CN117487727A - Method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application - Google Patents

Method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application Download PDF

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CN117487727A
CN117487727A CN202311299446.4A CN202311299446A CN117487727A CN 117487727 A CN117487727 A CN 117487727A CN 202311299446 A CN202311299446 A CN 202311299446A CN 117487727 A CN117487727 A CN 117487727A
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gene
escherichia coli
olep
deoxycholic acid
enzyme
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赵鑫锐
孙驰翔
周景文
王敏浩
堵国成
陈坚
李江华
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Jiangnan University
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Abstract

The invention discloses a method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application thereof, belonging to the technical field of genetic engineering. The invention enhances the capability of catalyzing the hydroxylation of deoxycholic acid 6 beta by improving the soluble expression of P450 enzyme Olep, modifying heme synthesis path, modifying redox partner engineering and optimizing a whole-cell catalytic system, and finally the conversion rate of catalyzing the synthesis of the deoxycholic acid 6 beta-OH deoxycholic acid reaches 99.1 percent. The method provides a new strategy for improving biosynthesis of hydroxylated steroid substances.

Description

Method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application
Technical Field
The invention relates to a method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application thereof, belonging to the technical field of genetic engineering.
Background
Cytochrome P450 enzymes are a class of monooxygenases capable of oxidizing the inert hydrocarbon bonds in a substrate under mild conditions, and are widely found in organisms such as animals, plants, archaea, bacteria, eukaryotes and the like. In prokaryotes, cytochrome P450 enzymes are free in the cytoplasm and are a soluble protein; in eukaryotes, cytochrome P450 enzymes are a membrane-bound protein that is predominantly distributed on the endoplasmic reticulum, microsomes, and mitochondrial inner membranes. Currently more than 50% of drug metabolism is oxidized by cytochrome P450 enzymes, playing an important role in drug metabolism and detoxification. The P450 enzyme can also catalyze a series of catalytic reactions such as regional or stereo hydroxylation, epoxidation, dealkylation, dehalogenation and the like of natural products (such as antibiotics, steroids, terpenes, vitamins, fatty acids and the like). By virtue of having a broad substrate spectrum and catalyzing a variety of reaction types, P450 enzymes are increasingly being used in the microbiology field to synthesize valuable natural products or drugs.
Steroid drugs are the second largest class of drugs next to antibiotics. Deoxycholic acid is a bile acid steroid medicine, has surface activity, is a safe and effective emulsifier in cosmetics and medicaments, has antifungal and anti-inflammatory effects, and can be used for treating tooth root diseases. Can be used for treating sebaceous gland hypersecretion in skin surgery. The product can be used for removing excessive sebum and sweat stain without giving dry feeling to skin. However, deoxycholate has poor water solubility and instability, which limits its use in medicine. The hydroxylation modification not only can improve the solubility and stability of deoxycholic acid, but also can enhance the biological activity of deoxycholic acid.
Compared with chemical hydroxylation reaction, the biological method is a more efficient and environment-friendly method by catalyzing hydroxylation reaction with P450 enzyme, has the advantages of mild reaction condition, high catalytic efficiency, strong selectivity and the like, and can make up for the shortages of chemical synthesis. Coli is the most commonly used expression system and efficient whole cell transformation system for recombinant proteins, and P450 enzymes involved in hydroxylation reaction have the defects of difficult soluble expression, insufficient heme supply, low electron transfer efficiency, insufficient cofactor supply and the like in escherichia coli. It has been found that 7. Alpha. -HSDH (Zhang, xuan., bioprocess and Biosystems Engineering,42 (9), 1537-1545.2019), 7. Beta. -HSDH (Li, hai. Peng., ACS Sustainable Chemistry) are expressed in E.coli&Engineering,10.456-463.2022)、CYP107D1(Grobe,Sascha.,Angewandte Chemie,60(2),753-757.2021)、CYP3A(MaglioccoEnzymes such as J Pers Med,11 (2), 57-58.2021) and CYP102A1 (Hayes Martin A., drug Metab Dispos,44 (9), 1480-9.2016) can hydroxylate specific bile acids, but have problems such as low conversion rate. Therefore, the development of a method for synthesizing the hydroxylated deoxycholic acid by using the whole cell catalysis of the escherichia coli P450 enzyme has important significance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for synthesizing hydroxylated deoxycholic acid by using the whole cell catalysis of Escherichia coli P450 enzyme and application thereof, and aims to solve the technical problems that the P450 enzyme participating in hydroxylation reaction in the prior art has difficult soluble expression, insufficient heme supply, low electron transfer efficiency, insufficient cofactor supply and low deoxycholic acid hydroxylation rate in Escherichia coli.
The first technical scheme of the invention is a genetically engineered bacterium, which takes escherichia coli as a chassis strain to express cytochrome P450 enzyme Olep from streptomycete (Streptomyces antibioticus), and the genetically engineered bacterium also expresses a redox partner gene; the redox partner gene encodes a redox partner for transferring electrons from an electron donor NAD (P) H to a P450 enzyme active site.
The amino acid sequence of the cytochrome P450 enzyme Olep is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the cytochrome P450 enzyme Olep is shown in SEQ ID NO. 9.
In certain embodiments, the redox partner genes include a combination of the gene encoding a synechocystis (Synechocystis PCC 6803) -derived ferredoxin reductase PetH and a ferredoxin PetF. The amino acid sequence of the ferredoxin reductase PetH is shown as SEQ ID NO.7, and the amino acid sequence of the ferredoxin PetF is shown as SEQ ID NO. 8.
In certain embodiments, the redox partner gene is selected from any one of the following (1), (2), (3): (1) genes encoding ferredoxin reductase CamA and ferredoxin CamB derived from Pseudomonas putida Pseudomonas putida, (2) genes encoding reductase domain of BM3 derived from Bacillus megaterium Bacillus megatherium, and (3) genes encoding ferredoxin reductase Fdr and ferredoxin Fdx derived from Synechococcus elongatus Synethococcus elongatus PCC 7942. The amino acid sequences of the ferredoxin reductase CamA, the ferredoxin CamB, the reductase domain of BM3, the ferredoxin reductase Fdr and the ferredoxin Fdx are respectively shown in SEQ ID NO. 2-SEQ ID NO.6, and the nucleotide sequences of the encoding ferredoxin reductase CamA, the ferredoxin CamB, the reductase domain of BM3, the ferredoxin reductase Fdr and the ferredoxin Fdx genes are respectively shown in SEQ ID NO. 10-SEQ ID NO. 14.
In certain embodiments, the cytochrome P450 enzyme Olep is fused at the N-terminus to the pro-lytic tag MBP or TF.
In certain embodiments, the E.coli co-expresses the gene encoding the cytochrome P450 enzyme Olep and the redox partner gene using an expression vector.
Further, the expression vector is pRSFDuet-1 plasmid, pETDuet plasmid or pACYCDuet plasmid.
In certain embodiments, the E.coli includes, but is not limited to E.coli BL21 (DE 3), C41 (DE 3) or C43 (DE 3).
In certain embodiments, the genetically engineered bacterium also expresses both a glutamyl tRNA reductase gene hemA and a glutamate-1-semialdehyde aminotransferase gene hemL derived from E.coli (Escherichia coli). The synthesis pathway of heme is divided into ALA synthesis pathway and downstream heme synthesis pathway, wherein key genes of ALA synthesis pathway include hemA (NCBI accession No. NP-415728.1) and hemL (NCBI accession No. NP-414696.1).
In certain embodiments, the genetically engineered bacterium also expresses NAD derived from Escherichia coli + Kinase gene NadK and membrane-bound transhydrogenase gene PntAB.
The second technical scheme provided by the invention is the construction method of the genetically engineered bacterium of the first technical scheme, comprising the following steps:
s1, using escherichia coli as a chassis strain, expressing a cytochrome P450 enzyme Olep from streptomyces (Streptomyces antibioticus);
s2, expressing a redox partner gene in the recombinant escherichia coli obtained in the step S1.
In certain embodiments, the redox partner gene is a combination of the gene encoding a synechocystis (Synechocystis PCC 6803) -derived ferredoxin reductase PetH and a ferredoxin PetF.
In certain embodiments, the redox partner gene is selected from any one of the following (1), (2), (3): (1) genes encoding ferredoxin reductase CamA and ferredoxin CamB derived from Pseudomonas putida Pseudomonas putida, (2) genes encoding reductase domain of BM3 derived from Bacillus megaterium Bacillus megatherium, and (3) genes encoding ferredoxin reductase Fdr and ferredoxin Fdx derived from Synechococcus elongatus Synethococcus elongatus PCC 7942.
In certain embodiments, the cytochrome P450 enzyme Olep is fused at the N-terminus to the pro-lytic tag MBP or TF.
In certain embodiments, the E.coli co-expresses the gene encoding the cytochrome P450 enzyme Olep and the redox partner gene using an expression vector.
Further, the expression vector is pRSFDuet-1 plasmid, pETDuet plasmid or pACYCDuet plasmid.
In certain embodiments, the E.coli includes, but is not limited to E.coli BL21 (DE 3), C41 (DE 3), or C43 (DE 3).
In some embodiments, the method further comprises step S3, specifically as follows: expressing a glutamyl tRNA reductase gene hemA and a glutamic acid-1-semialdehyde transaminase gene hemL derived from Escherichia coli (Escherichia coli) in the recombinant Escherichia coli obtained in step S2.
Further, the method also comprises a step S4, which is specifically as follows: expression of NAD derived from Escherichia coli (Escherichia coli) in the recombinant Escherichia coli obtained in step S3 + Kinase gene NadK and membrane-bound transhydrogenase gene PntAB.
The third technical scheme provided by the invention is a method for preparing stereoselectivity hydroxylation deoxycholic acid by whole cell catalysis, wherein the genetically engineered bacterium of the first technical scheme is used as a whole cell catalyst to catalyze the 6 beta hydroxylation of deoxycholic acid.
In certain embodiments, the whole cell catalyst is prepared by: culturing genetically engineered bacteria at 35-37deg.C to OD 600 Adding isopropyl-beta-D-thiogalactoside with the concentration of 0.1-1mM with the value of 0.6-0.8, and continuously culturing at 16-30 ℃ for 12-20h; and centrifuging at low temperature, collecting and washing thalli, and re-suspending thalli after washing is completed to obtain the whole cell catalyst.
Further, the washing uses potassium phosphate buffer with pH of 8.0.
In certain embodiments, the resuspension employs potassium glycerophosphate buffer at a pH of 8.0, 5% -10% v/v.
In certain embodiments, deoxycholic acid 6 beta is catalyzedThe hydroxylation reaction is carried out in a 50-100mM potassium phosphate buffer system, and the biomass OD of genetically engineered bacteria 600 10-35, deoxycholate concentration 0.5-2.0mg/mL, reaction temperature 20-37 ℃ and reaction time 1-24h.
The fourth technical scheme provided by the invention is the genetically engineered bacterium of the first technical scheme, the method of the second technical scheme or the application of the method of the third technical scheme in the production of 6 beta-OH deoxycholic acid.
The fifth technical scheme provided by the invention is the application of the genetically engineered bacterium described in the first technical scheme or the method described in the second technical scheme in the production of the deoxycholic acid.
In certain embodiments, the synthesis of murine deoxycholic acid (MDCA) is catalyzed by lithocholic acid (LCA) as a substrate.
Compared with the prior art, the beneficial effects are that:
(1) The invention can improve the soluble expression of the P450 enzyme Olep by 51.7 times and the catalytic efficiency by means of screening plasmids with different copy numbers, expressing hosts of escherichia coli, promoting dissolution labels, molecular chaperones, optimizing induction conditions and the like.
(2) Further, through in vitro addition of heme precursor and intracellular reinforcement of heme synthesis pathway, the heme binding rate of P450 enzyme Olep is improved to 67.7%, the hydroxylation capacity of catalytic deoxycholic acid (DCA) 6 beta is enhanced, and the conversion rate of deoxycholic acid is improved to 40.7%.
(3) Further, by designing a novel redox partner sensor, the redox partners PetH and PetF matched with the P450 enzyme Olep are selected, the capability of catalyzing the hydroxylation of deoxycholic acid 6 beta is enhanced, and the conversion rate of deoxycholic acid is improved to 89.2%.
(4) Still further, through optimizing the whole-cell catalytic system of the P450 enzyme Olep and constructing a cofactor circulation system, the capability of catalyzing the 6 beta hydroxylation of deoxycholic acid is enhanced, and finally, the conversion rate of synthesizing 6 beta-OH deoxycholic acid by catalyzing the deoxycholic acid reaches 99.1 percent.
Drawings
FIG. 1 is a graph showing analysis of the expression and catalytic efficiency of the P450 enzyme Olep in E.coli; a is a full wavelength scan of purified Olep in three states; b is SDS-PAGE analysis of Olep enzyme, lane 1: soluble expressed Olep, lane 2: inclusion bodies of Olep; lane 3: purifying the Olep enzyme; m: a marker; c is an analysis chart of an Olep catalytic deoxycholic acid hydroxylation product, a is a control reaction catalyzed by E.coli C43 (DE 3) carrying pET28a empty plasmid, and b recombinant strain E.coli O1 catalyzes DCA hydroxylation.
FIG. 2 is a graph showing the enhancement of soluble expression of the P450 enzyme Olep in E.coli; a is screening of plasmids; b is screening of host cells; c is the screening of the dissolution promoting label; d is the screening of plasmids with different copy numbers; e is the screening of the optimal induction temperature; f is the screening of IPTG concentration.
FIG. 3 shows the effect of different chaperones on the expression of the P450 enzyme Olep in E.coli, wherein lanes 1, 3, 5, 7, 9 are respectively the intracellular enzyme-broken supernatant C41-pRSFDuet-camA-camB-olesP and pGro7, pKJE7, pGKJE8, pTf, pTf2; lanes 2, 4, 6, 8, 10 are supernatant C41-pRSFDuet-camA-camB-oleP and pGro7, pKJE7, pG-KJE8, pTf, pG-Tf2, respectively, of the pellet after inclusion body-wall disruption, resuspended with 8M urea.
FIG. 4 is a graph showing enhancement of heme supply in E.coli to increase the heme binding rate of the P450 enzyme Olep; a is heme binding rate of wild Olep, MBP-Olep and TF-Olep; b is the biosynthesis path of colibacillus heme; c is the effect of the extender on Olep catalysis; d is the color of the engineering strain and the pure enzyme reaction solution, 1: coli O1 strain; 2: coli O2 strain; 3: with ALA and FeCl 3 A cultured e.coli O2 strain; 4: e.coli AL-BCDH strain; 5: olep enzyme purified from E.coli AL-BCDH, bottom values represent intracellular heme content of different engineering strains.
FIG. 5 is a schematic diagram showing the construction of a novel sfGFP sensor to screen for redox partners that are compatible with the P450 enzyme Olep; a is a protocol for constructing an sfGFP sensor; b is Olep and Fdx self-assembly based on sfGFP three-dimensional structure; c is screening of redox partner genes of different sources; d is the catalytic conversion of recombinant strains R2 to R5.
FIG. 6 is a schematic illustration of the construction of a highly efficient E.coli whole cell catalytic system; a is biomass OD 600 For allEffects of cell catalysis; b is the influence of substrate concentration on whole cell catalysis; c is the influence of the catalysis time on whole cell catalysis; d is the effect of NADPH on whole cell activity.
FIG. 7 shows the preparation of murine deoxycholate from whole cell catalytic lithocholic acid: a is biocatalytic reaction of LCA, a is E.coli C41 (DE 3) strain carrying pRSFDuet-1 empty plasmid as control group, b is recombinant strain E.coli O1 catalyzes hydroxylation of LCA, C is recombinant strain E.coli C4 catalyzes hydroxylation of LCA, black and red arrows point to substrate (LCA) and hydroxylation product (MDCA), respectively; b is mass spectrometry of a substrate LCA; c is mass spectrometry analysis of the product MDCA.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Materials and methods
LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, and sterilizing at 121deg.C for 15min.
TB medium: 12g/L peptone, 5g/L glycerol, 24g/L yeast extract, 17mM potassium dihydrogen phosphate, 72mM dipotassium hydrogen phosphate, and sterilizing at 121deg.C for 15min.
Plasmid and cell: strains Escherichia coli BL (DE 3), escherichia coli C (DE 3) and Escherichia coli C43 (DE 3), plasmid pET28a, pACYCDuet, pRSFDuet, pRSFDuet-1 is a commercial strain, plasmid.
Primer sequence synthesis, reagent purchase and gene sequencing verification are all purchased and completed by Shanghai Biotechnology company.
Whole cell catalysis:
(1) Culturing recombinant Escherichia coli at 35-37deg.C to OD 600 The value is 0.6-0.8, and the culture is continued for 12-20h at 16-30 ℃ after adding isopropyl-beta-D-thiogalactoside. Wherein the concentration of isopropyl-beta-D-thiogalactoside is 0.1-1mM.
(2) Centrifuging the fermentation broth obtained in the step (1) at 4 ℃ and 8000rpm for 10-20min, collecting thalli, and washing the thalli with potassium phosphate buffer with pH of 8.0. After the washing, the washed cells were resuspended in potassium phosphate (containing 5% -10% v/v glycerol) at pH 8.0.
(3) Carrying out hydroxylation reaction of deoxycholic acid by using the bacterial suspension obtained in the step (2), wherein a whole-cell reaction system comprises (based on the final concentration): 50g/L of bacterial cells and 1mg/mL of deoxycholic acid. The whole cell reacts at 20-37 ℃ for at least 1-12h, after the reaction is finished, a proper amount of reaction liquid is taken out, the ethyl acetate with the same volume is added for extraction, and after 3 times of extraction and separation, 6 beta-OH deoxycholic acid is obtained and analyzed and detected by high performance liquid chromatography.
High performance liquid chromatography:
(1) Mobile phase: phase a is ultrapure water containing 0.1% trifluoroacetic acid, and phase B is methanol containing 0.1% trifluoroacetic acid.
(2) Chromatographic column: reverse phase chromatography column ZORBAX Eclipse XDB-C18 (5 μm, 4.6X1250 mm, agilent, USA); column temperature is 40 ℃; flow rate: 0.8mL/min; elution procedure: 0-1min,10% B;1-10min,10% -40% B;10-20min,40% -90% B;20-23min,90% B;23-25min,90% -10% B;25-27min,10% B.
(3) The temperature of the drift tube was 40℃using an evaporative light scattering detector, nitrogen 350kPa, gain 6.
Calculation of conversion: the concentration of the product (mg/mL)/the concentration of the initial addition substrate (mg/mL). Times.100%.
Example 1: the soluble expression of the P450 enzyme Olep in escherichia coli is enhanced, and the capability of catalyzing the 6 beta hydroxylation of deoxycholic acid is improved.
P450 enzyme CYP107D1 (the amino acid sequence of which is shown as SEQ ID NO. 1) derived from streptomyces (Streptomyces antibioticus) is selected, abbreviated as Olep, a histidine tag is added at the C end through escherichia coli codon optimization, the gene (the nucleotide sequence of which is shown as SEQ ID NO. 9) is synthesized, and the gene is subcloned between the cleavage sites NcoI and XhoI of the plasmid pET28a, so that the recombinant plasmid pET28a-oleP is obtained.
Since the CYP107 family is a typical three-component P450 enzyme, requiring a reduction chaperonin to transfer electrons from an electron donor NAD (P) H to the active center of the P450 enzyme, ferredoxin reductase CamA (amino acid sequence shown in SEQ ID NO. 2) and ferredoxin CamB (amino acid sequence shown in SEQ ID NO. 3) derived from Pseudomonas putida (Pseudomonas putida) are selected as reduction chaperonins. Synthesizing a ferredoxin reductase gene CamA and a ferredoxin gene CamB gene (the nucleotide sequences are shown as SEQ ID NO. 10-11) with optimized codons, and subcloning the CamA and the CamB gene onto a plasmid pACYCDuet to obtain a recombinant plasmid pACYCDuet-camA-camB.
Then the recombinant plasmid pET28a-oleP, pACYCDuet-camA-camB is transformed into an escherichia coli expression host C43 (DE 3) to obtain 1 recombinant strain which is named as E.coli O1.
Shake flask fermentation of E.coli O1 recombinant strain using TB culture when recombinant strain E.coli O1 is cultured to OD at 37 ℃ 600 At a value of 0.6-0.8, 0.5mM isopropyl-. Beta. -D-thiogalactoside was added and the culture was continued at 25℃for 20 hours. After the fermentation was completed, the fermentation broth was centrifuged at 8000rpm at 4℃for 10min, and the cells were collected and washed with potassium phosphate buffer at pH 8.0.
On the other hand, after the completion of the cell washing, the cells were resuspended in potassium phosphate buffer (containing 500mM sodium chloride, 20mM imidazole) at pH 8.0, and disrupted on ice by an ultrasonic disrupter. The procedure of the ultrasonic breaker is as follows: power,38%;2s on/3s off;5min. The cell disruption solution is centrifuged for 10min at 4 ℃ and 8000rpm, and the supernatant is collected to obtain Olep crude enzyme. SDS-PAGE of Olep crude enzyme (cell disruption solution) showed that Olep expressed in E.coli formed a large number of inclusion bodies, only 32.4% of soluble protein. The Olep pure enzyme was obtained by Ni-NTA affinity chromatography, and the elution buffer was potassium phosphate buffer (containing 500mM sodium chloride, 200mM imidazole) at pH 8.0.
Purified Olep was desalted using an Amicon Ultra 30K ultrafiltration centrifuge tube. CO spectral experiments show that the pure enzyme Olep has a characteristic absorption peak at the wavelength of 450 nm.
On the other hand, after the cell washing was completed, the washed cell was resuspended in potassium phosphate (containing 10% v/v glycerol) at pH 8.0 to obtain a whole cell catalyst. The whole cell catalyzed reaction system is (based on final concentration): strain cell OD 600 =30, deoxycholic acid 1mg/mL, NADPH 1mM, glucose dehydrogenase 1U/mL. The results are shown in FIG. 1, recombinant strain E.coli O1 (containing recombinant plasmids pET28 a-oliP and pACYCDuet-camA-camB) can generate 0.048mg/mL 6 beta-OH deoxycholic acid (conversion rate 4.8%). Therefore, there is a need to increase the soluble expression of Olep in E.coli, thereby increasing the efficiency of Olep catalyzed preparation of 6β -OH deoxycholic acid.
The soluble expression of the P450 enzyme is further improved by screening different copy number plasmids (low copy plasmid pACYCDuet, medium copy plasmid pETDuet, high copy plasmid pRSFDuet), escherichia coli expression hosts (Escherichia coli BL (DE 3), escherichia coli C (DE 3) and Escherichia coli C (DE 3)), a dissolution promoting tag (SUMO, GST, MBP, TF, trx, nus), molecular chaperones (pGro 7, pKJE7, pG-KJE8, pTf16, pG-Tf 2), optimizing induction conditions and other strategies. When plasmids with different copy numbers are selected, pET28a-camA-camB-oleP plasmids are used as templates, a camA, camB, oleP sequence is amplified by a PCR program, and camA, camB, oleP sequences are respectively connected with pACYCDuet, pETDuet and pRSFDuet plasmids by a one-step cloning method to construct different recombinant plasmids.
When different dissolution promoting labels are screened, pACYCDuet-SUMO, pACYCDuet-GST, pACYCDuet-MBP, pACYCDuet-TF and pACYCDuet-Trx, pACYCDuet-Nus plasmids are used as templates, different dissolution promoting label SUMO, GST, MBP, TF, trx, nus sequences are amplified by a PCR program, and then the SUMO, GST, MBP, TF, trx, nus sequences are respectively connected with pRSFDuet-camA-camB-oleP plasmids by a one-step cloning method to construct complete plasmids containing different dissolution promoting labels. When screening different molecular chaperones, plasmids pG-KJE, pGro7, pKJE7, pG-Tf2 and pTf16 are respectively transferred into competent pRSFDuet-ca mA-cam B-oleP-C41 (DE 3) cells to construct strains containing different molecular chaperones. Finally, the recombinant strain E.coli O2 is obtained, and the results are shown in fig. 2 and 3, wherein the expression system of the E.coli O2 is as follows: a high copy of the recombinant plasmid pRSFDuet-camA-camB-oleP was selected, and instead of the ligation of the fusion promoting tag and the co-expression chaperone, E.coli C41 (DE 3) was selected as the expression host, the induction temperature was 25℃and the inducer IPTG concentration was 0.5mM, the soluble expression of Olep reached 67.1%, under which conditions 0.348mg/mL 6. Beta. -OH deoxycholic acid was produced using deoxycholic acid at a final concentration of 1mg/mL (conversion 34.8%). Compared with the initial strain E.coli O1, the soluble expression of Olep is improved by 51.7%, and the catalytic efficiency is improved by 6.25 times.
Example 2: the heme binding rate of the P450 enzyme Olep in escherichia coli is improved, and the capability of catalyzing deoxycholic acid 6 beta hydroxylation is improved.
In example 1, it was found that when the pro-lytic tag MBP or TF was fused at the N-terminus of Olep, the catalytic efficiency was reduced although the soluble expression of Olep was increased to 92.3%, 91.5%. When the heme binding rate was measured (FIG. 4A), it was found that the heme binding rates of MBP-Olep and TF-Olep were only 21.1% and 16.8%, respectively, which were lower than those of the heme binding rate without the solubilizing label Olep (39.4%). Insufficient heme supply may therefore be a key factor limiting the catalytic efficiency of Olep. There are currently mainly 2 ways to enhance the heme supply of P450 enzymes (fig. 4B). One strategy is to exogenously add the precursor 5-aminolevulinic acid (ALA) of Heme during P450 enzyme expression, and the other strategy is to intensify the synthesis pathway of Heme in E.coli.
First 3 different exogenous additive combinations were tested (direct addition of Heme, addition of precursor ala+feso 4 Addition of the precursor ALA+FeCl 3 ). The results showed (FIG. 4C) that 0.64mM ALA and 0.3mM FeCl were added during the induction of Olep 3 The heme binding rate of Olep was increased to 67.7% and the deoxycholic acid conversion rate was also increased to 40.7%. But the method of exogenously adding ALA increases the whole cell catalytic cost by 60%, so another strategy will be tested. The synthesis pathway of heme is divided into ALA synthesis pathway and downstream heme synthesis pathway. The key genes for the ALA synthesis pathway are hemA (NCBI accession No. NP-415728.1) and hemL (NCBI accession No. NP-414696.1), the key genes for the downstream heme synthesis pathway are hemB (NCBI accession No. NP-414903.4), hemC (NCBI accession number YP_ 026260.1), hemD (NCBI accession number NP_ 418248.1) and hemH (NCBI accession number NP_ 415008.1). Thus, first, recombinant plasmids pCDFDuet-hemA-hemH (subcloning hemA gene between NcoI and AflII sites of plasmid pCDFDuet, subcloning hemL gene between NdeI and PacI sites of plasmid pCDFDuet), pETDuet-hemB-hemC-hemD-hemH (subcloning hemB, hemC, hemD and hemH genes, respectively) were constructed, respectively3 recombinant strains were constructed from the above plasmid (E.coli AL strain (recombinant plasmid pCDFDuet-hemA-hemL was expressed in E.coli O2), E.coli BCDH strain (recombinant plasmid pETDuet-hemB-hemC-hemD-hemH was expressed in E.coli O2), E.coli AL-BCDH strain (recombinant plasmid pCDFDuet-hemA-hemH and pETDuet-hemB-hemC-hemD-hemH were expressed in E.coli O2) after the cleavage site NcoI and the AflII site of plasmid pETDuet were separated by RBS sequence GGATCCGAATTCGAGCTCATAAAAGGAGGAAAATAT).
Recombinant strain E.coli AL without ALA and FeCl addition 3 On the premise of (a) that, as shown in fig. 4D and 4E, the heme binding rate of Olep was increased to 53.9%, and the conversion rate of deoxycholic acid was also increased to 41.4%.
Example 3: and a fluorescence sensor is constructed to screen an oxidation-reduction partner optimally matched with the P450 enzyme Olep, so that the capability of catalyzing the 6 beta hydroxylation of deoxycholic acid is improved.
For three-component P450 enzymes, the efficiency of electron transfer is critical for whole cell catalysis. In order to screen out suitable redox partners, a P450 BM3 reduction domain part (the amino acid sequence of which is shown as SEQ ID NO.4, and the nucleotide sequence of which is shown as SEQ ID NO. 12) derived from bacillus megaterium (Bacillus megaterium) is selected as a group 1 redox partner; selecting a combination of a ferredoxin reductase CamA and a ferredoxin CamB from Pseudomonas putida (Pseudomonas putida) as a group 2 redox partner; selecting ferrioxacin reductase FdR _0978 (with amino acid sequence shown in SEQ ID NO.5 and nucleotide sequence shown in SEQ ID NO. 13) and ferrioxacin Fdx _1499 (with amino acid sequence shown in SEQ ID NO.6 and nucleotide sequence shown in SEQ ID NO. 14) derived from Synechococcus (Synechococcus elongates) as a group 3 redox partner combination; ferredoxin reductase PetH (with the amino acid sequence shown as SEQ ID NO.7, the nucleotide sequence shown as SEQ ID NO. 15) and ferredoxin PetF (with the amino acid sequence shown as SEQ ID NO.8, the nucleotide sequence shown as SEQ ID NO. 16) derived from synechocystis (Synechocystis PCC 6803) are selected as the group 4 redox partner combination.
For rapid screening of redox partners optimally adapted to Olep, the technique is based on bimolecular fluorescence complementationA novel redox partner sensor was constructed to measure the interaction force between Olep and the redox partner. The sensor used a superfolder green fluorescent protein (sfGFP) which was split into two parts, sfGFP-1-10 (NCBI accession number 4W6R_A) and sfGFP-11 (NCBI accession number 4W6R_A) without fluorescence. When the interaction between sfGFP-1-10 and sfGFP-11 occurs, the spatial distance is shortened, and the refolding can emit green fluorescence. Thus, 4 kinds of iron redox proteins were fused to the N-terminus of sfGFP-1-10 and Olep was fused to the C-terminus of sfGFP-11, respectively, to give recombinant plasmid pRSFDuet-BM3-GFP-1- 10-GFP-11-olep，pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep，pRSFDuet-FdR0978-Fdx1499-GFP-1-10-GFP-11-olep，pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep(underline represents fusion gene). The above 4 recombinant plasmids were transformed into BL21 (DE 3) to obtain recombinant strains G2 strain to G5 strain, respectively. The recombinant strains G2 to G5 were subjected to shaking flask fermentation under the same conditions as in example 1, and after completion of the fermentation, 200ul of cells were added to a 96-well plate and the biomass (wavelength: 600 nm) and fluorescence values (excitation wavelength: 488nm, emission wavelength: 520 nm) were measured by an enzyme-labeled instrument. The fluorescence intensity (fluorescence value/biomass) of the strain was calculated. Recombinant strain G5 (containing recombinant plasmid pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep) The fluorescence intensity of (1.2X10) 6 ) Is a control strain G2 (containing recombinant plasmid pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep) Is 6 times the fluorescence intensity of (c).
In addition, 4 sets of redox partners were constructed on high copy plasmids pRSFDuet to obtain recombinant plasmids pRSFDuet-BM3-olep (fusion protein BM3-olep gene was subcloned between the NdeI and XhoI sites of the plasmid pRSFDuet), pRSFDuet-camA-camB-olep (camA, camB genes were subcloned between the NdeI and AflII sites of the plasmid pRSFDuet, and RBS sequence GGATCCGAATTCGAGCTCATAAAAGGAGGAAAATAT was used for the separation, olep gene was subcloned between the NdeI and XhoI sites of the plasmid pRSFDuet, pRSFDuet-FdR-0978-Fdx-1499-olep (FdR,351499 genes were subcloned between the NcoI and AflII sites of the plasmid pRSFDuet), RBS sequence GGATCCGAATTCGAGCTCATAAAAGGAGGAAAATAT was used for the separation, and the restriction of the gene was subcloned between the NdeI and XhoI sites of the plasmid pRSFDuet, and the NdeF-FdR-0978-Fdx-1499-olep genes were used for the separation between the NdeF and XpeDuet. And transformed into C41 (DE 3) to obtain recombinant strains R2 to R5. Recombinant strains G2 to G5 were subjected to shake flask fermentation experiments and whole cell catalytic system was as in example 1. As a result, as shown in FIG. 5, the conversion rate of recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-ole) was highest, and significantly increased from 41.4% to 89.2%. Therefore, the oxidation-reduction partner PetH/PetF from the synechocystis is successfully screened as the optimal oxidation-reduction partner of the P450 enzyme Olep, and the construction of the sfGFP sensor is verified, so that the oxidation-reduction partner matched with the P450 enzyme can be screened efficiently and accurately.
Example 4: the full-cell catalytic system with the highest efficiency of the P450 enzyme Olep is constructed, the capability of catalyzing the 6 beta hydroxylation of deoxycholic acid is improved, and the 6 beta hydroxylation of lithocholic acid is prepared by catalysis.
The three strategies of example 1, example 2 and example 3 were combined to give a highly efficient recombinant strain E.coli C1 (containing recombinant plasmids pRSFDuet-petH-petF-olep and pCDFDuet-hemA-hemL). The strain C1 is free from ALA and FeCl during induction 3 In the case of (2), the conversion rate reached 89.7%. The whole cell catalytic condition optimization was then performed on the C1 strain. When the substrate concentration at the time of the reaction was set to 1mg/mL and the catalytic time was set to 12 hours, the biomass OD was examined 600 (10, 15, 20, 25, 30, 35) effect on whole cell catalysis; when biomass OD 600 When the catalyst time was set to 30 and 12 hours, the effect of the substrate concentration (0.5 mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL, 3.5 mg/mL) on whole cell catalysis was examined; when biomass OD 600 When the substrate concentration was set to 30 and 2.0mg/mL, the effect of the catalytic time (0 h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24 h) on whole cell catalysis was examined. Other reaction conditions were the same as in example 1. The results are shown in FIG. 6, where the optimal reaction conditions for strain C1 areIs biomass OD 600 The concentration of the substrate was set at 30, the concentration of the substrate was set at 2.0mg/mL, and the catalytic time was set at 12h. Under the reaction conditions, the whole-cell catalyst prepared by the C1 strain can generate 1.85mg/mL 6 beta-OH deoxycholic acid (the conversion rate is 92.5%).
The P450 enzyme catalyzes the substrate by requiring the redox partner to transfer the electrons of NADPH to heme. The intracellular NADPH content of the escherichia coli is low, and the requirement of self-growth can be met only. And the cost of externally adding NADPH is higher, and the catalytic efficiency is lower. By constructing a cofactor circulatory system, the effect of NADPH on whole cell activity was examined. NAD (NAD) + The kinases NadK (NCBI accession NC_ 000913.3) and membrane-bound transhydrogenase PntAB (NCBI accession NP_416120.1, NP_416119.1) can be used to enhance the circulation of NADPH. Thus, 3 recombinant plasmids pACYCDuet-nadK (subcloning the gene encoding NadK between the cleavage sites NcoI and AflII of plasmid pACYCDuet), pACYCDuet-pntAB (subcloning the gene encoding PntAB between the cleavage sites NcoI and AflII of plasmid pACYCDuet), pACYCDuet-pntAB-nadK (subcloning the gene encoding NadK and the gene encoding PntAB between the cleavage sites NcoI and AflII of plasmid pACYCDuet, respectively) and E.coli C2 (recombinant plasmid pACDuet-nadK is expressed in E.coli C1), E.coli C4 (recombinant plasmid pACDuet-pnaK is expressed in E.coli C1) were constructed using the above recombinant plasmids. Under the condition of no addition of NADPH, the conversion rate of the whole cell catalyst prepared by the E.coli C4 strain is as high as 99.1 percent.
Recombinant strain e.coli C4 was subjected to shake flask fermentation using TB medium under the same conditions as in example 1, and a whole cell catalyst was prepared in the same manner as in example 1. And (3) taking lithocholic acid with the final concentration of 0.5mg/mL as a substrate to perform whole-cell catalysis. The results are shown in FIG. 7, and the conversion rate of the E.coli C4 strain for catalyzing cholic acid to generate the 6-beta-hydroxylation product of the deoxycholic acid is improved from 1.6% to 42.7% compared with the control strain E.coli O1.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A genetic engineering bacterium is characterized in that a cytochrome P450 enzyme Olep derived from streptomycete Streptomyces antibioticus is expressed by taking escherichia coli as a chassis strain, and a redox partner gene is also expressed by the genetic engineering bacterium; the redox partner encoded by the redox partner gene is used to transfer electrons from an electron donor NAD (P) H to the active center of the P450 enzyme.
2. The genetically engineered bacterium of claim 1, wherein the redox partner gene comprises a combination of genes encoding a ferredoxin reductase PetH and a ferredoxin PetF from synechocystis Synechocystis PCC 6803;
or the redox partner gene is selected from any one of the following (1), (2) and (3): (1) genes encoding ferredoxin reductase CamA and ferredoxin CamB derived from Pseudomonas putida Pseudomonas putida, (2) genes encoding reductase domain of BM3 derived from Bacillus megaterium Bacillus megatherium, and (3) genes encoding ferredoxin reductase Fdr and ferredoxin Fdx derived from Synechococcus elongatus Synethococcus elongatus PCC 7942.
3. Genetically engineered bacterium according to claim 1 or 2, wherein the cytochrome P450 enzyme Olep has an N-terminal fusion with the pro-lytic tag MBP or TF.
4. The genetically engineered bacterium of any one of claims 1 to 3, wherein the genetically engineered bacterium expresses both a glutamyl tRNA reductase gene hemA and a glutamate-1-semialdehyde transaminase gene hemL derived from Escherichia coli.
5. The genetically engineered bacterium of any one of claims 1 to 4, wherein the substrateBecause the engineering bacteria also express NAD derived from Escherichia coli + Kinase gene NadK and membrane-bound transhydrogenase gene PntAB.
6. The construction method of the genetically engineered bacterium is characterized by comprising the following steps:
s1, expressing a cytochrome P450 enzyme Olep from streptomycete Streptomyces antibioticus by taking escherichia coli as a chassis strain;
s2, expressing a redox partner gene in the recombinant escherichia coli obtained in the step S1; the redox partner encoded by the redox partner gene is used to transfer electrons from an electron donor NAD (P) H to the active center of the P450 enzyme;
s3, expressing a glutamyl tRNA reductase gene hemA and a glutamic acid-1-semialdehyde transaminase gene hemL derived from Escherichia coli in the recombinant Escherichia coli obtained in the step S2;
s4, expressing NAD derived from Escherichia coli in the recombinant Escherichia coli obtained in the step S3 + Kinase gene NadK and membrane-bound transhydrogenase gene PntAB.
7. A method for preparing stereoselective hydroxylated deoxycholic acid by using whole cell catalysis, which is characterized in that the genetically engineered bacterium according to any one of claims 1 to 5 is used as a whole cell catalyst for catalyzing 6 beta hydroxylation of deoxycholic acid.
8. The method according to claim 7, wherein the reaction for catalyzing the hydroxylation of deoxycholic acid 6 beta is carried out in a 50-100mM potassium phosphate buffer system, and the biomass OD of genetically engineered bacteria 600 10-35, deoxycholate concentration 0.5-2.0mg/mL, reaction temperature 20-37 ℃ and reaction time 1-24h.
9. Use of the genetically engineered bacterium of any one of claims 1 to 5, the method of claim 6 or the method of any one of claims 7 to 8 for the production of 6β -OH deoxycholic acid.
10. Use of the genetically engineered bacterium of any one of claims 1 to 5 or the method of claim 6 for the production of deoxycholic acid, characterized in that it uses lithocholic acid as substrate for the catalytic synthesis of the deoxycholic acid.
CN202311299446.4A 2023-10-09 2023-10-09 Method for synthesizing hydroxylated deoxycholic acid by using escherichia coli P450 enzyme whole cell catalysis and application Pending CN117487727A (en)

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