Engineering bacterium for de novo biosynthesis of mendalin as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to an engineering bacterium for de novo biosynthesis of mendalin as well as a construction method and application of the engineering bacterium. Glucose is taken as a substrate, key enzyme genes are expressed in saccharomyces cerevisiae cells, and the mendalin is de novo synthesized through a microbial fermentation process; according to the constructed engineering strain Med21, the yield of the mediodarin is 180 mg / L; the yield of the constructed optimized strain Med25 can be increased to 450 mg / L after fed-batch fermentation; compared with a traditional chemical synthesis method and a method for extracting from natural plants, the method for synthesizing the medioren does not depend on exogenously added precursor substances, has the advantages of being low in cost, high in production efficiency, small in environmental pollution, high in product yield and purity and the like, provides possibility for large-scale industrial production of the medioren, and has a wide application prospect. The method has important application value and market prospect, and opens up a new way for production and application of the mendalin.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and relates to a natural active ingredient of a plant, in particular to an engineered bacterium for de novo biosynthesis of meditartin. Background Art

[0002] Medipterocarpus indica (chemical formula is C 16 H 14 O4) is an isoflavone compound distributed in the rhizomes of 31 legumes, including Glycyrrhiza glabra and Sophora japonica. It has a unique benzopyran-benzofuran tetracyclic structure skeleton and significant pharmacological activity. As an estrogen receptor (ER) agonist, medipterostilbene promotes osteoblast differentiation through ERβ, helping to achieve peak bone mass; its non-uterine estrogenicity makes it potentially useful in the treatment of bone diseases such as osteoporosis. In addition, medipterostilbene can also inhibit inflammation, improve oxidative stress, and relieve fatigue. The traditional way to obtain medipterostilbene is mainly to extract it from licorice plants, but this method is prone to damage to wild plant resources such as licorice, and is affected by climate and growth cycles. By identifying key enzyme genes through multi-omics data mining, researchers have successfully achieved heterologous synthesis of medipterostilbene from licorice in Saccharomyces cerevisiae, opening up a new path for the sustainable production of medipterostilbene.

[0003] Synthetic biology, a cutting-edge interdisciplinary field, has experienced rapid growth in recent years. Through the rational design and construction of microbial cell factories, this discipline offers humanity a novel, editable biomanufacturing technology. The advantage of synthetic biology lies in its high degree of customizability and precision. This technology allows for the design and construction of biological components and systems with targeted functions tailored to specific needs, enabling precise biomanufacturing and regulation within microbial cell factories. Furthermore, synthetic biology offers the advantages of high efficiency and environmental friendliness, reducing energy consumption and pollution during the synthesis process, thereby minimizing negative environmental impacts.

[0004] As a commonly used cell factory, the genome sequencing of Saccharomyces cerevisiae was completed in 1996, making it the first eukaryotic organism to complete genome sequencing. After sequencing, it was found that the genome of Saccharomyces cerevisiae consists of a nucleotide sequence of 12068kb, with a short intergenic region and few introns in the genes. Saccharomyces cerevisiae has the advantages of a clear genetic background, mature research on genetic manipulation tools, and a high-efficiency and high-fidelity homologous recombination system, which enables it to achieve rapid construction and optimization of the biosynthetic pathway of target compounds, making it an important tool for exploring new biosynthetic pathways of natural products. Patent 202210926480.9 discloses a method for heterologously synthesizing the natural high-value active compound mediptertin in Saccharomyces cerevisiae using synthetic biology technology, but its synthesis pathway relies on multiple exogenously added precursor substances (such as isoliquiritigenin, liquiritigenin, daidzein, etc.), which increases production costs and process complexity, and lacks potential for industrial application.

[0005] Therefore, in the future, we can explore strategies for de novo synthesis of medipterostilbene, reduce dependence on precursor substances, focus on optimizing the fermentation process, develop efficient product separation and purification technologies, reduce production costs, and promote the industrial production of medipterostilbene. Summary of the Invention

[0006] To solve the above problems, the present invention proposes an engineered bacterium for de novo biosynthesis of meditartin and a construction method and application thereof.

[0007] The technical solution of the present invention is achieved as follows:

[0008] The present invention performed gene fusion and multi-copying on a total of 32 genes, including heterologous genes and endogenous genes of Saccharomyces cerevisiae, in order to construct 25 engineered Saccharomyces cerevisiae strains. By reconstructing the central carbon metabolism of Saccharomyces cerevisiae cells and optimizing the key precursor synthesis metabolic pathways, the efficient synthesis of medipterostilbene was achieved.

[0009] On the one hand, the present application provides a method for constructing an engineered bacterium for de novo biosynthesis of medipterostilbene: ARO1, ARO2, ARO3, PHA2, CYB5, ACC1, ARO4 K229L 、ARO7 G141S, AtPAL2, AtATR2, AtC4H, At4CL1, GmCHR5, GmCHS8, GmCHI1B2, GmCPR, Gu4CL1, GgCHS, GgCHR, GgCHI, FjTAL, BbXfpk, CkPta, MtPDH1, Gg2-HIS, GgHID, GgI4'OMT, GgIFR4, GgI2'H5, GgVR, GePTS1 and EGFP genes were introduced into the Saccharomyces cerevisiae genome by in vitro cloning and recombination technology, thereby constructing an engineered bacterium for de novo biosynthesis of meditartin.

[0010] Preferably, the steps of the above construction method are selected from any one of the following:

[0011] (1) Build a file containing ARO7 G141S and ARO4 K229L an expression cassette M1 comprising the AtPAL2, AtATR2 and AtC4H genes, an expression cassette M6 comprising the GePTS1, PHA2, CYB5, ARO2 and GgVR genes, an expression cassette M10 comprising the At4CL1, GmCHS8, GmCHR5 and GmCHI1B2 genes, an expression cassette M12 comprising the ARO1, ARO3 and GmCHI1B2 genes, an expression cassette M13 comprising the CkPta, BbXfpk, MtPDH1 and FjTAL genes, an expression cassette M16 comprising the Gg2-HIS, GgHID, GmCPR and GgI4'OMT genes, an expression cassette M17 comprising the Gg2-HIS, GmCPR, GgI2'H5 genes, and an expression cassette M18 comprising the ACC1 and GgIFR4 genes;

[0012] The expression cassette M6 was transferred into the Saccharomyces cerevisiae strain to construct the Med6 strain, the expression cassette M7 was transferred into the Med6 strain to construct the Med7 strain, the expression cassette M10 was transferred into the Med7 strain to construct the Med10 strain, the expression cassette M16 was transferred into the Med10 strain to construct the Med16 strain, the expression cassette M1 was transferred into the Med16 strain to construct the Med17 strain, the expression cassette M12 was transferred into the Med17 strain to construct the Med18 strain, the expression cassette M13 was transferred into the Med18 strain to construct the Med19 strain, the expression cassette M17 was transferred into the Med19 strain to construct the Med20 strain, and the expression cassette M18 was transferred into the Med20 strain to construct the Med21 strain;

[0013] (2) Construct a system including ARO1, ARO2, ARO3, and ARO4 K229L 、ARO7 G141S, PHA2, CYB5, BbXfpk, CkPta, MtPDH1, FjTAL, GmCHS8, GmCHR5, GmCHI1B2, AtPAL2, AtC4H, AtATR2, At4CL1, ACC1, Gg2-HIS, GgHID, GmCPR, GgI4'OMT, GgI2'H5, GgIFR4, GePTS1 and GgVR genes; the expression cassette M21 was transferred into the Saccharomyces cerevisiae strain to construct the Med25 strain.

[0014] Preferably, the above ARO1, ARO2, ARO3, ARO4 K229L 、ARO7 G141S , PHA2, CYB5 and ACC1 genes are all from Saccharomyces cerevisiae; AtPAL2, AtATR2, AtC4H and At4CL1 genes are all from Arabidopsis thaliana; GmCHR5, GmCHS8, GmCHI1B2 and GmCPR genes are all from soybean; Gu4CL1 gene is from Glycyrrhiza uralensis; FjTAL gene is from Flavobacterium johnsonii; BbXfpk gene is from Bifidobacterium breve; CkPta gene is from Clostridium krusei; MtPDH1 gene is from Medicago truncatula; GgCHS, GgCHR, GgCHI, Gg2-HIS, GgHID, GgI4'OMT, GgI2'H5, GgIFR4 and GgVR genes are all from Glycyrrhiza glabra; GePTS1 gene is from Glycyrrhiza spinulosa.

[0015] Preferably, the gene encoding 4-coumaryl-CoA ligase is derived from the At4CL1 gene of Arabidopsis thaliana, and the synthetic sequence of the codon-optimized At4CL1 gene is shown in SEQ ID No. 1; the gene encoding chalcone reductase is derived from the GmCHR5 gene of Arabidopsis thaliana, and the synthetic sequence of the codon-optimized GmCHR5 gene is shown in SEQ ID No. 2; the gene encoding chalcone synthase is derived from the GmCHS8 gene of Arabidopsis thaliana, and the codon-optimized GmCHS8 gene is shown in SEQ ID No. 3; the gene encoding chalcone isomerase is derived from the GmCHI1B2 gene of Arabidopsis thaliana, and the codon-optimized GmCHI1B2 gene is shown in SEQ ID No. 4;

[0016] The above-mentioned FjTAL gene encoding tyrosine ammonia lyase is derived from Flavobacterium johnsonii, and the codon-optimized FjTAL gene has a synthetic sequence shown in SEQ ID No. 5; the BbXfpk gene encoding phosphoketolase is derived from Bifidobacterium breve, and the codon-optimized BbXfpk gene has a synthetic sequence shown in SEQ ID No. 6; the CkPta gene encoding phosphotransacetylase is derived from Clostridium krusei, and the codon-optimized CkPta gene has a synthetic sequence shown in SEQ ID No. 7; the MtPDH1 gene encoding prephenate dehydrogenase is derived from alfalfa, and the codon-optimized MtPDH1 gene has a synthetic sequence shown in SEQ ID No. 8; the codon-optimized synthetic EGFP gene has a sequence shown in SEQ ID No. 9.

[0017] Using Saccharomyces cerevisiae as the host cell, the key enzyme genes were introduced into the yeast to construct a gene expression module. In the Saccharomyces cerevisiae cell factory, the gene expression module enabled the de novo biosynthesis of medipterostilbene. Bioinformatics analysis was used to align the key enzyme genes and verify their in vitro enzymatic activity and function. The genes were then integrated into the Saccharomyces cerevisiae genome through in vitro cloning and recombination techniques, and stably expressed in the yeast, thereby establishing the biosynthesis pathway for medipterostilbene.

[0018] Preferably, the promoter of the above expression cassette is any one of pTPI1, pENO2, pTEF1, pPGK1, pINO1, pARO7, pTDH3 and pFBA1; the terminator is any one of tCYC1, tHSP26, tADH1, tCPS1, tTPS1 and tIDP1.

[0019] Preferably, the integration site of the above expression cassette is the Delta site.

[0020] Preferably, the above-mentioned Saccharomyces cerevisiae is the Saccharomyces cerevisiae IMX581 strain; and the gRNA plasmid used for transfer is pMEL10-Delta.

[0021] In a second aspect, the engineered bacteria constructed by the above construction method are engineered strain Med21 or engineered strain Med25.

[0022] The third aspect is the application of the above-mentioned engineered bacteria in the de novo biosynthesis of medipterostilbene.

[0023] The fermentation conditions of Saccharomyces cerevisiae were optimized: in the first stage of fermentation, Saccharomyces cerevisiae was fermented at 30°C, 20% or 40% glucose solution was added every 24 hours, and the glucose consumption was monitored. When glucose was no longer consumed, low-temperature culture was switched to 28°C to provide conditions for Saccharomyces cerevisiae to produce the target substance. Fermentation was stopped after 48 hours of low-temperature culture to improve the synthesis efficiency and purity of medipterostilbene: the seed liquid of the engineered strain Med21 or the engineered strain Med25 was inoculated into YPD fermentation medium and fermented at 28-30°C and 180-200rpm in a shaker for 120-144 hours. After the fermentation was completed, the cells were broken and medipterostilbene was extracted.

[0024] Preferably, the OD value of the fermentation broth is 25-30.

[0025] Furthermore, the extracted medipterostilbene is separated and purified to obtain high-purity and high-activity medipterostilbene.

[0026] The present invention has the following beneficial effects:

[0027] The present application constructed 25 engineered strains in Saccharomyces cerevisiae by means of mutation and overexpression of heterologous genes and endogenous genes of Saccharomyces cerevisiae, gene fusion and multi-copy; using Saccharomyces cerevisiae IMX581 strain as the initial strain, p-coumaric acid was synthesized de novo in engineered strain Med7 with a maximum yield of 250 mg / L; using a strain that produces p-coumaric acid as the initial strain, glycyrrhizin was synthesized de novo in engineered strain Med13 with a maximum yield of 58.2 mg / L; using p-coumaric acid as the substrate, after adding glycyrrhizin, engineered strain Med15 synthesized daidzein with a maximum yield of 27.5 mg / L, and after additional addition of coumaric acid, engineered strain Med18 synthesized daidzein with a maximum yield of 27.5 mg / L. The highest yield of synthesized daidzein was 29.3 mg / L; using glycyrrhizin and p-coumaric acid as substrates, formononetin was synthesized in the engineered strain Med18 with a maximum yield of 33.6 mg / L; an engineered strain Med21 was constructed to synthesize mediptertin from scratch using glucose as a substrate. After adding 20% ​​glucose solution to optimize the fermentation conditions, the yield of mediptertin was 180 mg / L; the strain Med21 was optimized, and on the basis of the original optimized fermentation conditions, the 20% glucose solution was replaced with a 40% glucose solution for supplementary fermentation. The yield of mediptertin in the engineered strain Med25 finally constructed can be increased to 450 mg / L. The method of the present application has the advantages of low cost, high production efficiency, low environmental pollution, high product yield and purity, and has opened up a new path for the production and application of mediptertin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 p-Coumaric acid production in Med6 and Med7 engineered yeast strains.

[0030] Figure 2 This is the liquid chromatogram of glycyrrhizin in the Med13 strain.

[0031] Figure 3 The yield of liquiritigenin in Med10-Med13 engineered yeast strains.

[0032] Figure 4 This is the liquid chromatogram of daidzein, glycyrrhizin and formononetin in the Med18 strain.

[0033] Figure 5 The yield of daidzein in Med14, Med15 and Med18 engineered yeast strains.

[0034] Figure 6 The yield of formononetin in Med15 and Med18 engineered yeast strains.

[0035] Figure 7 This is the liquid chromatogram of Med21 strain synthesizing medipterostilbene.

[0036] Figure 8 is the content of mediptertin in Med21 and Med25 engineered yeast strains. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0039] The present invention mines and verifies key enzyme genes related to the biosynthesis of medipterostilbene, specifically including endogenous five-functional aromatic protein ARO1 (GenBank No.: NM_001180435.1), chorismate synthase ARO2 (GenBank No.: NM_001181013.1), 3-deoxy-D-arabinoheptulose-7-phosphate synthase ARO3 (GenBank No.: NM_001180343.3), prephenate dehydrogenase PHA2 (GenBank No.: NM_001183154.1), yeast natural cytochrome P450 enzyme CYB5 (GenBank No.: NM_001182949.3), acetyl-CoA carboxylase ACC1 (GenBank No.: NM_001183193.1), 3-deoxy-D-arabinoheptulose-7-phosphate synthase ARO4 (GenBank No.: NM_00118343.3), prephenate dehydrogenase PHA2 (GenBank No.: NM_001183154.1), yeast natural cytochrome P450 enzyme CYB5 (GenBank No.: NM_001182949.3), acetyl-CoA carboxylase ACC1 (GenBank No.: NM_001183193.1), 3-deoxy-D-arabinoheptulose-7-phosphate synthase ARO5 (GenBank No.: NM_00118343.3), K229L (GenBank No.: NM_001178597.1) and chorismate mutase ARO7 G141S (GenBank No.: NM_001184157.1) and other genes; genes encoding phenylalanine ammonia lyase AtPAL2 (GenBank No.: AY133595.1), P450 reductase AtATR2 (GenBank No.: AF325101.1), cinnamate hydroxylase AtC4H (GenBank No.: NM_128601.3), 4-coumaryl-CoA ligase At4CL1 from Arabidopsis thaliana, wherein the synthetic sequence of the codon-optimized At4CL1 gene is shown in SEQ ID No. 1; genes encoding chalcone reductase (GmCHR5), chalcone synthase GmCHS8, chalcone isomerase GmCHI1B2 and P450 oxidoreductase GmCPR from soybean (GenBank No.: AY170374.1), wherein the synthetic sequence of the codon-optimized GmCHR5 gene is shown in SEQ ID No. No.2; the synthetic sequence of the codon-optimized GmCHS8 gene is shown in SEQ ID No.3; the synthetic sequence of the codon-optimized GmCHI1B2 gene is shown in SEQ ID No.4.

[0040] The gene encoding 4-coumaryl-CoA ligase Gu4CL1 (GenBank No.: MK341782.1) from Glycyrrhiza uralensis; the gene encoding tyrosine ammonia lyase FjTAL from Flavobacterium johnsonii, the codon-optimized FjTAL gene, and its synthetic sequence is shown in SEQ ID No. 5; the gene encoding phosphoketolase BbXfpk from Bifidobacterium breve, the codon-optimized BbXfpk gene, and its synthetic sequence is shown in SEQ ID No. 6; the gene encoding phosphotransacetylase CkPta from Clostridium krusei, the codon-optimized CkPta gene, and its synthetic sequence is shown in SEQ ID No. 7; the gene encoding prephenate dehydrogenase MtPDH1 from Medicago truncatula, the codon-optimized MtPDH1 gene, and its synthetic sequence is shown in SEQ ID No. ID No. 8; from Glycyrrhiza glabra, encoding chalcone synthase GgCHS (GenBank No.: OQ067102.1), chalcone reductase GgCHR (GenBank No.: OQ067259.1) and chalcone isomerase GgCHI (GenBank No.: OQ067258.1), 2-hydroxyisoflavone synthase Gg2-HIS (GenBank No.: OQ102530.1), 2-hydroxyisoflavone dehydratase GgHID (GenBank No.: OQ102529.1), isoflavone-4'-O-methyltransferase The invention also includes genes encoding pterostilbene synthase GgI4'OMT (GenBank No.: OQ067265.1), isoflavone reductase GgIFR4 (GenBank No.: OQ067263.1), isoflavone-2'-hydroxylase GgI2'H5 (GenBank No.: OQ067262.1), and estrogen reductase GgVR (GenBank No.: OQ067264.1); a gene encoding pterostilbene synthase GePTS1 (GenBank No.: LC121822.1) from Glycyrrhiza uralensis; and a codon-optimized EGFP gene, the sequence of which is shown in SEQ ID No. 9.

[0041] Using Saccharomyces cerevisiae as the host cell, the key enzyme genes were introduced into the yeast to construct a gene expression module. In the Saccharomyces cerevisiae cell factory, the gene expression module was used to achieve the de novo biosynthesis of medipterostilbene.

[0042] The culture medium and culture method required in each embodiment of the present invention, the method for constructing the gene expression cassette, the method for constructing the engineered Saccharomyces cerevisiae strain, and the method for preparing the engineered strain test sample are the same, as follows:

[0043] 1. Culture medium and culture method: The culture medium is YPD and YNB medium. The culture medium formula is as follows:

[0044] YPD liquid medium: 22 g / L glucose, 10 g / L yeast extract powder, 20 g / L peptone; YPD solid medium: Add 20 g / L agar powder to the YPD liquid medium formula. Make up the volume of either solid or liquid YPD medium to 1 L with ddH2O and sterilize at 121°C for 20 min.

[0045] YNB liquid medium: 6.7 g / L amino-free yeast nitrogen source, 0.77 g / L DO Summplement-Ura, 22 g / L glucose. YNB solid medium: Based on the liquid medium formula, add 20 g / L agar powder. YPD medium (solid or liquid) was diluted to 1 L with ddH2O. The solid medium was adjusted to 6-7°C with NaOH solution and sterilized at 121°C for 20 min.

[0046] The Saccharomyces cerevisiae strain used in this invention is cultured as follows: a Saccharomyces cerevisiae liquid is streaked onto a YPD or YNB solid plate and incubated in a 30°C incubator for 2-3 days, until a single colony of appropriate size appears on the YPD or YNB solid plate. In a clean bench, the single colony from the YPD or YNB plate is transferred to YPD or YNB liquid medium and incubated in a constant temperature shaker at 30°C, 200 rpm for 12-16 hours for Saccharomyces cerevisiae genome extraction or competent cell preparation.

[0047] 2. Gene expression cassette construction method:

[0048] Extraction of the Saccharomyces cerevisiae genome: Centrifuge the bacterial suspension in a sterile EP tube at 15,000 × g for 3 minutes and discard the supernatant. Add 100 μL of LiAc + 1% SDS cell lysis buffer to the cells, gently pipette to mix, and incubate in a 70°C water bath for 8 minutes. After the water bath, add 300 μL of 96% ethanol solution to the bacterial suspension and pipette to mix. After mixing, centrifuge at 15,000 × g for 3 minutes and discard the supernatant. Add another 500 μL of 70% ethanol solution to the cells and centrifuge at 15,000 × g for 3 minutes and discard the supernatant. Add 20-40 μL of sterile ddH2O to the pellet and gently pipette to mix. Centrifuge at 15,000 × g for 1 minute and transfer the supernatant to another clean, sterile 1.5 mL EP tube. This solution is the Saccharomyces cerevisiae genome and store at -20°C.

[0049] PCR amplification of genes, terminators, promoters, and site homology arms: For endogenous Saccharomyces cerevisiae genes, promoters, terminators, and site homology arms, PCR amplification was performed using DNA polymerase using an extracted Saccharomyces cerevisiae genome as a template. The reaction conditions were: 25 μL of 2× Phanta Max Buffer, 1 μL of dNTP Mix, 2 μL of the F-terminal primer of the amplified sequence, 2 μL of the R-terminal primer of the amplified sequence, 1 μL of template, and 18 μL of sterile ddH2O, for a total reaction volume of 50 μL (operated on ice). The reaction program was as follows: first stage, 95°C, 3 min, 1 cycle; second stage, 95°C, 15 s, 55°C, 15 s, 72°C, 30-60 s / kb, 35 cycles; third stage, 72°C, 5 min, 1 cycle. The amplified PCR products were subjected to electrophoresis and compared with DNA Marker after the electrophoresis. The remaining PCR products with correct bands were recovered using FastPure Gel DNA Extraction MiniKit and stored at -20°C.

[0050] In vitro assembly of gene fragments: The recovered gene, promoter, terminator, and site homology arms were ligated and amplified using PrimeSTAR Max DNA Polymerase and Phanta Max Super-Fidelity DNA Polymerase. The PrimeSTAR Max DNA Polymerase reaction system was 25 μL, with 10-15 μL of sequence fragment added. The remaining system was then filled in with PrimeSTAR Max DNA Polymerase (operated on ice). The enzyme program was as follows: Phase 1, 95°C, 3 min, 1 cycle; Phase 2, 98°C, 10 s, 58°C, 15 s, 72°C, 60 s / kb, 15 cycles; Phase 3, 72°C, 10 min, 1 cycle. The PCR product amplified with PrimeSTAR Max DNA Polymerase was used as a template for reamplification using Phanta Max Super-Fidelity DNA Polymerase. The product was purified and stored at -20°C.

[0051] 3. Methods for constructing engineered Saccharomyces cerevisiae strains:

[0052] Saccharomyces cerevisiae transformation gene expression cassette: Streak the Saccharomyces cerevisiae culture liquid on a YPD plate and culture it in a 30℃ incubator for 2-3 days until a single colony of appropriate size grows. Pick a single colony of appropriate size and place it in 5mL of YPD liquid medium. Culture it in a constant temperature shaker at 30℃ and 200rpm for 16-18h. In a sterile conical flask, add fresh YPD liquid medium with a pipette, take an appropriate amount of the culture liquid that has been cultured for 16-18h and transfer it to fresh YPD liquid medium, and measure its OD using a UV spectrophotometer. 600value, so that the initial OD 600 The value is 0.15-0.2.

[0053] The transferred YPD bacterial solution was cultured in a constant temperature shaker at 30°C and 200 rpm until the bacterial solution OD 600 The value is 0.4-0.6. Centrifuge the bacterial solution at 3000 rpm for 5 minutes. Discard the supernatant after centrifugation. Add 20-30 mL of sterile ddH2O to the cells and mix thoroughly by pipetting. Centrifuge at 3000 rpm for 5 minutes. Discard the supernatant and add 100 mM lithium acetate solution to the cells and mix thoroughly by pipetting.

[0054] In a clean bench, take 240 μL of the prepared PEG 3350 buffer into a sterile EP tube, 36 μL of 1 M lithium acetate solution, 10 μL of 2 mg / mL SS-carrier DNA (bath in a 95°C water bath for 5-10 minutes in advance, and immediately cool on ice after the end), 1000 ng of DNA splicing fragments, 100 ng of sgRNA plasmid, and fill to 351 μL with ddH2O.

[0055] Transfer the premix to the bacterial cells and gently pipette to mix. Heat shock the mixture in a 42°C water bath for 30 minutes, gently inverting the mixture every 10 minutes. Add 200 μL of sterile ddH2O to the cells and gently pipette to mix. After mixing, spread 200 μL of the mixture onto a YNB plate and incubate in a 30°C incubator until transformants emerge.

[0056] After transformants emerge on the YNB plates, pick them and transfer them to 5 mL of YNB liquid medium. Incubate at 30°C, 200 rpm for 18-24 hours to extract their genomes. Using the extracted transformant genomes as templates, perform PCR amplification using 2× Magic Green Taq SuperMix enzyme and primers for the gene being verified. The protocol is as follows: Phase 1, 95°C, 3 min, 1 cycle; Phase 2, 95°C, 10 s, 55°C, 10 s, 72°C, 60 s / kb, 30-40 cycles; Phase 3, 72°C, 5 min, 1 cycle. After the PCR cycle is complete, verify the PCR products by electrophoresis and sequencing.

[0057] Culture the transformants that have successfully integrated the gene in a shaker at 30°C, 200 rpm for 18-24 hours. Repeat the same method for 3-5 consecutive transfers to verify plasmid removal. Store the plasmid-free strain in 50% glycerol and store at -80°C.

[0058] 4. Preparation of engineered strain test samples: Ferment each engineered strain continuously for 5-6 days in three parallels to obtain a fermentation broth with an OD value of 25-30. The specific steps are: streak the strain on a YPD plate, culture in a 30°C incubator for 2-3 days, grow a single colony of appropriate size, and then pick a single colony for activation. Pipette 1mL of activated bacterial solution and transfer it to fresh YPD liquid culture medium, culture at 30°C and 200rpm, add 20% or 40% glucose solution every 24h, and monitor the glucose consumption. When glucose is no longer consumed, switch to low-temperature culture at 28°C. Stop fermentation after 48h of low-temperature culture. Add chromatographic-grade methanol to the yeast extract and filter it through a 0.22μm filter membrane. Collect and test.

[0059] Example 1: Construction of an engineered Saccharomyces cerevisiae strain that synthesizes p-coumaric acid and glycyrrhizin

[0060] (1) Type of strain, required culture medium and culture method

[0061] The present invention is constructed using Saccharomyces cerevisiae IMX581 as the chassis strain, and the gRNA plasmid used is pMEL10.

[0062] The culture medium and culture method used were the same as above.

[0063] (2) Design and construction of key enzyme gene expression cassettes in engineered strains

[0064] The genes used in the present invention to construct the engineered Saccharomyces cerevisiae strain for synthesizing p-coumaric acid and glycyrrhizin are the endogenous genes ARO1, ARO2, ARO3, and ARO4 of Saccharomyces cerevisiae. K229L 、ARO7 G141S , PHA2, and CYB5; heterologous genes BbXfpk, CkPta, MtPDH1, FjTAL, GmCHS8, GmCHR5, GmCHI1B2, AtPAL2, AtC4H, AtATR2, At4CL1, Gu4CL1, GgCHS, GgCHR, and GgCHI. Promoters used for gene expression were pTPI1, pENO2, pTEF1, pPGK1, pINO1, pARO7, pTDH3, and pFBA1; terminators used were tCYC1, tHSP26, tADH1, tCPS1, tTPS1, and tIDP1; and the locus homology arm used was Delta.

[0065] The gene expression cassette was constructed in the same manner as above.

[0066] The experimental results are as follows:

[0067] ARO7 G141S and ARO4 K229LGenes were constructed in expression cassette M1; EGFP, ARO7 G141S and ARO4 K229L The gene was constructed into expression cassette M2; PHA2 and CYB5 genes were constructed into expression cassette M3; AtPAL2 gene was constructed into expression cassette M4; AtPAL2 and AtATR2 genes were constructed into expression cassette M5; AtPAL2, AtATR2 and AtC4H genes were constructed into expression cassette M6; GePTS1, PHA2, CYB5, ARO2 and GgVR genes were constructed into expression cassette M7; GgCHS, GgCHR and GgCHI genes were constructed into expression cassette M8; AtC4H and Gu4CL1 genes were constructed into expression cassette M9. The genes were constructed into expression cassette M9; At4CL1, GmCHS8, GmCHR5 and GmCHI1B2 genes were constructed into expression cassette M10; EGFP, AtPAL2, AtATR2, AtC4H, At4CL1, GmCHS8, GmCHR5 and GmCHI1B2 genes were constructed into expression cassette M11; ARO1, ARO3 and GmCHI1B2 genes were constructed into expression cassette M12; CkPta, BbXfpk, MtPDH1 and FjTAL genes were constructed into expression cassette M13.

[0068] (3) Construction of engineered Saccharomyces cerevisiae strains

[0069] The gene expression cassette constructed in (2) was integrated into the Saccharomyces cerevisiae chromosome via the CRISPR / Cas9 system to construct the Med1-Med13 engineered strains. The method for constructing the engineered Saccharomyces cerevisiae strains was the same as above.

[0070] The experimental results are as follows:

[0071] Table 1 Genotypes of p-coumaric acid and glycyrrhizin engineered yeast strains

[0072]

[0073] As shown in Table 1, using IMX581 as the initial strain, M1, M2, M4, M5 and M6 were integrated respectively to obtain Med1, Med2, Med4, Med5 and Med6 strains; M3 was integrated into the Med1 strain to obtain the Med3 strain; M7 was integrated into the Med6 strain to obtain the Med7 strain; M8 was integrated into the Med5 strain to obtain the Med8 strain; M9 was integrated into the Med8 strain to obtain the Med9 strain; M10 was integrated into the Med7 strain to obtain the Med10 strain; M11 was integrated into the IMX581 strain to obtain the Med11 strain; M12 was integrated into the Med10 strain to obtain the Med12 strain; and M13 was integrated into the Med12 strain to obtain the Med13 strain.

[0074] (4) Detection of p-coumaric acid and glycyrrhizin synthesis by engineered Saccharomyces cerevisiae strains

[0075] After constructing the synthesis pathway of p-coumaric acid and glycyrrhizin in a Saccharomyces cerevisiae strain, the p-coumaric acid and glycyrrhizin of the relevant strain were detected. The preparation method of the engineered strain test sample was the same as above.

[0076] The experimental results are as follows:

[0077] The samples of the prepared engineered strains were subjected to HPLC testing, and the results showed that the Med6 and Med7 strains were able to produce p-coumaric acid, of which the Med7 strain had the highest p-coumaric acid content. After fermentation according to the fermentation conditions in the instructions, the p-coumaric acid production of the Med6 strain was 120 mg / L, and the p-coumaric acid production of the Med7 strain was 250 mg / L ( Figure 1 ). Med10-Med13 strains are able to synthesize glycyrrhizin ( Figure 2 After fermentation according to the fermentation conditions in the manual, the glycyrrhizin production of Med10, Med11, Med12 and Med13 engineered strains were 31.9 mg / L, 29.0 mg / L, 43.1 mg / L and 58.2 mg / L, respectively ( Figure 3 ).

[0078] Example 2: Construction of an engineered Saccharomyces cerevisiae strain for synthesizing daidzein and formononetin

[0079] (1) Type of strain, required culture medium and culture method

[0080] The strains used in constructing the engineered Saccharomyces cerevisiae strain for synthesizing daidzein and formononetin in the present invention are the Med9 and Med10 strains constructed in Example 1, and the culture medium and culture method used are the same as above.

[0081] (2) Design and construction of key enzyme gene expression cassettes in engineered strains

[0082] The genes used in the construction of the engineered Saccharomyces cerevisiae strain for synthesizing daidzein and formononetin are the endogenous genes ARO1, ARO3, and ARO7 of Saccharomyces cerevisiae. G141S and ARO4 K229L The heterologous genes included Gg2-HIS, GgHID, GmCPR, GgI4'OMT, and GmCHI1B2. The promoters used for gene expression were pTPI1, pENO2, pTEF1, and pTDH3; the terminators were tCYC1, tHSP26, and tADH1; and the locus homology arm was Delta. The gene expression cassette was constructed using the same method as above.

[0083] The experimental results are as follows:

[0084] ARO7 G141Sand ARO4 K229L The genes were constructed into expression cassette M1; ARO1, ARO3 and GmCHI1B2 genes were constructed into expression cassette M12; Gg2-HIS and GgHID genes were constructed into expression cassette M14; GmCPR and GgI4'OMT genes were constructed into expression cassette M15; Gg2-HIS, GgHID, GmCPR and GgI4'OMT genes were constructed into expression cassette M16.

[0085] (3) Construction of engineered Saccharomyces cerevisiae strains

[0086] The gene expression cassette constructed in (2) was integrated into the chromosome of Saccharomyces cerevisiae through the CRISPR / Cas9 system to construct the Med14-Med18 engineered strain.

[0087] The experimental results are as follows:

[0088] Table 2 Genotypes of yeast strains engineered for daidzein and formononetin

[0089]

[0090] As shown in Table 2, Med9 was used as the initial strain, and M14 was integrated into the strain to obtain the Med14 strain; M15 was integrated into the Med14 strain to obtain the Med15 strain; M16 was integrated into the Med10 strain to obtain the Med16 strain; M1 was integrated into the Med16 strain to obtain the Med17 strain; and M12 was integrated into the Med17 strain to obtain the Med18 strain.

[0091] (4) Detection of the synthesis of daidzein and formononetin by engineered Saccharomyces cerevisiae strains

[0092] The method for preparing engineered strain test samples is the same as above.

[0093] The experimental results are as follows:

[0094] The samples of the prepared engineered strains were subjected to HPLC detection. The results showed that, using liquiritigenin as substrate, the Med14 and Med15 engineered strains synthesized daidzein. Using p-coumaric acid as substrate, the Med18 engineered strain synthesized daidzein ( Figure 4 After adding liquiritin substrate, Med14 and Med15 strains were able to synthesize daidzein at a yield of 14.6 mg / L and 27.5 mg / L, respectively; after adding p-coumaric acid, Med18 strain was able to synthesize daidzein at a yield of 29.3 mg / L ( Figure 5 The strains Med15 and Med18, which use liquiritigenin as substrate and p-coumaric acid as substrate, were able to produce formononetin with yields of 21.5 mg / L and 33.6 mg / L, respectively. Figure 6 ).

[0095] Example 3: De novo synthesis of medipterostilbene by engineered yeast

[0096] (1) Type of strain, required culture medium and culture method

[0097] The strains used in constructing the engineered Saccharomyces cerevisiae strain for de novo synthesis of medipterostilbene in the present invention are the Med15 and Med18 strains constructed in Example 2, and the culture medium and culture method used are the same as above.

[0098] (2) Design and construction of key enzyme gene expression cassettes in engineered strains

[0099] The endogenous Saccharomyces cerevisiae gene used in constructing an engineered Saccharomyces cerevisiae strain for de novo synthesis of medipterostilbene is ACC1; the heterologous genes include Gg2-HIS, GgI2'H5, GmCPR, GgIFR4, GePTS1, GgVR, CkPta, FjTAL, MtPDH1, and BbXfpk. Promoters used for gene expression include pTPI1, pENO2, pTEF1, pINO1, and pTDH3; terminators include tCYC1, tHSP26, tADH1, tCPS1, and tTPS1; and the locus homology arm is Delta. The gene expression cassette was constructed using the same method as described above.

[0100] The experimental results are as follows:

[0101] The CkPta, BbXfpk, MtPDH1 and FjTAL genes were constructed into expression cassette M13; the Gg2-HIS, GmCPR, GgI2'H5 genes were constructed into expression cassette M17; the ACC1 and GgIFR4 genes were constructed into expression cassette M18; the GgI2'H5, GgIFR4 and GgVR genes were constructed into expression cassette M19; and the GePTS1 gene was constructed into expression cassette M20.

[0102] (3) Construction of engineered Saccharomyces cerevisiae strains

[0103] The gene expression cassette constructed in (2) was integrated into the Saccharomyces cerevisiae chromosome via the CRISPR / Cas9 system to construct the Med19-Med24 engineered strain. The method for constructing the engineered Saccharomyces cerevisiae strain was the same as above.

[0104] The experimental results are as follows:

[0105] Table 3 Genotypes of yeast strains engineered for medipterostilbene

[0106]

[0107] As shown in Table 3, the Med15 and Med18 engineered yeast strains were used as the initial strains, and M13 was integrated into the Med18 strain to obtain the Med19 strain; M17 was integrated into the Med19 strain to obtain the Med20 strain; M18 was integrated into the Med20 strain to obtain the Med21 strain; M19 was integrated into the Med15 strain to obtain the Med22 strain; M17 was integrated into the Med22 strain to obtain the Med23 strain; and M20 was integrated into the Med23 strain to obtain the Med24 strain.

[0108] (4) Detection of mediptertin in engineered Saccharomyces cerevisiae strains

[0109] The method for preparing engineered strain test samples is the same as above.

[0110] The experimental results are as follows:

[0111] The samples of the prepared engineered strain were subjected to HPLC testing. The results showed that after fermentation according to the fermentation conditions in the instructions, the Med21 engineered strain was able to detect medipterostilbene ( Figure 7 ), the yield of de novo synthesis of medipterostilbene using glucose as substrate was 180 mg / L ( Figure 8 ).

[0112] Example 4: Optimization of the yield of mediptertin in engineered Saccharomyces cerevisiae

[0113] (1) Type of strain, required culture medium and culture method

[0114] The strain used in the present invention for optimizing the yield of medipterostilbene in engineered cerevisiae is the IMX581 strain, the culture medium used is the same as above, and the culture method is to increase the amount of glucose solution added during the fermentation process from 20% to 40%.

[0115] (2) Design and construction of key enzyme gene expression cassettes in engineered strains

[0116] When constructing an optimized medipterostilbene synthesizing strain, the present invention integrated the key enzymes for medipterostilbene synthesizing in Examples 1, 2, and 3 into the Delta multi-copy locus, enabling multi-copy expression of the key enzyme genes. This enhanced the conversion of a series of key precursors, thereby increasing medipterostilbene yield. The gene expression cassette was constructed in the same manner as above.

[0117] The experimental results are as follows:

[0118] 32 key enzyme genes related to medipterostilbene synthesis were integrated into the Delta site of Saccharomyces cerevisiae to construct the expression cassette M21.

[0119] (3) Construction of engineered Saccharomyces cerevisiae strains

[0120] The gene expression cassette constructed in (2) was integrated into the chromosome of Saccharomyces cerevisiae using the CRISPR / Cas9 system to construct the Med25 engineered strain. The method for constructing the engineered Saccharomyces cerevisiae strain was the same as above.

[0121] The experimental results are as follows:

[0122] The M21 expression cassette was integrated into the IMX581 chassis strain to obtain the engineered Saccharomyces cerevisiae strain Med25.

[0123] (4) Detection of mediptertin in engineered Saccharomyces cerevisiae strains

[0124] The method for preparing engineered strain test samples is the same as above.

[0125] The experimental results are as follows:

[0126] The samples of the engineered strain were subjected to HPLC testing. The results showed that after adding 40% glucose solution during the fermentation period, the Med25 engineered strain was able to detect 450 mg / L of meditartin ( Figure 8 ).

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an engineered bacterium for de novo biosynthesis of medipterostilbene, characterized by: In the Saccharomyces cerevisiae chassis strain, ARO1, ARO2, ARO3, PHA2, CYB5, ACC1, and ARO4 were expressed. K229L 、ARO7 G141S , AtPAL2, AtATR2, AtC4H, At4CL1, GmCHR5, GmCHS8, GmCHI1B2, GmCPR, Gu4CL1, GgCHS, GgCHR, GgCHI, FjTAL, BbXfpk, CkPta, MtPDH1, Gg2-HIS, GgHID, GgI4'OMT, GgIFR4, GgI2'H5, GgVR, GePTS1 and EGFP genes were introduced into the Saccharomyces cerevisiae genome by in vitro cloning and recombination technology, thereby constructing an engineered bacterium for de novo biosynthesis of meditartin.

2. The method for constructing an engineered bacterium for de novo biosynthesis of medipterostilbene according to claim 1, characterized in that: The steps of the construction method are selected from any one of the following: (1) Build including ARO7 G141S and ARO4 K229L an expression cassette M1 comprising the AtPAL2, AtATR2 and AtC4H genes, an expression cassette M6 comprising the GePTS1, PHA2, CYB5, ARO2 and GgVR genes, an expression cassette M10 comprising the At4CL1, GmCHS8, GmCHR5 and GmCHI1B2 genes, an expression cassette M12 comprising the ARO1, ARO3 and GmCHI1B2 genes, an expression cassette M13 comprising the CkPta, BbXfpk, MtPDH1 and FjTAL genes, an expression cassette M16 comprising the Gg2-HIS, GgHID, GmCPR and GgI4'OMT genes, an expression cassette M17 comprising the Gg2-HIS, GmCPR, GgI2'H5 genes, and an expression cassette M18 comprising the ACC1 and GgIFR4 genes; The expression cassette M6 was transferred into the Saccharomyces cerevisiae strain to construct the Med6 strain, the expression cassette M7 was transferred into the Med6 strain to construct the Med7 strain, the expression cassette M10 was transferred into the Med7 strain to construct the Med10 strain, the expression cassette M16 was transferred into the Med10 strain to construct the Med16 strain, the expression cassette M1 was transferred into the Med16 strain to construct the Med17 strain, the expression cassette M12 was transferred into the Med17 strain to construct the Med18 strain, the expression cassette M13 was transferred into the Med18 strain to construct the Med19 strain, the expression cassette M17 was transferred into the Med19 strain to construct the Med20 strain, and the expression cassette M18 was transferred into the Med20 strain to construct the Med21 strain; (2) Construct a system including ARO1, ARO2, ARO3, and ARO4 K229L 、ARO7 G141S , PHA2, CYB5, BbXfpk, CkPta, MtPDH1, FjTAL, GmCHS8, GmCHR5, GmCHI1B2, AtPAL2, AtC4H, AtATR2, At4CL1, ACC1, Gg2-HIS, GgHID, GmCPR, GgI4'OMT, GgI2'H5, GgIFR4, GePTS1 and GgVR genes; the expression cassette M21 was transferred into the Saccharomyces cerevisiae strain to construct the Med25 strain.

3. The method for constructing an engineered bacterium for de novo biosynthesis of medipterocarpin according to claim 2, characterized in that: ARO1, ARO2, ARO3, ARO4 K229L 、ARO7 G141S , PHA2, CYB5 and ACC1 genes are all from Saccharomyces cerevisiae; AtPAL2, AtATR2, AtC4H, At4CL1, GmCHR5, GmCHS8, GmCHI1B2 and GmCPR genes are all from Arabidopsis thaliana; Gu4CL1 gene is from Glycyrrhiza uralensis; FjTAL gene is from Flavobacterium johnsonii; BbXfpk gene is from Bifidobacterium breve; CkPta gene is from Clostridium krusei; MtPDH1 gene is from Medicago truncatula; GgCHS, GgCHR, GgCHI, Gg2-HIS, GgHID, GgI4'OMT, GgI2'H5, GgIFR4 and GgVR genes are all from Glycyrrhiza glabra; GePTS1 gene is from Glycyrrhiza spinulosa.

4. The method for constructing an engineered bacterium for de novo biosynthesis of medipterocarpin according to claim 3, characterized in that: The promoter of the expression cassette is any one of pTPI1, pENO2, pTEF1, pPGK1, pINO1, pARO7, pTDH3 and pFBA1; the terminator is any one of tCYC1, tHSP26, tADH1, tCPS1, tTPS1 and tIDP1.

5. The method for constructing an engineered bacterium for de novo biosynthesis of medipterocarpin according to claim 4, characterized in that: The integration site of the expression cassette is the Delta site.

6. The method for constructing an engineered bacterium for de novo biosynthesis of medipterocarpin according to claim 5, characterized in that: The cerevisiae yeast was the cerevisiae IMX581 strain; the gRNA plasmid used for transfer was pMEL10-Delta.

7. The engineered bacteria constructed by the construction method according to any one of claims 1 to 6, characterized in that: The engineered bacteria is the engineered strain Med21 or the engineered strain Med25.

8. Use of the engineered bacteria according to claim 7 in the de novo biosynthesis of meditartin.

9. The use according to claim 8, characterized in that The steps are: inoculating the seed liquid of the engineered strain Med21 or the engineered strain Med25 into YPD fermentation medium, fermenting at 28-30°C and 180-200 rpm in a shaker for 120-144 hours, crushing the cells after the fermentation, and extracting medipterostilbene.

10. The use according to claim 9, characterized in that: The OD value of the fermentation broth is 25-30.

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