Steroid compound 11 alpha-hydroxylase and application thereof

Abstract

The invention provides a steroid compound 11 alpha-hydroxylase and application of the steroid compound 11 alpha-hydroxylase. The coexpression vector provided by the invention can simultaneously express the steroid compound 11alpha-hydroxylase and cytochrome P450 reductase in escherichia coli or mycobacteria, a genetic engineering strain containing the coexpression vector can be suitable for 11alpha-hydroxyl conversion of various steroid substrates, the specificity is relatively strong, the highest conversion rate can reach 90.39%, and the coexpression vector can be applied to the conversion of 11alpha-hydroxyl of various steroid substrates. And the production technology is simple, efficient and controllable, and has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biotransformation technology, specifically relating to steroid compound 11α-hydroxylase and its uses. Background Technology

[0002] Steroid drugs are the second largest class of drugs used clinically, after antibiotics. They possess a variety of therapeutic effects, including anti-inflammatory, immunosuppressive, antithrombotic, antitumor, anticoagulant, and anti-shock effects, and occupy a significant share in the modern pharmaceutical industry. Currently, the main methods for preparing steroid drugs include traditional chemical synthesis and microbial transformation. Among these, microbial transformation is receiving increasing attention due to its significant advantages, such as specific and efficient reactions, low cost, mild conditions, and safety and environmental friendliness.

[0003] Hydroxyl groups are essential for the physiological activity of many steroid drugs, and 11α-hydroxylated steroid compounds play an irreplaceable role in drug synthesis as important intermediates for corticosteroids. As early as 1952, Murray and Peterson pioneered the use of *Rhizopus arrhizus* and *Rhizopus nigricans* to convert progesterone, successfully generating 11α-OH progesterone. This achievement not only ingeniously solved the hydroxylation problem in corticosteroid production but also significantly reduced the subsequent production cost of corticosteroids, opening a new path for the production of steroid drugs.

[0004] However, the development of microbial transformation techniques for preparing 11α-hydroxylated steroidal drug intermediates has remained relatively primitive for many years. Currently, it mainly relies on whole-cell transformation using native fungi or fungi overexpressing hydroxylase, but this method has many drawbacks. For example, the transformation cycle is long, leading to low production efficiency; at the same time, there are many byproducts, making it difficult to achieve ideal product purity. These factors greatly limit the widespread application of this technology in industrial production.

[0005] Identifying and isolating independent hydroxylases from fungal cells and functionally expressing them in suitable heterologous hosts is an important research direction for the full utilization of fungal steroid hydroxylases. However, while *Escherichia coli*, the preferred bacterial host for heterologous protein expression, has advantages such as a clear genetic background, ease of genetic manipulation, rapid reproduction, high density, and low cost, its cells lack typical organelle membranes. This makes it difficult for membrane-anchored fungal hydroxylases to fold, locate, and form active proteins correctly in *E. coli*. Furthermore, *E. coli* lacks a compatible electron transport system, further hindering the hydroxylation reaction and severely impacting the activity of fungal steroid compound 11α-hydroxylases and the efficient production of 11α-hydroxylated steroid compounds. Mycobacteria, as highly efficient steroid metabolizers, possess powerful steroid transport systems and an internal environment that maintains the functionalization of various endogenous steroid metabolism-related hydroxylases, making them advantageous hosts for steroid compound hydroxylation. However, their cells also lack typical organelle membranes, thus making it difficult to achieve efficient expression of fungal hydroxylases.

[0006] Therefore, the core problem that urgently needs to be solved in this field is that the fungal steroid compound 11α-hydroxylase has low activity in Escherichia coli and mycobacteria and is difficult to achieve efficient heterologous expression, thus failing to meet the industrial demand for efficient production of 11α-hydroxylated steroid compounds. Summary of the Invention

[0007] Therefore, in view of the shortcomings of the prior art, the purpose of this invention is to provide a steroid compound 11α-hydroxylase, its expression vector and expression strain, and their use in the preparation of 11α-hydroxysteroid compounds.

[0008] The results of this invention demonstrate that the steroid compound 11α-hydroxylase and its bicistronic expression vector provided by this invention have, for the first time, achieved functional expression of fungal steroid compound 11α-hydroxylase and hydroxylation of steroid compounds in Escherichia coli and Mycobacterium. This breakthrough overcomes the difficulties of low activity and heterologous expression of fungal steroid compound hydroxylases. It not only enhances the activity of the 11α-hydroxylase but also reveals that it has 11α-hydroxylation effects on a variety of steroid compounds, with a conversion rate as high as 90.39%. This lays an important foundation for the industrial application of steroid compound 11α-hydroxylase.

[0009] The steroidal compound 11α-hydroxylase used in this invention is derived from CYP509C12 of the fungus *Rhizopus oryzae*. CYP509C12 is one of the few reported steroidal compound 11α-hydroxylases, but previous researchers only verified its basic expression characteristics in eukaryotic hosts; there are no publicly available reports on its expression in *Escherichia coli* or its application in steroidal transformation. This invention successfully modified CYP509C12 using genetic engineering techniques to enable its expression in prokaryotic hosts. Furthermore, by constructing a co-expression vector of the modified CYP509C12 and cytochrome P450 reductase using *Escherichia coli* or mycobacteria as hosts, the activity and reaction efficiency of the 11α-hydroxylase were further improved.

[0010] The encoding genes for steroid compound 11α-hydroxylase and cytochrome P450 reductase ATR2 of the present invention can be co-expressed using the same or different promoters, preferably by constructing a bicistronic expression vector. The definition of the term "bicistronic expression vector" in this invention is consistent with its generally accepted and followed definition in the art. The bicistronic expression vector described in this invention refers to a vector that shares a single promoter with the encoding genes for steroid compound 11α-hydroxylase and cytochrome P450 reductase ATR2 of the present invention, and each gene has a ribosome binding site (RBS) preceding it to ensure efficient translation initiation. This design allows both genes to be transcribed and translated simultaneously in the host cell. The steroid compound 11α-hydroxylase used in this invention belongs to the cytochrome P450 oxidase family and requires electrons provided by cytochrome P450 reductase to complete the hydroxylation reaction. The cytochrome P450 reductase used in this invention is cytochrome P450 reductase (ATR2) derived from Arabidopsis thaliana. It can transfer electrons to P450 oxidase via NADPH, thereby activating the active site of the enzyme and enabling it to catalyze the hydroxylation reaction of the substrate. By co-expressing both enzymes in the same host cell, it is ensured that both enzymes coexist and function within the cell, synergistically enhancing the activity and reaction efficiency of 11α-hydroxylase, thus achieving highly efficient conversion of 11α-hydroxysteroid compounds.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] In a first aspect, the present invention provides a steroidal compound 11α-hydroxylase, the amino acid sequence of which consists of the amino acid sequence shown in SEQ ID NO. 1.

[0013] In a second aspect, the present invention provides a gene encoding the steroid compound 11α-hydroxylase according to the first aspect of the present invention.

[0014] According to some embodiments of the present invention, the nucleotide sequence of the gene encoding the steroid compound 11α-hydroxylase consists of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.

[0015] According to some embodiments of the present invention, the nucleotide sequence of the gene encoding the steroid compound 11α-hydroxylase may consist of the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[0016] Thirdly, the present invention provides a co-expression vector having steroid compound 11α-hydroxylase activity, which comprises the gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase as described in the second aspect of the present invention.

[0017] According to some embodiments of the present invention, the amino acid sequence of the cytochrome P450 reductase consists of the amino acid sequence shown in SEQ ID NO. 3.

[0018] According to some embodiments of the present invention, 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.

[0019] According to some embodiments of the present invention, 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.

[0020] The present invention does not impose any particular limitation on the manner in which the gene encoding the steroid compound 11α-hydroxylase and the gene encoding the cytochrome P450 reductase are introduced and expressed according to the present invention, including but not limited to introduction via expression plasmids or genomic insertion.

[0021] According to some embodiments of the present invention, the gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase are integrated into the chromosome of the host bacterium or located on a free plasmid outside the chromosome of the host bacterium.

[0022] According to some embodiments of the present invention, the gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase are controlled by the same or different promoters.

[0023] According to some embodiments of the present invention, the promoter is a eukaryotic promoter or a prokaryotic promoter.

[0024] Preferably, the promoter can initiate gene expression in Escherichia coli or mycobacteria.

[0025] More preferably, the E. coli expression promoter is selected from one or more of the T7 promoter, Lac promoter, and Tac promoter.

[0026] More preferably, the mycobacterial expression promoter is one or both of the Hsp60 and G13 promoters.

[0027] According to some embodiments of the present invention, the initial vector of the co-expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0028] Preferably, the initial vector for the co-expression vector is an Escherichia coli expression vector or a mycobacterial expression vector.

[0029] More preferably, the Escherichia coli expression vector is selected from one or more of the following plasmids: pET26b (+), pET28a (+), pET32a (+), pETDuet, pRSFDuet, and pCDFDuet.

[0030] More preferably, the mycobacterial expression vector is the pMV261 plasmid.

[0031] According to some embodiments of the present invention, the upstream nucleotide sequences of the gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase respectively contain nucleotide sequences encoding ribosome binding sites.

[0032] Fourthly, the present invention provides a genetically engineered strain for preparing 11α-hydroxysteroid compounds, which expresses the steroid compound 11α-hydroxylase according to the first aspect of the present invention, a gene encoding the steroid compound 11α-hydroxylase according to the second aspect of the present invention, and / or a co-expression vector according to the third aspect of the present invention.

[0033] The initial strain of the genetically engineered strain described in 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 yeast.

[0034] According to some embodiments of the present invention, the initial strain of the genetically engineered strain is preferably Escherichia coli or Mycobacterium.

[0035] More preferably, the initial strain of the genetically engineered Escherichia coli strain is Escherichia coli BL21 (DE3), Escherichia coli JM109 or Escherichia coli C43 (DE3), and even more preferably Escherichia coli BL21 (DE3).

[0036] More preferably, the initial strain of the mycobacterial genetically engineered strain is Mycobacterium smegmatis, Mycobacterium virginianum, or Mycobacterium sporadicum, and even more preferably Mycobacterium virginianum.

[0037] Fifthly, the present invention provides a method for preparing 11α-hydroxysteroid compounds, comprising using a steroid compound 11α-hydroxylase according to the first aspect of the present invention, a gene encoding the steroid compound 11α-hydroxylase according to the second aspect of the present invention, a co-expression vector according to the third aspect of the present invention, and / or a genetically engineered strain according to the fourth aspect of the present invention to convert a steroid compound into an 11α-hydroxysteroid compound.

[0038] According to some embodiments of the present invention, the transformation is carried out in a reaction solution containing steroidal compounds using genetically engineered strains of the present invention in a growing or resting state, as described in the fourth aspect of the invention.

[0039] According to some embodiments of the present invention, the steroidal compound is a steroidal drug intermediate, preferably one or more of androstenedione (AD), androstenedione (ADD), bis(2-hydroxyprogesterone) (BA), progesterone (PG), and 17α-hydroxyprogesterone.

[0040] According to some embodiments of the present invention, the 11α-hydroxysteroid compound is an 11α-hydroxylated product generated by converting a steroidal drug intermediate through the 11α-hydroxylase of the steroidal compound described in the first aspect of the present invention; preferably one or more of 11α-hydroxyandrostenedione (11α-OH AD), 11α-hydroxyandrostenedione (11α-OH ADD), 11α-hydroxydihydroxyprogesterone (11α-OH BA), 11α-hydroxyprogesterone (11α-OH PG), and 11α,17α-dihydroxyprogesterone.

[0041] This invention unexpectedly discovered that removing the N-terminal hydrophobic region of the steroid compound 11α-hydroxylase CYP509C12 from the fungus *Rhizopus oryzae* enhances its expression in prokaryotic hosts. Furthermore, by co-cloning *Arabidopsis thaliana* cytochrome P450 reductase, as an electron transport protein, with the N-terminally truncated *Rhizopus oryzae* steroid 11α-hydroxylase into the same bicistronic expression vector, both were co-expressed in *Escherichia coli* or mycobacteria, thus forming a highly efficient hydroxylase system. This hydroxylase system is applicable to the 11α-hydroxyl conversion of various steroid substrates, exhibiting high specificity and a conversion rate of up to 90.39%. Moreover, the production technology is simple, efficient, and controllable, possessing significant industrial application value.

[0042] In a sixth aspect, the present invention provides the use of the steroid compound 11α-hydroxylase according to the first aspect of the present invention, the gene encoding the steroid compound 11α-hydroxylase according to the second aspect of the present invention, the co-expression vector according to the third aspect of the present invention, and / or the genetically engineered strain according to the fourth aspect of the present invention in the preparation of 11α-hydroxysteroid compounds by transforming steroid compounds.

[0043] According to some embodiments of the present invention, the steroidal compound is selected from one or more of androstenedione, androsadienedione, dihydroxyprogesterone, progesterone, and 17α-hydroxyprogesterone, and the corresponding 11α-hydroxylated product of the steroidal compound is selected from one or more of 11α-hydroxyandrostenedione (11α-OH AD), 11α-hydroxyandrosadienedione (11α-OH ADD), 11α-hydroxydihydroxyprogesterone (11α-OH BA), 11α-hydroxyprogesterone (11α-OH PG), and 11α,17α-dihydroxyprogesterone. Attached Figure Description

[0044] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0045] Figure 1 The image shows the plasmid of the Escherichia coli bicistronic co-expression vector pET26b-11AOH-C1-ATR2 prepared in Example 1.

[0046] Figure 2 The HPLC detection results of the engineered Escherichia coli strain in Example 2 after 48 h of transformation with AD substrate are shown.

[0047] Figure 3 The diagram illustrates the process of 11α-hydroxylation of AD substrate by the engineered strain of Escherichia coli in Example 2 or the engineered strain of Mycobacterium in Example 4, where 11AOH-C1-ATR2 represents the co-expressed steroid compound 11α-hydroxylase and cytochrome P450 reductase.

[0048] Figure 4 The HPLC results of the engineered Escherichia coli strain in Example 2 after 48 h of transformation with ADD substrate are presented.

[0049] Figure 5 The diagram illustrates the process of 11α-hydroxylation of ADD substrate by the engineered strain of Escherichia coli in Example 2 or the engineered strain of Mycobacterium in Example 4, where 11AOH-C1-ATR2 represents the co-expressed steroid compound 11α-hydroxylase and cytochrome P450 reductase.

[0050] Figure 6The HPLC results of the engineered Escherichia coli strain in Example 2 after 48 h of transformation of PG substrate are presented.

[0051] Figure 7 The diagram illustrates the 11α-hydroxylation reaction of the PG substrate by the engineered Escherichia coli strain in Example 2 or the engineered Mycobacterium strain in Example 4, where 11AOH-C1-ATR2 represents the co-expressed steroid compound 11α-hydroxylase and cytochrome P450 reductase.

[0052] Figure 8 The image shows the plasmid of the bicistronic co-expression vector pMV261-11AOH-C1-ATR2 for mycobacteria prepared in Example 3.

[0053] Figure 9 The HPLC detection results of the engineered mycobacterial strain in Example 4 after 72 h of transformation of AD substrate are shown.

[0054] Figure 10 The HPLC results of the engineered mycobacterial strain in Example 4 after 72 h of transformation with ADD substrate are presented.

[0055] Figure 11 The HPLC results of the engineered mycobacterium strain in Example 4 after 72 h of transformation of PG substrate are presented. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0057] The gene manipulation techniques used in this invention are mainly gene expression techniques. Molecular biology manipulation methods are performed in accordance with the "Molecular Cloning Laboratory Manual". Other experimental methods without specific steps and conditions are performed in accordance with conventional steps and conditions or the manufacturer's recommendations.

[0058] The following are the experimental materials used in the examples:

[0059] The expression host, Mycobacterium neogoldensis strain MN-L01, was deposited on January 23, 2019, at the China General Biotechnology Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 17227. This strain is a mutant of Mycobacterium neogoldensis DSM44074 and mainly accumulates AD.

[0060] The cloning host *Escherichia coli* DH5α, the expression host *Escherichia coli* BL21(DE3), the *Escherichia coli* expression plasmid pET26b(+), and the mycobacterial expression plasmid pMV261 were purchased from Wuhan Miaoling Biotechnology Co., Ltd.; molecular biology reagents were purchased from Thermo Fisher Scientific and Nanjing Novizan Biotechnology Co., Ltd.; and the synthesis of genes and primers was completed by Suzhou Genewise Biotechnology Co., Ltd.

[0061] LB liquid medium: peptone 10.0 g / L, yeast extract 5.0 g / L and NaCl 10.0 g / L;

[0062] LB solid medium: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L and agar powder 15.0 g / L;

[0063] Resting cell reaction solution: Na2HPO4 12.8 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, glucose 4.0 g / L;

[0064] NA solid medium: peptone 10.0 g / L, beef meal 1.0 g / L, NaCl 5.0 g / L, agar powder 15.0 g / L, adjust pH to 7.2-7.4;

[0065] BPYG seed culture medium: beef extract 0.3 g / L, peptone 1.0 g / L, yeast extract 0.3 g / L, glycerol 1.5 g / L, pH adjusted to 7.0;

[0066] Transformation fermentation medium: soybean peptone 3.0 g / L, yeast extract 1.2 g / L, glucose 1.2 g / L, citric acid 0.2 g / L, ferric ammonium citrate 0.03 g / L, K2HPO4 1.2 g / L, MgSO4·7H2O 0.05 g / L, NH4NO3 0.3 g / L, pH adjusted to 7.0.

[0067] All other experimental materials were standard biochemical reagents, purchased from the sales company.

[0068] Example 1: Construction of an Escherichia coli strain co-expressing steroid compound 11α-hydroxylase and cytochrome P450 reductase

[0069] 1. Total synthesis of steroid compound 11α-hydroxylase (11AOH-C1) gene

[0070] The fungal steroid compound 11α-hydroxylase used was derived from the fungus *Rhizopus oryzae*, specifically the protein CYP509C12 (locus tag: RO3G_05077.1). Structural analysis revealed that the first 49 amino acids of CYP509C12 constitute a hydrophobic transmembrane region. This amino acid sequence anchors to the endoplasmic reticulum membrane in fungi; however, due to the absence of the endoplasmic reticulum in prokaryotes, it can easily lead to protein misfolding, aggregation, or degradation. To achieve expression of CYP509C12 in a prokaryotic host, this region was deleted, and the resulting CYP509C12 was named 11AOH-C1, the amino acid sequence of which is shown in SEQ ID NO. 1. Further optimization of the 11AOH-C1 amino acid sequence according to the codon bias of *E. coli* yielded the nucleotide sequence shown in SEQ ID NO. 2. The commissioned sequencing company synthesized the complete 11AOH-C1 gene de novo through artificial chemical synthesis, namely the fully synthesized steroid compound 11α-hydroxylase (11AOH-C1) gene.

[0071] SEQ ID NO. 1:

[0072] MVPKRFQHLPKLSYFPSAKSIFNNEPIYDRYKRLVFPVIKENNGIYVSKISFDWTVYIANPVAAKHVLLKADLYPKSHDFLKMLGSNSPVVQFLGYDSVGLTNGHVWKNQRKLMNPAFH RSMPINTMSTVIPDLFFVIEKENGTIAVPSVMRDFTLDVLGLTVFGFDFKALKGDPDEWTKTFTLANEGLFDPILNIFGPFSFILTAIFPKRREQLKAVAKLNGKLEQLIHQKRMEIENG AYSNTPENEKDLVALMLEAEKRGEGLTNDLELRHNIAGFFLAGHDTTANALSFCFYNLAKNKHVQNKLRQEIISVLGDDPKDVVPTLDQLKEMPYLNLVLKENLRLNGPADNILPRVAA KDMVVDGTFIPKGATVNIDIYGIHHNPKFWNNPDDFIPERLDENGEQDSHDGLTWLPFGNGARQCLGMNFSLTEQRLLLVMMIRKYEIDVPKDSIHYERVIFGSETTPPNSLELTFKKRY

[0073] SEQ ID NO. 2:

[0074]

[0075] 2. Construction of pET26b-11AOH-C1 intermediate plasmid

[0076] A. PCR amplification was performed using plasmid pUC57-11AOH-C1 (a 11AOH-C1 synthetic gene carrier plasmid optimized according to E. coli codon preferences returned by the sequencing company) as a template and 11AOH-C1-F1 / 11AOH-C1-R1 as primers. 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, followed by a final extension at 72℃ for 5 min. The PCR products were subjected to agarose gel electrophoresis and recovered by gel excision.

[0077] The primer sequences for 11AOH-C1 (optimized codon sequence for E. coli) are as follows:

[0078] 11AOH-C1-F1:

[0079] AAAGAAGGAGATATACATATGGTCCCGAAGGCCTTCAG (SEQ ID NO. 5)

[0080] 11AOH-C1-R1:

[0081] TCGAGTGCGGCCGCAAGCTTTTAGTAGCCGCTTTTTAAAGG (SEQ ID NO. 6)

[0082] B. The pET26b(+) plasmid was double-digested with Nde I / Hind III, and the products were subjected to agarose gel electrophoresis and recovered. Using a CloneExpress II One Step Cloning Kit, the recovered plasmid digestion products and the PCR product of 11AOH-C1 were ligated, and the ligation product was transformed into *E. coli* DH5α competent cells. After recovery, the cells were plated on LB agar containing 50 μg / mL kanamycin, and positive transformants were picked to extract the plasmid, obtaining the intermediate plasmid pET26b-11AOH-C1.

[0083] 3. Total synthesis of cytochrome P450 reductase (ATR2) gene

[0084] The gene for cytochrome P450 reductase, ATR2 (accession number: NP_194750.1), is an electron transport protein derived from Arabidopsis thaliana. Since the prokaryotic system lacks an electron transport system, 11AOH-C1 and ATR2 were co-expressed in a bicistronic expression vector. The amino acid sequence of ATR2 (as shown in SEQ ID NO. 3) was optimized according to the codon preference of *E. coli* to obtain the nucleotide sequence (as shown in SEQ ID NO. 4), and the ATR2 gene was fully synthesized by a sequencing company.

[0085] SEQ ID NO. 3:

[0086] MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNA ARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVL CDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAV PYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW

[0087] SEQ ID NO. 4:

[0088]

[0089] 4. Obtain ATR2 with ribosome binding sites (RBS) by PCR amplification.

[0090] Using plasmid pUC57-ATR2 (an ATR2 synthetic gene carrier plasmid optimized according to E. coli codon preference returned by the sequencing company) as a template, PCR amplification was performed using ATR2-F1 / ATR2-R1 primers. The PCR reaction conditions were: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 2 min, 30 cycles, and a final extension at 72℃ for 5 min.

[0091] The primer sequences for ATR2 (E. coli codon optimization sequence) are as follows:

[0092] ATR2-F1:

[0093] TTAAAAGCGCTACTAAGCTTAAGGAGATATAAATATGAGTAGCTCCTCATCC (SEQ ID NO. 7)

[0094] ATR2-R1:

[0095] GTGGTGGTGGTGGTGCTCGAGTTACCACACATCGCGCAG (SEQ ID NO. 8)

[0096] The RBS sequence (AAGGAGA) was added to the primer ATR2-F1 to enable 11AOH-C1 and ATR2 to be expressed in bicistronic mode.

[0097] 5. Construct the bicistronic co-expression vector pET26b-11AOH-C1-ATR2

[0098] The pET26b-11AOH-C1 intermediate plasmid was double-digested with Hind III / Xho I, and the products were subjected to agarose gel electrophoresis and recovered. Using a CloneExpress II One Step Cloning Kit, the recovered plasmid digestion products were ligated with the ATR2 PCR product from step 4. The ligation product was transformed into *E. coli* DH5α competent cells, and after recovery, the cells were plated on LB agar containing 50 μg / mL kanamycin. Positive transformants were picked, plasmids were extracted, and sequenced. Sequencing results showed that the plasmid sequence was consistent with the theoretical nucleotide sequence, thus successfully constructing the bicistronic co-expression vector pET26b-11AOH-C1-ATR2 (see...). Figure 1 ).

[0099] 6. The plasmid pET26b-11AOH-C1-ATR2 was transformed into Escherichia coli BL21(DE3) competent cells. After recovery, the cells were plated on LB solid medium containing 50 μg / mL kanamycin. The positive transformants obtained were the engineered Escherichia coli strains that co-expressed the steroid compound 11α-hydroxylase and cytochrome P450 reductase.

[0100] Example 2: Application of engineered Escherichia coli strains in the transformation of various steroidal compounds

[0101] 1. Select a single colony of the engineered Escherichia coli strain prepared in Example 1 and inoculate it into LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C for 16 h to obtain seed culture.

[0102] 2. The obtained seed culture was inoculated into LB liquid medium containing 50 μg / mL kanamycin at an inoculation rate of 1%. When the culture reached the logarithmic growth phase, IPTG inducer at a final concentration of 0.5 mM and 5-aminolevulinic acid (5-ALA) were added. After induction at 25℃ for 24 h, the cells were collected by centrifugation at 3500 rpm for 10 min at 4℃.

[0103] 3. Resuspend the bacterial cells in an equal volume of resting cell reaction solution, and add the substrates androstenedione and androstenedione to a final concentration of 50 mg / L and the substrate progesterone to a final concentration of 100 mg / L, respectively. React at 28°C for 48 h.

[0104] 4. Take 500 μL of the reaction solution and extract twice with two volumes of ethyl acetate. Combine the supernatants and dry them. Reconstitute the sample with 200 μL of chromatographic grade methanol. Quantitative analysis of the product was performed by HPLC using an Agilent Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm), a detection wavelength of 241 nm, and a column temperature of 40℃. Gradient elution was used in HPLC.

[0105] The chromatographic conditions for detecting the converted samples of AD and ADD were as follows: mobile phase acetonitrile (B)-water (A), flow rate 1 mL / min, injection volume 10 μL, pre-equilibration time 5 min, and run time 20 min. The mobile phase gradient conditions were 32%-65% B for 0-15 min; and 65%-90% B for 15-20 min.

[0106] The specific chromatographic conditions for detecting PG in converted samples were as follows: mobile phase methanol (B)-water (A), flow rate 1 mL / min, injection volume 10 μL, pre-equilibration time 5 min, and run time 25 min. The mobile phase gradient conditions were 40%-90% B, 0-20 min; 90% B, 20-25 min.

[0107] 5. Experimental Results

[0108] (1) AD substrate transformation results

[0109] HPLC analysis results after 48 h of transformation of AD substrate by engineered bacteria (see...) Figure 2 The final concentration of 11α-OH AD in the reaction solution was 4.10 mg / L. Based on this, the conversion rate of the engineered strain to the AD substrate was calculated to be 7.78%. The specific conversion process is detailed below. Figure 3 .

[0110] (2) ADD substrate transformation results:

[0111] HPLC analysis results were obtained 48 h after the engineered bacteria transformed the ADD substrate (see [link]). Figure 4 The final concentration of 11α-OH ADD in the reaction solution was 3.71 mg / L. Based on this, the conversion rate of the engineered strain to the ADD substrate was calculated to be 7.02%. The specific conversion process is detailed below. Figure 5 .

[0112] (3) PG substrate transformation results:

[0113] HPLC analysis results after 48 h of engineered bacteria transforming PG substrate (see [link to HPLC analysis]). Figure 6 The final concentration of 11α-OH PG in the reaction solution was 57.66 mg / L. The conversion rate of the engineered strain to the PG substrate was calculated to be 54.86%. The detailed reaction process is shown in [link to reaction procedure]. Figure 7 .

[0114] Example 3 Construction of a mycobacterial strain co-expressing steroid compound 11α-hydroxylase and cytochrome P450 reductase

[0115] 1. Total synthesis of steroid compound 11α-hydroxylase (11AOH-C1) gene

[0116] The amino acid sequence of 11AOH-C1 was further optimized according to the codon preference of mycobacteria to obtain the nucleotide sequence, which is shown in SEQ ID NO. 9. The sequencing company was commissioned to synthesize the complete 11AOH-C1 gene de novo through artificial chemical synthesis, namely the fully synthesized steroid compound 11α-hydroxylase (11AOH-C1) gene.

[0117] SEQ ID NO. 9:

[0118]

[0119] 2. Construction of pMV261-11AOH-C1 intermediate plasmid

[0120] A. PCR amplification was performed using plasmid pUC57-11AOH-C1 (a 11AOH-C1 synthetic gene carrier plasmid optimized according to mycobacterial codon preferences returned by the sequencing company) as a template and 11AOH-C1-F2 / 11AOH-C1-R2 as primers. 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, followed by a final extension at 72℃ for 5 min. The PCR products were subjected to agarose gel electrophoresis and recovered from the gel.

[0121] The primer sequences for 11AOH-C1 (mycobacterial codon optimized sequence) are as follows:

[0122] 11AOH-C1-F2:

[0123] GGCCAAGACAATTGCGGATCCATGGTGCCGAAGCGCTTCC (SEQ ID NO. 11)

[0124] 11AOH-C1-R2:

[0125] ACATCGATAAGCTTCGAATTCTTAGTAGCGCTTCTTGAAGG (SEQ ID NO. 12)

[0126] B. The pMV261 plasmid was double-digested with BamHI / EcoRI, and the products were subjected to agarose gel electrophoresis and recovered. Using a CloneExpress II One Step Cloning Kit, the recovered plasmid digestion products were ligated with the PCR product of 11AOH-C1, and the ligation product was transformed into *E. coli* DH5α competent cells. After recovery, the cells were plated on LB agar containing 50 μg / mL kanamycin, and positive transformants were picked to extract the plasmid, obtaining the intermediate plasmid pMV261-11AOH-C1.

[0127] 3. Total synthesis of cytochrome P450 reductase (ATR2) gene

[0128] The amino acid sequence of ATR2 (as shown in SEQ ID NO. 3) was optimized according to the codon preference of mycobacteria to obtain the nucleotide sequence (as shown in SEQ ID NO. 10), and the ATR2 gene was fully synthesized by a sequencing company.

[0129] SEQ ID NO. 10:

[0130]

[0131] 4. Obtain ATR2 with ribosome binding sites (RBS) by PCR amplification.

[0132] Using plasmid pUC57-ATR2 (an ATR2 synthetic gene carrier plasmid optimized according to mycobacterial codon preferences returned by the sequencing company) as a template, PCR amplification was performed using ATR2-F2 / ATR2-R2 primers. The PCR reaction conditions were: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 2 min, 30 cycles, and a final extension at 72℃ for 5 min.

[0133] The primer sequences for ATR2 (Mycobacterial codon optimization sequence) are as follows:

[0134] ATR2-F2:

[0135] CAAGAAGCGCTACTAAGAATTCAAGGAGATATAAATATGTCGTCGTCGTCGTCCTC (SEQ ID NO.13)

[0136] ATR2-R2:

[0137] CTACGTCGACATCGATAAGCTTTCACCAGACGTCGCGCAG (SEQ ID NO. 14)

[0138] The RBS sequence was added to the primer ATR2-F2 to enable 11AOH-C1 and ATR2 to be expressed in bicistronic mode.

[0139] 5. Construct the bicistronic co-expression vector pMV261-11AOH-C1-ATR2

[0140] The pMV261-11AOH-C1 intermediate plasmid was digested with EcoR I / Hind III, and the products were subjected to agarose gel electrophoresis and recovered. Using a CloneExpress II One Step Cloning Kit, the recovered plasmid digestion products were ligated with the ATR2 PCR product from step 4. The ligation product was transformed into *E. coli* DH5α competent cells, and after recovery, the cells were plated on LB agar containing 50 μg / mL kanamycin. Positive transformants were picked, plasmids were extracted, and sequenced. Sequencing results showed that the sequence was consistent with the theoretical nucleotide sequence, thus successfully constructing the bicistronic co-expression vector pMV261-11AOH-C1-ATR2 (see...). Figure 8 ).

[0141] 6. Preparation of new Mycobacterium goldi competent cells and plasmid transformation

[0142] The plate-activated Mycobacterium MN-L01 was inoculated at a rate of 1% into 10 mL of LB liquid medium and cultured overnight at 32°C. The next morning, 20 mL of LB liquid medium was added and cultured until the OD600 was about 0.8. The bacterial cells were collected by centrifugation, washed twice with 2 volumes of deionized double-distilled water, and finally suspended in 10% glycerol. The cells were then dispensed into 100 μL containers.

[0143] 1 μL of the constructed bicistronic co-expression vector pMV261-11AOH-C1-ATR2 was added to 100 μL of competent cells. After mixing by pipetting and aspiration, the mixture was added to a 2 mm electroporation cuvette for electroporation. Electroporation conditions: 2500 V, 25 μF, 600 Ω.

[0144] 7. Screening and validation of recombinants

[0145] Immediately add 1 mL of pre-cooled LB liquid medium to the electroporation product, revive at 32°C for 3-4 h, and spread on NA solid medium containing 50 μg / mL kanamycin. The positive transformants obtained are the mycobacterial engineered strains that co-express the steroid compound 11α-hydroxylase and cytochrome P450 reductase.

[0146] Example 4: Application of engineered mycobacterial strains in the transformation of various steroidal compounds

[0147] 1. Select single colonies of the mycobacterial engineered strain prepared in Example 3 and perform primary and secondary seed culture sequentially: Inoculate the single colonies into seed culture medium BPYG and culture at 32°C for 3 days to obtain primary seed solution. Transfer the primary seed solution to seed culture medium BPYG at an inoculation rate of 1% and continue to culture at 32°C for 3 days to obtain secondary seed solution.

[0148] 2. The obtained secondary seed culture was inoculated into the transformation fermentation medium at an inoculation rate of 5%, and the substrates androstenedione, androstenedione and progesterone were added to a final concentration of 200 mg / L, respectively. The transformation was carried out at 32℃ for 72 h.

[0149] 3. Take 500 μL of the reaction solution and extract twice with two volumes of ethyl acetate. Combine the supernatants and dry them. Reconstitute the sample with 200 μL of chromatographic grade methanol and perform quantitative analysis of the product using HPLC. The specific method is consistent with the quantitative analysis method of the transformation product of the engineered E. coli strain in Example 2.

[0150] 4. Experimental Results

[0151] (1) AD substrate transformation results

[0152] HPLC analysis results after 72 h of transformation of AD substrate by engineered bacteria (see...) Figure 9 The final concentration of 11α-OH AD in the reaction solution was 139.20 mg / L. Based on this, the conversion rate of the engineered strain to the AD substrate was calculated to be 65.91%. The specific conversion process is detailed below. Figure 3 .

[0153] (2) ADD substrate transformation results:

[0154] HPLC analysis results after 72 h of transformation of ADD substrate by engineered bacteria (see...) Figure 10 The final concentration of 11α-OH ADD in the reaction solution was 61.88 mg / L. Based on this, the conversion rate of the engineered strain to the ADD substrate was calculated to be 29.29%. The specific conversion process is detailed below. Figure 5 .

[0155] (3) PG substrate transformation results:

[0156] HPLC analysis results after 72 h of transformation of PG substrate by engineered bacteria (see...) Figure 11 The final concentration of 11α-OH PG in the reaction solution was 189.99 mg / L. The conversion rate of the engineered strain to the PG substrate was calculated to be 90.39%. The detailed reaction process is shown in [link to reaction procedure]. Figure 7 .

[0157] Comparative Example 1

[0158] 11AOH-C1 was combined with other reported fungal cytochrome P450 reductases, TcCPR (accession number: AVW80114.1), RoCPR1 (accession number: EIE89541.1), and ClCPR1 (accession number: ABW86977.1), respectively. The amino acid sequence identity of these three cytochrome P450 reductases with ATR2 was 34.06%, 36.49%, and 34.30%, respectively. A bicistronic co-expression vector and an *E. coli* expression strain were constructed according to the method in Example 1, and substrate transformation was detected according to the method in Example 2. The results showed that no significant product was generated when the expression strains transformed AD and ADD substrates. Although products were generated when transforming PG substrate, the transformation rates were reduced. Specifically, the final product concentrations of PG transformed by the expression strains 11AOH-C1 with TcCPR, RoCPR1 and ClCPR1 were 46.84 mg / L, 19.27 mg / L and 14.63 mg / L, respectively, with transformation rates of 44.59%, 18.36% and 13.92%, respectively.

[0159] Comparative Example 2

[0160] The original CYP509C12 strain (without deletion of the first 49 amino acids) and Arabidopsis thaliana ATR2 (accession number: NP_194750.1) were used to construct a bicistronic co-expression vector and an Escherichia coli expression strain according to the method in Example 1. Substrate transformation was then performed according to the method in Example 2. The results showed that the final product concentrations after transformation of AD, ADD, and PG by the expression strain were 3.25 mg / L, 2.78 mg / L, and 48.02 mg / L, respectively, with transformation efficiencies of 6.16%, 5.26%, and 45.69%.

[0161] Comparative Example 3

[0162] 11AOH-C1 was combined with other reported fungal cytochrome P450 reductases TcCPR, RoCPR1 and ClCPR1 to construct bicistronic co-expression vectors and mycobacterial expression strains according to the method in Example 3, and substrate transformation was detected according to the method in Example 4. The results showed that although all expression strains produced products upon transformation of AD, ADD, and PG substrates, the transformation rates were all reduced. Specifically, the final product concentrations of AD transformed by the 11AOH-C1 strain combined with TcCPR, RoCPR1, and ClCPR1 were 120.85 mg / L, 90.52 mg / L, and 86.04 mg / L, respectively, with transformation rates of 57.22%, 42.86%, and 40.74%. The final product concentrations of ADD transformed by the 11AOH-C1 strain combined with TcCPR, RoCPR1, and ClCPR1 were 50.23 mg / L, 38.44 mg / L, and 38.56 mg / L, respectively, with transformation rates of 23.78%, 18.19%, and 18.25%. The final product concentrations of PG transformed by the 11AOH-C1 strain combined with TcCPR, RoCPR1, and ClCPR1 were 170.66 mg / L, 134.28 mg / L, and 134.28 mg / L, respectively. The conversion rates were 81.19%, 63.88%, and 59.50% for mg / L and 125.06 mg / L, respectively.

[0163] Comparative Example 4

[0164] The original CYP509C12 strain (without deleting the first 49 amino acids) was combined with Arabidopsis thaliana ATR2 to construct a bicistronic co-expression vector and a mycobacterial expression strain according to the method in Example 3. Substrate transformation was then performed according to the method in Example 4. The results showed that the final product concentrations after transformation of AD, ADD, and PG by the expression strain were 128.01 mg / L, 53.26 mg / L, and 178.50 mg / L, respectively, with transformation efficiencies of 60.61%, 25.21%, and 84.92%.

[0165] The data above show that, compared to the *E. coli* expression strain provided in Comparative Example 1, which only exhibits 11α-hydroxylation of progesterone, the *E. coli* engineered strain provided in Example 1 of this invention exhibits 11α-hydroxylation of various steroidal compounds, including androstenedione, androsadienedione, and progesterone. Furthermore, compared to the *E. coli* expression strain provided in Comparative Example 2, the *E. coli* engineered strain provided in Example 1 of this invention has higher concentrations and conversion rates of conversion products when transforming steroidal compounds such as androstenedione, androsadienedione, and progesterone, with a conversion rate of up to 54.86% for progesterone.

[0166] Compared with the mycobacterial expression strains provided in Comparative Examples 3 and 4, the mycobacterial engineered strain provided in Example 3 of this invention has higher concentration and conversion rate of transformation products when transforming steroidal compounds such as androstenedione, androstenedione and progesterone. The conversion rate of progesterone can reach 90.39%, which has high industrial production value.

[0167] Further comparison shows that the mycobacterial engineered strain constructed in this invention has a better ability to transform various steroidal compounds, including androstenedione, and progesterone, than the Escherichia coli engineered strain constructed in this invention.

[0168] While various aspects and embodiments of the invention have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and spirit of the invention are determined solely by the appended claims.

Claims

1. A steroid compound 11α-hydroxylase, characterized in that, Its amino acid sequence consists of the amino acid sequence shown in SEQ ID NO.

1.

2. A gene encoding the 11α-hydroxylase of the steroid compound of claim 1.

3. The gene encoding 11α-hydroxylase of steroidal compounds 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 possessing steroid compound 11α-hydroxylase activity, characterized in that, It contains the gene encoding steroid compound 11α-hydroxylase as described in claim 2 or 3 and the gene encoding cytochrome P450 reductase.

5. The co-expression vector according to claim 4, characterized in that, 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, characterized in that, The nucleotide sequence of the gene encoding cytochrome P450 reductase consists of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

3.

7. The co-expression vector according to claim 4, characterized in that, The genes encoding steroid compound 11α-hydroxylase and cytochrome P450 reductase are integrated into the host bacterium's chromosome or located on a free plasmid outside the host bacterium's chromosome.

8. The co-expression vector according to claim 4, characterized in that, The gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase are controlled by the same or different promoters.

9. The co-expression vector according to claim 8, characterized in that, The promoter is a eukaryotic promoter or a prokaryotic promoter.

10. The co-expression vector according to claim 4, characterized in that, The initial vector for the co-expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

11. The co-expression vector according to claim 4, characterized in that, The upstream nucleotide sequences of the gene encoding steroid compound 11α-hydroxylase and the gene encoding cytochrome P450 reductase respectively contain nucleotide sequences encoding ribosome binding sites.

12. A genetically engineered strain for preparing 11α-hydroxysteroid compounds, characterized in that, It expresses the steroid compound 11α-hydroxylase according to claim 1, the gene encoding the steroid compound 11α-hydroxylase according to claim 2 or 3, and / or the co-expression vector according to any one of claims 4-11.

13. The genetically engineered strain according to claim 12, characterized in that, The initial strains of the genetically engineered strains were selected from strains of the genera *Streptomyces*, *Bacillus*, *Pseudomonas*, *Corynebacterium*, *Arthrobacter*, *Rhodococcus*, *Mycobacterium*, *Escherichia coli*, and *Saccharomyces*.

14. The genetically engineered strain according to claim 13, characterized in that, The initial strain of the genetically engineered strain is Escherichia coli or Mycobacterium.

15. A method for preparing 11α-hydroxysteroid compounds, characterized in that, It includes using the steroid compound 11α-hydroxylase according to claim 1, the gene encoding the steroid compound 11α-hydroxylase according to claim 2 or 3, the co-expression vector according to any one of claims 4-11, and / or the genetically engineered strain according to any one of claims 12-14 to convert steroid compounds into 11α-hydroxysteroid compounds.

16. The method according to claim 15, characterized in that, The transformation is carried out in a reaction solution containing steroidal compounds using genetically engineered strains in either a growing or resting state according to any one of claims 12-14.

17. The method according to claim 15 or 16, characterized in that, The steroidal compound is a steroidal drug intermediate, and the 11α-hydroxysteroidal compound is an 11α-hydroxylated product generated from the steroidal drug intermediate by the steroidal compound 11α-hydroxylase.

18. The method according to claim 15 or 16, characterized in that, The steroidal compound is selected from one or more of androstenedione, androsadienedione, dihydroxyprogesterone, progesterone, and 17α-hydroxyprogesterone, and the 11α-hydroxylated product of the steroidal compound is selected from one or more of 11α-hydroxyandrostenedione, 11α-hydroxyandrosadienedione, 11α-hydroxydihydroxyprogesterone, 11α-hydroxyprogesterone, and 11α,17α-dihydroxyprogesterone.

19. Use of the steroid compound 11α-hydroxylase according to claim 1, the gene encoding the steroid compound 11α-hydroxylase according to claim 2 or 3, the co-expression vector according to any one of claims 4-11, and / or the genetically engineered strain according to any one of claims 12-14 in the preparation of 11α-hydroxysteroid compounds by transforming steroid compounds.

20. The use according to claim 19, characterized in that, The steroidal compound is selected from one or more of androstenedione, androsadienedione, dihydroxyprogesterone, progesterone, and 17α-hydroxyprogesterone, and the 11α-hydroxylated product of the steroidal compound is selected from one or more of 11α-hydroxyandrostenedione, 11α-hydroxyandrosadienedione, 11α-hydroxydihydroxyprogesterone, 11α-hydroxyprogesterone, and 11α,17α-dihydroxyprogesterone.

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