Genetic engineering strain for producing bicarpin and construction method
By replacing the bicatron synthesis gene into the yeast-like Kabatiella bupleuri strain, a genetically engineered strain with high yield of bicatron was constructed, solving the problem of lack of environmentally friendly genetically engineered strains in the prior art for the synthesis of bicatron, and achieving efficient production of bicatron.
Patent Information
- Application Number
- CN202311664682.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-06
- Publication Date
- 2025-06-06
AI Technical Summary
There has been no report on the use of yeast-like Kabatiella bupleuri as a chassis cell to produce bicatronectin after genetically modified, resulting in a lack of environmentally friendly genetically engineered strains for the synthesis of bicatronectin.
A genetically engineered strain for producing bicatronin was constructed by replacing the corresponding genes in the anthraquinone compound synthesis genes in the yeast-like Kabatiella bupleuri strain.
High yield of bicatronin was achieved, demonstrating that this genetically engineered strain produced higher bicatronin than wild bacteria.
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Figure CN120099061A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of genetic engineering, and in particular to a genetic engineering strain for producing biccardiac and a construction method thereof. Background Art
[0002] Bikaverin is a red tetracyclic polyketide compound with antibacterial, antifungal, antiprotozoal and antitumor effects, and has great application potential in agriculture, forestry and clinical medicine. As a secondary metabolite, Bikaverin is mainly produced by Fusarium in nature. Since Fusarium is a common plant pathogen, using it to produce Bikaverin is potentially dangerous to environmental vegetation protection, so the use of environmentally friendly genetically engineered strains to synthesize Bikaverin has important application value.
[0003] At present, the research has confirmed the bicardin biosynthesis gene cluster, which contains 6 proteins, namely bik1, bik2, bik3, bik4, bik5, and bik6. Among them, bik1, bik2, and bik3 enzymes are predicted to be responsible for the synthesis of bicardin, bik4 and bik5 are predicted to be transcriptional regulators, and bik6 is speculated to be a permease or transporter. Bik1 responsible for the synthesis of bicardin is a PKS polyketide synthase, bik2 is a monooxygenase, and bik3 is a methyltransferase.
[0004] The yeast-like Kabatiella bupleuri is an endophyte of the traditional Chinese medicinal plant Bupleurum. Previous studies have found that this strain can synthesize a red anthraquinone compound. Gene knockout has confirmed that the gene cluster that synthesizes this anthraquinone compound includes PKS polyketide synthase, monooxygenase and laccase. Currently, there are no reports on the production of bicamectin using yeast-like Kabatiella bupleuri as a chassis cell after genetic modification. Summary of the invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a genetically engineered strain for producing bicardin.
[0006] The second object of the present invention is to provide a method for constructing a genetically engineered strain for producing biccardin.
[0007] The third object of the present invention is to provide an application of a genetically engineered strain for producing bicamectin by fermentation.
[0008] The technical solution of the present invention is summarized as follows:
[0009] A method for constructing a genetically engineered strain for producing bicamectin comprises the following steps: sequentially replacing the gene KbPKS1, the gene KbMO1 and the gene KbLC1 in the anthraquinone compound synthesis gene cluster in the genome of the Kabatiella bupleuri strain with the deposit number of CGMCC No. 25421 with the synthetic gene Bik1, the synthetic gene Bik2 and the synthetic gene Bik3 of bicamectin, to obtain a genetically engineered strain for producing bicamectin;
[0010] The nucleotide sequence of the gene Bik1 is shown in SEQ ID NO.1;
[0011] The nucleotide sequence of the gene Bik2 is shown in SEQ ID NO.2;
[0012] The nucleotide sequence of the gene Bik3 is shown in SEQ ID NO.3;
[0013] The nucleotide sequence of the gene KbPKS1 is shown in SEQ ID NO.4;
[0014] The nucleotide sequence of the gene KbMO1 is shown in SEQ ID NO.5;
[0015] The nucleotide sequence of the gene KbLC1 is shown in SEQ ID NO.6.
[0016] The above construction method constructs a genetically engineered strain for producing biccardiac.
[0017] The application of the above genetically engineered strain in the fermentation production of bicardin.
[0018] Advantages of the present invention:
[0019] Experiments have shown that the yield of bicamectin produced by the genetically engineered strain of the present invention is higher than that of wild bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a graph of the bicamectin production of a genetically engineered strain (referred to as bicamectin in the figure). DETAILED DESCRIPTION
[0021] The original bacteria of the present invention are classified and named Kabatiella bupleuri strain number TSYW-49, and were deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on July 27, 2022, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.25421.
[0022] The above-mentioned strain has been patented and published. The name of the invention is: A Kabatiella bupleuri strain producing anthraquinone compounds and its application. The patent application number is: 2022116450474.
[0023] The strain number TSYW-49 Kabatiella bupleuri selected in each embodiment of the present invention is referred to as Kabatiella bupleuri strain or Bika.
[0024] The present invention will be further described below in conjunction with specific embodiments.
[0025] The synthetic gene Bik1 of bicamectin was obtained from the NCBI database (NCBI No. CP130300.1);
[0026] The synthetic gene Bik2 of bicamectin was obtained from the NCBI database (NCBI No. CP052901.1);
[0027] The synthetic gene Bik3 of biccardin was derived from the NCBI database (NCBI No. CP052889.1).
[0028] Example 1 Construction of Kabatiella bupleuri strain-specific gene editing vector
[0029] First, the sequences of genes KbPKS1 (SEQ ID NO.4), KbMO1 (SEQ ID NO.5) and KbLC1 (SEQ ID NO.6) in the anthraquinone compound synthesis gene cluster in the genome of Kabatiella bupleuri strain of CGMCC No.25421 were input into the input box of gRNA prediction software (CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ) and uploaded to the server to obtain the corresponding gRNA nucleotide sequences, as shown in Table 1.
[0030] Table 1 gRNA nucleotide sequence list
[0031]
[0032] The Golden Gate vector construction method was used to replace the three sequences of gRNA with the Eco31I restriction site sequence in the gRNA expression cassette of the Kabatiella bupleuri strain gene editing vector to obtain the Kabatiella bupleuri strain-specific gene editing vectors THKbPKS1 (SEQ ID NO.7), THKbMO1 (SEQ ID NO.8) and THKbLC1 (SEQ ID NO.9); all gRNA gene sequences were synthesized by Suzhou GeneWeizhi Co., Ltd.
[0033] Example 2 Obtaining a donor DNA vector with homologous end repair
[0034] 1) The Fusarium oxysporum biccardin synthesis genes Bik1 (SEQ ID NO.1), Bik2 (SEQ ID NO.2) and Bik3 (SEQ ID NO.3) were synthesized by Suzhou Jinweizhi Co., Ltd.
[0035] 2) PCR amplification of the sequences 500 bp upstream and downstream of the target sequences of KbPKS1, KbMO1 and KbLC1 (can also be any value in the range of 450 bp-500 bp), combined with the genes synthesized in step 1) using seamless cloning to obtain donor DNA (DKbbik1 (SEQ ID NO.10), DKbbik2 (SEQ ID NO.11) and DKbbik3 (SEQ ID NO.12)) vectors for homologous end repair;
[0036] The primers of the donor DNA are shown in Table 2.
[0037] Table 2 Primer sequence list
[0038]
[0039]
[0040] More specifically, the genome of the yeast-like Kabatiella bupleuri strain was amplified by primer pairs DKbPKS1-5F / 5R, DKbMO1-5F / 5R and DKbLC1-5F / 5R, and the upstream homology arm fragments of KbPKS1, KbMO1 and KbLC1, KbPKS1 5arm, KbMO1 5arm and KbLC1 5arm, were recovered;
[0041] The genome of yeast-like Kabatiella bupleuri strain was amplified by primer pairs DKbPKS1-3F / 3R, DKbMO1-3F / 3R and DKbLC1-3F / 3R, and the downstream homology arm fragments of KbPKS1, KbMO1 and KbLC1, KbPKS13arm, KbMO1 3arm and KbLC1 3arm, were recovered;
[0042] The synthetic genes Bik1, Bik2 and Bik3 were amplified by primer pairs Bik1-F / R, Bik2-F / R and Bik3-F / R, and the fragments were recovered to obtain dBik1, dBik2 and dBik3.
[0043] The reaction system and reaction conditions are shown in Table 3 and Table 4.
[0044] Table 3 PCR reaction system
[0045]
[0046] Table 4 PCR reaction conditions
[0047]
[0048] The upstream and downstream homology arms of KbPKS1, KbMO1 and KbLC1 were seamlessly cloned and assembled with dBik1, dBik2 and dBik3, respectively, to obtain the homologous end-repaired donor DNA (DKbbik1, DKbbik2 and DKbbik3) vectors. The seamless cloning kit was purchased from Novozymes Biotech Co., Ltd., catalog number C116-01. For detailed operations, please refer to the instruction manual.
[0049] Example 3 Genetic transformation of strain Kabatiella bupleuri
[0050] The present invention adopts protoplast transformation method to genetically transform the strain, and the protoplast and transformation method are as follows:
[0051] 1) Inoculate Kabatiella bupleuri strain into 50mL GMM liquid medium (20×Nitrate Salts 50mL, Trace Elements 1mL, glucose 10g, sterilize at 115℃ for 30 minutes. (20×Nitrate Salts is NaNO 3 120g, KCl 10.4g, MgSO 4 .7H2O 10.4g,KH 2 PO 430.4 g dissolved in 1 L distilled water, sterilized at 121 °C for 20 minutes; Trace Elements is ZnSO 4 .7H 2 O 2.2g,H 3 BO 3 1.1g,MnCl 2 .4H 2 O 0.5g,FeSO 4 .7H 2 O0.16g,CuSO 4 .5H 2 O0.16g, (NH 4 ) 6 MoO 24 .4H 2 O 0.11g,Na 4 5 g of EDTA was dissolved in 100 mL of distilled water and sterilized at 121 °C for 20 min)) overnight culture, the bacterial precipitate was collected by centrifugation and washed three times with sterile deionized water;
[0052] 2) Prepare enzymatic solution: Dissolve the mixed enzyme of mass ratio of 45 mg of lyase (45 mg) to 105 mg of lyase (105 mg) = 3:7 (catalyst number: D861435, lyase number: 89833) in 10 mL of sorbitol-trisodium citrate buffer (pH = 5.8) containing 0.41 g of sodium chloride;
[0053] 3) Add appropriate amount of bacterial precipitate, incubate at 30°C, 220 rpm, and perform enzymatic hydrolysis for 3.5 h;
[0054] 4) The protoplast pellet was centrifuged and suspended in an ice-bathed STC aqueous solution (1 M sorbitol, 10 mM Tris-Cl, 50 mM CaCl 2 , pH = 7.5), repeated once, and finally suspended in an appropriate amount of STC aqueous solution, adjusting the protoplasts to 10 8 Pieces / mL;
[0055] 5) 10 μg of the Kabatiella bupleuri strain-specific gene editing vector obtained in Example 1, 10 μg of the homologous end-repaired donor DNA vector obtained in Example 2, and 50 μL of a 25% PEG6000 aqueous solution were sequentially added to 100 μL of protoplasts, mixed, and placed on ice for 20 min;
[0056] 6) Add 1 mL of 25% PEG6000 aqueous solution and 2 mL of STC aqueous solution in sequence, mix well, and apply 160 μL on a GMM plate containing hygromycin Hyg resistance;
[0057] 7) After 2-3 days of incubation at 30°C, transformants were selected for identification, and a successfully transformed genetically engineered strain for producing biccardiac was obtained and stored in a frozen state at -80°C.
[0058] Example 4 Detection of Bicardin Content
[0059] 1) After thawing a genetically engineered strain producing bicamectin and a wild strain (WT, Kabatiella bupleuri strain number TSYW-49) frozen at -80°C, streak inoculation on YPD solid culture medium, and static activation culture at 30°C for 2 days; picking a single clone with uniform size and normal color, inoculating it into YPD liquid culture medium, and shaking culture at 30°C and 220 rpm for 3 days to obtain a fresh seed liquid of the genetically engineered strain producing bicamectin;
[0060] 2) Preparation of fermentation medium: sucrose 104.26 g / L, peanut powder 8.49 g / L, ammonium chloride 0.34 g / L, disodium hydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate 6 mg / L, calcium chloride 15 mg / L;
[0061] 3) inoculating the fresh seed liquid obtained in step 1) into a fermentation medium for fermentation at a speed of 220 r / min and a temperature of 30° C. for 5 days to obtain a biccardin fermentation liquid;
[0062] 4) Ethyl acetate and the biccardin fermentation broth were uniformly mixed at a volume ratio of 1:1, and the solid and liquid were separated, and the supernatant was collected for the production detection of biccardin.
[0063] 5) Using 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution as mobile phase, the absorption peak of the sample at 310nm was detected to determine the content of biccardin. Figure 1 .
Claims
1. A method for constructing a genetically engineered strain for producing biccardiac. Its characteristics are The method comprises the following steps: replacing the gene KbPKS1, the gene KbMO1 and the gene KbLC1 in the anthraquinone compound synthesis gene cluster in the genome of the Kabatiella bupleuri strain with the deposit number of CGMCC No. 25421 with the synthesis gene Bik1 of biscardin, the synthesis gene Bik2 of biscardin and the synthesis gene Bik3 of biscardin in sequence, so as to obtain a genetic engineering strain for producing biscardin; The nucleotide sequence of the gene Bik1 is shown in SEQ ID NO.1; The nucleotide sequence of the gene Bik2 is shown in SEQ ID NO.2; The nucleotide sequence of the gene Bik3 is shown in SEQ ID NO.3; The nucleotide sequence of the gene KbPKS1 is shown in SEQ ID NO.4; The nucleotide sequence of the gene KbMO1 is shown in SEQ ID NO.5; The nucleotide sequence of the gene KbLC1 is shown in SEQ ID NO.
6.
2. A genetically engineered strain for producing biccardiac constructed by the construction method of claim 1.
3. Use of the genetically engineered strain of claim 2 in fermentation production of bicardin.