CN116904328A

CN116904328A - Engineering bacterium for high-expression of pyripyropene A and fermentation medium

Info

Publication number: CN116904328A
Application number: CN202310862163.XA
Authority: CN
Inventors: 卞小莹; 孙健鹏; 代广知; 孙志恒; 张友明
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-07-13
Filing date: 2023-07-13
Publication date: 2023-10-20

Abstract

The application relates to engineering bacteria for high-expression pyripyropene A and a fermentation medium. In the current microbial synthesis method of pyripyropene A, the defects of insufficient yield, more secondary metabolites and the like exist. The application aims to provide engineering bacteria suitable for industrial production, and based on the purpose, aspergillus oryzae is used as an initial strain, and genes nef1-nef9 related to pyripyropene A synthesis in Neosartorya fischeri DSM 3700 are introduced to obtain engineering bacteria A.oryzae-nef1-9. Furthermore, the application also provides a culture medium suitable for fermentation of the engineering bacteria, the yield of pyripyropene A is obviously improved, the fermentation time can reach 332.9mg/L in 3-5 days, secondary metabolites in fermentation products are fewer, and the requirements of industrial fermentation are met.

Description

Engineering bacterium for high-expression of pyripyropene A and fermentation medium

Technical Field

The application belongs to the technical field of engineering microorganisms, and particularly relates to engineering bacteria for high-expression of pyripyropene A, a construction method of the engineering bacteria, a culture medium for high-efficiency fermentation of pyripyropene A based on the strain and a biosynthesis method of pyripyropene A.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Pyripyropene is a mixed source sesquiterpene compound with pyridine ring structure and alpha-pyrone structure generated by fungi. Wherein pyripyropene A is a high-efficiency, selective SOAT2/ACAT2 inhibitor capable of reducing hypercholesterolemia and atherosclerosis in vivo, and IC thereof ₅₀ The value is 0.07 mu M, and the compound is expected to become a powerful cardiovascular therapeutic drug. In addition, the pyripyropene A has a similar structure to the hydroprene, and the hydroprene can be synthesized by a quick and efficient chemical semisynthesis method (shown in the following formula I). The hydroprene is a novel pesticide with a brand new action mechanism. Thus, pyripyropene a is a high value compound.

The pyripyropene A is mainly produced by fungi, according to the prior literature report, the original strain with the highest pyripyropene A yield is Penicillium griseofulvum F1959, the yield is about 20mg/L, but secondary metabolites of the original strain are numerous, the target product is not easy to separate and have potential toxicity, due to the excellent activity of the pyripyropene A, many researchers search for a chemical synthesis method, but according to the literature report, the current chemical synthesis method for the pyripyropene A needs 17 reaction steps, the total yield of the reaction is only 5.3%, the steps are numerous and complex, and the cost is high.

Furthermore, as a biosynthetic pathway of pyripyropene a, a synthetic pathway from niacin to desacetylpyripyropene E is elucidated in Nature Chemistry (2010), 2, 858-864. In The Journal of Antibiotics (2011) 64,221-227 and Biotechnology & Biotechnological Equipment (2014) 28,5,818-826, the function of the remaining two P450 genes and two acyltransferases, respectively, was elucidated by feeding acetylpyripyropene E as substrate.

Disclosure of Invention

The application predicts the function of the Open Reading Frame (ORF) coding protein of the strain of Fischer-Tropsch sarium (Neosartorya fischeri) DSM 3700 by homology analysis, locks the gene cluster related to pyripyropene A biosynthesis in the strain and is named nef. The gene cluster comprises 9 genes for encoding proteins involved in the biosynthesis of pyripyropene A, and the genes are specifically as follows:

(1) The coenzyme A ligase gene has a nucleotide sequence shown in SEQ ID No.1 and is named nef1;

(2) Polyketide synthase gene, the nucleotide sequence is shown in SEQ ID No.2, named nef2;

(3) Cytochrome P450 enzyme gene with nucleotide sequence shown in SEQ ID No.3 and named nef3;

(4) The nucleotide sequence of the cyclase gene is shown as SEQ ID No.4 and is named nef4;

(5) The FAD-dependent monooxygenase gene has a nucleotide sequence shown in SEQ ID No.5 and is named nef5;

(6) The nucleotide sequence of the isopentenyl transferase gene is shown as SEQ ID No.6 and is named nef6;

(7) The nucleotide sequence of the acetyltransferase gene is shown as SEQ ID No.7 and is named nef7;

(8) The nucleotide sequence of the acetyltransferase gene is shown in SEQ ID No.8 and is named nef8;

(9) The cytochrome P450 enzyme gene has a nucleotide sequence shown in SEQ ID No.9 and is named nef9.

The application adopts Aspergillus oryzae as host bacteria, and introduces the gene cluster to construct engineering bacteria, and provides engineering bacteria capable of efficiently synthesizing pyripyropene A, which is named as A.oryzae-nef1-9. Proved by verification, the synthetic gene can be successfully expressed in exogenous in engineering bacteria A.oryzae-nef1-9. According to the technical achievements, the application provides the following technical scheme:

in a first aspect, an engineering bacterium with high expression of pyripyropene A is provided, wherein an original strain of the engineering bacterium is Aspergillus oryzae, and compared with the original strain, the engineering bacterium is modified to have pyripyropene A biosynthesis genes nef1-nef9.

The engineering bacteria have the following preferable technical scheme:

a starting strain, the starting strain being aspergillus oryzae (Aspergillus oryzae) NSAR1.

nef1-nef9, the engineering bacterium is modified to have nef1-nef9 genes, and the genes can express coded proteins in host bacteria and exert enzyme-like activity. Any gene of nef1-nef9 can be one or more copies, and a technician can adjust according to the synthesis effect of pyripyropene A and the metabolic condition of the original strain.

The engineering bacteria may be modified by introducing the exogenous gene by ligating the desired nucleotide sequence into a vector, introducing the vector into a host bacterium, and the "vector" may include a construct having the desired nucleotide sequence and a regulatory sequence capable of replicating or functioning independently of the genome of the host bacterium after transferring into the host bacterium, and integrating the vector into the host genome itself, wherein the regulatory sequence includes a promoter for initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. Vectors such as recombinant plasmids, cosmids, viruses or phages that fulfil the above functions. In one embodiment of the present application, five vectors pTAex3, pUSA, pAdeA, pBARI and pUNA are used and are linked to the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.9, respectively, to construct recombinant plasmids, and the engineering bacteria are constructed.

Therefore, in a second aspect of the present application, there is provided a method for constructing the engineering bacteria according to the first aspect, comprising the steps of:

adding primers to amplify nef1-nef9 genes, introducing homology arms at two ends of the genes, and sequentially connecting the genes to a vector by using a Gibson assembly mode, wherein the connection mode is as follows:

two ends of the nef1 gene are introduced with homologous arms and connected to a pUSA vector to form a pUSA-nef1 recombinant plasmid;

introducing homologous arms at two ends of the nef2 gene, and connecting the homologous arms to the pTAex3 vector to form a pTAex3-nef2 plasmid;

designing primers to sequentially amplify gene fragments of an amyB promoter-nef 4/nef5/nef 6-terminator, introducing homologous arms at two ends, constructing three gene fragments on the same pAdeA vector, and constructing a recombinant plasmid pAdeA-PAmyB-nef4-TAmyB-PAmyB-nef6-TAmyB-PAmyB-nef5-TAmyB;

designing primers to sequentially amplify gene fragments of an amyB promoter-nef 3/nef 7-terminator, introducing homologous arms at two ends, constructing two gene fragments on the same pBARI vector, and constructing a recombinant plasmid pBARI-PAmyB-nef3-TAmyB-PAmyB-nef7-TAmyB;

designing primers to sequentially amplify gene fragments of an amyB promoter-nef 8/nef 9-terminator, introducing homologous arms at two ends, constructing two gene fragments on the same pUNA vector, and constructing a recombinant plasmid pUNA-PAmyB-nef8-TAmyB-PAmyB-nef9-TAmyB;

and sequentially introducing the five recombinant plasmids into an original strain by adopting a protoplast transformation method, and screening positive clones by using an MM screening plate to obtain the engineering strain.

A common culture medium of Aspergillus oryzae is a Chlamydia medium, and provides substrates such as sucrose and nitrate for mold such as Penicillium and Aspergillus. The application adopts the Chlamydia culture medium to culture the engineering bacteria, and can realize the synthesis of pyripyropene A, but the compound yield is less than 5mg/L, and the yield requirement of industrial fermentation is difficult to meet. Therefore, the application optimizes the fermentation conditions of engineering bacteria, and the PCM culture medium is adopted to remarkably improve the yield of pyripyropene A and meet the requirements of industrial production of pyripyropene A, and the culture medium is remarkably characterized by containing 0.5-2% of potato extract powder and 0.1-0.3% of nicotinic acid, and experiments prove that the composition and optimization can remarkably improve the fermentation level and efficiency of pyripyropene A.

In a third aspect, a fermentation medium of the engineering bacteria of the first aspect is provided, wherein the components and the contents of the medium are as follows:

sodium nitrate 0.2-0.6%, dipotassium hydrogen phosphate 0.1-0.3%, yeast extract 0.1-0.5%, magnesium sulfate 0.04-0.1%, potassium chloride 0.04-0.1%, ferrous sulfate 0.001-0.005%, calcium chloride 0.03-0.06%, sucrose 1-3%, maltose 1-3%, glucose 1-5%, potato extract powder 0.5-2%, nicotinic acid 0.1-0.3%, pH 6.0-7.5, and water in balance.

In an example of the better effect of the fermentation medium, the content of each component in the medium is as follows: sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.15%, yeast extract 0.3%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.002%, calcium chloride 0.04%, sucrose 2%, maltose 2%, glucose 4%, potato extract 1%, nicotinic acid 0.2%, pH 7.0, and water in balance.

In yet another example, the contents of the components in the medium are as follows: sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, yeast extract 0.1%, magnesium sulfate 0.04%, potassium chloride 0.04%, ferrous sulfate 0.001%, calcium chloride 0.03%, sucrose 1%, maltose 1%, glucose 1%, potato extract 0.5%, nicotinic acid 0.1%, pH 6.0, and the balance water.

In yet another example, the contents of the components in the medium are as follows: sodium nitrate 0.6%, dipotassium hydrogen phosphate 0.3%, yeast extract 0.5%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.005%, calcium chloride 0.06%, sucrose 3%, maltose 3%, glucose 5%, potato extract 2%, nicotinic acid 0.3%, pH7.5, and water in balance.

In a fourth aspect, there is provided a method for biosynthesis of pyripyropene A, comprising shake flask fermentation using the fermentation medium of the third aspect at 100-200rpm and 25-28℃for 3-5 days.

Further, the above biosynthesis method comprises the following specific steps:

activating engineering bacteria A.oryzae-nef1-9 onto a DPY solid plate, and culturing for 3-5d at 25-28 ℃; selecting small fungus blocks, transferring the small fungus blocks into a shake flask of the liquid fermentation culture medium, and fermenting for 3-5 days at 100-200rpm and 25-28 ℃; the flask was taken out, an equal volume of ethyl acetate was added thereto for repeated extraction, and ethyl acetate was partially dried to obtain.

Furthermore, the above ethyl acetate dried product also has corresponding purification processes, such as filtration, recrystallization, column chromatography, etc., and the purification method can be selected by those skilled in the art according to the actual situation.

The beneficial effects of the above technical scheme are:

1. the original host adopted by the application, namely the Fishcet's sartolomyces (Neosartorya fischeri) DSM 3700 strain, has the capacity of metabolizing to produce pyripyropene A, but the yield is extremely low, the yield of pyripyropene A in a general culture medium (a Chlamydomonas's medium) is about 0.2mg/L, and secondary metabolites are various, so that the strain is not beneficial to the separation and purification of pyripyropene A and cannot meet the application of industrial production. Aspergillus oryzae strains have the ability to autonomously metabolize to produce farnesyl diphosphate (FPP), a precursor for terpenoid synthesis, and thus Aspergillus oryzae is an heterologous expression host for terpenoid expression, including hetero-terpenoids, with significant advantages. Meanwhile, the aspergillus oryzae is an important production strain in the food industry in China, has the characteristics of fewer secondary metabolite types, no secondary metabolic toxin, green and environment-friendly production process, and is favorable for industrial production as a heterologous host. The application uses aspergillus oryzae as engineering bacteria constructed by chassis bacteria, does not generate secondary metabolic toxin, and has the characteristic that pyripyropene A in metabolic products is easy to separate and purify, thereby meeting the requirements of industrial production.

2. According to the application, the pyripyropene A biosynthesis gene cluster is transferred into aspergillus oryzae for heterologous expression, so that an engineering strain capable of producing pyripyropene A is constructed. Furthermore, the fermentation process of the engineering bacteria is optimized, and the culture medium (PCM culture medium) of the pyripyropene A provided by the application has good effect, and in a fermentation mode, the yield of the pyripyropene A reaches 332.9mg/L; aspergillus oryzae is used as a culture medium (Chlamydia medium) for fermentation, and the yield is 1.6mg/L. The fermentation method provided by the application improves the yield of pyripyropene A by 208 times, and compared with the highest yield of pyripyropene A reported in the current literature, the yield of pyripyropene A is improved by 16.6 times by 20 mg/L; and the fermentation time is short, and in the optimal scheme, the yield of pyripyropene A can reach the highest level after 4 days of fermentation.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.

FIG. 1 is a schematic diagram showing the structure of pTAex3-nef2 recombinant vector described in example 1;

FIG. 2 is a schematic structural diagram of the pUSA-nef1 recombinant vector described in example 1;

FIG. 3 is a schematic diagram showing the structure of pAdeA-pAmyB-nef4-tAmyB-pAmyB-nef6-tAmyB-pAmyB-nef5-tAmyB recombinant vector described in example 1;

FIG. 4 is a schematic diagram of the structure of the pBARI-pAmyB-nef3-tAmyB-pAmyB-nef7-tAmyB recombinant vector described in example 1;

FIG. 5 is a schematic diagram showing the structure of pUNA-pAmyB-nef8-tAmyB-pAmyB-nef9-tAmyB recombinant vector described in example 1;

FIG. 6 is a graph of UV peaks at Aspergillus oryzae nef1-9 in example 2;

wherein 1 is the pyripyropene A compound peak.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

Term interpretation:

PCM medium: the culture medium for preparing the pyripyropene A by fermenting the aspergillus oryzae for heterologously expressing the pyripyropene A gene, namely the culture medium used by fermenting engineering bacteria A.oryzae-nef1-9, is called PCM culture medium for short, the description is adopted in the document of the application, and the components and the dosage of the culture medium are shown as a third aspect of the application, and are not necessarily related to the PCM culture medium in the market products.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.

EXAMPLE 1 construction of engineering Strain A. Oryzae-nef1-9

1. Extraction of genome of the strain Fischer-Tropsch sarium (Neosartorya fischeri) DSM 3700

The Neosartorya fischeri DSM 3700 strain was inoculated into PDA solid plates from-80℃and cultured at 30℃for 3 days, and the scraped cells were inoculated into a 50mL/250mL Erlenmeyer flask in YPD (10 g yeast extract, 10g polypeptone, 10g casein hydrolysate, 20g sucrose, 1000mL water added and sterilized at high temperature) liquid medium and cultured at 30℃for 2 days at 150rpm to give cells in a pellet shape. The bacterial cells were dried by suction with filter paper, sub-packed in 1.8mL to 2mL centrifuge tubes, immersed in liquid nitrogen, then ground with a grinding rod, and after grinding, 500. Mu.L of lysate (400 mM Tris-HCl buffer pH8.0, 60mM EDTA pH8.0, 150mM sodium chloride and 1% sodium dodecyl sulfate) was added to each tube, mixed with 150. Mu.L of 3M sodium acetate in a water bath at 65℃for 20 minutes, centrifuged at 12000rpm for 10 minutes, 500. Mu.L of supernatant was placed in a new 1.5M centrifuge tube, mixed with 500. Mu.L of isopropanol, centrifuged at 12000rpm for 5 minutes, the supernatant was removed, washed with 1mL of 70% ethanol, sucked out of ethanol, dried naturally, and dissolved in 200. Mu.L of water.

2. Construction of recombinant vectors

The E.coli strain containing pTAex3 was inoculated from a glycerol tube deposited at-80℃onto LB plates containing 100. Mu.g/mL ampicillin, three-fold streaked, and inverted and cultured overnight in an incubator at 37 ℃. Single colonies were picked using a 2mL ep tube into 1.8mL liquid LB medium containing ampicillin, and incubated at 950rpm for about 8h at 37 ℃. The centrifuge tube was then removed and centrifuged at 12000rpm for 1min. The LB medium was removed from the supernatant, 200. Mu.L of buffer P1 (RNase was added in advance to a final concentration of 0.1 mg/mL) was added, and the mixture was placed on a shaker at 1300rpm for 10 minutes. 200. Mu.L of buffer P2 was added. 1300rpm for 5 seconds, left to stand for 5 seconds, and repeated 3 times. 200. Mu.L of buffer P3 was added thereto, and the mixture was shaken at 1300rpm for 5 seconds, allowed to stand for 5 seconds, and then repeated 3 times, followed by mixing upside down. Centrifuge at 14000rpm for 20 minutes and take the supernatant into a clean 1.5mL ep tube. 600. Mu.L of isopropanol was added to the tube, mixed upside down, and centrifuged at 14000rpm for 20 minutes. The supernatant was discarded, the interior of the ep tube wall was wiped dry with absorbent paper and washed once with 1mL of 70% absolute ethanol. The plasmid concentration was determined by pouring out 70% absolute ethanol, inverting and air-drying, then adding 25. Mu.L of sterile deionized water, and after complete dissolution. The pTAex3 plasmid was digested with KpnI endonuclease, and the specific reaction system is shown in Table 1:

TABLE 1 Endonuclease cleavage System

The enzyme digestion system is placed in a 37 ℃ incubator and kept stand for 2 hours. To this system, 20. Mu.L of 3M sodium acetate solution was added and thoroughly mixed. 400. Mu.L of pre-cooled absolute ethyl alcohol is added, and the mixture is placed in a refrigerator at the temperature of minus 20 ℃ for 20 minutes after being fully and evenly mixed. Centrifuge at 14000rpm for 10 minutes, discard supernatant and wash once with 1mL of 70% absolute ethanol. The concentration of the linearized fragment was determined after pouring out 70% absolute ethanol, inverting and air-drying, then adding 20. Mu.L of sterile deionized water, and fully dissolving. And then placed in a 4 c refrigerator for later use.

According to the sequencing result of the pyripyropene synthetic gene cluster in the Neosartorya fischeri DSM 3700 strain, the primer 1F and the primer 1R are designed. The nef2 gene in the genome is amplified, two ends of the primer are respectively provided with homologous arms of 20bp pTAex3, and the specific reaction system is shown in Table 2:

TABLE 2PCR amplified Gene System

PCR procedure: pre-denaturing for 2min at 98 ℃ and denaturing for 10s at 98 ℃; annealing at 55 ℃ for 15s; extending at 72 ℃ for 1min;34 cycles; extending at 72℃for 10min.

And (3) performing agarose gel electrophoresis on the PCR product, then cutting the glue at the position of the target strip, and recovering the target strip by using a glue recovery kit of the root organism. At the same time, 1.8mL of E.coli GB2005 (hereinafter referred to as GB 05) containing liquid LB medium was cultured overnight at 37℃in 2mL of ep tube on the previous day, 30. Mu.L was transferred to 1.5mL of ep tube containing 1.3mL of liquid LB medium, and the culture was performed at 37℃for 2 hours. The pTAex3 plasmid was digested with KpnI, and the digested product was recovered by ethanol precipitation (200. Mu.L of 3M sodium acetate and 500. Mu.L of pre-chilled absolute ethanol were added to the digested system, the mixture was allowed to stand in a refrigerator at-20℃for 10min, centrifuged at 12000rpm for 1min, the supernatant was poured, and 1mL of 70% absolute ethanol was added to wash twice, and dried, and 20. Mu.L of sterile water was added) to prepare a linearized pTAex3 fragment. The recovered product was assembled with a linearized fragment of pTAex3 by Gibson, and the specific reaction system is shown in table 3:

TABLE 3Gibson Assembly System

PCR procedure: the reaction was carried out at 55℃for 30min.

The reaction product was subjected to a desalting treatment using a desalting membrane for 40 minutes. Centrifuging the transferred and grown GB05 at 10000rpm for 1min, pouring out the supernatant, washing twice with sterile water, washing off culture medium components, then supplementing sterile water to 30 mu L, adding all reaction products after desalting, mixing uniformly, and placing into an electric rotating cup for 1350V electric shock. The cells in the electrorotor were resuspended in 1mL of antibiotic-free liquid LB and transferred to a 1.5mL ep tube at 950rpm for 1h at 37 ℃. The bacterial solution was centrifuged at 10000rpm for 1min, the supernatant was removed, a volume of 30. Mu.L was left, and the solution was spread on LB solid plates containing ampicillin resistance, and incubated overnight at 37 ℃. The monoclonal was picked up and inoculated into 1.8mL of ampicillin-containing LB liquid medium, and cultured at 950rpm and 37℃for 8 hours, followed by extraction of the plasmid in the same manner as in the above step. Enzyme digestion verification is carried out by using corresponding restriction enzymes, and the reaction system is shown in table 4:

TABLE 4 Endonuclease cleavage System

The reaction system was placed in a constant temperature incubator at 37℃for 2 hours, and then 5. Mu.L of the reaction product was subjected to agarose gel electrophoresis. The plasmid was extracted from the corresponding colony with the correct electrophoresis band to obtain pTAex3-nef2 plasmid. Stored in a refrigerator at 4 ℃ for standby.

The E.coli strain containing pUSA was subjected to strain activation by the method for obtaining pTAex3 plasmid as described above, to extract plasmids, and the pUSA plasmid was digested with KpnI endonuclease was selected as well. According to the sequencing result of the pyripyropene synthetic gene cluster in the Neosartorya fischeri DSM 3700 strain, a primer 2F and a primer 2R are designed, the nef1 gene in the genome is amplified, and two ends of the primer are respectively provided with homologous arms of pUSA of 20 bp. The pUSA-nef1 recombinant plasmid was constructed by the same method as described above for the construction of pTAex3-nef2 plasmid.

E.coli strains containing pAdeA were subjected to strain activation and plasmid extraction by the same method as described above for pTAex3 plasmid, except that the pAdeA plasmid was digested with SpeI endonuclease. According to the sequencing result of pyripyropene synthetic gene cluster in Neosartorya fischeri DSM 3700 strain, designing a primer 3F, a primer 3R, a primer 4F, a primer 4R, a primer 5F and a primer 5R, respectively amplifying nef4, nef5 and nef6 genes in the genome, wherein two ends of the primer are respectively provided with homology arms of 20bp pTAex 3. pTAex3-nef4, pTAex3-nef5 and pTAex3-nef6 recombinant plasmids were constructed using previously linearized pTAex3, respectively, by the method for constructing pTAex3-nef2 plasmid as described above. Because the pTAex3 plasmid contains an amyB promoter and terminator sequence and a KpnI cleavage site is arranged between the two sequences, a primer 6F, a primer 6R, a primer 7F, a primer 7R, a primer 8F and a primer 8R can be designed, and two ends of the primer are provided with 20bp homologous arms which are mutually overlapped, so that the three gene fragments can be connected with a linearization vector together. The amyB promoter and terminator and the gene in the middle are amplified together by PCR to obtain three fragments containing amyB promoter and terminator and nef4, nef6 and nef5 genes respectively, and the two ends of the fragments are respectively provided with 20bp homologous arms which are mutually overlapped. The three gene fragments and the linearized pAdeA fragment are connected through Gbison to construct pAdeA-PAmyB-nef4-TAmyB-PAmyB-nef6-TAmyB-PAmyB-nef5-TAmyB recombinant plasmid.

E.coli strains containing pBARI were subjected to strain activation and plasmid extraction by the same method as described above for pTAex3 plasmid, except that the plasmid pBARI was digested with SacI endonuclease. Placing in a refrigerator at 4 ℃ for standby. According to the sequencing result of the pyripyropene synthetic gene cluster in the Neosartorya fischeri DSM 3700 strain, a primer 9F, a primer 9R, a primer 10F and a primer 10R are designed to amplify nef3 and nef7 genes in the genome respectively, and the two ends of the primer are respectively provided with homologous arms of 20bp pTAex 3. pTAex3-nef3 and pTAex3-nef7 recombinant plasmids were constructed by the same method as described above for constructing pTAex3-nef 2. The primer 11F, the primer 11R, the primer 12F and the primer 12R are designed, and two ends of the primer are provided with 20bp homologous arms which are mutually overlapped, so that two gene fragments can be connected together with a linearization vector. PCR amplification gives two fragments containing the amyB promoter and terminator and containing nef3 and nef7 genes respectively, and the two ends of the fragments are provided with 20bp homologous arms which are mutually overlapped. The two gene fragments are connected with the pBARI fragment of the enzyme tangentially through Gbison to construct the pBARI-PAmyB-nef3-TAmyB-PAmyB-nef7-TAmyB recombinant plasmid.

The pUNA-containing E.coli strain was subjected to strain activation by the method of obtaining pTAex3 plasmid as described above, followed by plasmid extraction, and the pUNA plasmid was digested with SpeI endonuclease. Placing in a refrigerator at 4 ℃ for standby. According to the sequencing result of the pyripyropene synthetic gene cluster in the Neosartorya fischeri DSM 3700 strain, a primer 13F, a primer 13R, a primer 14F and a primer 14R are designed to amplify nef8 and nef9 genes in the genome respectively, and the two ends of the primer are respectively provided with homologous arms of 20bp pTAex 3. pTAex3-nef8 and pTAex3-nef9 recombinant plasmids were constructed using pTAex3 linearized previously by the same method as described above for constructing pTAex3-nef 2. The primer 15F, the primer 15R, the primer 16F and the primer 16R are designed, and two ends of the primer are provided with 20bp homologous arms which are mutually overlapped, so that two gene fragments can be connected together with a linearization vector. PCR amplification gives two fragments containing the amyB promoter and terminator and containing nef8 and nef9 genes respectively, and the two ends of the fragments are provided with 20bp homologous arms which are mutually overlapped. The two gene fragments are connected with the pUNA fragment which is tangential to the enzyme through Gbison to construct recombinant plasmid pUNA-PAmyB-nef8-TAmyB-PAmyB-nef9-TAmyB.

The primers and plasmids involved in the above construction are shown in Table 1 below:

TABLE 1 oligonucleotide sequences used in engineering bacteria construction

3. Construction of engineering Strain

PEG-mediated protoplast transformation was used to construct the heterologously expressed engineered strain. Wild type A.oryzae (Aspergillus oryzae) NSAR1 was first activated in YPD plates and this strain was given benefit from the teachings of K.Gomi, japan, a strain commonly studied in the art. The activated aspergillus oryzae is inoculated into 200mL/500mL of YPD liquid culture medium, cultured at 30 ℃ at 125rpm overnight, then kept still for precipitation, the supernatant is poured off, the thallus is broken up by using a breaking cup, and the thallus after treatment is placed into a 50mL centrifuge tube and centrifuged at 3950rpm for 25min. An enzyme solution (lying Enzymes 20mg/mL, drisease 5mg/mL, yatalase 5mg/mL, dissolved using MN buffer, and sterilized by filtration) 25mL was prepared. Preparation of MN buffer: 7.849g of magnesium sulfate, 1.752g of sodium chloride, and water to a volume of 100mL. After centrifugation, the supernatant was removed, and the bacterial pellet was put into an enzyme solution and lysed at 30℃and 125rpm for 2 hours. The lysed protoplasts were then filtered into 15mL tubules using sterile gauze, 1850g, centrifuged for 11min, the supernatant removed, the pellet was taken, 5mL washed with STC buffer (21.86 g sorbitol, 0.55g calcium chloride, 802. Mu.l 1M Tris-HCl, 196. Mu.l 1MTris fixed to 100mL using double distilled water), 1850g, centrifuged for 11min, the pellet was taken, 800. Mu.l STC was added, blown down evenly, split into 1.5mL centrifuge tubes, 100. Mu.l per tube, 20. Mu.l pTAex3-nef2 recombinant plasmid (500 ng/mL) was added per tube, 50. Mu.l PEG3350 (4.478 g potassium chloride, 0.55g calcium chloride, 1mL 1M Tris-HCl,25g PEG3350, fixed to 100 mL), blown down evenly, 25min ice-bath, 1mL PEG3350 was added per tube, blowing uniformly, standing at room temperature for 25min, pouring the mixture into a MM screening culture medium (2 g of ammonium chloride, 1g of ammonium sulfate, 0.5g of potassium chloride, 0.5g of sodium chloride, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 0.02g of ferrous sulfate, 20g of glucose, 0.1g of adenine, 1.5g of methionine, 1L of double distilled water to a constant volume, adding 58.28g of mannitol into 400mL of MM liquid component, culturing overnight, pouring the mixture into a MM solid screening plate (adding 3% of agar into MM liquid culture medium component), standing at 30 ℃ for 3d, picking up a monoclonal to extract genome, carrying out sequencing verification on a target band of PCR amplification, and selecting a correct strain for subsequent transformation.

Then, pUSA-nef1 recombinant plasmid was transformed into A.oryzae-nef2 strain using the same method as the above-described transformation of pTAex3-nef2 recombinant plasmid, and positive selection was performed using MM selection medium without methionine. Selecting a monoclonal to extract a genome, amplifying a target gene by PCR, carrying out sequencing verification on a target strip, selecting a correct strain for subsequent transformation, and naming the strain as A.oryzae-nef2-nef1.

Similarly, pAdeA-pAmyB-nef4-tAmyB-pAmyB-nef6-tAmyB-pAmyB-nef5-tAmyB recombinant plasmid is transferred into an A.oryzae-nef2-nef1 strain to obtain an A.oryzae-nef2-nef1-nef4-nef6-nef5 strain; continuing to transfer into pBARI-pAmyB-nef3-tAmyB-pAmyB-nef7-tAmyB recombinant plasmid to obtain an A.oryzae-nef2-nef1-nef4-nef6-nef5-nef3-nef7 strain; continuing to transfer into

pUNA-pAmyB-nef8-tAmyB-pAmyB-nef9-tAmyB recombinant plasmid was positively selected using MM selection medium without methionine and adenine, with ammonium chloride and ammonium sulfate replaced with sodium nitrate components. And (3) selecting a monoclonal to extract a genome, amplifying a target gene by PCR, and carrying out sequencing verification on a target strip, wherein a target strip-containing strain is the engineering strain A.oryzae-nef1-9. The AmyB promoter and terminator sequences used in the application exist in the pTAex3 vector, and the sequences are respectively shown as SEQ ID No.10 and SEQ ID No. 11.

Example 2 engineering bacteria fermentation Process

In this example, the yield of the engineering strain A. Oryzae-nef1-9 under different fermentation conditions was measured as follows:

quantitative analysis is carried out on the sample by using a pyripyropene A standard substance through a high performance liquid chromatograph, and the detection conditions are as follows: the chromatographic column is Agilent ZPRBAX SB-C ₁₈ (9.5X105 nm,5 μm), mobile phase A is deionized water, B is acetonitrile, sample injection amount is 10 μL, flow rate is 2mL/min, ultraviolet absorption wavelength is 310nm, elution procedure is 0-25min 20-100% B,25.01-27min 100% B, and 27.01-30min 20% B. The peak time of pyripyropene A was 17min. And calculating the yield of the pyripyropene A according to the concentration relation curve of the absorption peak area and the pyripyropene A.

The PCM culture medium optimized in this example comprises the following components: sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.15%, yeast extract 0.3%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.002%, calcium chloride 0.04%, sucrose 2%, maltose 2%, glucose 4%, potato extract 1%, nicotinic acid 0.2%, pH 7.0, 125rpm, 28 ℃ for 4 days.

And (3) fermenting by using a PCM fermentation medium, and activating the engineering strain A.oryzae-nef1-9 onto a new DPY solid plate, wherein the culture temperature is 28 ℃ and the culture is 2d. The small pieces were picked and transferred to 250mL shake flasks containing 50mL of liquid PCM fermentation medium. Fermenting at 28 deg.C at 125rpm for 4 days. The shake flask was taken out, an equal volume of ethyl acetate was added thereto for extraction, and the extraction was repeated 3 times using a separating funnel. The extracted ethyl acetate was evaporated to dryness using a rotary evaporator, and the sample was then dissolved using 1mL of chromatographic grade methanol. The dissolved sample was centrifuged at 14000rpm for 5min, the supernatant was removed of insoluble impurities, the sample was then filtered using a 0.22 μm filter, and the filtered sample was placed in a 1.5mL liquid vial, and the results of three sets of experiments were averaged. The yield of pyripyropene A was found to be 332.9mg/L.

Example 3

In this example, a fermentation culture method of an engineering strain A. Oryzae-nef1-9 is provided, which is different from example 2 in that the components and contents of the culture medium are as follows: sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, yeast extract 0.1%, magnesium sulfate 0.04%, potassium chloride 0.04%, ferrous sulfate 0.001%, calcium chloride 0.03%, sucrose 1%, maltose 1%, glucose 1%, potato extract 0.5%, nicotinic acid 0.1%, pH 6.0, 100rpm, and fermentation at 26℃for 3 days.

The PCM medium was used for fermentation, which was different from example 2 in that the engineering strain a. Oryzae-nef1-9 was activated onto a new DPY solid plate, and cultured at 26 ℃ for 2d; after transfer to shake flask, fermentation conditions: fermentation was carried out at 100rpm at 26℃for 3 days, and the other settings were the same as in example 2. The yield of pyripyropene A was found to be 137.0mg/L.

Example 4

In this example, a fermentation culture method of an engineering strain A. Oryzae-nef1-9 is provided, which is different from example 2 in that the components and contents of the culture medium are as follows: sodium nitrate 0.6%, dipotassium hydrogen phosphate 0.3%, yeast extract 0.5%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.005%, calcium chloride 0.06%, sucrose 3%, maltose 3%, glucose 5%, potato extract 2%, nicotinic acid 0.3%, pH7.5, 200rpm, and fermentation at 26℃for 5 days.

The PCM medium was used for fermentation, which was different from example 2 in that the engineering strain a. Oryzae-nef1-9 was activated onto a new DPY solid plate, and cultured at 26 ℃ for 2d; after transfer to shake flask, fermentation conditions: the culture was carried out at 26℃and 200rpm for 5 days, and the other conditions were the same as in example 2. The yield of pyripyropene A was 253.5mg/L.

Comparative example 1

In this example, engineering bacteria were fermented using a common medium for Aspergillus oryzae-a Morse medium.

The components of the culture medium are as follows: maltose 2%, sucrose 3%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.132%, yeast extract 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate heptahydrate 0.001%, and calcium chloride 0.055%.

Fermentation was performed using a pergola fermentation medium and the engineering strain a. Oryzae-nef1-9 was activated onto a new DPY solid plate. Culturing at 30 ℃ for 2d. The small pieces were picked and transferred to 250mL shake flasks containing 50mL of liquid kohlrabi fermentation medium. Culturing at 30℃and 220rpm for 5d. The rest of the arrangement is the same as that of the example 2, the samples are quantitatively analyzed by a high performance liquid chromatograph by using a pyripyropene A standard substance, and the results of the three groups of experiments are averaged. The yield of pyripyropene A was 1.6mg/L.

Comparative example 2

In this example, the original strain Neosartorya fischeri DSM 3700 was fermented using a common pergola medium.

The components of the culture medium are as follows: maltose 2%, sucrose 3%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.132%, yeast extract 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate heptahydrate 0.001%, and calcium chloride 0.055%. Original strain Neosartorya fischeri DSM 3700 was inoculated to a fermentation medium for fermentation, and the rest was set as in comparative example 1. The yield of pyripyropene A was detected to be 0.2mg/L.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. The engineering bacterium for high expression of pyripyropene A is characterized in that an original strain of the engineering bacterium is aspergillus oryzae, and compared with the original strain, the engineering bacterium is modified to have pyripyropene A biosynthesis genes nef1-nef9:

(1) nef1 is coenzyme A ligase gene, and the nucleotide sequence is shown as SEQ ID No. 1;

(2) nef2 is polyketide synthase gene, and the nucleotide sequence is shown as SEQ ID No. 2;

(3) nef3 is a cytochrome P450 enzyme gene, and the nucleotide sequence is shown as SEQ ID No. 3;

(4) nef4 is a cyclase gene, and the nucleotide sequence is shown as SEQ ID No. 4;

(5) nef5 is a 5FAD dependent monooxygenase gene, and the nucleotide sequence is shown as SEQ ID No. 5;

(6) nef6 is an isopentenyl transferase gene, and the nucleotide sequence is shown as SEQ ID No. 6;

(7) nef7 is an acetyltransferase gene, and the nucleotide sequence is shown as SEQ ID No. 7;

(8) nef8 is an acetyltransferase gene, and the nucleotide sequence is shown as SEQ ID No. 8;

(9) nef9 is cytochrome P450 enzyme gene, and the nucleotide sequence is shown as SEQ ID No. 9.

2. The engineered bacterium that highly expresses pyripyropene a of claim 1, wherein the starting strain is aspergillus oryzae (Aspergillus oryzae) NSAR1.

3. The engineered bacterium that highly expresses pyripyropene a according to claim 1, wherein said pyripyropene a biosynthesis genes nef1-nef9 are derived from fischer-tropsch bacteria (Neosartorya fischeri) DSM 3700.

4. The method for constructing the engineering bacteria with high pyripyropene A expression as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:

the nef1-nef9 genes were amplified by addition of primers, homology arms were introduced at both ends of each gene, and then the genes were sequentially ligated to the vector using Gibson assembly.

5. The method for constructing engineering bacteria according to claim 4, wherein the connection mode is as follows:

the five recombinant plasmids are sequentially introduced into an original strain by adopting a protoplast transformation method, positive clones are screened by an MM screening plate, and an engineering strain is obtained.

6. A fermentation medium for engineering bacteria highly expressing pyripyropene a according to any one of claims 1 to 3, wherein the components and contents of the medium are as follows:

7. The fermentation medium of engineering bacteria highly expressing pyripyropene A according to claim 6, wherein the contents of the components in the medium are as follows: sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.15%, yeast extract 0.3%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.002%, calcium chloride 0.04%, sucrose 2%, maltose 2%, glucose 4%, potato extract 1%, nicotinic acid 0.2%, pH 7.0, and water in balance;

or the content of each component in the culture medium is as follows: sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, yeast extract 0.1%, magnesium sulfate 0.04%, potassium chloride 0.04%, ferrous sulfate 0.001%, calcium chloride 0.03%, sucrose 1%, maltose 1%, glucose 1%, potato extract 0.5%, nicotinic acid 0.1%, pH 6.0, and water in balance;

or the content of each component in the culture medium is as follows: sodium nitrate 0.6%, dipotassium hydrogen phosphate 0.3%, yeast extract 0.5%, magnesium sulfate 0.1%, potassium chloride 0.1%, ferrous sulfate 0.005%, calcium chloride 0.06%, sucrose 3%, maltose 3%, glucose 5%, potato extract 2%, nicotinic acid 0.3%, pH7.5, and water in balance.

8. A process for the biosynthesis of pyripyropene A, characterized in that it comprises shaking the flask with the fermentation medium according to claim 5 or 6 at 100-200rpm and 25-28℃for 3-5 days.

9. The method for biosynthesis of pyripyropene A according to claim 8, comprising the following steps:

10. The method for the biosynthesis of pyripyropene a according to claim 9, wherein the ethyl acetate-dried product is further provided with a corresponding purification process.