Application of gene in synthesis of polyester, plasmid and engineering bacterium

Abstract

The invention relates to the technical field of microorganisms, in particular to application of genes in synthesis of polyester, a plasmid and an engineering bacterium, the genes comprise a gene 1 and a gene 2, the gene 1 comprises a gene TCAP03220 with a nucleotide sequence as shown in SEQ ID No.1 or a homologous gene with the homology greater than 70% of the gene TCAP03220, and the gene 2 comprises a gene TCAP03221 with a nucleotide sequence as shown in SEQ ID No.2 or a homologous gene with the homology greater than 70% of the gene TCAP03221. According to the method, biosynthetic genes TCAP03221 and TCAP03220 derived from Tolypocladium capitatum are subjected to heterologous expression in aspergillus nidulans with high fermentation performance, the bio-based polyester A is successfully synthesized, the polyester A is subjected to separation and identification to determine the structure of the polyester A, and in addition, the yield of the polyester A is increased to 1.2 g / L by optimizing a culture medium. The constructed engineering bacterium provides a good expression system for industrial production of polyester.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, in particular to the application of genes in synthetic polyester, plasmids and engineering bacteria. Background Art

[0002] Polyesters are a class of synthetic polymers made up of multiple chemical repeating units linked by ester (CO-O) groups. Polyesters have a wide range of properties and practical applications; permanent ironing fabrics, disposable soft drink bottles, compact discs, rubber tires, and enamel coatings are just a few of the products in this category. Polyesters are typically made by a condensation reaction between an organic alcohol (containing a hydroxyl [OH] group) and a carboxylic acid (containing a carboxyl [COOH] group). The two functional groups react to form a characteristic ester bond, a chemical group with the following structure: .

[0003] Currently, almost all of the materials used to make polyester come from the extraction of fossil fuels, which is an immediate red flag for the environment. From extraction to transportation to combustion, every stage of fossil fuel use causes harm, resulting in the majority of greenhouse gas emissions, various types of pollution, oil spills, health threats, and more. In addition, polyester is a major culprit for microfiber and microplastic pollution. Every time we wear and wash polyester clothes, they shed tiny microplastics, which inevitably make their way into our waterways and food chain. Just washing synthetic fibers such as polyester, nylon, or acrylic results in approximately half a million tons of plastic microfibers being discharged into the ocean each year.

[0004] The development of sustainable and environmentally friendly polymer materials is one of the most pressing contemporary challenges facing polymer science and engineering. Bio-based polyesters hold great promise because they use renewable raw materials and can be rapidly degraded by microorganisms. Bio-based polyesters have attracted increasing interest as a platform to meet this demand due to their adjustable properties and wide range of applications. In recent years, as the preparation technology of bio-based monomers has become more mature, a large number of bio-based polyesters have emerged in the rapidly developing bio-based industry. Among them, the fastest growing market will be materials such as polyhydroxyalkanoates (PHA), polylactic acid PLA and bio-ethylene used to produce bioplastics. The development of new bio-based polyester materials has significant market value.

[0005] However, currently used are short-chain fatty acids such as polyhydroxyalkanoates (PHA), polylactic acid PLA and bio-ethylene. The development of long-chain fatty acid polyesters as new materials has significant application value in the industrial and aerospace fields. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an application of genes in the synthesis of polyester, plasmids and engineered bacteria, Tolypocladium capitatumThe biosynthetic genes TCAP_03221 and TCAP_03220 were heterologously expressed in Aspergillus nidulans, and the resulting engineered bacteria were able to synthesize long-chain bio-based polyester A with a yield of up to 1.2 g / L.

[0007] In order to achieve the above-mentioned object and other related objects, the present invention provides the following technical solutions: The first aspect of the present invention provides the application of co-expression of two genes in synthesizing polyester, comprising: Gene 1, including gene TCAP_03220 with a nucleotide sequence as shown in SEQ ID No. 1 or a homologous gene with a homology greater than 70% thereto; Gene 2 includes gene TCAP_03221 whose nucleotide sequence is shown in SEQ ID No. 2 or a homologous gene with a homology greater than 70%.

[0008] Among them, gene TCAP_03220 and gene TCAP_03221 are both derived from Toxoplasma capitis Tolypocladium capitatum CBS 113982 strain.

[0009] In one embodiment of the present invention, genes TCAP_03220 and TCAP_03221 are Tolypocladium capitatum The CBS 113982 genome was used as a template and amplified by PCR.

[0010] The primers used for amplification are as follows:

[0011] In one embodiment of the present invention, Tolypocladium capitatum The extraction method of CBS 113982 genome includes strain culture, bacterial collection, bacterial cell disruption, DNA extraction, DNA precipitation, washing and dissolution.

[0012] Further, the method specifically comprises the following steps: Tolypocladium capitatum The CBS 113982 strain was inoculated into a PDA solid plate at -80°C for culture, and the cells were scraped and inoculated into a YPD liquid medium for culture, and the cells were clumped; the water of the cells was absorbed by filter paper, and the cells were divided into centrifuge tubes and immersed in liquid nitrogen; the cells were then ground with a grinding rod, and lysis solution was added to each tube after grinding, water bathed, 3M sodium acetate was added to mix, centrifuged, the supernatant was taken and put into a new centrifuge tube, isopropanol was added to mix, centrifuged, the supernatant was removed, washed with 70% ethanol, the ethanol was sucked out, naturally dried, and dissolved in water.

[0013] Furthermore, the YPD liquid culture medium includes 10 g yeast extract, 10 g polypeptone, 10 g casein hydrolysate, 20 g sucrose and 1000 mL water.

[0014] Furthermore, the lysis solution includes 400 mM Tris-HCl buffer (pH 8.0), 60 mM EDTA (pH 8.0), 150 mM sodium chloride and 1% sodium dodecyl sulfate.

[0015] In a second aspect of the present invention, a recombinant plasmid comprising gene 1 or gene 2 is provided.

[0016] In one embodiment of the present invention, the recombinant plasmid is pIM 1 containing gene 1, and its plasmid map is as follows: Figure 1 shown.

[0017] Furthermore, the construction method of the recombinant plasmid pIM 1 includes: constructing gene 1 onto pANR vector by using yeast homologous recombination method, and introducing a constitutive or inducible strong promoter in front of gene 1, and finally obtaining the recombinant plasmid pIM 1.

[0018] Furthermore, the gene 1 is gene TCAP_03220.

[0019] Furthermore, the strong promoter is selected from gpdA or glaA .

[0020] In one embodiment of the present invention, the recombinant plasmid is pIM 2 containing gene 2, and its plasmid map is as follows: Figure 2 shown.

[0021] Furthermore, the construction method of the recombinant plasmid pIM 2 includes: constructing gene 2 onto the pANU vector using a yeast homologous recombination method, and introducing a constitutive or inducible strong promoter in front of gene 2, and finally obtaining the recombinant plasmid pIM 2.

[0022] Furthermore, the gene 2 is gene TCAP_03221.

[0023] Specifically, the method for yeast homologous recombination includes: simultaneously introducing gene 1 and pANR vector (or gene 2 and pANU vector) into yeast competent cells, extracting plasmids after obtaining yeast mutants and transforming them into Escherichia coli, extracting plasmids and verifying them through PCR, and recording the recombinant plasmid containing gene 1 with the correct sequence as pIM 1 (or the recombinant plasmid containing gene 2 as pIM 2).

[0024] The primers used for verification are as follows:

[0025] The third aspect of the present invention provides an engineered bacterium co-transformed with the recombinant plasmid pIM 1 and the recombinant plasmid pIM 2.

[0026] In one embodiment of the present invention, the base fungus includes Aspergillus nidulans, yeast or other microbial bases.

[0027] In one embodiment of the present invention, the recombinant plasmid pIM 1 and the recombinant plasmid pIM 2 are co-transformed into A. nidulans In the A1145 protoplasts, positive transformants were screened using a nutrient-deficient medium, which were the engineered bacteria.

[0028] Furthermore, the co-transformation method includes: resuspending the protoplasts with STC Buffer, mixing the recombinant plasmid and the protoplast resuspension, placing on ice for a period of time, then adding a PEG solution, placing at room temperature for a period of time, coating on a regeneration medium containing hygromycin B, and culturing, and picking a single clone.

[0029] Furthermore, the preparation method of the protoplasts comprises: inoculating Aspergillus nidulans spore liquid into CD liquid culture medium for culturing, germination, centrifugation, discarding the supernatant to collect the bacterial bodies, blowing and washing the bacterial bodies with Osmotic medium solution, centrifugation, discarding the supernatant, and repeating this step twice; adding Osmotic medium solution containing Lysing Enzymes and Yatalase, resuspending the bacterial bodies, and then transferring to a sterile conical flask for enzymolysis overnight, transferring the bacterial liquid to a sterile centrifuge tube, adding Trapping Buffer, centrifuging, and a layer of white floccules can be seen on the middle interface, which is the protoplasts.

[0030] Furthermore, the nutrient-deficient culture medium includes at least one of riboflavin, uridine and uracil nutrient-deficient culture medium.

[0031] The fourth aspect of the present invention provides the use of the recombinant plasmids pIM 1 and pIM 2 or the engineered bacteria in the synthesis of polyester.

[0032] The fifth aspect of the present invention provides a method for efficiently synthesizing polyester, comprising fermenting the engineered bacteria, collecting the fermentation liquid, and purifying it.

[0033] Specifically, the method comprises the following steps: inoculating the positive transformant into the corresponding nutrient-deficient CD-ST liquid fermentation medium, and fermenting and culturing for 5 days; extracting with an equal volume of ethyl acetate, concentrating to dryness under reduced pressure, dissolving with chromatographic methanol, fractionating the crude residual oil by liquid chromatography, and further purifying and separating the individual components of the fractions by CH3CN / H2O semi-preparative high performance liquid chromatography to obtain polyester A.

[0034] Furthermore, the structural formula of the polyester A is as follows: .

[0035] Wherein, the bio-based polyester A belongs to long-chain fatty acid polyester.

[0036] Furthermore, the fractionation process is: 25% MeOH for 10 min, 25-50% linear gradient MeOH for 15 min, 50% MeOH for 15 min, 50-80% linear gradient MeOH for 15 min, 80% linear gradient MeOH for 20 min, 80-100% linear gradient MeOH for 15 min, and 100% MeOH for 20 min.

[0037] In a sixth aspect of the present invention, a compound with a new structure is provided, wherein the compound is polyester A, and the structural formula of the polyester A is as follows: .

[0038] As described above, the application of the gene, plasmid and engineered bacteria in the synthesis of polyester has the following beneficial effects: Tolypocladium capitatum The biosynthetic genes TCAP_03221 and TCAP_03220 were heterologously expressed in Aspergillus nidulans with high fermentation performance, and bio-based polyester A was successfully synthesized. Polyester A was isolated and identified to determine its structure. In addition, the optimized culture medium of the present invention increased the yield of polyester A to 1.2 g / L. The engineered bacteria constructed by the present invention provide a good expression system for the industrial production of polyester. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a molecular structure diagram of the bio-based polyester A disclosed in Example 5 of the present invention.

[0040] Figure 2 This is the plasmid map of the recombinant plasmid pIM 1 disclosed in the present invention.

[0041] Figure 3 This is the plasmid map of the recombinant plasmid pIM 2 disclosed in the present invention. DETAILED DESCRIPTION

[0042] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0043] Example 1 Tolypocladium capitatum Extraction of CBS 113982 genome: Will Tolypocladium capitatumThe CBS 113982 strain was inoculated from -80°C to a PDA solid plate, cultured at 30°C for 3 days, and the cells were scraped and inoculated into YPD (10 g yeast extract, 10 g polypeptone, 10 g casein hydrolysate, 20 g sucrose, sterilized by high temperature with 1000 mL water) liquid medium, cultured at 30°C, 150 rpm for 2 days, and the cells formed clumps. Use filter paper to absorb the moisture of the bacteria, divide 1.8mL into 2mL centrifuge tubes, immerse in liquid nitrogen, and then use a grinding rod to grind the bacteria. After grinding, add 500μL of lysis solution (400mM Tris-HCl buffer pH8.0, 60mM EDTApH8.0, 150mM sodium chloride and 1% sodium dodecyl sulfate) to each tube, bathe at 65℃ for 20min, add 150μL of 3M sodium acetate and mix, centrifuge at 12000rpm for 10 minutes, take 500μL of supernatant and put it into a new 1.5m centrifuge tube, add 500μL of isopropanol and mix, centrifuge at 12000rpm for 5 minutes, remove the supernatant, wash with 1mL of 70% ethanol, aspirate the ethanol, dry naturally, and dissolve in 200μL of water.

[0044] Example 2 Construction of recombinant plasmids pIM 1 and pIM 2: by Tolypocladium capitatum Using the CBS 113982 genome as a template, two gene fragments TCAP_03220 and TCAP_03221 were obtained by PCR amplification. The primers used for amplification are as follows:

[0045] The reaction system and cycle parameters are as follows:

[0046] Using yeast homologous recombination, gene TCAP_03220 was constructed into pANR vector, and gene TCAP_03221 was constructed into pANU vector. A strong promoter was introduced in front of each gene, and finally two recombinant plasmids pIM1 and pIM 2 were obtained. The plasmid maps are shown in Figure 2. Figure 2 and 3 shown.

[0047] Among them, the 2×Hieff™ PCR Master Mix (With Dye) kit (Shanghai Yisheng Biotechnology Co., Ltd.) was used for PCR verification of recombinant transformants. The positive recombinant transformants of Escherichia coli were verified by bacterial liquid PCR. A single colony of Escherichia coli was picked and cultured in LB liquid medium (containing antibiotics) at 37°C and 220 rpm for 3-4 hours. 1 μL of bacterial liquid was taken as a PCR verification template, and the denaturation time was 10 minutes.

[0048] The PCR reaction system and cycle parameters are as follows (10 μL amplification system):

[0049] The primers used for verification are as follows:

[0050] Example 3 A. nidulans Preparation of A1145 protoplasts: Inoculate the Aspergillus nidulans spores into 40 mL CD liquid medium (supplemented with riboflavin, pyridoxineHCl, uridine and uracil), and culture at 37 ℃, 220 rpm for about 9 h for germination; then centrifuge at 3750 rpm, 4 ℃ for 5 min, and discard the supernatant to collect the bacteria. Wash the bacteria with 15 mL Osmotic medium solution, centrifuge at 3750rpm, 4 ℃ for 5 min, discard the supernatant, and repeat this step twice. Add 10 mL Osmotic medium solution containing 30 mg Lysing Enzymes and 20 mg Yatalase, resuspend the bacteria, and then transfer to a 50 mL sterile conical flask, 28 ℃, 80 rpm, and enzymolysis overnight. Transfer the bacterial solution to a 50 mL sterile centrifuge tube, slowly add 10 mL Trapping Buffer, and centrifuge at 5000 rpm, 4 ℃ for 20 min. A layer of white floccules can be seen at the middle interface, which is the protoplast layer.

[0051] Example 4 Construction of engineered bacteria: Pipette the protoplasts prepared in Example 3 into a 10 mL sterile centrifuge tube, add 2 volumes of pre-cooled STC Buffer, gently invert to mix, centrifuge at 5000 rpm, 4 °C for 8 min, and discard the supernatant. Add an appropriate amount of STC Buffer, resuspend the protoplasts, and dispense 100 µL into 1.5 mL sterile centrifuge tubes; Then add the recombinant plasmids pIM 1 and pIM 2, place on ice for 30 min, slowly add 600 µL of 60% PEG solution from bottom to top, place at room temperature for 30 min, spread on the regeneration medium containing 200 mg / mL hygromycin B, culture at 25 °C for 3-4 days, pick out single clones; then use nutrient-deficient medium to screen for positive transformants.

[0052] Example 5 Separation of Bio-based Polyester A: The positive transformants obtained in Example 4 were inoculated into 5 L of the corresponding auxotrophic CD-ST liquid fermentation medium and fermented at 25°C and 220 rpm for three days. A. nidulans A1145 wild-type strain was used as a control. The samples were extracted with an equal volume of ethyl acetate, concentrated to dryness under reduced pressure, and then dissolved in methanol for chromatography.

[0053] The crude residue was fractionated by liquid chromatography at a flow rate of 25 mL / min, and the fractionation process was as follows: 25% MeOH for 10 min, 25-50% linear gradient MeOH for 15 min, 50% MeOH for 15 min, 50-80% linear gradient MeOH for 15 min, 80% linear gradient MeOH for 20 min, 80-100% linear gradient MeOH for 15 min, and 100% MeOH for 20 min. A 50% / 50%-MeOH / H2O fraction containing compound 1 (125 mg) was obtained.

[0054] Then, the isolated individual components were further purified by semi-preparative HPLC using 40% (v / v) CH3CN / H2O to afford compound 1 (9 mg, tR=10.5 min).

[0055] Compound 1 was tested by HMBC, and the test results are shown in the following table:

[0056] After analysis, the structure of compound 1 (i.e. bio-based polyester A) is as follows Figure 1 As shown, polyester A is a compound with a new structure.

[0057] In summary, the present invention will come from Tolypocladium capitatum The biosynthetic genes TCAP_03220 and TCAP_03221 of the present invention were heterologously expressed in Aspergillus nidulans with high fermentation performance, and bio-based polyester A was successfully synthesized. Polyester A was isolated and identified to determine its structure. In addition, the present invention optimized the culture medium to increase the yield of polyester A to 1.2 g / L. The engineered bacteria constructed by the present invention provide a good expression system for the industrial production of polyester. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0058] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Application of co-expression of two genes in polyester synthesis, characterized in that: include: Gene 1, including gene TCAP_03220 with a nucleotide sequence as shown in SEQ ID No. 1 or a homologous gene with a homology greater than 70% thereto; Gene 2 includes gene TCAP_03221 whose nucleotide sequence is shown in SEQ ID No. 2 or a homologous gene with a homology greater than 70%.

2. The use according to claim 1, characterized in that: Genes TCAP_03220 and TCAP_03221 are Tolypocladium capitatum The CBS 113982 genome was used as a template and amplified by PCR.

3. Recombinant plasmid containing gene 1 or gene 2.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid is pIM 1 containing gene 1. The construction method of the recombinant plasmid pIM 1 includes: constructing gene 1 onto a pANR vector by using a yeast homologous recombination method, and introducing a constitutive or inducible strong promoter in front of gene 1, and finally obtaining the recombinant plasmid pIM 1; The recombinant plasmid is pIM 2 containing gene 2. The construction method of the recombinant plasmid pIM 2 includes: constructing gene 2 onto a pANU vector using a yeast homologous recombination method, and introducing a constitutive or inducible strong promoter in front of gene 2, thereby finally obtaining the recombinant plasmid pIM 2.

5. The recombinant plasmid according to claim 4, characterized in that The strong promoter is selected from gpd or glaA .

6. An engineered bacterium co-transformed with the recombinant plasmid pIM 1 and the recombinant plasmid pIM 2 according to any one of claims 3 to 5.

7. The engineered bacteria according to claim 6, characterized in that The recombinant plasmids pIM 1 and pIM 2 were co-transformed into A. nidulans Positive transformants were screened in A1145 protoplasts using nutrient-deficient medium.

8. The engineered bacteria according to claim 7, characterized in that The preparation method of protoplasts comprises: inoculating Aspergillus nidulans spore liquid into CD liquid culture medium for culturing, germination, centrifugation, discarding supernatant to collect bacterial bodies, blowing and washing bacterial bodies with Osmotic medium solution, centrifugation, discarding supernatant, and repeating the steps twice; adding Osmotic medium solution containing Lysing Enzymes and Yatalase, resuspending bacterial bodies, and then transferring to a sterile conical flask for enzymolysis overnight, transferring bacterial liquid to a sterile centrifuge tube, adding Trapping Buffer, centrifuging, and a layer of white flocculent can be seen at the middle interface, which is the protoplast.

9. A method for efficiently synthesizing polyester, characterized in that: The method comprises fermenting the engineering bacteria according to any one of claims 6 to 8, collecting the fermentation liquid, purifying the liquid, and obtaining polyester.

10. A polyester, characterized in that The structural formula of the polyester is as follows: 。

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