Recombinant Escherichia coli capable of efficiently synthesizing furocoumarin isodecursolactone

Abstract

The invention discloses recombinant Escherichia coli capable of efficiently synthesizing furocoumarin isodecursorlactone, and belongs to the technical field of gene engineering. According to the invention, high-activity isopentenyl transferase PpPT1 and high-activity peucedanum praeruptorum lactone synthase PpDC are screened through activity analysis, and are used for constructing a synthetic route of furocoumarin peucedanum praeruptorum lactone in escherichia coli; the activity of the PpPT1 and the PpDC is improved through a dissolution promoting label and redox partner engineering; and the synthesis level of the recombinant Escherichia coli isopraeruptolide is improved by modifying an endogenous MEP (Methyl Ester Phosphate) pathway and enhancing the supply of dimethylallyl pyrophosphate through modular optimization. The recombinant escherichia coli MAR-18 constructed by the invention can be used for synthesizing 203.7 mg / L of isodecursorlactone at the level of a fermentation tank, the molar conversion rate of umbelliferone reaches 81.4%, and a foundation is laid for green biosynthesis of other complex high-value furocoumarin.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli for efficiently synthesizing furanocoumarin isopructose lactone, belonging to the technical field of genetic engineering. Background Art

[0002] Furocoumarins are a special class of coumarin compounds found in plants of the Apiaceae, Moraceae, Rutaceae and Leguminosae families. They have a wide range of biological activities and show great application prospects in antiviral, antifungal, anti-inflammatory, antiallergic and anticancer aspects. Due to their phototherapeutic properties, linear furanocoumarins such as psoralen, zanthoxylum and bergamot have been used to treat severe vitiligo, psoriasis and fungal diseases. Isoprugated purpurogenolide is a dihydrofuranocoumarin and a key intermediate in the synthesis of complex linear furanocoumarins. In addition to its anti-inflammatory, anticancer and anti-angiogenic properties, isoprugated purpurogenolide can also inhibit the growth of β-heme crystals, which helps to improve the antimalarial activity.

[0003] At present, furanocoumarins are mainly obtained by extraction or chemical synthesis from plants. However, due to the low content of furanocoumarins in plants and the need for complex separation and purification processes, the extraction method is limited (Limones-Mendez et al., Plant Sci.292, 110392.2020; Vialart et al., Plant J.70, 460-470.2012). In particular, as a key intermediate in the synthesis of complex furanocoumarins, isoprene lactone is easily metabolized into other types of final products, so its content in plants is insufficient. Although many chemical synthesis methods have been reported for the synthesis of furanocoumarins, their industrial production is limited due to the long reaction steps and low yield (Kommera et al., Synth. Commun.50, 3204-3211.2020). Therefore, a more efficient and environmentally friendly method for producing furanocoumarins should be developed.

[0004] Compared with chemical synthesis and plant extraction methods, biosynthesis of furanocoumarins in heterologous microorganisms is a renewable and sustainable alternative ( Figure 1). Recently, researchers have studied the use of microorganisms to heterologously synthesize isoprugated peucedanum (Bu et al., ACS Synth. Biol. 9, 2282-2290.2020; Rodrigues et al., Molecules 27, 7230.2022; Wang et al., ACS Synth. Biol. 12, 2922-2933.2023). By introducing PcPT1 and ximD into Escherichia coli and Streptomyces xiamenensis, 3.6 mg / L and 5.2 mg / L of isoprugated peucedanum can be synthesized by adding the precursor 7-demethylcarboxylic acid or umbelliferone, respectively (Bu et al., ACS Synth. Biol. 9, 2282-2290.2020). However, the FAD-dependent monooxygenase XimD shows a high degree of heterozygosity in catalyzing the formation of the pyran ring, indicating that it is not the enzyme that is truly responsible for the cyclization in the coumarin biosynthesis process (He et al., ACS Catal. 9, 5291-5399.2019). Although higher titers of isopyruvate lactone (27.7 mg / L; Wang et al., ACS Synth. Biol. 12, 2922-2933.2023) can be obtained using Saccharomyces cerevisiae, studies have reported that coumarin compounds will be further converted into dihydrocoumarins in Saccharomyces cerevisiae, indicating that Saccharomyces cerevisiae is not an ideal host for the production of furanocoumarins (Haeser et al., J. Agric. Food Chem. 54, 6236-6240.2006). Other studies have shown that isopentenyl transferases from Petroselinum crispum, Pastinaca sativa, and Ficus carica cannot be functionally expressed in Saccharomyces cerevisiae (Karamat et al., Plant J. 77, 627-638. 2014; Munakata et al., New Phytol. 211, 332-344. 2016; Munakata et al., New Phytol. 225, 2166-2182. 2019). Compared with Streptomyces (stability: 26.4%), isopause lactone is more stable in Escherichia coli (stability: 99.7%), and the fermentation time of Escherichia coli is shorter, indicating that Escherichia coli is more suitable for the heterologous biosynthesis of furanocoumarins (Bu et al., ACSSynth.Biol.9, 2282-2290.2020), but the recombinant Escherichia coli that efficiently synthesizes isopause lactone has not been reported. Therefore, it is of great significance to construct a recombinant Escherichia coli that can efficiently synthesize the furanocoumarin isopause lactone, which lays the foundation for the green biosynthesis of other complex high-value furanocoumarins. Summary of the invention

[0005] The invention provides a recombinant Escherichia coli. Based on a starting strain, isopentenyl transferase and peucedanum lactone synthase are expressed; the isopentenyl transferase is selected from PcPT (GenBank: BAO31627.1) derived from Petroselinum crispum, or PsPT1 (GenBank: AJW31563.1) derived from Pastinaca sativa, or PpPT1 (GenBank: WIL06374.1) derived from Peucedanum praeruptorum; the peucedanum lactone synthase is selected from CYP76F112 (GenBank: QVT76730.1) and PpDC (amino acid sequence such as SEQ ID NO.7) with 2nd to 29th amino acids truncated.

[0006] In one embodiment, the starting strain includes but is not limited to Escherichia coli BL21 (DE3) or Escherichia coli C41 (DE3).

[0007] In one embodiment, the nucleotide sequence of the PcPT is as shown in SEQ ID NO.1, the nucleotide sequence of the PsPT1 is as shown in SEQ ID NO.2, the nucleotide sequence of the PpPT1 is as shown in SEQ ID NO.4, the nucleotide sequence of the CYP76F112 is as shown in SEQ ID NO.5, and the nucleotide sequence of the PpDC with the 2nd to 29th amino acids truncated is as shown in SEQ ID NO.6.

[0008] In one embodiment, the recombinant Escherichia coli further comprises at least one of the characteristics (a) to (c):

[0009] (a) fusing a solubility-promoting tag, such as MBP, Sumo, GST, TF, TrxA or NusA, to the N-terminus of the isopentenyl transferase;

[0010] (b) expressing a redox chaperone; the redox chaperone includes Fdx_1499 / FdR_0978 from Synechococcus sp., PetF / PetH from Synechocystis sp., or SmCPR from Silybum marianum. I453V , or flavodoxin reductase Fpr of Escherichia coli and flavodoxin Fld / FldA / FldB derived from Escherichia coli;

[0011] (c) expressing one or more of the endogenous MEP synthesis pathway enzymes DXS, IDI and IspF.

[0012] In one embodiment, the recombinant Escherichia coli expresses the isopentenyl transferase PsPT1 fused with the lytic tag MBP, and co-expresses the isoprenolactone synthase PpDC and the redox chaperone SmCPR I453V .

[0013] In one embodiment, the nucleotide sequence of the lytic tag MBP is shown as SEQ ID NO.16.

[0014] In one embodiment, the redox partner comprises Fdx_1499 (SEQ ID NO.9) / FdR_0978 (SEQ ID NO.10) from Synechococcus sp. or PetF (SEQ ID NO.11) / PetH (SEQ ID NO.12) from Synechocystis sp. or SmCPR (SEQ ID NO.13) from Silybum marianum. I453V (SEQ ID NO. 8) or a combination of flavodoxin reductase Fpr of Escherichia coli (GenBank: QJZ14319.1) and any one of flavodoxin Fld (GenBank: QJZ13227.1), FldA (GenBank: QJZ11404.1), and FldB (GenBank: QJZ13309.1) derived from Escherichia coli.

[0015] In one embodiment, the gene encoding the MEP synthesis pathway enzyme DXS is shown in Gene ID: 945060, the gene encoding IDI is shown in Gene ID: 949020; and the gene encoding IspF is shown in Gene ID: 945057.

[0016] In one embodiment, the recombinant Escherichia coli uses one or more expression vectors selected from the group consisting of pRSFDuet-1, pETDuet-1, pCDFDuet-1, and pACYCDuet-1.

[0017] The invention also provides a method for synthesizing the furanocoumarin isopructose lactone, and utilizes recombinant Escherichia coli to ferment and produce isopructose lactone.

[0018] In one embodiment, the recombinant E. coli is inoculated into a fermentation medium and cultured at 35-37°C until the OD 600 When the value is 0.6-0.8, the inducer isopropyl-β-D-thiogalactoside and the substrate umbelliferone are added and fermented at 20-37°C for 12-96h.

[0019] In one embodiment, the final concentration of the inducer isopropyl-β-D-thiogalactoside is 0.1-1.0 mM; the final concentration of the substrate umbelliferone is 100-500 mg / L.

[0020] In one embodiment, the fermentation medium includes but is not limited to TB, LB, M9 or MR medium.

[0021] The invention also provides a method for improving the ability of recombinant Escherichia coli to synthesize isoprucidin lactone.

[0022] In one embodiment, the method comprises performing one or more of the improvements (1) to (4) on the starting strain:

[0023] (1) expressing isopentenyl transferase and peucedanum lactone synthase; the isopentenyl transferase is selected from PcPT (GenBank: BAO31627.1) derived from Petroselinum crispum, or PsPT1 (GenBank: AJW31563.1) derived from Pastinaca sativa, or PpPT1 (GenBank: WIL06374.1) derived from Peucedanum praeruptorum; the peucedanum lactone synthase is selected from CYP76F112 and PpDC with 2-29 amino acids truncated;

[0024] (2) fusing a solubility-promoting tag, such as MBP, Sumo, GST, TF, TrxA or NusA, to the N-terminus of the isopentenyl transferase;

[0025] (3) expressing a redox chaperone; the redox chaperone includes Fdx_1499 / FdR_0978 from Synechococcus sp., PetF / PetH from Synechocystis sp., or SmCPR from Silybum marianum. I453V , or flavodoxin reductase Fpr of Escherichia coli and flavodoxin Fld / FldA / FldB derived from Escherichia coli;

[0026] (4) Expression of one or more of the endogenous MEP synthesis pathway enzymes DXS, IDI and IspF.

[0027] The invention also provides application of the genetically engineered bacteria in producing furanocoumarin isopructose peucedanum lactone and its derivatives.

[0028] In one embodiment, the derivatives include but are not limited to psoralen, xanthoxylum bungeanum, bergamot lactone, isoanisole, and the like.

[0029] Beneficial effects:

[0030] (1) The present invention screened out highly active isopentenyl transferases PcPT and PpPT1 and peucedanum praeruptolide synthases CYP76F112 and PpDC through activity comparison, and used them to construct a synthetic pathway of the furanocoumarin peucedanum praeruptolide in Escherichia coli.

[0031] (2) The present invention significantly improves the activities of PcPT, PpPT1, CYP76F112 and PpDC through solubilizing tags and redox chaperone engineering; and enhances the supply of dimethylallyl pyrophosphate by transforming the endogenous MEP pathway and modular optimization, thereby increasing the synthesis level of purpurogenolide in recombinant Escherichia coli.

[0032] (3) The present invention obtains recombinant Escherichia coli MAR-18 after optimization. MAR-18 can synthesize 203.7 mg / L of isoprugated peucedanum lactone at the fermentation tank level, and the molar conversion rate of umbelliferone reaches 81.4%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 For the furanocoumarin biosynthetic pathway in recombinant Escherichia coli.

[0034] Figure 2 To screen for isopentenyl transferases that efficiently catalyze the synthesis of 7-demethylxanthin from umbelliferone.

[0035] Figure 3 This is the HPLC chromatogram of the synthesis of 7-demethylcork pepper from umbelliferone.

[0036] Figure 4 The effect of dimethylallyl pyrophosphate concentration on the activity of isopentenyl transferase.

[0037] Figure 5 Improving the catalytic efficiency of peucedanum argentea lactone synthase for reductive chaperone engineering.

[0038] Figure 6 This is the HPLC chromatogram of the synthesis of isopructose lactone by whole cell catalysis of 7-demethyl cypermethrin.

[0039] Figure 7 To transform the endogenous MEP pathway, enhance the supply of dimethylallyl pyrophosphate and improve the synthesis of peucedanum lactone.

[0040] Figure 8 Modular optimization for enhanced synthesis of isoprugated peucedanum lactone in recombinant Escherichia coli MAR-18.

[0041] Fig. 9 Optimization of fermentation conditions at shake flask level for recombinant Escherichia coli MAR-18.

[0042] Fig.10 To efficiently synthesize isoprugated peucedanum lactone at the fermentation tank level in recombinant Escherichia coli MAR-18. DETAILED DESCRIPTION

[0043] Materials and Methods

[0044] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, sterilized at 121°C for 15 min.

[0045] TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM potassium dihydrogen phosphate, sterilized at 121°C for 15 min.

[0046] MR culture medium: 6.67 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 0.8 g / L citric acid, 0.8 g / L magnesium sulfate heptahydrate, 5 mL TMS buffer, sterilized at 121°C for 15 min.

[0047] TMS buffer: 10mL hydrochloric acid, 10g / L ferrous sulfate heptahydrate, 2.25g / L zinc sulfate heptahydrate, 0.58g / L manganese sulfate pentahydrate, 1g / L copper sulfate pentahydrate, 0.1g / L ammonium molybdate tetrahydrate, 0.02g / L borax decahydrate, 2g / L calcium chloride dihydrate.

[0048] Feed medium: 700 g / L glucose, 8 g / L MgSO 4 7H 2 O, 5 g / L yeast extract and 5 g / L (NH 4 ) 2 SO.

[0049] Fermentation conditions:

[0050] (1) Cultivate the recombinant E. coli at 35-37°C until OD 600 The value is 0.6-0.8, and after adding isopropyl-β-D-thiogalactoside, the culture is continued at 20-30°C for 12-20 hours. The concentration of isopropyl-β-D-thiogalactoside is 0.1-1.0mM.

[0051] (2) The fermentation broth obtained in step (1) was centrifuged at 4°C and 8000 rpm for 10-20 min to collect the bacterial cells, and the bacterial cells were washed with a potassium phosphate buffer at pH 8.0. After washing, the cells were used for crude enzyme solution reaction or whole cell catalysis.

[0052] Crude enzyme solution reaction:

[0053] The washed cells in step (2) were resuspended in a potassium phosphate buffer at pH 8.0 so that the OD values ​​of the different recombinant bacterial suspensions were all 15, and the crude enzyme solution was obtained by ultrasonic disruption. The reaction system of the crude enzyme solution was 100 μL potassium phosphate buffer: containing 50 μL crude enzyme solution, 100 mg / L umbelliferone, 0.1 mM-0.5 mM dimethylallyl pyrophosphate, MgCl 20.2mM. The crude enzyme solution was reacted at 20-37°C for at least 1-12h. After the reaction, an equal volume of methanol was added for mixing. The reaction mixture was centrifuged at 14000rpm for 10-20min, and the supernatant was passed through a membrane for high performance liquid chromatography analysis.

[0054] Whole cell catalysis:

[0055] The washed bacteria in step (2) are resuspended in potassium phosphate (containing 5%-10% v / v glycerol) at pH 8.0. The bacterial suspension is obtained for whole-cell catalysis of 7-demethylated cypermethrin to synthesize isoprugated peucedanum lactone. The whole-cell reaction system includes (by final concentration): 50 g / L bacterial cells and 115 mg / L 7-demethylated cypermethrin. The whole-cell reaction is carried out at 20-37°C for at least 1-12 hours. After the reaction is completed, an appropriate amount of the reaction solution is taken out and added with an equal volume of methanol for mixing. The reaction mixture is centrifuged at 14000 rpm for 10-20 minutes, and the supernatant is passed through a membrane for high performance liquid chromatography analysis.

[0056] Synthesis of isoprugated peucedanum lactone by shake flask level:

[0057] When the recombinant E. coli was cultured at 37°C until OD 600 When the value is 0.6-0.8, add isopropyl-β-D-thiogalactoside and substrate umbelliferone, and ferment at 20-37℃ for 72h. The concentration of isopropyl-β-D-thiogalactoside is 0.1-1.2mM; the concentration of substrate umbelliferone is 100-500mg / L. Take samples every 24h, take out an appropriate amount of fermentation liquid and add an equal volume of methanol to mix. The mixed solution is centrifuged at 14000rpm for 10-20min, and the supernatant is used for high performance liquid chromatography analysis after passing through the membrane.

[0058] Synthesis of isoprugated peucedanum lactone by fermentation tank level:

[0059] When the recombinant bacteria were cultured at 37°C until OD 600 When the value is 5, add 0.4mM isopropyl-β-D-thiogalactoside and umbelliferone (100mg / L or 200mg / L) and ferment at 30℃ for 96h. During the fermentation process, the dissolved oxygen in the fermentation liquid is controlled at 30% by adjusting the speed change (400-800rpm), and the pH of the fermentation liquid is controlled to be maintained at 7.0±0.02 using 50% ammonia water. pH-star is used for feeding, and the feeding medium is 700g / L glucose, 8g / L magnesium sulfate heptahydrate, 5g / L yeast extract and 5g / L ammonium sulfate. Samples are taken every 8h, and an appropriate amount of fermentation liquid is taken out and mixed with an equal volume of methanol. The mixed solution is centrifuged at 14000rpm for 10-20min, and the supernatant is used for high performance liquid chromatography analysis after passing through the membrane.

[0060] HPLC analysis:

[0061] (1) Mobile phase: Phase A is ultrapure water containing 0.1% trifluoroacetic acid, and phase B is methanol containing 0.1% trifluoroacetic acid.

[0062] (2) Chromatographic column: Reverse phase column ZORBAX Eclipse XDB-C18 (5 μm, 4.6×250 mm, Agilent, USA); column temperature was 30°C; flow rate was 0.8 mL / min; elution program was: 0-1 min, 10% B; 1-10 min, 10%-50% B; 10-20 min, 50%-80% B; 20-26 min, 80% B; 26-28 min, 80%-10% B; 28-30 min, 10% B.

[0063] (3) Use an ultraviolet detector with a detection wavelength of 330 nm.

[0064] Calculation of molar conversion rate: product molar concentration (mM) / initial substrate molar concentration (mM) × 100%.

[0065] Product molar concentration (mM): product concentration (mg / L) / product molar mass (g / mol)

[0066] Initial substrate substance concentration (mM): substrate concentration (mg / L) / substrate molar mass (g / mol)

[0067] Example 1: Screening for an isopentenyl transferase that efficiently catalyzes the synthesis of 7-demethylxanthin from umbelliferone

[0068] PcPT from Petroselinum crispum (GenBank: BAO31627.1), PsPT1 from Pastinacasativa (GenBank: AJW31563.1), FcPT1 from Ficus carica (GenBank: BBC82715.1), and PpPT1 from Peucedanum praeruptorum (GenBank: WIL06374.1) were selected, and the above genes (nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively) were synthesized through Escherichia coli codon optimization, and the above synthetic genes were subcloned into the Nde I and Xho I sites of pRSFDuet-1, respectively, to obtain four recombinant plasmids pRSF-PcPT, pRSF-PsPT1, pRSF-FcPT1 and pRSF-PpPT1. Further, in order to study whether the solubility-promoting tag can improve the activity of isopentenyl transferase, the MBP tag (amino acid sequence such as SEQ ID NO.16, nucleotide sequence such as SEQ ID NO.17) was fused to the N-terminus of each isopentenyl transferase through a connecting peptide GSS, and four recombinant plasmids containing MBP tags, pRSF-MBP-PcPT, pRSF-MBP-PsPT1, pRSF-MBP-FcPT1 and pRSF-MBP-PpPT1, were obtained. The above 8 recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and Escherichia coli C41 (DE3), respectively, to obtain 16 recombinant strains (Table 1).

[0069] Table 1 Strains and characteristics

[0070]

[0071]

[0072] The 16 recombinant strains constructed above were fermented in TB medium by shaking flask fermentation. When the recombinant E. coli was cultured at 37°C to OD 600 When the value was 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1mM and continue to culture at 30°C for 20h. After the fermentation is completed, the fermentation broth is centrifuged at 4°C and 8000rpm for 10min, the bacteria are collected, and the bacteria are washed with potassium phosphate buffer at pH 8.0. Then, the bacteria are resuspended with potassium phosphate buffer at pH 8.0 so that the OD value of the different recombinant bacterial suspensions is controlled to 15, and the crude enzyme solution is obtained after ultrasonic disruption.

[0073] The obtained crude enzyme solution was used to catalyze the preparation of 7-demethylcork pepper, and the reaction system was 100 μL potassium phosphate buffer: according to the final concentration, it contained 50 μL crude enzyme solution, 100 mg / L substrate umbelliferone, 100 mg / L MgCl 2 0.2m M, and dimethylallyl pyrophosphate with final concentrations of 0.1mM, 0.3mM, and 0.5mM respectively. The reaction was carried out at 30℃ for 10 hours. The results are as follows Figure 2 As shown in the figure, when the added concentration of dimethylallyl pyrophosphate in the enzyme solution reaction system was 0.1 mM, the recombinant strain C41-MBP-PpPT1 could catalyze umbelliferone to produce 20.3 mg / L 7-demethyl cypermethrin, which was 2.1 and 2.1 times the yield of 7-demethyl cypermethrin produced by the recombinant strains C41-MBP-PcPT and C41-MBP-PsPT1, respectively ( Figure 2 and Figure 3 When the concentration of dimethylallyl pyrophosphate in the enzyme solution reaction system was increased to 150 mg / L (0.5 mM), the recombinant strain C41-MBP-PpPT1 could catalyze umbelliferone to synthesize 79.2 mg / L 7-demethyl cypermethrin ( Figure 4 ).

[0074] Example 2: Reductive chaperone engineering to improve the catalytic efficiency of peucedanum sutchuenensis lactone synthase

[0075] At present, only Ficus carica-derived isoprenate lactone synthase CYP76F112 (GenBank: QVT76730.1) and Peucedanum praeruptorum-derived isoprenate lactone synthase PpDC (GenBank: WIL06380.1) have been reported to catalyze 7-demethyl cypermethrin to synthesize isoprenate lactone. Through codon optimization in Escherichia coli, the above-mentioned genes encoding isoprenate lactone synthase CYP76F112 (shown in SEQ ID NO.5) and PpDC with 2-29 amino acids truncated (shown in SEQ ID NO.6) were synthesized, and the synthesized genes were subcloned into the Nco I and Sac I sites of plasmid pETDuet-1, respectively, to obtain two recombinant plasmids pET-CYP76F112 and pET-PpDC, respectively. Further, to study whether the solubility-promoting tag can improve the activity of peucedanum arborescens synthase, the MBP tag was fused to the N-terminus of CYP76F112 and PpDC through the connecting peptide GSS, and the recombinant plasmids pET-MBP-CYP76F112 and pET-MBP-PpDC containing the MBP tag were obtained respectively. The above four recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and Escherichia coli C41 (DE3), respectively, and 8 recombinant strains BL21-CYP76F112, BL21-MBP-CYP76F112, BL21-PpDC, BL21-MBP-PpDC, C41-CYP76F112, C41-MBP-CYP76F112, C41-PpDC and C41-MBP-PpDC were obtained (Table 2).

[0076] The 8 recombinant strains constructed above were subjected to shake flask fermentation experiments. The fermentation conditions were the same as those in Example 1. After the fermentation was completed, the fermentation broth was centrifuged at 4°C and 8000rpm for 10 minutes, the cells were collected, and the cells were washed with a potassium phosphate buffer at pH 8.0. The washed cells were resuspended in potassium phosphate at pH 8.0 (containing 10% v / v glycerol) to obtain the whole-cell catalyst. The whole-cell reaction system included (by final concentration): 50 g / L of bacterial cells and 115 mg / L of 7-demethylcork pepper. The reaction was carried out at 30°C for 12 hours. The results are as follows: Figure 5 As shown, in the whole-cell catalytic system containing 10% v / v glycerol, C41-CYP76F112 (containing recombinant plasmid pET-CYP76F112) and C41-PpDC (containing recombinant plasmid pET-PpDC) can catalyze 7-demethylcarboxylic acid to synthesize 4.3 mg / L and 6.5 mg / L of isoprene lactone, respectively.

[0077] Since CYP76F112 and PpDC are P450 enzymes, the efficiency of electron transfer is crucial for the whole-cell catalysis of P450 enzymes. In order to enhance the catalytic efficiency of CYP76F112 and PpDC on the substrate 7-demethylcorticosterol, ferredoxin Fdx_1499 (GenBank: WP_011244794) and ferredoxin reductase FdR_0978 (GenBank: WP_011242878) from Synechococcus elongates were selected as the first group of reduction partner combinations; ferredoxin PetF (GenBank: BAA10197.1) and ferredoxin reductase PetH (GenBank: BAA18459.1) from Synechocystis sp. PCC 6803 were selected as the second group of reduction partner combinations; flavodoxin Fld (GenBank: QJZ13227.1), FldA (Gene ID: 945293) and FldB (Gene ID: The 3rd to 5th groups of reduction partner combinations were obtained by combining the flavodoxin reductase Fpr (Gene ID: 948414) from Escherichia coli with the flavodoxin reductase Fpr (Gene ID: 947361) from Escherichia coli; the SmCPR mutated on the basis of the SmCPR from Silybum marianum was selected. I453V (SEQ ID NO.8) as the sixth group of reduction partners; the above-mentioned reduction partner encoding genes were synthesized respectively, and the six groups of reduction partners were assembled into the Hind III and Tat I sites of plasmids pET-CYP76F112 and pET-PpDC to obtain 12 recombinant plasmids pET-CYP76F112-Fdx_1499-FdR_0978, pET-CYP76F112-PetF-PetH, pET-CYP76F112-Fld-Fpr, pET-CYP76F112-FldA-Fpr, pET-CYP76F112-FldB-Fpr, pET-CYP76F112-SmCPR I453V , pET-PpDC-Fdx_1499-FdR_0978, pET-PpDC-PetF-PetH, pET-PpDC-Fld-Fpr, pET-PpDC-FldA-Fpr, pET-PpDC-FldB-Fpr and pET-PpDC-SmCPR I453V The above 12 recombinant plasmids were transformed into C41 (DE3) respectively to obtain 12 recombinant strains (C41-CYP76F112-1 to C41-CYP76F112-6; C41-PpDC-1 to C41-PpDC-6).

[0078] Table 2 Strains and characteristics

[0079] strain feature BL21-CYP76F112 BL21(DE3) contains recombinant plasmid pET-CYP76F112 BL21-MBP-CYP76F112 BL21(DE3) containing recombinant plasmid pET-MBP-CYP76F112 BL21-PpDC BL21(DE3) containing recombinant plasmid pET-PpDC BL21-MBP-PpDC BL21(DE3) containing recombinant plasmid pET-MBP-PpDC C41-CYP76F112 C41(DE3) contains recombinant plasmid pET-CYP76F112 C41-MBP-CYP76F112 C41(DE3) contains recombinant plasmid pET-MBP-CYP76F112 C41-PpDC C41(DE3) contains recombinant plasmid pET-PpDC C41-MBP-PpDC C41(DE3) contains recombinant plasmid pET-MBP-PpDC C41-CYP76F112-1 C41(DE3) containing recombinant plasmid pET-CYP76F112-Fdx_1499-FdR_0978 C41-CYP76F112-2 C41(DE3) contains recombinant plasmid pET-CYP76F112-PetF-PetH C41-CYP76F112-3 C41(DE3) contains recombinant plasmid pET-CYP76F112-Fld-Fpr C41-CYP76F112-4 C41(DE3) contains recombinant plasmid pET-CYP76F112-FldA-Fpr C41-CYP76F112-5 C41(DE3) contains recombinant plasmid pET-CYP76F112-FldB-Fpr C41-CYP76F112-6 <![CDATA[C41(DE3) containing recombinant plasmid pET-CYP76F112-SmCPR I153V > C41-PpDC-1 C41(DE3) contains recombinant plasmid pET-PpDC-Fdx_1499-FdR_0978 C41-PpDC-2 C41(DE3) contains recombinant plasmid pET-PpDC-PetF-PetH C41-PpDC-3 C41(DE3) contains recombinant plasmid pET-PpDC-Fld-Fpr C41-PpDC-4 C41(DE3) contains recombinant plasmid pET-PpDC-FldA-Fpr C41-PpDC-5 C41(DE3) contains recombinant plasmid pET-PpDC-FldB-Fpr C41-PpDC-6 <![CDATA[C41(DE3) containing recombinant plasmid pET-PpDC-SmCPR I153V >

[0080] The 12 recombinant strains constructed above were subjected to shake flask fermentation, and the fermentation conditions were the same as those in Example 1. After the fermentation was completed, the fermentation broth was centrifuged at 4°C, 8000rpm for 10 minutes, the bacteria were collected, and the bacteria were washed with potassium phosphate buffer at pH 8.0. The washed bacteria were resuspended in potassium phosphate at pH 8.0 (containing 10% v / v glycerol), and the washed bacteria were the whole-cell catalyst. The whole-cell reaction system included (by final concentration): 50 g / L bacterial cells, 115 mg / L 7-demethylcork pepper. The reaction was carried out at 30°C for 12 hours. The results are as follows: Figure 5 As shown, in the whole-cell catalytic system containing 10% v / v glycerol, the recombinant strains C41-CYP76F112-1 (containing the recombinant plasmid pET-CYP76F112-Fdx_1499-FdR_0978), C41-CYP76F112-2 (containing the recombinant plasmid pET-CYP76F112-PetF-PetH), C41-CYP76F112-4 (containing the recombinant plasmid pET-CYP76F112-FldA-Fpr), C41-CYP76F112-5 (containing the recombinant plasmid pET-CYP76F112-FldB-Fpr), C41-CYP76F112-6 (containing the recombinant plasmid pET-CYP76F112-SmCPR I453V ) The production of isoprugated peucedanum lactone was 2.0, 5.5, 1.7, 1.8 and 6.6 times that of the control strain C41-CYP76F112 (containing recombinant plasmid pET-CYP76F112; 4.3 mg / L). The recombinant strains C41-PpDC-1 (containing recombinant plasmid pET-PpDC-Fdx_1499-FdR_0978), C41-PpDC-2 (containing recombinant plasmid pET-PpDC-PetF-PetH), C41-PpDC-4 (containing recombinant plasmid pET-PpDC-FldA-Fpr), C41-PpDC-5 (containing recombinant plasmid pET-PpDC-FldB-Fpr), C41-PpDC-6 (containing recombinant plasmid pET-PpDC-SmCPR I453V ) the yields of isoprugated peucedanum lactone were 2.3, 2.6, 1.4, 1.5 and 19.1 times that of the control strain C41-PpDC (containing the recombinant plasmid pET-PpDC; 6.5 mg / L). I453V ) can completely convert 7-demethyl cypermethrin in the reaction system into isoprene lactone ( Figure 5 and Figure 6 ).

[0081] Example 3: Enhancing the intracellular dimethylallyl pyrophosphate supply to improve the synthesis capacity of isoprugated peucedanum lactone

[0082] In order to realize the conversion of the readily available and inexpensive umbelliferone into isoprugated peucedanum lactone, the recombinant plasmid pRSF-MBP-PpPT1 constructed in Example 1 and the recombinant plasmid pET-PpDC-SmCPR constructed in Example 2 were I153V The recombinant strain MAR-1 was transformed into Escherichia coli C41 (DE3). In order to enhance the synthesis and supply of intracellular dimethylallyl pyrophosphate, the endogenous 2-methyl-D-erythritol-4-phosphate pathway (MEP pathway) was enhanced or the exogenous mevalonate pathway (MVA pathway) was introduced.

[0083] Using the genome of Escherichia coli C41 (DE3) as a template, the genes encoding the key enzymes DXS (Gene ID: 945060) and IDI (Gene ID: 949020) in the MEP pathway were inserted into the Nde I / Xho I and Nco I / BamH I restriction sites of plasmid pCDFDuet-1, respectively, to obtain the recombinant plasmid pCDF-DXS-IDI. At the same time, the effect of overexpression of genes in other synthetic pathways of the MEP pathway on the synthesis and supply of dimethylallyl pyrophosphate was studied. Using the genome of Escherichia coli C41 (DE3) as a template, the sequences encoding IspC (Gene ID: 945019), IspD (Gene ID: 948269), IspE (Gene ID: 945774), IspF (GeneID: 945057), IspG (Gene ID: 946991) and IspH (Gene ID: 944777) were inserted into the Sac I / Not I restriction site of the recombinant plasmid pCDF-DXS-IDI, respectively, to obtain the recombinant plasmids pCDF-DXS-IDI-ispC, pCDF-DXS-IDI-ispD, pCDF-DXS-IDI-ispE, pCDF-DXS-IDI-ispF, pCDF-DXS-IDI-ispG and pCDF-DXS-IDI-ispH. The seven recombinant plasmids constructed above were respectively transferred into the recombinant strain MAR-1 to obtain the recombinant strains MAR-2, MAR-5 to MAR-10 (Table 3).

[0084] By introducing exogenous MVA pathway to enhance the synthesis and supply of intracellular dimethylallyl pyrophosphate, the genes encoding endogenous atoB (Gene ID: 946727) of Escherichia coli C41 (DE3), MvaS (GenBank: AAG02439, the nucleotide sequence after codon optimization is shown in SEQ ID NO.14) and MvaE (GenBank: AAG02438, the nucleotide sequence after codon optimization is shown in SEQ ID NO.15) from Enterococcus faecalis were respectively inserted into the NcoI / BamH I, Sac I / Not I and Nde I / Xho I sites of plasmid pACYCDuet-1 to obtain the recombinant plasmid pACYC-upper-MVA; the genes encoding Nco I / BamH I, Sac I / Not I and Nde I / Xho I sites of plasmid pRSFDuet-1 were respectively inserted into the Nco I / BamH I, Sac I / Not I and Nde I / Xho I sites to obtain the recombinant plasmid pRSF-upper-MVA. The endogenous idi gene of Escherichia coli C41 (DE3), the endogenous ERG12 (Gene ID: 855248), ERG8 (Gene ID: 855260) and ERG19 (Gene ID: 855779) genes of Saccharomyces cerevisiae CEN.PK2-1C were respectively inserted into the Nco I / BamH I, Sac I / Not I, Nde I / Bgl II and Kpn I / Xho I sites of plasmid pCDFDuet-1 to obtain the recombinant plasmid pCDF-lower-MVA. The recombinant plasmid pACYC-MBP-PpPT1 was constructed in the same manner as in Example 1. The recombinant plasmids pACYC-upper-MVA and pCDF-lower-MVA were co-transferred into the recombinant strain MAR-1 constructed above to obtain the recombinant strain MAR-3; the recombinant plasmids pRSF-upper-MVA, pCDF-lower-MVA, pET-PpDC-SmCPR I153V and pACYC-MBP-PpPT1 were co-transformed into Escherichia coli C41(DE3) to obtain the recombinant strain MAR-4 (Table 3).

[0085] Table 3 Strains and characteristics

[0086]

[0087] The recombinant strains MAR-1 to MAR-10 were fermented in shake flasks in TB medium at 37°C until OD 600When the value is 0.6-0.8, add 1mM IPTG and 100mg / L substrate umbelliferone according to the final concentration, and continue fermentation at 30℃ for 72h. Take samples every 24h, take out an appropriate amount of reaction solution and add an equal volume of methanol to mix. The reaction mixture is centrifuged at 14000rpm for 20-30min, and the supernatant is passed through the membrane for high performance liquid chromatography analysis. The results are as follows Figure 7 As shown in the results, compared with the control strain MAR-1 (6.7 mg / L), the recombinant strain MAR-2 with modified endogenous MEP pathway could synthesize 21.7 mg / L of isoprugated peucedanum lactone in 72 h, which was better than the recombinant strain MAR-3 with introduced exogenous MVA pathway (18.6 mg / L). By further optimizing the endogenous MEP pathway, the recombinant strain MAR-8 (containing recombinant plasmids pRSF-MBP-PpPT1, pET-PpDC-SmCPR I153V and pCDF-DXS-IDI-ispF) could synthesize 24.4 mg / L of isoprene lactone in 72 h.

[0088] Example 4: Modular optimization to improve the ability to synthesize isopructose

[0089] The recombinant strain MAR-8 (containing the recombinant plasmids pRSF-MBP-PpPT1, pET-PpDC-SmCPR) constructed in Example 3 was I153V The synthesis of isoprugated peucedanum lactone was divided into three modules: a DMAPP supply module containing DXS, IDI and IspF; an umbelliferone catalytic module containing MBP-PpPT1; and a catalytic module containing PpDC and SmCPR. I453V The three modules were expressed using high-copy number plasmid pRSFDuet-1, medium-copy number plasmid pETDuet-1 or pCDFDuet-1, and low-copy number plasmid pACYCDuet-1, respectively, and combined to obtain 24 recombinant strains MAR-8 and MAR-11 to recombinant strains MAR-33 (Table 4).

[0090] Table 4 Strains and characteristics

[0091]

[0092] The recombinant strains MAR-8 and MAR-11 to MAR-33 were fermented in shake flasks under the same fermentation conditions as in Example 3, and sampling and HPLC analysis were performed according to the method in Example 3. Figure 8 As shown, the recombinant strain MAR-18 (containing the recombinant plasmids pET-MBP-PpPT1, pCDF-PpDC-SmCPR I153Vand pRSF-DXS-IDI-ispF) can synthesize 37.1 mg / L of isoprugated peucedanum lactone in 72 h, which is a strain MAR-8 (containing recombinant plasmids pRSF-MBP-PpPT1, pET-PpDC-SmCPR I153V and 1.5 times that of pCDF-DXS-IDI-ispF).

[0093] Example 5: Optimization of fermentation conditions of recombinant strain MAR-18

[0094] The fermentation conditions of the recombinant strain MAR-18 constructed in Example 4 were optimized at the shake flask level. Based on the fermentation conditions of Example 3, when the induction temperature was set to 30°C and the umbelliferone addition concentration was 100 mg / L, the effect of the addition amount of the inducer IPTG (0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM and 1.2 mM) on the synthesis of isoprugated peucedanum by MAR-18 was investigated; when the addition amount of the inducer IPTG was set to 0.4 mM and the umbelliferone addition concentration was 100 mg / L, the effect of the addition amount of the inducer IPTG (0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM and 1.2 mM) on the synthesis of isoprugated peucedanum by MAR-18 was investigated. The effects of induction temperature (20℃, 25℃, 30℃ and 37℃) on the synthesis of isoprugated peucedanum by MAR-18; when the addition amount of inducer isopropyl-β-D-thiogalactoside was set to 0.4mM and the induction temperature was set to 30℃, the effects of umbelliferone addition concentration (100mg / L, 200mg / L, 300mg / L, 400mg / L and 500mg / L) on the synthesis of isoprugated peucedanum by MAR-18 were investigated. The results are shown in Fig. 9 As shown, the optimal fermentation conditions of the recombinant strain MAR-18 at the shake flask level were: the addition amount of the inducer isopropyl-β-D-thiogalactoside was 0.4 mM; the induction temperature was 30°C and the addition concentration of umbelliferone was 100 mg / L.

[0095] Example 6: Fermentation-level synthesis of isoprugated peucedanum lactone by recombinant strain MAR-18

[0096] The recombinant strain MAR-18 constructed in Example 4 was fermented in a 5 L fermenter containing MR medium with a final concentration of 20 g / L glucose and 5 g / L yeast extract. When the recombinant strain was cultured at 37°C until OD 600 When the pH value was 5, 0.4 mM IPTG and umbelliferone (200 mg / L) were added and fermentation was continued at 30°C for 96 hours. During the fermentation process, the dissolved oxygen in the fermentation liquid was controlled to be maintained at 30% by adjusting the rotation speed (400-800 rpm). Feeding was controlled by pH-star. When the pH exceeded 7.02, feed medium was added. When the pH dropped to below 6.98, 50% ammonia water was added to control the pH of the fermentation liquid to be maintained at 7.0±0.02. Samples were taken every 8 hours and the titer of isoprugated peucedanum was analyzed according to the method in Example 3. The results are shown in the figure. Fig.10As shown, in the batch fermentation with 100 mg / L umbelliferone added to the MR medium, the recombinant strain MAR-18 synthesized 10.3 mg / L of isoprugated peucedanum lactone in 48 hours; in the fed-batch fermentation, the recombinant strain MAR-18 synthesized 203.7 mg / L of isoprugated peucedanum lactone in 88 hours, and the molar conversion rate of umbelliferone reached 81.4%.

[0097] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Recombinant Escherichia coli, based on the starting strain, expressing isopentenyl transferase and peucedanum lactone synthase; the isopentenyl transferase is any one of (a1) to (a3): (a1) PcPT from Petroselinum crispum; (a2) PsPT1 from Pastinaca sativa; (a3) PpPT1 from Peucedanum praeruptorum; The peucedanum arvense lactone synthase is an enzyme with an amino acid sequence such as Genbank accession number: QVT76730.1 or a PpDC truncation with an amino acid sequence such as SEQ ID NO.

7.

2. The recombinant Escherichia coli according to claim 1, characterized in that The starting strain includes but is not limited to Escherichia coli BL21 (DE3) or Escherichia coli C41 (DE3).

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The recombinant Escherichia coli also includes at least one of the characteristics (a) to (c): (a) fusing a solubility-promoting tag, such as MBP, Sumo, GST, TF, TrxA or NusA, to the N-terminus of the isopentenyl transferase; (b) expressing a redox chaperone; the redox chaperone includes Fdx_1499 / FdR_0978 from Synechococcus sp., PetF / PetH from Synechocystis sp., or SmCPR from Silybum marianum. I453V , or flavodoxin reductase Fpr of Escherichia coli and flavodoxin Fld / FldA / FldB derived from Escherichia coli; (c) expressing one or more enzymes in the endogenous MEP synthesis pathway, including but not limited to: DXS, IDI or IspF.

4. The recombinant Escherichia coli according to claim 1 or 2, characterized in that Expression of the isopentenyl transferase PsPT1 fused with the solubility-promoting tag MBP, and co-expression of the isoprenolactone synthase PpDC and the redox chaperone SmCPR I453V .

5. The recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that The plasmid for expressing the exogenous gene is selected from one or more of pRSFDuet-1, pETDuet-1, pCDFDuet-1, and pACYCDuet-1.

6. A method for synthesizing furanocoumarin isopructose peucedanum lactone, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 5 is used to ferment and produce isoprugated peucedanum lactone.

7. The method according to claim 6, characterized in that The recombinant E. coli was cultured in a fermentation medium at 35-37°C until OD 600 The value is 0.6-0.

8. After adding the inducer isopropyl-β-D-thiogalactoside and the substrate umbelliferone, fermentation is carried out at 20-37°C for 12-96h.

8. The method according to claim 7, characterized in that The final concentration of the inducer isopropyl-β-D-thiogalactoside is 0.1-1.0 mM; the final concentration of the substrate umbelliferone is 100-500 mg / L.

9. A method for improving the ability of recombinant Escherichia coli to synthesize isoprugated peucedanum lactone, characterized in that: The method comprises performing one or more of the following improvements (1) to (4) on the starting strain: (1) expressing isopentenyl transferase and peucedanum lactone synthase; the isopentenyl transferase is selected from PcPT derived from Petroselinum crispum, PsPT1 derived from Pastinaca sativa, or PpPT1 derived from Peucedanumpraeruptorum; the peucedanum lactone synthase is CYP76F112 or PpDC with 2-29 amino acids truncated; (2) fusing a solubility-promoting tag, such as MBP, Sumo, GST, TF, TrxA or NusA, to the N-terminus of the isopentenyl transferase; (3) expressing a redox chaperone; the redox chaperone includes Fdx_1499 / FdR_0978 from Synechococcus sp., PetF / PetH from Synechocystis sp., or SmCPR from Silybum marianum. I453V , or flavodoxin reductase Fpr of Escherichia coli and flavodoxin Fld / FldA / FldB derived from Escherichia coli; (4) Expression of one or more of the endogenous MEP synthesis pathway enzymes DXS, IDI and IspF.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 5, or the method according to any one of claims 6 to 9 in the production of furanocoumarin isopructose lactone or its derivatives.

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