CN122012425A

CN122012425A - 11α-hydroxylase of steroidal compounds and its uses

Info

Publication number: CN122012425A
Application number: CN202610449801.9A
Authority: CN
Inventors: 常尊学; 隋靓; 张祎; 孟欣达
Original assignee: Shenyang Botai Biopharmaceutical Co ltd
Current assignee: Shenyang Botai Biopharmaceutical Co ltd
Priority date: 2026-04-08
Filing date: 2026-04-08
Publication date: 2026-05-12

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.

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 important foundation for realizing the industrialized application of the steroid 11 alpha-hydroxylase.

The steroid 11 alpha-hydroxylase used in the present invention is derived from CYP509C12 of the fungus Rhizopus oryzae. CYP509C12 is one of the few steroid 11 alpha-hydroxylases reported at present, but previous researchers only verify the basic expression characteristics of the enzyme in eukaryotic hosts, and have not been reported in public about the expression of the enzyme in escherichia coli and the application of the enzyme in steroid conversion. The invention successfully modifies CYP509C12 by genetic engineering means to ensure that the CYP509C12 has the expression capability in a prokaryotic host, and further improves the activity and the reaction efficiency of 11 alpha-hydroxylase by constructing a co-expression vector of the modified CYP509C12 and cytochrome P450 reductase taking escherichia coli or mycobacterium as the host.

The coding genes of the steroid 11 alpha-hydroxylase and the cytochrome P450 reductase ATR2 can be expressed together by adopting the same or different promoters, and are preferably carried out in a mode of constructing a bicistronic expression vector. The term "bicistronic expression vector" is defined in the present invention consistent with its commonly accepted and adhered to definitions in the art. The bicistronic expression vector disclosed by the invention refers to a design that the coding genes of the steroid 11 alpha-hydroxylase and the cytochrome P450 reductase ATR2 share one promoter and each gene is preceded by a Ribosome Binding Site (RBS) so as to ensure efficient translation initiation, and the design enables two genes to be transcribed and translated simultaneously in a host cell. The steroid 11 alpha-hydroxylase used in the present invention belongs to the family of cytochrome P450 oxidase enzymes, which require electrons provided by cytochrome P450 reductase to complete the hydroxylation reaction. The cytochrome P450 reductase used in the present invention is an Arabidopsis-derived cytochrome P450 reductase (ATR 2) which can transfer electrons to the P450 oxidase via NADPH, thereby activating the active center of the enzyme and allowing it to catalyze hydroxylation of a substrate. Through co-expression of the two in the same host cell, the two enzymes can be ensured to exist and act in the cell simultaneously, and the activity and the reaction efficiency of 11 alpha-hydroxylase are synergistically improved, so that the efficient conversion of 11 alpha-hydroxy steroid compounds is realized.

The above object of the present invention is achieved by the following technical solutions:

In a first aspect, the present invention provides a steroid 11 alpha-hydroxylase having an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO. 1.

In a second aspect, the present invention provides a gene encoding a steroid 11 alpha hydroxylase according to the first aspect of the invention.

According to some embodiments of the invention, wherein the nucleotide sequence of the gene encoding the steroid 11 a-hydroxylase consists of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1.

According to some embodiments of the invention, wherein the nucleotide sequence of the gene encoding the steroid 11 a-hydroxylase may consist of the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 9.

In a third aspect, the present invention provides a co-expression vector having steroid 11 a-hydroxylase activity comprising a gene encoding steroid 11 a-hydroxylase and a gene encoding cytochrome P450 reductase according to the second aspect of the invention.

According to some embodiments of the invention, wherein the amino acid sequence of the cytochrome P450 reductase consists of the amino acid sequence shown in SEQ ID No. 3.

According to some embodiments of the invention, 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.

According to some embodiments of the invention, wherein the nucleotide sequence of the gene encoding cytochrome P450 reductase may consist of the nucleotide sequence shown in SEQ ID No. 4 or SEQ ID No. 10.

The present invention is not particularly limited in the manner of introducing and expressing the gene encoding steroid 11 a-hydroxylase and the gene encoding cytochrome P450 reductase according to the present invention, and includes, but is not limited to, introduction by an expression plasmid or genomic insertion, etc.

According to some embodiments of the invention, the gene encoding the steroid 11 alpha hydroxylase and the gene encoding the 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.

According to some embodiments of the invention, wherein the gene encoding the steroid 11 a-hydroxylase and the gene encoding the cytochrome P450 reductase are controlled by the same or different promoters.

According to some embodiments of the invention, wherein the promoter is a eukaryotic promoter or a prokaryotic promoter.

Preferably, the promoter may promote gene expression in E.coli or Mycobacterium.

More preferably, the E.coli expression promoter is selected from one or more of T7 promoter, lac promoter and Tac promoter.

More preferably, the mycobacterial expression promoter is one or both of an Hsp60 and a G13 promoter.

According to some embodiments of the invention, wherein the initial vector of the co-expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

Preferably, the initial vector of the co-expression vector is an E.coli expression vector or a Mycobacterium expression vector.

More preferably, the E.coli expression vector is selected from one or more of pET26b (+), pET28a (+), pET32a (+), pETDuet, pRSFDuet and pCDFDuet plasmids.

More preferably, the mycobacterial expression vector is a pMV261 plasmid.

According to some embodiments of the invention, wherein the nucleotide sequences of the gene encoding steroid 11 a-hydroxylase and the gene encoding cytochrome P450 reductase comprise nucleotide sequences encoding ribosome binding sites, respectively, upstream of each other.

In a fourth aspect, the present invention provides a genetically engineered strain for the preparation of 11 a-hydroxysteroids expressing a steroid 11 a-hydroxylase according to the first aspect of the invention, comprising a gene encoding a steroid 11 a-hydroxylase according to the second aspect of the invention and/or comprising a co-expression vector according to the third aspect of the invention.

The initial strain of the genetically engineered strain according to the fourth aspect of the present invention is not particularly limited, and includes, but is not limited to, strains derived from Streptomyces, bacillus, pseudomonas, corynebacterium, arthrobacter, rhodococcus, mycobacterium, escherichia coli or Saccharomyces, and the like.

According to some embodiments of the invention, wherein the initial strain of the genetically engineered strain is preferably E.coli or Mycobacterium.

More preferably, the initial strain of the E.coli genetically engineered strain is E.coli BL21 (DE 3), E.coli JM109 or E.coli C43 (DE 3), and still more preferably E.coli BL21 (DE 3).

More preferably, the initial strain of the mycobacterium genetically engineered strain is mycobacterium smegmatis, mycobacterium neogold or mycobacterium fortuitum, even more preferably mycobacterium neogold.

In a fifth aspect, the present invention provides a process for preparing 11 a-hydroxysteroids comprising converting a steroid into an 11 a-hydroxysteroid using a steroid 11 a-hydroxylase according to the first aspect of the invention, a gene encoding a steroid 11 a-hydroxylase according to the second aspect of the invention, a co-expression vector according to the third aspect of the invention and/or a genetically engineered strain according to the fourth aspect of the invention.

According to some embodiments of the invention, wherein the transformation is achieved by using the genetically engineered strain according to the fourth aspect of the invention in a growing or resting state in a reaction solution containing a steroid.

According to some embodiments of the invention, wherein the steroid is a steroid drug intermediate, preferably one or more of Androstenedione (AD), androstenedione (ADD), bisnorol (BA), progesterone (PG), and 17 a-hydroxy progesterone.

According to some embodiments of the invention, wherein the 11 alpha-hydroxy steroid is an 11 alpha-hydroxylation product of a steroid 11 alpha-hydroxylase conversion of the steroid intermediate according to the first aspect of the invention, preferably one or more of 11 alpha-hydroxyandrostenedione (11 alpha-OH AD), 11 alpha-hydroxyandradienedione (11 alpha-OH ADD), 11 alpha-hydroxybisnoralcohol (11 alpha-OH BA), 11 alpha-hydroxy progesterone (11 alpha-OH PG) and 11 alpha, 17 alpha-dihydroxyprogesterone.

According to the research of the invention, the removal of the N-terminal hydrophobic region amino acid fragment of the steroid 11 alpha-hydroxylase CYP509C12 from the fungus rhizopus oryzae can improve the expression capacity of the amino acid fragment in a prokaryotic host, and meanwhile, the cytochrome P450 reductase of arabidopsis thaliana is further used as an electron transfer protein to be cloned together with the rhizopus oryzae steroid 11 alpha-hydroxylase with the N-terminal truncation on the same bicistronic expression vector, so that the two are expressed together in escherichia coli or mycobacterium, and a high-efficiency hydroxylase system is formed. The hydroxylase system can be suitable for 11 alpha-hydroxyl conversion of various steroid substrates, has strong specificity, the highest conversion rate can reach 90.39%, and the production technology is simple, efficient and controllable, and has important industrial application value.

In a sixth aspect, the present invention provides the use of a steroid 11 a-hydroxylase according to the first aspect of the invention, a gene encoding a steroid 11 a-hydroxylase according to the second aspect of the invention, a co-expression vector according to the third aspect of the invention and/or a genetically engineered strain according to the fourth aspect of the invention in the preparation of 11 a-hydroxysteroids by transformation of a steroid.

According to some embodiments of the invention, 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 corresponding steroid is selected from one or more of 11 a-hydroxy androstenedione (11 a-OH AD), 11 a-hydroxy androstenedione (11 a-OH ADD), 11 a-hydroxy bisnoralcohol (11 a-OH BA), 11 a-hydroxy progesterone (11 a-OH PG), and 11 a, 17 a-dihydroxy progesterone.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a plasmid map of E.coli bicistronic co-expression vector pET26b-11AOH-C1-ATR2 prepared in example 1;

FIG. 2 shows the HPLC detection result of the E.coli engineering strain in example 2 when converting AD substrate 48 h;

FIG. 3 shows a schematic representation of the 11 alpha hydroxylation of AD substrates by an E.coli engineered strain in example 2 or by a Mycobacterium engineered strain in example 4, wherein 11AOH-C1-ATR2 represents a co-expressed steroid 11 alpha-hydroxylase and cytochrome P450 reductase;

FIG. 4 shows the HPLC detection result of the E.coli engineering strain in example 2 when converting ADD substrate 48 h;

FIG. 5 shows a schematic representation of the 11 alpha hydroxylation of an ADD substrate by an E.coli engineered strain in example 2 or by a Mycobacterium engineered strain in example 4, wherein 11AOH-C1-ATR2 represents a co-expressed steroid 11 alpha-hydroxylase and cytochrome P450 reductase;

FIG. 6 shows the HPLC detection result of the E.coli engineering strain in example 2 when converting PG substrate 48 h;

FIG. 7 shows a schematic representation of the 11. Alpha. -hydroxylation reaction of the E.coli engineered strain in example 2 or the Mycobacterium engineered strain in example 4 on PG substrates, wherein 11AOH-C1-ATR2 represents the co-expressed steroid 11. Alpha. -hydroxylase and cytochrome P450 reductase;

FIG. 8 is a plasmid map of the Mycobacterium bicistronic co-expression vector pMV261-11AOH-C1-ATR2 prepared in example 3;

FIG. 9 shows the HPLC detection result of Mycobacterium engineering strain in example 4 when converting AD substrate 72 h;

FIG. 10 shows the HPLC detection result of Mycobacterium engineering strain in example 4 when converting ADD substrate 72 h;

FIG. 11 shows the HPLC detection results of Mycobacterium engineering strain in example 4 when converting PG substrate 72 h.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.

The gene operation technology adopted by the invention is mainly gene expression technology, the molecular biology operation method is carried out according to the manual of molecular cloning laboratories, and other experimental methods without specific steps and conditions are carried out according to the conventional steps and conditions or the suggestions of manufacturers.

The following are the experimental materials used in the examples:

The expression host mycobacterium new gold MN-L01 strain is preserved in China general microbiological culture Collection center (CGMCC) for 1 month and 23 days in 2019, the preservation unit address is the institute of microbiological study, china academy of sciences, no.1 and 3 of the West way, north Star, the Korean area of Beijing, and the preservation number is CGMCC No.17227, and the strain is a mutant strain of mycobacterium new gold DSM44074 and mainly accumulates AD.

Cloning host E.coli DH5 alpha, expression host E.coli BL21 (DE 3), E.coli expression plasmid pET26b (+) and mycobacterium expression plasmid pMV261 were purchased from Wuhan vast Biotechnology Inc., molecular biological reagents were purchased from Simer Feichi Tech Co., and Nanjinovirginia Biotechnology Co., ltd, and gene and primer synthesis was completed by Suzhou Jin Weizhi Biotechnology Co., ltd.

LB liquid medium, peptone 10.0 g/L, yeast powder 5.0 g/L and NaCl 10.0 g/L;

LB solid culture medium, peptone 10.0 g/L, yeast powder 5.0 g/L, naCl 10.0 g/L and agar powder 15.0 g/L;

resting cell reaction liquid, na ₂HPO₄ 12.8 g/L,KH₂PO₄ 3.0.0 g/L, naCl 0.5 g/L and glucose 4.0 g/L;

NA solid culture medium, peptone 10.0 g/L, beef powder 1.0 g/L, naCl 5.0 g/L, agar powder 15.0 g/L, and pH value adjusted to 7.2-7.4;

BPYG seed culture medium, beef extract 0.3 g/L, peptone 1.0 g/L, yeast powder 0.3 g/L, glycerol 1.5 g/L, and adjusting pH to 7.0;

The transformation fermentation medium comprises 3.0 g/L soybean peptone, 1.2 g/L yeast extract, 1.2 g/L glucose, 0.2 g/L citric acid and 0.03 g/L,K₂HPO₄ 1.2 g/L,MgSO₄·7H₂O 0.05 g/L,NH₄NO₃ 0.3 g/L, ferric ammonium citrate, and the pH is adjusted to 7.0.

The rest experimental materials are conventional biochemical reagents, and are purchased by a marketing company.

EXAMPLE 1 construction of E.coli Strain coexpressing steroid 11 alpha-hydroxylase and cytochrome P450 reductase

1. Total synthesis of steroid 11 alpha-hydroxylase (11 AOH-C1) gene

The fungal steroid 11 alpha-hydroxylase used is derived from the fungus Rhizopus oryzae, a protein known as CYP509C12 (locus tag: RO3G 05077.1). Structural analysis of this found that the first 49 amino acids of CYP509C12 are hydrophobic transmembrane domains. This stretch of amino acid sequence is anchored to the endoplasmic reticulum membrane in fungi, but since the prokaryotic system is absent from the endoplasmic reticulum, it is instead prone to misfolding, aggregation or degradation of the protein. In order to achieve expression of CYP509C12 in a prokaryotic host, the region is deleted and the amino acid sequence of CYP509C12 deleted from the region is designated 11AOH-C1,11AOH-C1 as shown in SEQ ID NO. 1. The amino acid sequence of the 11AOH-C1 is further optimized according to the codon preference of the escherichia coli to obtain a nucleotide sequence, and the nucleotide sequence is shown as SEQ ID NO. 2. The sequencing-by-order company synthesizes the complete 11AOH-C1 gene from the head by means of artificial chemical synthesis, namely the total synthesis of the steroid 11 alpha-hydroxylase (11 AOH-C1) gene.

SEQ ID NO. 1:

MVPKRFQHLPKLSYFPSAKSIFNNEPIYDRYKRLVFPVIKENNGIYVSKISFDWTVYIANPVAAKHVLLKADLYPKSHDFLKMLGSNSPVVQFLGYDSVGLTNGHVWKNQRKLMNPAFHRSMPINTMSTVIPDLFFVIEKENGTIAVPSVMRDFTLDVLGLTVFGFDFKALKGDPDEWTKTFTLANEGLFDPILNIFGPFSFILTAIFPKRREQLKAVAKLNGKLEQLIHQKRMEIENGAYSNTPENEKDLVALMLEAEKRGEGLTNDLELRHNIAGFFLAGHDTTANALSFCFYNLAKNKHVQNKLRQEIISVLGDDPKDVVPTLDQLKEMPYLNLVLKENLRLNGPADNILPRVAAKDMVVDGTFIPKGATVNIDIYGIHHNPKFWNNPDDFIPERLDENGEQDSHDGLTWLPFGNGARQCLGMNFSLTEQRLLLVMMIRKYEIDVPKDSIHYERVIFGSETTPPNSLELTFKKRY

SEQ ID NO. 2:

ATGGTCCCGAAGCGCTTTCAGCATCTGCCGAAGCTGAGTTATTTCCCGAGCGCCAAAAGCATCTTCAACAATGAACCGATTTACGATCGTTATAAGCGTCTTGTGTTTCCGGTGATTAAGGAAAACAATGGCATTTATGTTTCGAAGATCTCCTTCGATTGGACGGTCTATATCGCCAATCCGGTCGCCGCGAAACATGTACTGCTGAAAGCCGATCTTTACCCTAAATCTCACGACTTCCTCAAGATGTTGGGTTCGAACTCTCCGGTCGTACAGTTCCTGGGATATGATTCAGTGGGTCTGACGAATGGTCACGTGTGGAAAAATCAGCGCAAATTAATGAATCCGGCCTTCCACCGCTCTATGCCTATTAATACAATGAGCACCGTGATTCCGGACTTGTTTTTTGTGATTGAAAAAGAGAACGGTACCATTGCCGTGCCGTCAGTTATGCGTGACTTTACCTTAGATGTGTTAGGTTTGACTGTATTTGGGTTTGATTTCAAAGCGCTGAAGGGTGACCCGGATGAATGGACCAAGACCTTTACCCTTGCGAATGAAGGCCTGTTCGACCCGATCCTGAATATTTTTGGCCCTTTCAGCTTTATTCTGACCGCCATCTTTCCGAAACGCCGCGAGCAATTAAAAGCGGTTGCGAAACTGAACGGCAAGCTGGAACAGCTGATCCATCAAAAACGCATGGAGATCGAAAATGGCGCCTACTCGAATACGCCTGAAAACGAAAAAGACCTGGTTGCACTGATGCTGGAGGCGGAAAAACGCGGCGAGGGTTTGACTAATGACCTTGAACTCCGCCATAACATCGCAGGCTTCTTTTTGGCCGGTCACGATACAACTGCCAACGCCCTGAGCTTCTGCTTCTACAACCTGGCGAAAAATAAACATGTCCAGAACAAATTACGTCAAGAAATCATTAGCGTGTTGGGGGATGACCCAAAAGATGTTGTTCCGACCCTGGACCAACTCAAAGAAATGCCGTACCTGAACTTGGTATTGAAAGAGAATCTGCGCCTGAACGGCCCTGCAGATAACATTCTGCCGCGCGTCGCGGCGAAAGATATGGTGGTCGATGGTACCTTTATTCCGAAAGGTGCTACGGTTAATATCGACATCTACGGTATCCATCACAATCCGAAATTCTGGAATAACCCCGACGACTTCATTCCGGAACGCTTAGACGAAAATGGCGAGCAGGATTCGCATGATGGCCTGACTTGGCTGCCGTTTGGCAATGGCGCCCGCCAGTGTCTTGGCATGAACTTTTCGCTCACTGAACAACGTTTACTGTTAGTGATGATGATTCGCAAATACGAAATCGACGTACCTAAGGACTCAATCCACTATGAACGCGTGATCTTTGGCAGCGAAACCACCCCGCCCAACTCCCTTGAACTGACCTTTAAAAAGCGCTACTAA

2. Construction of pET26b-11AOH-C1 intermediate plasmid

A. PCR amplification was performed using the plasmid pUC57-11AOH-C1 (11 AOH-C1 synthetic gene carrying plasmid returned by sequencing Co, optimized for E.coli codon preference) as a template and 11AOH-C1-F1/11AOH-C1-R1 as primers, wherein the PCR reaction conditions were 98℃pre-denaturation for 30 s,98℃denaturation for 10 s,60℃annealing for 10 s,72℃extension for 75 s,30 cycles, and 72℃full extension for 5 min. And (5) agarose gel electrophoresis and gel cutting recovery are carried out on the PCR product.

The primer sequences for 11AOH-C1 (E.coli codon optimized sequence) were as follows:

11AOH-C1-F1:

AAGAAGGAGATATACATATGGTCCCGAAGCGCTTTCAG(SEQ ID NO. 5)

11AOH-C1-R1:

TCGAGTGCGGCCGCAAGCTTTTAGTAGCGCTTTTTAAAGG(SEQ ID NO. 6)

B. The pET26b (+) plasmid was digested with NdeI/HindIII, and the product was subjected to agarose gel electrophoresis and cut for recovery. The recovered plasmid enzyme cleavage product and the PCR product of 11AOH-C1 are connected by using a kit (CloneExpress II One Step Cloning Kit), the connection product is transformed into DH5 alpha competence of the escherichia coli, the mixture is coated on LB solid medium containing 50 mug/mL kanamycin after resuscitating, positive transformant is picked up to extract plasmid, and intermediate plasmid pET26b-11AOH-C1 is obtained.

3. Total synthesis of cytochrome P450 reductase (ATR 2) gene

The gene name of cytochrome P450 reductase is ATR2 (accession number: NP-194750.1), which is a protein having an electron-transporting function derived from Arabidopsis thaliana (Arabidopsis thaliana). 11AOH-C1 and ATR2 were co-expressed in the form of a constructed bicistronic expression vector due to the lack of electron transfer systems within the prokaryotic system. The amino acid sequence of ATR2 (shown as SEQ ID NO. 3) is optimized according to the codon preference of the escherichia coli to obtain a nucleotide sequence (shown as SEQ ID NO. 4), and the sequencing company is entrusted to fully synthesize the ATR2 gene.

SEQ ID NO. 3:

MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW

SEQ ID NO. 4:

ATGAGTAGCTCCTCATCCTCATCAACCTCAATGATTGACCTGATGGCAGCAATCATCAAAGGTGAACCGGTCATTGTAAGTGACCCAGCCAACGCGAGCGCTTACGAGAGCGTCGCGGCGGAACTGAGCAGCATGCTGATTGAAAACCGCCAGTTTGCAATGATTGTCACCACCAGCATCGCAGTTCTTATCGGGTGTATTGTGATGTTGGTGTGGCGTCGCAGCGGTAGCGGCAACTCAAAACGCGTAGAACCTTTGAAACCGCTCGTGATTAAACCTCGCGAGGAAGAAATCGATGATGGCCGCAAAAAAGTTACCATCTTTTTTGGCACCCAGACGGGTACCGCGGAAGGCTTCGCGAAAGCCCTCGGCGAAGAGGCCAAGGCGCGCTATGAAAAAACGCGCTTTAAGATCGTGGATCTGGATGACTACGCTGCAGATGACGATGAATACGAAGAAAAGCTGAAGAAAGAAGATGTAGCGTTCTTTTTTCTGGCGACTTATGGGGATGGCGAACCGACCGATAACGCAGCGCGCTTCTACAAATGGTTCACAGAAGGGAATGACCGCGGTGAGTGGCTGAAGAACCTGAAATATGGTGTGTTTGGTCTTGGAAATCGTCAGTATGAGCATTTCAACAAGGTTGCTAAAGTGGTGGATGACATCCTGGTTGAACAGGGCGCGCAGCGTCTGGTCCAAGTAGGCCTCGGCGATGATGATCAGTGCATTGAGGACGACTTCACTGCATGGCGCGAGGCTCTGTGGCCAGAATTGGACACCATCCTGCGTGAAGAAGGTGATACCGCTGTAGCTACCCCGTATACCGCTGCAGTGCTGGAATATCGCGTAAGCATTCACGACTCCGAAGACGCAAAATTTAATGATATTAACATGGCAAACGGCAACGGCTACACTGTGTTTGACGCACAGCACCCATATAAGGCGAATGTAGCCGTGAAACGTGAATTACACACCCCGGAATCAGACCGCAGCTGTATCCATCTGGAATTCGATATTGCCGGGAGTGGTCTGACCTATGAAACCGGCGACCATGTTGGCGTGCTCTGTGATAACCTGAGCGAAACCGTCGATGAGGCCCTCCGCCTGCTTGATATGTCGCCGGATACGTATTTCAGTTTGCACGCCGAAAAAGAGGACGGAACGCCGATTTCTAGTAGCCTGCCTCCGCCATTTCCGCCGTGTAATCTGCGTACCGCGCTGACTCGCTATGCCTGCTTGTTATCCAGCCCTAAGAAATCTGCCCTCGTAGCATTAGCTGCGCACGCCAGCGATCCGACAGAAGCAGAGCGTCTGAAACACCTGGCCTCCCCTGCCGGCAAGGACGAGTATAGTAAGTGGGTAGTAGAATCGCAGCGCTCCCTGTTGGAAGTGATGGCTGAATTTCCGTCCGCAAAACCACCGCTGGGTGTTTTCTTCGCCGGCGTGGCCCCTCGCTTACAGCCGCGTTTTTACAGCATTAGCTCCAGCCCGAAAATTGCTGAGACCCGCATTCATGTGACGTGCGCCCTGGTTTACGAAAAAATGCCGACAGGCCGCATTCATAAGGGAGTATGCTCTACCTGGATGAAAAATGCAGTGCCGTACGAAAAGAGCGAGAACTGTAGTAGCGCGCCGATCTTTGTGCGCCAGAGTAATTTTAAGTTGCCGTCAGACTCCAAAGTCCCAATTATCATGATCGGTCCAGGTACCGGCCTGGCGCCGTTTCGCGGCTTTCTGCAGGAACGTTTAGCTCTGGTCGAAAGCGGCGTTGAACTGGGGCCGAGTGTTCTGTTCTTCGGGTGCCGCAACCGCCGCATGGATTTTATTTACGAGGAAGAACTGCAGCGTTTTGTCGAATCCGGCGCTCTGGCCGAACTCTCCGTTGCATTTAGCCGTGAAGGCCCTACGAAAGAGTACGTCCAACATAAAATGATGGATAAAGCGAGCGATATCTGGAACATGATCTCACAGGGCGCGTATCTTTACGTGTGTGGAGACGCGAAAGGCATGGCTCGTGATGTTCACCGCTCACTCCACACCATTGCGCAGGAGCAGGGCTCAATGGATAGCACCAAGGCGGAAGGTTTCGTGAAAAACTTACAGACTTCAGGGCGCTATCTGCGCGATGTGTGGTAA

4. ATR2 with Ribosome Binding Site (RBS) obtained by PCR amplification

PCR amplification was performed using plasmid pUC57-ATR2 (ATR 2 synthetic gene-carrying plasmid returned by sequencing Co, optimized for E.coli codon preference) as a template and ATR2-F1/ATR2-R1 as primers, wherein the PCR reaction conditions were 98℃pre-denaturation for 30s, 98℃denaturation for 10s, 60℃annealing for 10s, 72℃extension for 2min, 30 cycles, 72℃full extension for 5 min.

The primer sequences for ATR2 (e.coli codon optimised sequence) are as follows:

ATR2-F1:

TTAAAAAGCGCTACTAAGCTTAAGGAGATATAAATATGAGTAGCTCCTCATCC(SEQ ID NO. 7)

ATR2-R1:

GTGGTGGTGGTGGTGCTCGAGTTACCACACATCGCGCAG (SEQ ID NO. 8)

RBS sequence (AAGGAGA) was added to the above primer ATR2-F1 to allow expression of 11AOH-C1 and ATR2 in a bicistronic mode.

5. Construction of a bicistronic Co-expression vector pET26b-11AOH-C1-ATR2

PET26b-11AOH-C1 intermediate plasmid was digested with HindIII/Xho I, and the product was subjected to agarose gel electrophoresis and cut for recovery. The recovered plasmid cleavage product and the PCR product of ATR2 in step 4 were ligated using a kit (CloneExpress II One Step Cloning Kit), the ligation product was transformed into E.coli DH 5. Alpha. Competence, and after resuscitating, it was spread on LB solid medium containing 50. Mu.g/mL kanamycin, and positive transformants were picked up to extract the plasmid and sent to sequencing, which showed that it was identical to the theoretical nucleotide sequence, thereby successfully constructing the bicistronic co-expression vector pET26b-11AOH-C1-ATR2 (see FIG. 1).

6. The plasmid pET26b-11AOH-C1-ATR2 is transformed into the competent of the escherichia coli BL21 (DE 3), and is coated on an LB solid culture medium containing 50 mug/mL kanamycin after resuscitating, so that a positive transformant is the escherichia coli engineering strain for coexpression of steroid 11 alpha-hydroxylase and cytochrome P450 reductase.

EXAMPLE 2 transformation application of E.coli engineering Strain to various steroid Compounds

1. Single colonies of the E.coli engineering strain prepared in example 1 were picked up and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃for 16 h to obtain seed liquid.

2. The seed solution obtained was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin at an inoculum size of 1%, and cultured until the logarithmic phase, 0.5 mM final concentration of inducer IPTG and 0.6 mM of 5-aminolevulinic acid (5-ALA) were added, 24h was induced at 25℃and then cells were collected by centrifugation at 10min at 3500 rpm at 4 ℃.

3. The cells were resuspended in an equal volume of resting cell reaction solution, and the substrates androstenedione and androstenedione were added at a final concentration of 50 mg/L and the substrate progesterone at a final concentration of 100 mg/L, respectively, and reacted at 28℃for 48 h.

4. 500. Mu.L of the reaction mixture was extracted twice with diploid ethyl acetate, and the supernatants were combined and dried. The sample was reconstituted with 200. Mu.L of chromatographic grade methanol and the product was quantitatively analyzed by HPLC with a chromatography column AGILENT ECLIPSE XDB-C18 (4.6X1250 mm,5 μm) at a detection wavelength of 241 nm and a column temperature of 40 ℃. HPLC employed gradient elution.

The chromatographic conditions of the converted samples for detecting AD and ADD are specifically acetonitrile (B) -water (A) as a mobile phase, the flow rate is 1 mL/min, the sample injection amount is 10 mu L, the pre-equilibrium time is 5 min, and the running time is 20 min. The gradient condition of the mobile phase is 32-65% of B,0-15 min, 65-90% of B and 15-20 min.

The chromatographic conditions of the conversion sample for detecting PG are specifically mobile phase methanol (B) -water (A), the flow rate is 1 mL/min, the sample injection amount is 10 mu L, the pre-equilibrium time is 5min, and the running time is 25 min. The gradient condition of the mobile phase is 40% -90% B,0-20 min, 90% B,20-25 min.

5. Experimental results

(1) AD substrate conversion results

After the engineering bacteria converts the AD substrate 48 h, the HPLC analysis result (see FIG. 2) shows that the final concentration of 11 alpha-OH AD in the reaction solution is 4.10 mg/L. The conversion rate of the engineering strain to the AD substrate is calculated to be 7.78 percent. The specific transformation process is shown in FIG. 3.

(2) ADD substrate conversion results:

After the engineering bacteria converts ADD substrate 48 h, the HPLC analysis result (see FIG. 4) shows that the final concentration of 11 alpha-OH ADD in the reaction solution is 3.71: 3.71 mg/L. The conversion rate of the engineering strain to the ADD substrate is calculated to be 7.02%. The specific transformation process is shown in FIG. 5.

(3) PG substrate conversion results:

After converting the engineering bacteria into PG substrate 48 h, the HPLC analysis result (see FIG. 6) shows that the final concentration of 11 alpha-OH PG in the reaction solution is 57.66 mg/L. The conversion rate of the engineering strain to PG substrate is 54.86 percent through calculation. The specific reaction process is shown in FIG. 7.

EXAMPLE 3 construction of Mycobacterium Strain coexpression of steroid 11 alpha-hydroxylase and cytochrome P450 reductase

1. Total synthesis of steroid 11 alpha-hydroxylase (11 AOH-C1) gene

The amino acid sequence of the 11AOH-C1 is further optimized according to the codon preference of mycobacterium to obtain a nucleotide sequence shown in SEQ ID NO. 9. The sequencing-by-order company synthesizes the complete 11AOH-C1 gene from the head by means of artificial chemical synthesis, namely the total synthesis of the steroid 11 alpha-hydroxylase (11 AOH-C1) gene.

SEQ ID NO. 9:

ATGGTGCCGAAGCGCTTCCAGCACCTGCCGAAGCTGTCGTACTTCCCGTCGGCCAAGTCGATCTTCAACAACGAGCCGATCTACGACCGCTACAAGCGCCTGGTGTTCCCGGTGATCAAGGAGAACAACGGCATCTACGTCTCGAAGATCAGCTTCGACTGGACCGTGTACATCGCCAACCCGGTGGCCGCCAAGCACGTGCTGCTGAAGGCCGACCTGTACCCGAAGTCGCACGACTTCCTGAAGATGCTGGGTTCGAACTCGCCGGTGGTGCAGTTCCTGGGTTACGACTCGGTGGGTCTGACCAACGGTCACGTCTGGAAGAACCAGCGCAAGCTGATGAACCCGGCCTTCCACCGCTCGATGCCGATCAACACCATGTCGACCGTGATCCCGGACCTGTTCTTCGTGATCGAGAAGGAGAACGGTACCATCGCCGTCCCGTCGGTCATGCGCGACTTCACCCTGGACGTCCTGGGTCTGACCGTGTTCGGTTTCGACTTCAAGGCCCTGAAGGGCGACCCCGACGAGTGGACCAAGACCTTCACCCTGGCCAACGAGGGTCTGTTCGACCCGATCCTGAACATCTTCGGTCCGTTCTCGTTCATCCTGACCGCCATCTTCCCGAAGCGCCGCGAGCAGCTGAAGGCCGTGGCCAAGCTGAACGGTAAGCTGGAGCAGCTGATCCACCAGAAGCGCATGGAGATCGAGAACGGCGCCTACTCGAACACCCCGGAGAACGAGAAGGACCTGGTGGCCCTGATGCTGGAGGCCGAGAAGCGGGGTGAGGGTCTGACCAACGACCTGGAGCTGCGCCACAACATCGCGGGTTTCTTCCTGGCCGGTCACGACACCACCGCCAACGCCCTCAGCTTCTGCTTCTACAACCTGGCCAAGAACAAGCACGTGCAGAACAAGCTGCGCCAGGAGATCATCTCGGTGCTGGGCGACGACCCGAAGGACGTGGTGCCGACCCTGGACCAGCTGAAGGAGATGCCGTACCTGAACCTGGTGCTGAAGGAGAACCTGCGCCTGAACGGTCCGGCCGACAACATCCTGCCGCGCGTCGCCGCCAAGGACATGGTCGTGGACGGTACCTTCATCCCGAAGGGCGCCACCGTGAACATCGACATCTACGGTATCCACCACAACCCCAAGTTCTGGAACAACCCGGACGACTTCATCCCGGAGCGCCTGGACGAGAACGGCGAGCAGGACTCGCACGACGGTCTCACCTGGCTGCCCTTCGGTAACGGTGCCCGCCAGTGCCTCGGTATGAACTTCTCGCTGACCGAGCAGCGCCTGCTGCTGGTGATGATGATCCGCAAGTACGAGATCGACGTGCCGAAGGACTCGATCCACTACGAGCGCGTGATCTTCGGTTCGGAGACCACCCCGCCGAACTCGCTGGAGCTGACCTTCAAGAAGCGCTACTAA

2. Construction of pMV261-11AOH-C1 intermediate plasmid

A. PCR amplification was performed using the plasmid pUC57-11AOH-C1 (11 AOH-C1 synthetic gene carrying plasmid returned by sequencing Co, optimized for mycobacterial codon preference) as a template and 11AOH-C1-F2/11AOH-C1-R2 as primers, wherein the PCR reaction conditions were 98℃pre-denaturation for 30 s,98℃denaturation for 10 s,60℃annealing for 10 s,72℃extension for 75 s,30 cycles, and 72℃full extension for 5 min. And (5) agarose gel electrophoresis and gel cutting recovery are carried out on the PCR product.

The primer sequences for 11AOH-C1 (mycobacterial codon optimized sequence) are as follows:

11AOH-C1-F2:

GGCCAAGACAATTGCGGATCCATGGTGCCGAAGCGCTTCC(SEQ ID NO. 11)

11AOH-C1-R2:

ACATCGATAAGCTTCGAATTCTTAGTAGCGCTTCTTGAAGG(SEQ ID NO. 12)

B. The pMV261 plasmid was subjected to BamHI/EcoRI double digestion, and the product was subjected to agarose gel electrophoresis and cut out for recovery. The recovered plasmid enzyme cleavage product and the PCR product of 11AOH-C1 are connected by using a kit (CloneExpress II One Step Cloning Kit), the connection product is transformed into DH5 alpha competence of the escherichia coli, the mixture is coated on LB solid medium containing 50 mug/mL kanamycin after resuscitating, positive transformant is picked up to extract plasmid, and intermediate plasmid pMV261-11AOH-C1 is obtained.

3. Total synthesis of cytochrome P450 reductase (ATR 2) gene

The amino acid sequence of ATR2 (shown as SEQ ID NO. 3) is optimized according to the codon preference of mycobacteria to obtain a nucleotide sequence (shown as SEQ ID NO. 10), and the sequencing company is entrusted to fully synthesize the ATR2 gene.

SEQ ID NO. 10:

ATGTCGTCGTCGTCGTCCTCGTCGACCTCGATGATCGATCTGATGGCCGCCATCATCAAGGGTGAACCGGTCATCGTCTCGGACCCGGCCAACGCCTCGGCCTACGAATCGGTCGCCGCCGAACTGTCGTCGATGCTGATCGAAAACCGCCAGTTTGCCATGATCGTCACCACCTCCATCGCCGTCCTGATCGGTTGCATCGTCATGCTGGTCTGGCGCCGCTCCGGTTCGGGTAACTCGAAGCGCGTCGAACCGCTGAAGCCGCTGGTCATCAAGCCGCGCGAAGAAGAAATCGACGACGGTCGCAAGAAGGTCACCATCTTTTTCGGCACCCAGACCGGCACCGCCGAAGGTTTTGCGAAGGCCCTGGGCGAAGAAGCCAAGGCCCGCTACGAAAAGACCCGCTTCAAGATCGTGGATCTGGACGATTACGCCGCCGACGACGACGAATACGAAGAGAAGCTGAAGAAGGAGGACGTCGCCTTCTTCTTCCTGGCCACCTACGGCGACGGCGAACCGACCGATAACGCCGCCCGCTTTTACAAGTGGTTCACCGAAGGTAACGATCGCGGCGAATGGCTGAAGAACCTGAAGTACGGCGTCTTTGGTCTGGGTAACCGCCAGTACGAGCACTTTAACAAGGTCGCCAAGGTCGTCGACGATATCCTGGTCGAACAGGGCGCCCAGCGCCTGGTGCAGGTCGGTCTGGGCGACGACGATCAGTGCATCGAAGACGATTTTACCGCCTGGCGCGAAGCCCTGTGGCCGGAACTGGATACCATCCTGCGCGAAGAAGGCGATACCGCCGTCGCCACCCCGTACACCGCCGCCGTCCTGGAATACCGCGTCTCGATCCACGATTCGGAAGATGCCAAGTTCAACGACATCAACATGGCCAACGGTAACGGTTACACCGTCTTTGACGCCCAGCACCCGTACAAGGCCAACGTCGCCGTCAAGCGCGAACTGCACACCCCGGAATCGGATCGCAGCTGCATCCACCTGGAATTTGATATCGCCGGTTCGGGTCTGACCTACGAAACCGGCGATCACGTCGGCGTCCTGTGCGATAACCTGTCGGAAACCGTCGACGAAGCCCTGCGCCTGCTGGATATGTCGCCGGATACCTACTTTTCCCTGCACGCCGAAAAGGAAGACGGCACCCCGATCTCGTCGTCGCTGCCGCCGCCGTTTCCGCCCTGCAACCTGCGCACCGCCCTGACCCGCTACGCCTGCCTGCTGTCGTCGCCGAAGAAGTCGGCCCTGGTCGCCCTGGCCGCCCACGCCTCGGACCCGACCGAAGCCGAACGCCTGAAGCACCTGGCCTCGCCGGCCGGTAAGGACGAATACAGCAAGTGGGTCGTCGAATCGCAGCGCTCGCTGCTGGAAGTCATGGCGGAATTTCCCTCGGCCAAGCCGCCGCTGGGCGTCTTCTTCGCCGGTGTCGCCCCGCGCCTCCAGCCGCGCTTTTACTCGATCTCGTCGTCGCCGAAGATCGCCGAAACCCGCATCCACGTCACCTGCGCCCTGGTCTACGAAAAGATGCCGACCGGTCGCATCCACAAGGGCGTCTGCTCGACCTGGATGAAGAACGCCGTCCCGTACGAAAAGTCCGAAAACTGCTCGTCGGCCCCGATCTTTGTCCGCCAGTCGAACTTTAAGCTGCCGTCGGATTCGAAGGTCCCGATCATCATGATCGGTCCGGGCACCGGCCTGGCCCCGTTTCGCGGTTTTCTCCAGGAGCGCCTGGCCCTGGTCGAATCGGGCGTCGAACTGGGTCCGTCGGTCCTGTTCTTCGGCTGCCGCAACCGCCGCATGGATTTCATCTACGAAGAGGAACTCCAGCGCTTTGTCGAATCGGGTGCCCTGGCGGAACTGTCGGTCGCCTTTTCGCGCGAAGGTCCGACCAAGGAATACGTCCAGCACAAGATGATGGACAAGGCCTCCGATATCTGGAACATGATCTCGCAGGGCGCCTACCTGTACGTCTGCGGTGACGCCAAGGGTATGGCCCGCGACGTCCACCGCTCGCTGCACACCATCGCCCAGGAACAGGGTTCGATGGATTCGACCAAGGCCGAAGGTTTCGTCAAGAACCTCCAGACCTCGGGTCGCTACCTGCGCGACGTCTGGTGA

4. ATR2 with Ribosome Binding Site (RBS) obtained by PCR amplification

PCR amplification was performed using plasmid pUC57-ATR2 (ATR 2 synthetic gene-carrying plasmid returned by sequencing Co, optimized for mycobacterial codon preference) as a template and ATR2-F2/ATR2-R2 as primers, wherein the PCR reaction conditions were 98℃pre-denaturation for 30s, 98℃denaturation for 10s, 60℃annealing for 10s, 72℃extension for 2min, 30 cycles, 72℃full extension for 5 min.

The primer sequences for ATR2 (mycobacterial codon optimized sequence) are as follows:

ATR2-F2:

CAAGAAGCGCTACTAAGAATTCAAGGAGATATAAATATGTCGTCGTCGTCGTCCTC(SEQ ID NO. 13)

ATR2-R2:

CTACGTCGACATCGATAAGCTTTCACCAGACGTCGCGCAG (SEQ ID NO. 14)

RBS sequences were added to the above primers ATR2-F2 to allow expression of 11AOH-C1 and ATR2 in a bicistronic mode.

5. Construction of the bicistronic Co-expression vector pMV261-11AOH-C1-ATR2

The pMV261-11AOH-C1 intermediate plasmid was digested with EcoRI/HindIII, and the product was subjected to agarose gel electrophoresis and cut for recovery. The recovered plasmid cleavage product and the PCR product of ATR2 in step 4 were ligated using a kit (CloneExpress II One Step Cloning Kit), the ligation product was transformed into E.coli DH 5. Alpha. Competence, and after resuscitating, it was spread on LB solid medium containing 50. Mu.g/mL kanamycin, and positive transformant-extracted plasmid was picked up and sent to sequencing, and the sequencing result showed that it was identical to the theoretical nucleotide sequence, thereby successfully constructing the bicistronic co-expression vector pMV261-11AOH-C1-ATR2 (see FIG. 8).

6. Preparation of novel Mycobacterium aureum competent cells and plasmid transformation

The plate activated new gold mycobacterium MN-L01 is inoculated in LB liquid medium of 10 mL with 1 percent of inoculum size for overnight culture at 32 ℃,20 mL of LB liquid medium is added in the morning for the next day for continuous culture until OD600 is about 0.8, the thalli are collected by centrifugation, the thalli are washed twice by 2 times of deionized double distilled water, and finally 10 percent of glycerol is used for suspending the thalli, and each 100 mu L of thalli are split-packed.

Adding the constructed bicistronic co-expression vector pMV261-11AOH-C1-ATR 21 mu L into 100 mu L competent cells, blowing, sucking, mixing uniformly, and adding into a 2mm electric rotating cup for electric conversion under 2500V, 25 mu F and 600 omega.

7. Screening and verification of recombinants

Immediately adding 1 mL precooled LB liquid culture medium into the product after electric conversion, resuscitating for 3-4h at 32 ℃, and coating on NA solid culture medium containing 50 mug/mL kanamycin to obtain positive transformant, namely mycobacterium engineering strain which co-expresses steroid 11 alpha-hydroxylase and cytochrome P450 reductase.

EXAMPLE 4 conversion application of Mycobacterium engineering Strain to various steroid Compounds

1. The single colony of the Mycobacterium engineering strain prepared in example 3 is picked up and sequentially subjected to primary seed culture and secondary seed culture, wherein the single colony is inoculated into a seed culture medium BPYG, the culture is carried out for 3 days at the temperature of 32 ℃ to obtain primary seed liquid, and the primary seed liquid is inoculated into a seed culture medium BPYG at the temperature of 32 ℃ in an inoculum size of 1% to be continuously cultured for 3 days to obtain secondary seed liquid.

2. The obtained secondary seed solution is inoculated into a transformation fermentation medium according to the inoculum size of 5 percent, and substrates androstenedione, androstenedione and progesterone with the final concentration of 200 mg/L are respectively added for transformation at 32 ℃ for 72 h.

3. 500. Mu.L of the reaction mixture was extracted twice with diploid ethyl acetate, and the supernatants were combined and dried. The sample was re-dissolved in 200. Mu.L of chromatographic grade methanol, and the product was quantitatively analyzed by HPLC, and the specific method was the same as the quantitative analysis method of the transformed product of E.coli engineering strain in example 2.

4. Experimental results

(1) AD substrate conversion results

After the engineering bacteria converts the AD substrate 72 h, the HPLC analysis result (see FIG. 9) shows that the final concentration of 11 alpha-OH AD in the reaction solution is 139.20 mg/L. The conversion rate of the engineering strain to the AD substrate is 65.91 percent. The specific transformation process is shown in FIG. 3.

(2) ADD substrate conversion results:

After converting the engineering bacteria into the ADD substrate 72 h, the HPLC analysis result (see FIG. 10) shows that the final concentration of 11 alpha-OH ADD in the reaction solution is 61.88 mg/L. The conversion of the engineering strain to the ADD substrate was calculated to be 29.29%. The specific transformation process is shown in FIG. 5.

(3) PG substrate conversion results:

After converting the PG substrate 72 h by engineering bacteria, the HPLC analysis result (see FIG. 11) shows that the final concentration of 11 alpha-OH PG in the reaction solution is 189.99 mg/L. The conversion rate of the engineering strain to PG substrate is calculated to be 90.39%. The specific reaction process is shown in FIG. 7.

Comparative example 1

11AOH-C1 was combined with reported cytochrome P450 reductase TcCPR (accession number: AVW 80114.1), roCPR1 (accession number: EIE 89541.1) and ClCPR1 (accession number: ABW 86977.1), respectively, of other fungal origin, the amino acid sequence identity of the three cytochrome P450 reductases to ATR2 was 34.06%, 36.49% and 34.30%, respectively. A bicistronic co-expression vector and an E.coli expression strain were constructed as in example 1 and substrate transformation assays were performed as in example 2. The detection results show that no obvious products are generated when the AD substrate and the ADD substrate are converted by each expression strain, the conversion rate is reduced although the products are generated by the conversion PG substrate, specifically, the final concentrations of the products of the conversion PG by the expression strains of 11AOH-C1 matched with TcCPR, roCPR1 and ClCPR are 46.84 mg/L, 19.27 mg/L and 14.63 mg/L, and the conversion rates are 44.59%, 18.36% and 13.92% respectively.

Comparative example 2

Original CYP509C12 with the first 49 amino acids not deleted and Arabidopsis ATR2 (accession number: NP-194750.1) were subjected to substrate conversion detection in accordance with the method of example 1 to construct a bicistronic co-expression vector and an E.coli expression strain. The results of the tests showed that the final concentrations of the products of the expressed strains transformed AD, ADD and PG were 3.25 mg/L, 2.78 mg/L and 48.02 mg/L, respectively, and the conversion rates were 6.16%, 5.26% and 45.69%, respectively.

Comparative example 3

11AOH-C1 was combined with the reported cytochrome P450 reductase TcCPR, roCPR1 and ClCPR, respectively, of other fungal origin, a bicistronic co-expression vector and a Mycobacterium expression strain were constructed as in example 3 and substrate transformation assays were performed as in example 4. The detection results show that although the conversion rates of the AD, ADD and PG substrates are reduced, the final concentration of the AD converted by the 11AOH-C1 with TcCPR, roCPR1 and ClCPR1 is 120.85 mg/L, 90.52 mg/L and 86.04 mg/L respectively, the conversion rates are 57.22%, 42.86% and 40.74% respectively, the final concentration of the ADD converted by the 11AOH-C1 with TcCPR, roCPR1 and ClCPR1 is 50.23 mg/L, 38.44 mg/L and 38.56 mg/L respectively, the conversion rates are 23.78%, 18.19% and 18.25% respectively, the final concentration of the PG converted by the 11AOH-C1 with CPR, roCPR1 and ClCPR1 is 170.66 mg/L, 134.28 mg/L and 125.06 mg/L respectively, and the conversion rates are 81.19%, 63.88% and 59.50% respectively.

Comparative example 4

Original CYP509C12 with the first 49 amino acids not deleted and Arabidopsis ATR2 were constructed as in example 3 to give a bicistronic co-expression vector and a Mycobacterium expression strain and substrate conversion detection was performed as in example 4. The results of the tests showed that the final concentrations of the products of the expressed strains transformed AD, ADD and PG were 128.01 mg/L, 53.26 mg/L and 178.50 mg/L, respectively, and the conversion rates were 60.61%, 25.21% and 84.92%, respectively.

From the above data, it is understood that the E.coli engineering strain provided in example 1 of the present invention has 11. Alpha. -hydroxylation to progesterone only, compared to the E.coli expression strain provided in comparative example 1, which has 11. Alpha. -hydroxylation to various steroid compounds including androstenedione, androstenedione and progesterone, the E.coli engineering strain provided in example 1 of the present invention has higher conversion product concentration and conversion rate, and the conversion rate to progesterone is up to 54.86% when converting steroid compounds such as androstenedione, androstenedione and progesterone, compared to the E.coli expression strain provided in comparative example 2.

Compared with the mycobacterium expression strains provided in comparative examples 3 and 4, the mycobacterium engineering strain provided in the embodiment 3 of the invention has higher concentration and conversion rate of conversion products, and has higher industrial production value for progesterone, wherein the conversion rate of conversion products can reach 90.39% when converting steroid compounds such as androstenedione, progesterone and the like.

Further, the capability of the mycobacterium engineering strain constructed by the invention for converting various steroid compounds including androstenedione, androstenedione and progesterone is better than that of the escherichia coli engineering strain constructed by the invention.

Although various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are presented for purposes of illustration only and not limitation. The scope and spirit of the invention are to be determined only by the appended claims.

Claims

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. A gene encoding the steroid 11 a-hydroxylase of claim 1.

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. 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. 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. 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. 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. 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. The co-expression vector of claim 8, wherein the promoter is a eukaryotic promoter or a prokaryotic promoter.

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. 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. 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. 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. The genetically engineered strain of claim 13, wherein the original strain of the genetically engineered strain is escherichia coli or mycobacterium.

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. 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. 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. 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. 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. 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.