Genetic engineering strain for producing triacetate lactone and construction method

Abstract

The invention discloses a genetic engineering strain for producing triacetate lactone and a construction method of the genetic engineering strain. The preparation method comprises the following steps: knocking out a KbPKS1 gene in a Kabatilla bupleuri strain genome with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.25421; the method comprises the following steps: respectively replacing a gene KbMO1 and a gene KbLC1 in an anthraquinone compound synthesis gene cluster in a Kabatilla bupleuri bacterial strain genome with a synthetic gene GhPS of triacetate lactone, so as to obtain a genetic engineering bacterial strain for producing triacetate lactone, wherein the gene KbMO1 and the gene KbLC1 are in the anthraquinone compound synthesis gene cluster in the Kabatilla bupleuri bacterial strain genome; experiments prove that the yield of the triacetate lactone of the genetic engineering strain for producing the triacetate lactone is higher than that of wild bacteria.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a genetic engineering strain for producing triacetic acid lactone and a construction method thereof. Background Art

[0002] Triacetic acid lactone is found in African daisy. It is a small molecule polyketide compound and a renewable platform chemical. In addition, triacetic acid lactone is a high-value fine chemical with a very wide application market. For example, under the action of microorganisms, triacetic acid lactone can be converted into new plastic materials and applied in the field of materials, etc.; as an intermediate, it can produce more high-value-added compounds, such as patchouli ketone and sorbic acid, which are applied in the food field, etc.; it can synthesize phloroglucinol and acetylacetone, which are applied in the chemical field. In addition, because triacetic acid lactone is non-toxic, harmless and low-corrosive, it is also used in the medical field. It can also be used to synthesize piperidine and lactam compounds.

[0003] At present, there are several methods for producing triacetic acid lactone: plant extraction, chemical synthesis, biological fermentation and other methods. The content of triacetic acid lactone in plants is low, separation and purification are difficult, and the planting of plants takes a long time and occupies a lot of land resources, which is difficult to meet the needs of industrial production. Relying on petroleum cracking or using acetic acid as a raw material to produce triacetic acid lactone will consume a large amount of petroleum resources and consume a lot of energy. In the process of using chemical synthesis to produce triacetic acid lactone, harmful catalysts need to be used, and toxic by-products will be produced, which will cause great environmental pollution. Therefore, it is urgent to use genetic engineering means, microorganisms as tools, and adopt environmentally friendly and low-cost biological synthesis methods to produce triacetic acid lactone. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a genetically engineered strain for producing triacetic acid lactone.

[0005] The second object of the present invention is to provide a method for constructing a genetically engineered strain for producing triacetic acid lactone.

[0006] The third object of the present invention is to provide an application of a genetically engineered strain for producing triacetic acid lactone by fermentation.

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

[0008] A method for constructing a genetically engineered strain for producing triacetic acid lactone comprises the following steps: knocking out the KbPKS1 gene in the genome of the Kabatiella bupleuri strain with a deposit number of CGMCC No. 25421; replacing the gene KbMO1 and the gene KbLC1 in the anthraquinone compound synthesis gene cluster in the genome of the Kabatiella bupleuri strain with the triacetic acid lactone synthesis gene GhPS, respectively, to obtain a genetically engineered strain for producing triacetic acid lactone;

[0009] The nucleotide sequence of the gene GhPS is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the KbPKS1 gene is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the gene KbMO1 is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the gene KbLC1 is shown in SEQ ID NO.4.

[0013] The above construction method constructs a genetic engineering strain for producing triacetic acid lactone.

[0014] The application of the above genetically engineered strain in the production of triacetic acid lactone by fermentation.

[0015] Advantages of the present invention:

[0016] Experiments have shown that the yield of triacetyl lactone produced by the genetically engineered strain of the present invention for producing triacetyl lactone is higher than that of wild bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of triacetyl lactone production by a genetically engineered strain (abbreviated as TAL in the figure). DETAILED DESCRIPTION

[0018] 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.

[0019] The above 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.

[0020] The strain number TSYW-49 Kabatiella bupleuri selected in each embodiment of the present invention is referred to as Kabatiella bupleuri strain or TAL.

[0021] The present invention will be further described below in conjunction with specific embodiments.

[0022] The synthetic gene GhPS of triacetic acid lactone is from NCBI database (NCBI No.P48391.2).

[0023] Example 1 Construction of Kabatiella bupleuri strain-specific gene editing vector

[0024] The sequences of KbPKS1 (SEQ ID NO.2), KbMO1 (SEQ ID NO.3) and KbLC1 genes (SEQ ID NO.4) were input into the input box of the gRNA prediction software (CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ), uploaded to the server, and the gRNA nucleotide sequences were obtained. The gRNA nucleotide sequences of the target genes KbPKS1, KbMO1 and KbLC1 are shown in Table 1.

[0025] Table 1 gRNA nucleotide sequence table

[0026]

[0027] The Golden Gate vector construction method was used to replace the sequence with the Eco31I restriction site sequence in the gRNA expression cassette in the Kabatiella bupleuri strain gene editing vector to obtain the Kabatiella bupleuri strain-specific gene editing vectors THKbPKS1 (SEQ ID NO.5), THKbMO1 (SEQ ID NO.6) and THKbLC1 (SEQ ID NO.7); all gRNA gene sequences were synthesized by Suzhou GeneWeizhi Co., Ltd.

[0028] Example 2 Obtaining donor DNA fragments for homologous end repair

[0029] 1) The triacetic acid lactone synthesis gene GhPS was synthesized by Suzhou Jinweizhi Co., Ltd.

[0030] The nucleotide sequence of the triacetate lactone synthesis gene GhPS is shown in SEQ ID NO.1;

[0031] 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) by seamless cloning to obtain donor DNA for homologous end repair, i.e., DKbPKS1 (SEQ ID NO.8), DKbGhPS2 (SEQ ID NO.9) and DKbGhPS3 (SEQ ID NO.10) vectors;

[0032] The primers of the donor DNA are shown in Table 2.

[0033] Table 2 Primer sequence list

[0034]

[0035] 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 KbPKS1 5arm, KbMO1 5arm and KbLC1 5arm of KbPKS1, KbMO1 and KbLC1 were recovered; the genome of the 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 KbPKS1 3arm, KbMO1 3arm and KbLC1 were recovered. 3arm; the synthetic gene GhPS was amplified by primer pairs GhPS2F / R and GhPS2-F / R, and the fragments were recovered to obtain GhPS2 and GhPS3.

[0036] The reaction system and reaction conditions are shown in Table 3 and Table 4.

[0037] Table 3 PCR reaction system

[0038]

[0039] Table 4 PCR reaction conditions

[0040]

[0041] The upstream and downstream homology arms of KbPKS1, as well as the homology arms of KbMO1, KbLC1, and GhPS2 and GhPS3 were seamlessly cloned and assembled to obtain the homologous end-repaired donor DNA (DKbPKS1, DKbGhPS2 and DKbGhPS3) vector. The seamless cloning kit was purchased from Novozymes Biotech Co., Ltd., catalog number C116-01. For specific operations, please refer to the instruction manual.

[0042] The KbPKS1 gene in the genome of the Kabatiella bupleuri strain with a deposit number of CGMCC No. 25421 was knocked out; the gene KbMO1 and the gene KbLC1 in the anthraquinone compound synthesis gene cluster in the genome of the Kabatiella bupleuri strain were replaced with the triacetic acid lactone synthesis gene GhPS.

[0043] Example 3 Genetic transformation of strain Kabatiella bupleuri

[0044] The present invention adopts protoplast transformation method to genetically transform the strain, and the protoplast and transformation method are as follows:

[0045] 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 4 30.4 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 45 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;

[0046] 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;

[0047] 3) Add appropriate amount of bacterial precipitate, incubate at 30°C, 220 rpm, and perform enzymatic hydrolysis for 3.5 h;

[0048] 4) The protoplasts were centrifuged and suspended in an ice-cold STC aqueous solution (1 M sorbitol, 10 mM Tris-Cl, 50 mM CaCl2, pH = 7.5). The mixture was repeated once and finally suspended in an appropriate amount of STC aqueous solution. The protoplasts were adjusted to 10 8 Pieces / mL;

[0049] 5) 10 μg of the specific gene editing vector obtained in Example 1, 10 μg of the donor DNA fragment obtained in Example 2 after homologous end repair, and 50 μL of a 25% PEG6000 aqueous solution were sequentially added to 100 μL of protoplasts, mixed, and placed in an ice bath for 20 min;

[0050] 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;

[0051] 7) After 2-3 days of incubation in a 30°C constant temperature incubator, transformants were selected for identification, and the successfully transformed Kabatiella bupleuri strain was obtained, which was a genetically engineered strain for producing triacetic acid lactone and was stored in a -80°C freezer.

[0052] Example 4 Detection of triacetic acid lactone content

[0053] 1) A genetically engineered strain producing triacetic acid lactone and a wild strain (WT, Kabatiella bupleuri strain number TSYW-49) frozen at -80°C were thawed, streaked on YPD solid culture medium, and statically activated and cultured at 30°C for 2 days; single clones with uniform size and normal color were picked and inoculated into YPD liquid culture medium, and shaken and cultured at 30°C and 220 rpm for 3 days to obtain fresh seed liquid of the triacetic acid lactone producing strain.

[0054] 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.

[0055] 3) The fresh seed liquid obtained in step (1) was inoculated into the fermentation medium for fermentation at a speed of 220 r / min and a temperature of 30°C for 5 days to obtain a triacetic acid lactone fermentation broth.

[0056] 4) Ethyl acetate and bacterial solution were uniformly mixed at a ratio of 1:1, and the solid and liquid were separated. The supernatant was collected for the production detection of triacetic acid lactone.

[0057] 5) Using 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution as mobile phase, detect the absorption peak of the sample at 310nm and determine the content of triacetic acid lactone. Figure 1 .

Claims

1. A method for constructing a genetically engineered strain for producing triacetic acid lactone, Its characteristics are The method comprises the following steps: knocking out the KbPKS1 gene in the genome of the Kabatiella bupleuri strain with a deposit number of CGMCC No. 25421; replacing the gene KbMO1 and the gene KbLC1 in the anthraquinone compound synthesis gene cluster in the genome of the Kabatiella bupleuri strain with the synthesis gene GhPS of triacetic acid lactone, respectively, to obtain a genetically engineered strain for producing triacetic acid lactone; The nucleotide sequence of the gene GhPS is shown in SEQ ID NO.1; The nucleotide sequence of the KbPKS1 gene is shown in SEQ ID NO.2; The nucleotide sequence of the gene KbMO1 is shown in SEQ ID NO.3; The nucleotide sequence of the gene KbLC1 is shown in SEQ ID NO.

4.

2. A genetically engineered strain for producing triacetic acid lactone constructed by the construction method of claim 1.

3. Use of the genetically engineered strain of claim 2 in fermentation to produce triacetic acid lactone.

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