxylase, which is a steroid of fungi, and efficient production of 11. Alpha. -hydroxylated steroid. Mycobacteria are used as high-efficiency steroid metabolism bacteria, have a strong steroid transport system and maintain the inner environment of the functionalization of various endogenous steroid metabolism related hydroxylases, are dominant hosts for the hydroxylation of steroid compounds, but have no typical organelle membranes in cells, so that the high-efficiency expression of fungal hydroxylases is difficult to realize. Thus, the core problem to be solved in the art is that the activity of 11 alpha-hydroxylase of the fungal steroid in escherichia coli and mycobacterium is low and efficient heterologous expression is difficult to realize, so that the industrial requirement of efficient production of 11 alpha-hydroxylase cannot be met. Disclosure of Invention Therefore, in view of the shortcomings of the prior art, the invention aims to provide a steroid 11 alpha-hydroxylase, an expression vector and an expression strain thereof and application thereof in preparing 11 alpha-hydroxy steroid. The research result of the invention shows that the steroid 11 alpha-hydroxylase and the bicistronic expression vector thereof provided by the invention realize the functional expression of the fungus steroid 11 alpha-hydroxylase and the hydroxylation of the steroid in escherichia coli and mycobacterium for the first time, break through the dilemma of low activity and difficult heterologous expression of the fungus steroid hydroxylase, not only strengthen the activity of the 11 alpha-hydroxylase, but also find that the steroid 11 alpha-hydroxylase has 11 alpha-hydroxylation effect on various steroid compounds, and the conversion rate can reach 90.39 percent at the highest, thereby laying an impo