Recombinant expression plasmid, recombinant escherichia coli strain and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant expression plasmid, a recombinant escherichia coli strain and application, and the recombinant expression plasmid comprises a sequence for coding CYP8B1 and a sequence for coding CPR. A recombinant escherichia coli strain constructed by the recombinant expression plasmid and an escherichia coli whole-cell catalytic system have excellent 12alpha-hydroxylase catalytic activity, and the conversion rate of the recombinant expression plasmid to 7alpha-HCO (7alpha-hydroxy-4-cholestene-3-ketone) can reach 80% or above.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to recombinant expression plasmids, recombinant Escherichia coli strains, and their applications. Background Technology

[0002] CYP8B1 is a key enzyme in the classical bile acid synthesis pathway. It regulates the balance of bile acids (CA) and chenodeoxycholic acid (CDCA) in the liver by catalyzing the conversion of the CDCA precursor 7α-hydroxy-4-cholesten-3-one (7α-HCO) into the CA biosynthetic intermediate 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4). Due to the significant differences between CA and CDCA in their physicochemical properties (such as polarity and hydrophilicity) and signaling pathway activation (such as FXR agonist activity), the activity of CYP8B1 directly affects cholesterol absorption, glucose and lipid metabolism, and liver function. Studies have confirmed that inhibiting CYP8B1 can predominate the primary bile acid pathway, thereby improving glucose homeostasis, insulin sensitivity, and hepatic steatosis. Therefore, CYP8B1 is considered a core drug target for the treatment of metabolic diseases such as type 2 diabetes, non-alcoholic fatty liver disease, and obesity.

[0003] Currently, CYP8B1 activity assays rely on systems such as purified recombinant proteins, permeated fission yeast, COS cells, or liver microsomes, but all have significant drawbacks: Purifying recombinant proteins requires additional purification of the redox chaperone protein CPR and the addition of NADPH, which is cumbersome and the enzyme activity is easily lost in vitro, limiting high-throughput screening; fission yeast cells, although capable of co-expressing CYP8B1 and CPR, have different post-translational modifications in eukaryotes compared to mammals, resulting in enzyme expression characteristics inconsistent with in vivo; COS cells require repeated transfection and cannot establish stable cell lines, leading to poor experimental reproducibility; liver microsomes contain a large number of endogenous P450 enzymes that interfere with assay specificity, and enzyme activity cannot be maintained long-term, making it difficult to meet the needs of large-scale experiments. These limitations severely restrict the development efficiency of CYP8B1 inhibitors.

[0004] Escherichia coli (E. coli) has been widely used in whole-cell activity assays for P450 enzymes (such as CYP1A2, 2A6, and 3A4) due to its rapid growth, low culture cost, clear genetic background, high expression levels of heterologous proteins, and lack of endogenous P450 genes. However, no E. coli heterologous expression systems targeting CYP8B1 have been reported, and current technological bottlenecks have slowed progress in CYP8B1 inhibitor research. Therefore, constructing a whole-cell activity assay system for CYP8B1 based on E. coli can effectively address the problems of insufficient specificity, operational complexity, and throughput limitations of existing methods, providing key technical support for efficient screening of CYP8B1 inhibitors and advancing the development of therapeutic drugs for metabolic diseases. Summary of the Invention

[0005] Based on this, the present invention provides a recombinant expression plasmid comprising a sequence encoding CYP8B1 and a sequence encoding CPR, a recombinant Escherichia coli strain, and its application.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In a first aspect, the present invention provides a recombinant expression plasmid comprising a sequence encoding CYP8B1 and a sequence encoding CPR.

[0007] Preferably, in the above recombinant expression plasmids, CYP8B1 is a truncated CYP8B1, which is formed by shortening the N-terminus of CYP8B1 by 25 transmembrane amino acids and replacing it with the MAKKTSS sequence, and / or fusing a 6×His tag to the C-terminus of CYP8B1; and / or CPR is a truncated CPR, which is formed by shortening the N-terminus of CPR by 27 transmembrane amino acids, and / or fusing a 3×Gly linker peptide and a 6×His tag to the C-terminus of CPR.

[0008] Preferably, in the above-mentioned recombinant expression plasmid, the sequence encoding CYP8B1 and the sequence encoding CPR are linked by a ribosome binding site and expressed under the drive of a single promoter; and / or the sequence encoding CYP8B1 and the sequence encoding CPR are inserted into multiple cloning site 1 and multiple cloning site 2, respectively, and are driven by independent promoters.

[0009] Secondly, the present invention provides a recombinant Escherichia coli strain, which includes the above-mentioned recombinant expression plasmid and a sequence encoding a molecular chaperone protein.

[0010] Preferably, in the above-mentioned recombinant Escherichia coli strain, the molecular chaperone protein is groES-groEL, which is expressed by plasmid pGro7 or pGro12.

[0011] Preferably, in the above-mentioned recombinant Escherichia coli strain, the host strain of the recombinant Escherichia coli strain is selected from C41 (DE3), C43 (DE3), BL21-CodonPlus (DE3)-RIPL, Rosetta (DE3) or GSsetta (DE3).

[0012] More preferably, in the above-mentioned recombinant Escherichia coli strain, the host strain of the recombinant Escherichia coli strain is selected as C43 (DE3); in the recombinant expression plasmid, CYP8B1 is a truncated CYP8B1, CPR is a truncated CPR, and the sequence encoding CYP8B1 and the sequence encoding CPR are inserted into multiple cloning site 1 and multiple cloning site 2, respectively, driven by independent promoters, and multiple cloning site 1 and multiple cloning site 2 are derived from the expression vector pRSFDuet-1.

[0013] Thirdly, the present invention provides a whole-cell catalytic system for Escherichia coli, wherein the Escherichia coli heterologous expression system includes the above-mentioned recombinant Escherichia coli strain and substrate stock solution, wherein the substrate stock solution contains 7α-HCO3.

[0014] Preferably, in the above-mentioned Escherichia coli whole-cell catalytic system, the substrate mother liquor further contains the solvents dimethyl sulfoxide and / or ethanol and / or 45% HPCD.

[0015] Fourthly, the present invention provides an application of the above-described recombinant expression plasmid or the above-described recombinant Escherichia coli strain or the above-described Escherichia coli heterologous expression system, the application being selected from any of the following applications: (i) application in the synthesis of 12α-hydroxylated steroidal compounds; (ii) application in high-throughput screening of CYP8B1 inhibitors.

[0016] The beneficial effects of this invention include: the recombinant Escherichia coli strain constructed based on the recombinant expression plasmids containing sequences encoding CYP8B1 and CPR provided by this invention, as well as the whole-cell catalytic system of Escherichia coli, have excellent 12α-hydroxylase catalytic activity, and their conversion rate of 7α-HCO (7α-hydroxy-4-cholesten-3-one) can reach more than 80%. Attached Figure Description

[0017] Figure 1 The results are for the recombinant plasmid colony PCR gel electrophoresis detection; lanes 1 and 2 (parallel experiments) are pET-17b-tCYP8B1-fCPR, lanes 3 and 4 (parallel experiments) are pET-17b-tCYP8B1-tCPR, and lanes 5 and 6 (parallel experiments) are pRSFDuet-1-tCYP8B1-tCPR. Figure 2 SDS-PAGE analysis of protein expression in recombinant strains; lanes 1-1~5, 2-1~5, and 3-1~2 represent 12 recombinant strains; "1~3" before "-" represent plasmids pET-17b-tCYP8B1-fCPR, pET-17b-tCYP8B1-tCPR, and pRSFDuet-1-tCYP8B1-tCPR, respectively; "1~5" after "-" represent host strains C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) that co-express the molecular chaperone protein groES-groEL, respectively. Figure 3The results are full-spectral scans; (A) is 7α-hydroxy-4-cholesten-3-one (7α-HCO); (B) is 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4); the maximum UV absorption wavelength of the two compounds is approximately 240 nm, and their retention times are shown in the red boxes, which are 9.569 min and 5.805 min, respectively. Figure 4 The whole-cell viability assays of the CYP8B1 recombinant strain are shown below. (A) shows the effect of polymyxin B, a penetration enhancer, on whole-cell viability. (B) shows the effect of solvents HPCD, DMSO, and EtOH on whole-cell viability. (C) shows the whole-cell viability assay of the CYP8B112 recombinant strain. Strains 1-5 represent host strains C41 (DE3), C43 (DE3), BL21-CodonPlus (DE3)-RIPL, Rosetta (DE3), and GSsetta (DE3), respectively. Plasmids 1-3 represent pET-17b-tCYP8B1-fCPR, pET-17b-tCYP8B1-tCPR, and pRSFDuet-1-tCYP8B1-tCPR, respectively. Data are expressed as Mean ± SEM (n=3), ##, ** indicates P < 0.01.

[0018] Figure 5 This study describes the construction of a whole-cell activity assay system for human CYP8B1 based on *Escherichia coli*. a) By comparing whole-cell activity, the optimal expression system for CYP8B1 was determined to be the recombinant strain C43-pRSF-tCYP8B1-tCPR. b) The 12α-hydroxylase activity of the obtained recombinant strain was determined using RP-HPLC, with no polymyxin B added to the reaction system and DMSO used as the solvent for the substrate 7α-HCO. * represents the recombinant strain with the highest catalytic efficiency. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] In a first aspect, embodiments of the present invention provide a recombinant expression plasmid, the recombinant expression plasmid including a sequence encoding CYP8B1 and a sequence encoding CPR.

[0022] In some specific examples, in the above recombinant expression plasmids, CYP8B1 is a truncated CYP8B1, which is formed by shortening the N-terminus of CYP8B1 by 25 transmembrane amino acids and replacing it with the MAKKTSS sequence; and / or CPR is a truncated CPR, which is formed by shortening the N-terminus of CPR by 27 transmembrane amino acids.

[0023] It should be noted that the truncated N-terminal transmembrane helix of CYP8B1, a hydrophobic sequence, usually increases expression levels and solubility; similarly, the truncated CPR can also increase expression levels and solubility.

[0024] In some specific examples, the C-terminus of CYP8B1 is fused with a 6×His tag in the recombinant expression plasmids described above; and / or the C-terminus of CPR is fused with a 3×Gly linker peptide and a 6×His tag.

[0025] In some specific examples, in the recombinant expression plasmids described above, the sequence encoding CYP8B1 and the sequence encoding CPR are linked by a ribosome binding site and expressed under the drive of a single promoter; and / or the sequence encoding CYP8B1 and the sequence encoding CPR are inserted into multiple cloning sites 1 and 2, respectively, and are each driven by an independent promoter.

[0026] In some specific examples, the recombinant expression plasmids described above contain a single T7 promoter derived from the expression vector pET-17b; and / or multiple cloning sites 1 and 2 derived from the expression vector pRSFDuet-1.

[0027] Secondly, embodiments of the present invention provide a recombinant Escherichia coli strain, which includes the above-mentioned recombinant expression plasmid and a sequence encoding a molecular chaperone protein.

[0028] It should be noted that a reliable, efficient, and cost-effective high-throughput system is currently lacking for activity screening of CYP8B1 (P450 enzyme) inhibitors. Evaluation of mammalian P450 enzyme activity is typically performed using microsomal analysis. However, compared to using subcellular components, whole-cell methods based on *E. coli* offer advantages such as time-saving, low cost, no need for exogenous NADPH addition, and avoidance of enzymatic degradation during microsomal preparation. Furthermore, *E. coli* lacks endogenous P450 enzymes and exhibits extremely low mammalian-like biotransformation capabilities. Therefore, *E. coli* is widely used in constructing whole-cell activity systems for cytochrome P450 enzymes.

[0029] In some specific examples, the molecular chaperone protein in the above-mentioned recombinant E. coli strains is groES-groEL, which is expressed by plasmids pGro7 or pGro12.

[0030] In some specific examples, the host strain of the recombinant Escherichia coli strain mentioned above is selected from C41 (DE3), C43 (DE3), BL21-CodonPlus (DE3)-RIPL, Rosetta (DE3) or GSsetta (DE3).

[0031] It should be noted that when co-expressing CYP8B1, CPR, and groES-groEL in *E. coli*, we found that the expression level of CYP8B1 was highly dependent on the host bacterial species. Compared to C41(DE3) and C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) all supplemented rare codons lacking in *E. coli*, which was more conducive to the expression of recombinant CYP8B1 protein. The expression level of CPR was vector-dependent, with pRSFDuet-1 being significantly superior to pET-17b. This may be because when constructing co-expression plasmids using bicistronic RBS, the expression level of the downstream ORF is relatively low compared to the upstream ORF (usually 10%–20% of the upstream ORF); while the key advantage of the dual expression vector pRSFDuet-1 is that the expression level of the downstream ORF is comparable to (or slightly lower than) that of the upstream ORF. The expression levels of the groES-groEL complex (groES ~ 10.4 kDa, groEL ~ 57.3 kDa) were inversely proportional to the expression level of CYP8B1, which may be because the high expression of CYP8B1 compressed the expression space of groES-groEL.

[0032] In some specific examples, the host strain of the recombinant Escherichia coli strain mentioned above is C43 (DE3); in the recombinant expression plasmid, CYP8B1 is a truncated CYP8B1, CPR is a truncated CPR, and the sequences encoding CYP8B1 and CPR are inserted into multiple cloning sites 1 and 2, respectively, driven by independent promoters. Multiple cloning sites 1 and 2 are derived from the expression vector pRSFDuet-1.

[0033] It should be noted that the above-mentioned construction method of recombinant Escherichia coli strain is well known in the art. For example, the construction method includes: (1) transforming a plasmid encoding a sequence of molecular chaperone protein into the host strain of recombinant Escherichia coli strain to obtain an intermediate strain; (2) introducing a recombinant expression plasmid into the intermediate strain to obtain a recombinant Escherichia coli strain.

[0034] Thirdly, the present invention provides an Escherichia coli whole-cell catalytic system in which the Escherichia coli heterologous expression system includes the above-mentioned recombinant Escherichia coli strain and substrate stock solution, wherein the substrate stock solution contains 7α-HCO3.

[0035] In some specific examples, the substrate stock solution in the above-mentioned E. coli whole-cell catalytic system also contains solvents dimethyl sulfoxide and / or ethanol and / or 45% HPCD.

[0036] It should be noted that, compared to HPCD and ethanol, choosing DMSO as the substrate solvent significantly improved the catalytic reaction efficiency, which may be related to the fact that DMSO simultaneously increases cell membrane permeability. Using optimized conditions, we found that the transformation efficiency of the recombinant strain was not positively correlated with the expression level of CYP8B1; the difference in the CPR:CYP ratio may be the reason for the different results. The stoichiometric ratio of CPR to CYP is a key factor in the catalytic activity of a specific CYP form in a particular tissue, subcellular component, or expression system, and it depends not only on the abundance of CYP but also on its electron transfer partner, CPR. This may be a key factor affecting the whole-cell activity of the recombinant strain.

[0037] In some specific examples, in the above-mentioned whole-cell catalytic system of Escherichia coli, the final concentration of 7α-HCO in the substrate mother liquor is 10 μmol / L; and / or the volume percentage of DMSO in the reaction system is 5%.

[0038] Fourthly, embodiments of the present invention provide an application of the above-described recombinant expression plasmid, the above-described recombinant Escherichia coli strain, or the above-described Escherichia coli heterologous expression system, the application being selected from any of the following applications: (i) application in the synthesis of 12α-hydroxylated steroidal compounds; (ii) application in high-throughput screening of CYP8B1 inhibitors.

[0039] In some specific examples, the 12α-hydroxylated steroidal compounds mentioned above include, but are not limited to, 7α12αC4 (7α,12α-dihydroxy-4-cholesten-3-one).

[0040] It should be noted that this invention constructs a whole-cell expression system for the Escherichia coli (ECG) of CYP8B1, a key enzyme in bile acid synthesis. E. coli offers advantages such as simple cultivation, low cost, high expression levels of heterologous proteins, and the absence of endogenous P450 enzymes. This system can be used to establish a reliable and efficient whole-cell activity assay system for CYP8B1, enabling the screening of CYP8B1 inhibitors from a compound library—a common practice in the preclinical stage of drug development. Furthermore, whole-cell expression systems based on E. coli P450 enzymes are frequently used as biocatalysts to achieve the biotransformation and synthesis of specific compounds. The recombinant E. coli expression system for human CYP11B1 can biosynthesize cortisol. The whole-cell expression system for human CYP7A1 can also be used to achieve the biosynthesis of 7α-HCO3. Therefore, the whole-cell expression system for CYP8B1 in E. coli can be used as a whole-cell biocatalyst to achieve the biosynthesis of 12α-hydroxylated steroid compounds. This provides a scalable, stereoselective, and environmentally friendly alternative for the production of steroid intermediates.

[0041] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0042] In the following example, the gene IDs of human CPR and human CYP8B1 were obtained by searching on NCBI as 5447 and 1582, respectively. The corresponding transcript numbers NM_001395413.1 and NM_004391.3 were provided to Wuhan Miaoling Biotechnology Co., Ltd.; Escherichia coli DH5α, C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL and Rosetta(DE3) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; GSsetta(DE3) competent cells were purchased from Beijing Jinsha Biotechnology Co., Ltd.; chaperone protein groES-groEL expression plasmids pGro7 (chloramphenicol resistance) and pGro12 (kanamycin resistance) and vector pET-17b were purchased from Wuhan Miaoling Biotechnology Co., Ltd.; and the dual expression vector pRSFDuet-1 was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0043] In the following examples, LB liquid medium, LB solid medium, and TB medium were all purchased from Beijing Solarbio; the working concentrations of carbenicillin, kanamycin, and chloramphenicol were 100 μg / mL, 50 μg / mL, and 37 μg / mL, respectively.

[0044] In the following examples, 7α-hydroxy-4-cholesten-3-one was from Shanghai Yuanye; 7α,12α-dihydroxy-4-cholesten-3-one was from Beijing Innocare; isopropyl-β-D-thiogalactoside, L-arabinose, and 5-aminolevulinate were from Shanghai Bide; agarose was from Biowest Agarose; 50×TAE buffer was from Beijing Solarbio; Gene Red nucleic acid dye, endotoxin-free plasmid medium-quantity extraction kit, and DNA marker were from Tiangen Biotech; direct PCR kit was from Shanghai Beyotime; 2×TaqMasterMix (DyePlus) polymerase and Clone Express Ultra One Step Cloning Kit were from Nanjing Novizan. PCR instrument, Thermo Fisher Scientific, USA; high-speed low-temperature centrifuge, Sigma, Germany; constant temperature incubator, Shanghai Yiheng; vertical full-temperature shaking incubator, Lepotra; Mini-Sub Cell GT Cell electrophoresis tank and vertical electrophoresis apparatus, Bio-Rad, USA; gel imaging system, Shanghai Qinxiang; water bath, Shandong Boke; ultra-micro UV spectrophotometer, Shanghai Baoyide; vertical autoclave, Yamato; Agilent high-performance liquid chromatograph, Agilent; Shimadzu Nexera LC-40D, Shimadzu.

[0045] Example 1 This invention provides a recombinant expression plasmid and a method for constructing it.

[0046] The genes encoding human CYP8B1 (NM_004391.3) and CPR (NM_001395413.1) were codon-optimized and synthesized by Wuhan Miaoling Biotechnology Co., Ltd. Additionally, the corresponding primer pairs used in the following examples are shown in Table 1 below.

[0047] Table 1 Primer pairs used in Example 1

[0048] (1) Remove 25 amino acids from the N-terminus of CYP8B1 and replace them with a sequence encoding MAKKTSS; in addition, a 6×His tag was added to the C-terminus; at the same time, CPRΔ1-27 (a truncated version of CPR, with 27 amino acids removed from the N-terminus, where Δ represents "deletion" and 1-27 refers to the amino acid position) and full-length CPR were used as electron transfer body proteins, respectively.

[0049] (2) Construction of CYP8B1 and CPR co-expression plasmids (two types) Refer to the literature "Shang QN, Huang JH, Qin S, Zhu HC, Chen RL, Yin LN, Hu QZ. Efficient whole-cell biocatalytic production of 7α-hydroxy-4-cholesten-3-one via human CYP7A1 expressed in..." Escherichia coli [J]. The Journal of Steroid Biochemistry and Molecular Biology, 2026, 255 106866. The recombinant expression plasmids pET-17b-tCYP8B1-fCPR, pET-17b-tCYP8B1-tCPR, and pRSFDuet-1-tCYP8B1-tCPR were constructed, and the detailed characteristics of the constructed plasmids are shown in Table 2; the specific construction process is as follows: The first method involves using the high-copy-number plasmid pET-17b and bicistronic expression elements to link CYP8B1 and CPR via a ribosome binding site (RBS) and express them under the drive of a single T7 promoter. Specifically, a ribosome binding site (RBS) is inserted between CYP8B1 and CPR, causing them to form two separate transcripts, which are then translated into proteins. Using pET-17b as a vector, the synthesized CYP8B1 gene is inserted into the restriction enzyme site Nde I / Hind III to obtain the recombinant plasmid pET-17b-CYP8B1. Then, the synthesized CPR gene (CPRΔ1-27 or full-length CPR) is subcloned into the restriction enzyme site Hind III / BamH of the plasmid pET-17b-CYP8B1. The plasmids pET-17b-CYP8B1-CPR (pET-17b-tCYP8B1-fCPR and pET-17b-tCYP8B1-tCPR) were obtained between I and II. The second method involves using the high-copy-number vector pRSFDuet-1 to insert CYP8B1 and CPR into multiple cloning sites 1 (MCS1) and 2 (MCS2), respectively, driven by independent T7lac promoters. Hu BD et al. used the high-copy-number vector pRSFDuet-1 and the medium-copy-number vector pETDuet-1 to construct expression plasmids of P450 BM3mut and P450 sca-2mut, respectively, which were then used to transform host bacteria BL21 (DE3), C41 (DE3), and C43 (DE3), respectively. After whole-cell activity tests, the results showed that strains C41-pRSF-BM3mut and C41-pRSF-sca-2mut had the highest catalytic activity. Based on this, co-expression of CYP8B1 and CPR was achieved using the high-copy dual expression vector pRSFDuet-1; that is, the vector pRSFDuet-1 has dual T7lac expression cassettes, and CYP8B1 and CPR are inserted after the two T7lac promoters through the restriction sites Nco I / EcoRI and Nde I / Avr II, respectively, to obtain the co-expression recombinant plasmid pRSFDuet-1-tCYP8B1-tCPR.

[0050] Table 2. Plasmids used in Example 1

[0051] Note: In the table above, tCYP8B1 represents CYP8B1 with 25 N-terminal transmembrane amino acids truncated and replaced with the MAKKTSS sequence and a 6×His tag added to the C-terminus; fCPR represents the full-length CPR with 3×Gly and 6×His tags added to the C-terminus; tCPR represents CPR with 27 N-terminal transmembrane amino acids truncated and 3×Gly and 6×His tags added to the C-terminus.

[0052] (3) The plasmid was transformed into Escherichia coli DH5α competent cells using the standard heat shock transformation method; recombinant colonies were screened on LB agar plates supplemented with the corresponding antibiotics and verified by colony PCR (PCR primers are shown in Table 3 below, PCR reaction system is shown in Table 4 below, and PCR amplification conditions are shown in Table 5 below); the products after colony PCR screening were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown in the figure, the CYP8B1 band appeared at 253 bp, which is consistent with the expected result, proving that the selected colony is a positive clone. After appropriate incubation on the plate, clones can be picked again at the marked position for amplification culture and plasmid extraction.

[0053] Table 3. Identification primers and product size

[0054] Table 4 PCR reaction system

[0055] Table 5 PCR reaction amplification conditions

[0056] (4) The selected recombinant colonies were confirmed as positive clones by DNA sequencing, and plasmids were prepared using an endotoxin-free plasmid extraction kit (China Tiangen Biotech Co., Ltd.).

[0057] Example 2 This invention provides a co-expression recombinant Escherichia coli strain based on the recombinant expression plasmid prepared in Example 1 and a construction method thereof.

[0058] The construction of the co-expression system of molecular chaperone proteins groES-groEL, CYP8B1, and redox chaperone protein CPR was carried out using a two-step method. Specific steps were described in the literature "Shang QN, Huang JH, Qin S, Zhu HC, Chen RL, Yin LN, Hu QZ. Efficient whole-cell biocatalytic production of 7α-hydroxy-4-cholesten-3-one via human CYP7A1 expressed in..." Escherichia coli [J]. The Journal of Steroid Biochemistry and Molecular Biology, 2026, 255, 106866.; The specific steps are as follows: (1) The molecular chaperone plasmids pGro7 (chloramphenicol resistance) or pGro12 (kanamycin resistance) encoding groES-groEL were transformed into Escherichia coli strains C41 (DE3), C43 (DE3), Rosetta (DE3), GSsetta (DE3) and BL21-CodonPlus (DE3)-RIPL using the heat shock transformation method to obtain transformed strains C41-pGro12, C43-pGro12, BL21-pGro12, Rosetta-pGro12 and GSsetta-pGro12, respectively. (2) The CYP8B1 / CPR co-expression plasmid (recombinant expression plasmid) constructed in Example 1 was introduced into the transformant strain expressing groES-groEL in (1) above to obtain double transformants (CYP8B1 recombinant strains). The CYP8B1 recombinant strains were C41-pET-tCYP8B1-fCPR, C43-pET-tCYP8B1-fCPR, BL21-pET-tCYP8B1-fCPR, Rosetta-pET-tCYP8B1-fCPR, GSsetta-pET-tCYP8B1-fCPR and C41-pET-tCYP8B1-tCPR, C43-pET-tCYP8B1-tCPR, BL21-pET-tCYP8B1-tCPR, Ro The plasmids setta-pET-tCYP8B1-tCPR and GSsetta-pET-tCYP8B1-tCPR were used. Additionally, because plasmid pRSFDuet-1-tCYP8B1-tCPR is kanamycin resistant, plasmid pGro7 (chloramphenicol resistant) was selected as the expression plasmid for groES-groEL. This plasmid was transformed into strains C41 (DE3) and C43 (DE3) to obtain strains C41-pGro7 and C43-pGro7. These strains were then transformed with plasmid pRSFDuet-1-tCYP8B1-tCPR to obtain two CYP8B1 expression strains using pRSFDuet-1 as the expression vector: C41-pRSF-tCYP8B1-tCPR and C43-pRSF-tCYP8B1-tCPR. (3) Screen cotransformants on LB agar plates containing appropriate antibiotics to maintain the presence of both plasmids (add ampicillin 100 µg / mL, chloramphenicol 34 µg / mL or kanamycin 50 µg / mL as needed); validated clones are stored in bacterial culture containing 20% ​​(v / v) glycerol at -80°C for subsequent experiments; obtain the co-expression strain.

[0059] In this invention, a total of 12 strains co-expressing groES-groEL, CYP8B1, and CPR were obtained. Among them, 10 recombinant strains were constructed using plasmid pET-17b and five E. coli hosts: C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3). Two recombinant strains were constructed using plasmid pRSFDuet-1 and C41(DE3) and C43(DE3), as shown in Table 6 below.

[0060] Table 6. Strains used in Example 2

[0061] Related tests In the following tests, SPSS 26.0 software was used for statistical analysis, and GraphPad Prism 8.0 software was used to create statistical graphs. ± SEM represents the variance. If the data follows a normal distribution, a homogeneity of variance test is used. For two groups of data that meet the homogeneity of variance test, a two-independent-samples t-test is used. For multiple groups of data that meet the homogeneity of variance test, a one-way ANOVA is used. The Tukey HSD method is used for post-hoc comparisons. For multiple groups of data with unequal variances, Dunnett's T3 method is used for pairwise comparisons. P < 0.05 is considered statistically significant.

[0062] (a) SDS-PAGE analysis of recombinant protein expression (1) Activation, inoculation, and induction of target protein expression of engineered strains: Twelve strains co-expressing groES-groEL, CYP8B1, and CPR were taken from a -80℃ freezer and activated on LB plates containing the corresponding screening antibiotics. The next day, positive single clones were picked with an autoclaved pipette tip and cultured overnight at 37℃ and 220 r / min to obtain seed culture. The obtained seed culture was inoculated into TB liquid medium containing the corresponding antibiotics at a volume ratio of 1:100 (seed culture: TB liquid medium containing the corresponding antibiotics) and cultured with shaking at 37℃ and 220 r / min. 600 When OD600 = 0.5, 1 mmol / L 5-aminolevulinic acid salt and 4 mg / mL arabinose were added as expression inducers for heme precursor and groES-groEL, respectively; when OD600 = 0.8, 1 mmol / L IPTG was added to induce CYP8B1 and CPR expression, and the shaker was adjusted to 27.5℃ and 180 r / min, and cultured for 24 h to obtain bacterial culture; (2) Preparation of protein samples and SDS-PAGE analysis: Take 50 μL of bacterial culture, centrifuge at 5000 r / min for 10 min at 4ºC to collect the bacterial cells, add 50 μL of PBS to resuspend the bacterial cells; then add 50 μL of 2× Loading buffer and mix well, boil in water for 10 min, centrifuge at 13000 r / min for 10 min, take 10 μL of supernatant and load it for 8% SDS-PAGE electrophoresis (10V, 30 min; 80V, stacking gel; 120V, separating gel). (3) Then Coomassie brilliant blue staining was performed to analyze the expression of the target protein.

[0063] The results are as follows Figure 2As shown, the results indicated that the expression levels of CYP8B1 (molecular weight ~57.1 kDa) in the host strains BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) were significantly higher than those in C41(DE3) and C43(DE3), and were not significantly related to whether the vector was pET-17b or pRSFDuet-1. Although all five strains were derived from BL21(DE3), the differences were that C41(DE3) and C43(DE3) facilitated the expression of toxic proteins, while BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) supplemented rare codons lacking in E. coli, increasing the expression of AT- or GC-rich eukaryotic proteins. The expression level of nuclear genes in prokaryotic systems is more favorable for the expression of recombinant CYP8B1 protein. Compared to CYP8B1, the expression level of CPR (molecular weight ~70-78 kDa) is significantly reduced, especially when using pET-17b as a vector, where the corresponding band is no longer clearly identifiable, while the corresponding band is still identifiable when using pRSFDuet-1 as a vector. The expression level of the groES-groEL complex (groES ~10.4 kDa, groEL ~57.3 kDa) is inversely proportional to the expression level of CYP8B1; high CYP8B1 expression levels lead to decreased groES-groEL expression levels, and low CYP8B1 expression levels lead to increased groES-groEL expression levels. This further confirms that the expression host has a significant impact on the expression level of exogenous genes. Next, whole-cell activity tests will be performed on the recombinant strain to determine the optimal expression system for the CYP8B1 enzyme.

[0064] (II) Whole-cell activity assay of human CYP8B1 Escherichia coli heterologous expression system The recombinant expression strain CYP8B1 (oxosterosterol 12α-hydroxylase) can specifically 12α-hydroxylate 7α-HCO to generate 7α12αC4. The following tests used 7α-HCO as a substrate and determined the whole-cell activity of the CYP8B1 recombinant strain using RP-HPLC. The RP-HPLC method (reversed-phase high-performance liquid chromatography) was established as follows: Standard stock solutions of 7α-HCO and 7α12αC4 were prepared using ethanol as the solvent, with a final concentration of 10 mmol / L; an acetonitrile-methanol mobile phase system was selected, and different ratios were set with equal gradients to investigate the RP-HPLC separation of the standards; simultaneously, a full-wavelength scan was performed to determine the maximum absorption wavelength of the above compounds; the full-wavelength scan results of 7α-HCO and 7α12αC4 are shown below. Figure 3As shown, the maximum UV absorption wavelength of both compounds is 240 nm, therefore 240 nm was determined as the detection wavelength. Furthermore, based on the results of investigating the ratio of acetonitrile to methanol in the mobile phase under isogradient conditions, at an acetonitrile:methanol ratio of 7:3 and a flow rate of 0.7 mL / min, the retention times of 7α-HCO and 7α12αC4 were 9.569 and 5.805 min, respectively, indicating good separation. Figure 3 Based on this, the RP-HPLC method conditions used are shown in Table 7 below.

[0065] Table 7 RP-HPLC Method Conditions

[0066] (1) Preparation of substrate stock solution: 45% (m / V) HP-β-CD (hydroxypropyl-β-cyclodextrin): Weigh an appropriate amount of HP-β-CD, add the corresponding volume of deionized water, and prepare a 45% (m / V) HP-β-CD aqueous solution; 7α-HCO solution: Dissolve 7α-HCO in 45% (m / V) HP-β-CD aqueous solution, DMSO (dimethyl sulfoxide) and ethanol respectively to prepare different substrate stock solutions. The concentration of the substrate stock solution is 200 μmol / L.

[0067] (2) Preparation of bacterial culture: The bacterial strain was activated and the target protein was induced to express according to the above "(I) SDS-PAGE analysis of recombinant protein expression". The bacterial cells were collected by centrifugation at 4℃, 6000r / min for 10min. The bacterial cells were washed twice with 50mmol / L potassium phosphate buffer (pH7.4). After centrifugation, the bacterial cells were collected again. A certain volume of reaction buffer (50mmol / L potassium phosphate buffer (pH7.4), 2% glycerol, 1mmol / L IPTG (isopropyl-β-D-thiogalactoside), 1mmol / L 5-aminolevulinate and 4mg / mL arabinose) was added to resuspend the cells. The cells were divided into two groups. One group was added with 32μg / mL polymyxin B and the other group was not added (no substance was added). Then the wet weight (wcw) of the cells was controlled at about 25g / L to obtain different bacterial cultures.

[0068] (3) Reaction system: Take 450 μL of different bacterial solutions prepared in (2) above, add 50 μL of substrate stock solution prepared in (1) above, the substrate reaction concentration is 10 μmol / L, 27.5℃, 180 r / min, and react for 24 h.

[0069] (4) Sample preparation: Collect the sample into a 1.5 mL centrifuge tube containing 1 mL of ethyl acetate to quench the reaction. After vigorous vortexing and mixing, centrifuge at 12000 r / min for 5 min at 25 °C. Transfer the supernatant organic phase to a clean centrifuge tube and vacuum dry overnight. Then redissolve with 50 μL of mobile phase and place at 4 °C for RP-HPLC analysis.

[0070] Measurement details as follows Figure 4 As shown, the results indicated that the addition of polymyxin B, a penetration enhancer, not only failed to improve the whole-cell activity of the recombinant strain, but also caused a significant loss of activity, with the substrate conversion rate decreasing from 85.20% to 4.33% (P<0.01) (see...). Figure 4 (A) Furthermore, the substrate solvent also had a certain impact on the whole-cell activity of CYP8B1. The strongest whole-cell activity was observed when the substrate stock solution was prepared using the organic solvent DMSO and added to the reaction system at a 5% volume ratio, resulting in a final substrate concentration of 10 μmol / L, with a conversion rate exceeding 80%. However, when using EtOH as the solvent (added to the reaction system at a 5% volume ratio, with a final substrate concentration of 10 μmol / L), the conversion rate was only 2.30%. And when 45% (m / V) HP-β-CD aqueous solution was added (added to the reaction system at a 5% volume ratio, with a final substrate concentration of 10 μmol / L), almost no substrate conversion was observed (P<0.01) (see [reference needed]). Figure 4 (B)); In addition, using optimized biotransformation reaction conditions (without adding the osmotic aid polymyxin B, using DMSO to prepare the substrate stock solution, DMSO volume ratio in the reaction system being 5%, and final substrate concentration being 10 μmol / L), the optimal expression system for CYP8B1 was screened from 12 recombinant Escherichia coli strains, and the results are as follows. Figure 4 As shown in (C), the results showed that strain C43-pRSF-tCYP8B1-tCPR had the highest whole-cell activity (P<0.01), which can be used to screen 12α-hydroxylase substrates or inhibitors. In addition, it also has the application prospect of synthesizing high-value-added catalytic products by 12α-hydroxylation.

[0071] In summary, this invention constructed a heterologous expression system for CYP8B1, a key enzyme in bile acid synthesis, using *E. coli* as the host. The whole-cell activity of the recombinant strain was tested by RP-HPLC, demonstrating its catalytic activity as a 12α-hydroxylase. By comparing the whole-cell activity of the recombinant strain, strain C43-pRSF-tCYP8B1-tCPR was selected as the optimal expression system for CYP8B1. By comparing the effects of osmotic aids and solubilizers on the whole-cell catalytic activity, the optimal reaction conditions for the CYP8B1 recombinant strain were determined to be: no osmotic aid polymyxin B added to the reaction system, DMSO as the substrate 7α-HCO3 solvent, achieving a conversion rate of 85.20%. Therefore, the application of the *E. coli*-based whole-cell expression system for CYP8B1 has two main aspects: first, it can be used to establish a medium-to-high throughput activity testing system for CYP8B1; second, it can serve as a whole-cell catalyst to achieve the 12α-hydroxylation of steroidal compounds, supplementing the shortcomings of chemical synthesis methods regarding stereoselectivity and chirality (e.g.,...). Figure 5 (As shown).

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A recombinant expression plasmid characterized in that, comprising a sequence encoding CYP8B1 and a sequence encoding CPR.

2. The recombinant expression plasmid of claim 1, wherein, CYP8B1 is truncated CYP8B1, truncated CYP8B1 is truncated at N-terminus of CYP8B1 by 25 transmembrane amino acids and replaced by MAKKTSS sequence, and / or CYP8B1 is fused at C-terminus with 6xHis tag; and / or CPR is truncated CPR, truncated CPR is truncated at N-terminus of CPR by 27 transmembrane amino acids, and / or CPR is fused at C-terminus with 3xGly linker and 6xHis tag.

3. The recombinant expression plasmid of claim 1 or 2, wherein, the sequence encoding CYP8B1 and the sequence encoding CPR are linked by a ribosome binding site and expressed under the drive of a single promoter; and / or the sequence encoding CYP8B1 and the sequence encoding CPR are inserted into the multiple cloning site 1 and the multiple cloning site 2, respectively, and driven by independent promoters.

4. A recombinant E. coli strain, characterized in that, the recombinant expression plasmid of any one of claims 1 to 3, and a sequence encoding a molecular chaperone protein.

5. The recombinant E. coli strain of claim 4, wherein, the molecular chaperone protein is groES-groEL, which is expressed from plasmid pGro7 or pGro12.

6. The recombinant E. coli strain of claim 4 or 5, characterized in that, the host strain of the recombinant E. coli strain is selected from C41 (DE3), C43 (DE3), BL21-CodonPlus (DE3)-RIPL, Rosetta (DE3) or GSsetta (DE3).

7. The recombinant E. coli strain of claim 6, wherein, the host strain of the recombinant E. coli strain is C43 (DE3); in the recombinant expression plasmid, CYP8B1 is truncated CYP8B1, CPR is truncated CPR, and the sequence encoding CYP8B1 and the sequence encoding CPR are inserted into the multiple cloning site 1 and the multiple cloning site 2, respectively, and driven by independent promoters, the multiple cloning site 1 and the multiple cloning site 2 are from expression vector pRSFDuet-1.

8. A whole-cell catalytic system of E. coli, characterized in that, the recombinant E. coli strain of any one of claims 4 to 7, and a substrate mother liquor, the substrate mother liquor comprising 7a-HCO.

9. The E. coli heterologous expression system of claim 8, wherein, the substrate mother liquor further comprises solvent dimethyl sulfoxide and / or ethanol and / or 45% HPCD.

10. Use of the recombinant expression plasmid of any one of claims 1 to 3 or the recombinant E. coli strain of any one of claims 4 to 7 or the E. coli whole-cell catalytic system of any one of claims 8 to 9, the use being selected from any one of the following uses: (i) use in synthesis of 12a-hydroxylated steroid compounds; (ii) use in high-throughput screening of CYP8B1 inhibitors.

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