阅读说明：本技术 一种产莫纳可林j的曲霉菌及其构建方法与应用 (Monascus for producing monacolin J and construction method and application thereof ) 是由 吕雪峰 黄雪年 杨勇 郑玲辉 滕云 于 2018-08-24 设计创作，主要内容包括：本发明公开了一种产莫纳可林J的曲霉菌及其构建方法与应用。本发明公开的一种产莫纳可林J的曲霉菌的构建方法,包括在曲霉菌中完全阻断聚酮合酶LovF表达的步骤。本发明通过完全敲除lovF基因阻断洛伐他汀合成而实现莫纳可林J的积累,再在此基础上用组成型启动子过表达转录调控因子lovE,使得构建得到的菌株的莫纳可林J产量进一步显著提高,并增强了该菌株的发酵稳定性。可以利用本发明的菌株直接发酵生产莫纳可林J,从而简化辛伐他汀的生产工艺,降低成本,提高生产效率,减少环境污染。(The invention discloses an aspergillus for producing monacolin J and a construction method and application thereof. The invention discloses a construction method of Monacolin J-producing aspergillus, which comprises the step of completely blocking polyketide synthase LovF expression in the aspergillus. According to the invention, the accumulation of the monacolin J is realized by completely knocking out the lovF gene to block the synthesis of lovastatin, and then the constitutive promoter is used for over-expressing the transcription regulation factor lovE on the basis, so that the yield of the monacolin J of the constructed strain is further obviously improved, and the fermentation stability of the strain is enhanced. The strain can be used for directly fermenting and producing monacolin J, so that the production process of simvastatin is simplified, the cost is reduced, the production efficiency is improved, and the environmental pollution is reduced.)

1. A method of constructing Monacolin J-producing Aspergillus comprising the step of completely blocking the expression of the polyketide synthase LovF in Aspergillus.

2. The method of claim 1, wherein: the method for completely blocking the expression of the polyketide synthase LovF is to knock out all the lovF gene copies of the aspergillus;

the Aspergillus is Aspergillus HZ01(Aspergillus sp.HZ01), which is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 10 and 17 days, and the preservation number is CGMCC No. 12970.

3. The method of claim 2, wherein: the method for knocking out the lovF gene copy is a double exchange method for lovF gene homologous recombination.

4. A method according to any one of claims 1-3, characterized in that: the method for homologous recombination and double exchange of the lovF gene comprises the following steps:

(1) knock out ku80 gene in the aspergillus HZ01, construct aspergillus HZ02(HZ01, delta ku80:: ptrA) with efficient gene targeting, further knock out pyrG gene of the aspergillus HZ02, and construct a Chassis cell strain aspergillus HZ03(HZ01, delta ku80:: ptrA, delta pyrG) which can be subjected to genetic transformation screening based on uracil auxotrophy;

(2) adding a lovF gene targeting element lovF-KO1 into the protoplast suspension of the aspergillus HZ03 to knock out the first lovF gene copy to obtain an engineering strain HZ-delta lovF 1;

preferably, the lovF-KO1 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of the lovF-KO1 is shown in SEQ ID No. 10;

further, pyrG of the engineered strain HZ-. DELTA.lovF 1 was used in this step_AnRemoving the screening marker to obtain an engineering strain HZ-delta lovF 1-delta pyrGAN;

preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO1.5 into the protoplast suspension of the engineering strain HZ-delta lovF 1; further preferably, the sequence of said pyrGAn-KO1.5 is as shown in SEQ ID No. 12;

(3) adding a lovF gene targeting element lovF-KO2 into the protoplast suspension of the engineering strain HZ-delta lovF 1-delta pyrGAN to knock out a second lovF gene copy to obtain an engineering strain HZ-delta lovF 2;

preferably, the lovF-KO2 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of the lovF-KO2 is shown in SEQ ID No. 19;

further, pyrG of the engineered strain HZ-. DELTA.lovF 2 was used in this step_AnRemoving the screening marker to obtain an engineering strain HZ-delta lovF 2-delta pyrGAN;

preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO2.5 into the protoplast suspension of the engineering strain HZ-delta lovF 2; further preferably, the sequence of said pyrGAn-KO2.5 is as shown in SEQ ID No. 21;

(4) adding a lovF gene targeting element lovF-KO3 into the protoplast suspension of the engineering strain HZ-delta lovF 2-delta pyrGAN to knock out a third lovF gene copy to obtain an engineering strain HZ-delta lovF 3;

preferably, the lovF-KO3 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of said lovF-KO3 is shown in SEQ ID No. 28.

5. The method according to any one of claims 1-4, wherein: the method further comprises the step of overexpressing the transcriptional regulator lovE.

6. The method of claim 5, wherein: the method for over-expressing the transcription regulatory factor lovE is a homologous recombination double exchange method for over-expressing the lovE at a fixed point.

7. The method of claim 6, wherein: the site-directed overexpression lovE is the site-directed overexpression of the transcription regulating factor lovE by using a constitutive promoter PgpdAt;

preferably, the site-directed overexpression lovE is the lovE expression element which takes a constitutive promoter PgpdAt as a promoter and is site-directed integrated at the ku80 site of the engineered strain HZ-delta lovF 3-delta pyrGAN, and finally the engineered strain HZ-delta lovF3-lovE is obtained;

the engineering strain HZ-delta lovF 3-delta pyrGAN is the engineering strain HZ-delta lovF3 excision pyrG_AnObtaining a screening marker; preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO3.5 into the protoplast suspension of the engineering strain HZ-delta lovF 3; further preferably, the sequence of said pyrGAn-KO3.5 is as shown in SEQ ID No. 30.

8. An Aspergillus constructed by the method of any one of claims 1-7;

preferably, the Aspergillus is the HZ- Δ lovF3 or HZ- Δ lovF3-lovE or HZ- Δ lovF3- Δ pyrGAN.

9. A process for preparing monacolin J comprising fermenting the aspergillus of claim 8.

10. Use of an Aspergillus and/or a bacterial suspension thereof and/or a fermentation broth thereof and/or a metabolite thereof according to claim 8 for the preparation of monacolin J.

Technical Field

The invention belongs to the technical field of biological pharmacy, and relates to an aspergillus for producing monacolin J, a construction method and application thereof; still further, the present invention relates to the production of monacolin J.

Background

Cardiovascular and cerebrovascular diseases seriously threaten human health, and the morbidity and mortality of the cardiovascular and cerebrovascular diseases are ranked first in many countries and regions. Hyperlipidemia (hypercholesterolemia) is an important cause of cardiovascular and cerebrovascular diseases, so that the prevention of hyperlipidemia, the regulation of blood lipid and the reduction of blood lipid are the keys to the prevention and treatment of cardiovascular and cerebrovascular diseases. For the above reasons, cholesterol lowering drugs have a great market prospect, and have been in the front of global marketing drugs for many years.

Simvastatin (Simvastatin), known as sulbactam (Zocor), is an important hypolipidemic drug developed by Merck, can effectively inhibit the in vivo synthesis of cholesterol, and is one of the first-choice drugs for treating hyperlipidemia. With the expiration of 2006 patent, the market competition of simvastatin is increasingly intense, and the process technology research and the production cost of simvastatin become important factors of product competition. Simvastatin is not a natural compound, but is chemically synthesized from lovastatin (lovastatin), which is a fermentation product of aspergillus terreus. Simvastatin and lovastatin differ only in that the side chain has one more methyl group, simvastatin is a 2, 2-dimethylbutyric acid side chain, and lovastatin is a 2-methylbutyric acid side chain. In the synthesis process of simvastatin, the 2-methylbutyric acid side chain at C8 position of lovastatin needs to be removed to obtain monacolin J (Monacolin J), and then the 2, 2-dimethylbutyric acid side chain is added to C8 position of monacolin J through a subsequent process, so as to obtain simvastatin. It follows that monacolin J is an important precursor for the synthesis of simvastatin. At present, in the synthesis route of simvastatin, lovastatin is hydrolyzed by a chemical method to prepare monacolin J, a large amount of acid and alkali, methylene dichloride, toluene, diethyl ether and other organic reagents are used in the whole preparation process, and the method has the disadvantages of complex process, long time consumption, low yield and high pollution pressure.

Two polyketide synthases (PKS) are involved in the biosynthesis process of lovastatin, wherein the first polyketide synthase LovB synthesizes intermediate monacolin J under the assistance of other modification enzymes, and a 2-methylbutyrate side chain is synthesized by another polyketide synthase LovF and transferred to a hydroxyl group at C8 position of monacolin J under the action of acyltransferase LovD to form the final product lovastatin. It can be seen that LovF and LovD are two key enzymes for synthesis of lovastatin by monacolin J. Thus, it is theorized that knockdown of lovD and lovF can block lovastatin synthesis by monacolin J, thereby accumulating monacolin J. Genetic engineering is an effective genetic breeding technique. If genetic modification can be carried out on lovastatin-producing aspergillus terreus by a genetic engineering modification method so that the lovastatin-producing aspergillus terreus can be directly fermented to produce monacolin J, the synthesis process of simvastatin can be simplified, the production cost can be reduced, and meanwhile, the environmental pollution can be reduced, thereby improving the market competitiveness of products. However, the synthesis regulation mechanism of the secondary metabolite is very complex, blocking the synthesis pathway may cause significant influence on the whole synthesis pathway, and other upstream intermediates cannot be effectively accumulated while the final product disappears. Moreover, some intermediates are unstable and easy to degrade, or the downstream routes of the intermediates are not unique and may be intermediates of multiple synthetic pathways, so that the deletion of a certain downstream gene does not necessarily achieve the accumulation of the intermediate.

The synthesis of secondary metabolites is usually significantly interfered by environmental factors, and small differences of some unknown environmental factors may have a large influence on the synthesis of secondary metabolites. Lovastatin is a polyketone secondary metabolite, and the difference of the lovastatin yield among batches in the industrial production process is large, so that the difficulty in controlling the production process is increased, and the production efficiency is influenced. lovE is a transcriptional regulator gene located in the lovastatin-synthesizing gene cluster of Aspergillus terreus, which is capable of specifically regulating the transcriptional level of the lovastatin-synthesizing gene cluster, thereby controlling the synthesis of lovastatin.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the aspergillus capable of directly fermenting and producing monacolin J and the construction method and application thereof.

In order to solve the above technical problems, the present invention provides a method for constructing Monacolin J-producing Aspergillus comprising the step of completely blocking the expression of polyketide synthase LovF in Aspergillus.

In the above method, the method for completely blocking the expression of the polyketide synthase LovF is to knock out all the lovF gene copies of the Aspergillus;

the Aspergillus is Aspergillus HZ01(Aspergillus sp.HZ01), which is preserved in China general microbiological culture Collection center (CGMCC) (address: No.3 of Xilu 1 of Beijing Kogyo-sunny district), the preservation number is CGMCC No.12970, and the Aspergillus is a strain of Aspergillus terreus (Aspergillus terreus) in 2016, 10.17.2016, and the classification name is Aspergillus sp.

In the method, the method for knocking out the lovF gene copy is a double exchange method for lovF gene homologous recombination.

In any of the above methods, the double crossover method for homologous recombination of the lovF gene comprises the following steps:

(1) knock out ku80 gene in the aspergillus HZ01, construct aspergillus HZ02(HZ01, delta ku80:: ptrA) with efficient gene targeting, further knock out pyrG gene of the aspergillus HZ02, and construct a Chassis cell strain aspergillus HZ03(HZ01, delta ku80:: ptrA, delta pyrG) which can be subjected to genetic transformation screening based on uracil auxotrophy;

preferably, the construction of the aspergillus HZ03 comprises the following steps: with reference to example 2 of the invention patent application having publication No. CN104894165A entitled "method and application of Gene targeting technique to Aspergillus terreus" treating protoplast suspension of Aspergillus HZ01 to knock out ku80 of HZ01 and construct Aspergillus terreus HZ02(HZ01, Δ ku80:: ptrA) having high efficiency of Gene targeting, and with further reference to example 5 of the patent application knocking out pyrG gene, an underplate cell strain Aspergillus HZ03(HZ01, Δ ku80: ptrA, Δ pyrG) that can be screened for genetic transformation based on uracil auxotrophy was constructed, which was completely identical to the procedure in the example of patent application CN104894165A except that the host bacterium was replaced with HZ01 from Aspergillus terreus CICC 40205;

(2) adding a lovF gene targeting element lovF-KO1 into the protoplast suspension of the aspergillus HZ03 to knock out the first lovF gene copy to obtain an engineering strain HZ-delta lovF 1;

preferably, the lovF-KO1 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of the lovF-KO1 is shown in SEQ ID No. 10;

further, pyrG of the engineered strain HZ-. DELTA.lovF 1 was used in this step_AnRemoving the screening marker to obtain an engineering strain HZ-delta lovF 1-delta pyrGAN;

preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO1.5 into the protoplast suspension of the engineering strain HZ-delta lovF 1; further preferably, the sequence of said pyrGAn-KO1.5 is as shown in SEQ ID No. 12;

(3) adding a lovF gene targeting element lovF-KO2 into the protoplast suspension of the engineering strain HZ-delta lovF 1-delta pyrGAN to knock out a second lovF gene copy to obtain an engineering strain HZ-delta lovF 2;

preferably, the lovF-KO2 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of the lovF-KO2 is shown in SEQ ID No. 19;

further, pyrG of the engineered strain HZ-. DELTA.lovF 2 was used in this step_AnRemoving the screening marker to obtain an engineering strain HZ-delta lovF 2-delta pyrGAN;

preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO2.5 into the protoplast suspension of the engineering strain HZ-delta lovF 2; further preferably, the sequence of said pyrGAn-KO2.5 is as shown in SEQ ID No. 21;

(4) adding a lovF gene targeting element lovF-KO3 into the protoplast suspension of the engineering strain HZ-delta lovF 2-delta pyrGAN to knock out a third lovF gene copy to obtain an engineering strain HZ-delta lovF 3;

preferably, the lovF-KO3 comprises, in order from 5 'to 3', the upstream homology arm of the lovF gene, pyrG_AnA selection marker, the downstream homology arm of the lovF gene; further preferably, the sequence of said lovF-KO3 is shown in SEQ ID No. 28.

In any of the methods described above, the method further comprises the step of overexpressing the transcriptional regulator lovE.

In the above method, the method for overexpressing the transcriptional regulatory factor lovE is a homologous recombination double-crossover method to overexpress lovE at a fixed point.

In the method, the site-directed overexpression lovE is the site-directed overexpression of the transcription regulatory factor lovE by using a constitutive promoter PgpdAt;

preferably, the site-directed overexpression lovE is the lovE expression element which takes a constitutive promoter PgpdAt as a promoter and is site-directed integrated at the ku80 site of the engineered strain HZ-delta lovF 3-delta pyrGAN, and finally the engineered strain HZ-delta lovF3-lovE is obtained;

the engineering strain HZ-delta lovF 3-delta pyrGAN is an engineering strain HZ-delta lovF3 excision pyrG_AnObtaining a screening marker; preferably, said pyrG_AnThe selection marker excision is obtained by adding a selection marker excision element pyrGAn-KO3.5 into the protoplast suspension of the engineering strain HZ-delta lovF 3; further preferably, the sequence of said pyrGAn-KO3.5 is as shown in SEQ ID No. 30.

In the above method, the site-specific integration of the lovE expression element with constitutive promoter PgpdAt as promoter at ku80 site comprises the following steps: adding a targeting element Uku80-pyrGAN-PgpdAt-lovE-TtrpC-Dku80 for over-expressing lovE into a protoplast suspension of the engineered strain HZ- Δ lovF3- Δ pyrGAN, wherein the sequence of the Uku80-pyrGAN-PgpdAt-lovE-TtrpC-Dku80 is shown as SEQ ID No. 42.

In order to solve the above technical problems, the present invention also provides an Aspergillus constructed by the method of any one of the above, preferably the Aspergillus is the HZ- Δ lovF3 or HZ- Δ lovF3-lovE or HZ- Δ lovF3- Δ pyrGAN;

the HZ-delta lovF3 is obtained by completely knocking out and constructing a lovF gene according to the method;

the HZ-delta lovF3-lovE is constructed by completely knocking out lovF gene according to the method and then overexpressing a transcription regulatory factor lovE according to the method;

the HZ-DeltalovF 3-DeltapyrGAN was obtained by excising pyrG from the engineered strain HZ-DeltalovF 3 as described above_AnScreening for markers.

In order to solve the above technical problems, the present invention also provides a method for preparing monacolin J comprising fermenting the above aspergillus.

In order to solve the technical problems, the invention also provides application of the aspergillus and/or bacterial suspension thereof and/or fermentation liquor thereof and/or metabolite thereof in preparation of monacolin J.

According to the invention, the accumulation of monacolin J is realized by completely knocking out the lovF gene of aspergillus to block the synthesis of lovastatin, and then the constitutive promoter is used for over-expressing the transcription regulation factor lovE on the basis, so that the yield of monacolin J of the constructed strain is further obviously improved, and the fermentation stability of the strain is enhanced. The strain can be used for directly fermenting and producing monacolin J, so that the production process of simvastatin is simplified, the cost is reduced, the production efficiency is improved, and the environmental pollution is reduced.

Drawings

FIG. 1 is a schematic diagram of the strategy for complete knock-out of the lovF gene.

FIG. 2 is a map of plasmids pXH2-1(A) and pXH-106 (B).

FIG. 3 shows the genomic PCR validation of the first strain with the lovF gene copy knocked out;

wherein, A is a genome PCR verification result of an engineering strain HZ-delta lovF1 obtained by knocking out the first lovF gene copy; b is pyrG cleaved from HZ-. DELTA.lovF 1_AnPCR validation of the marker was screened.

FIG. 4 shows the genomic PCR validation results for the continuation of the knock-out of the second strain of the lovF gene copy;

wherein, A is the genome PCR verification result of the engineering strain HZ-delta lovF2 obtained by continuously knocking out the second lovF gene copy; b is pyrG cleaved from HZ-. DELTA.lovF 2_AnPCR validation of the marker was screened.

FIG. 5 shows the genomic PCR validation results of a third strain with a continuous knock-out of the lovF gene copy;

wherein, A is the genome PCR verification result of the engineering strain HZ-delta lovF3 obtained by continuously knocking out the third lovF gene copy; b is pyrG cleaved from HZ-. DELTA.lovF 3_AnPCR validation of the marker was screened.

FIG. 6 is a schematic diagram of construction of an over-expressed lovE genetic engineering strain and a genome PCR verification result;

wherein A is a construction schematic diagram, B is a genome PCR verification result, a lovE expression element and pyrG_AnThe screen marker replaced the ptrA screen marker originally located at position ku 80.

FIG. 7 shows the HPLC analysis result of the fermentation liquid of the incomplete knockout lovF genetically engineered strain.

FIG. 8 shows the fermentation results of statins produced by Aspergillus engineering strains;

wherein A is the HPLC analysis result of part of engineering strain fermentation liquor; b is a statistical chart of fermentation results of each strain, wherein HZ-delta lovF3-1 and HZ-delta lovF3-2 are two HZ-delta lovF3 engineering strains, and HZ-delta lovF3-lovE-1 and HZ-delta lovF3-lovE-2 are two HZ-delta lovF3-lovE engineering strains.

FIG. 9 is a schematic representation of the lovastatin biosynthetic pathway of A.terreus;

wherein, the polyketide synthase LovF is responsible for synthesizing the 2-methylbutyrate side chain of lovastatin, and the acyltransferase LovD is responsible for transferring the 2-methylbutyrate side chain to monacolin J to generate the lovastatin.

FIG. 10 shows the results of fermentation of engineered Aspergillus strains to produce monacolin J in two media;

wherein the culture medium A is lovastatin fermentation culture medium A, the culture medium B is lovastatin fermentation culture medium B, HZ-DeltalovF 3-1 and HZ-DeltalovF 3-2 are two HZ-DeltalovF 3 engineering strains, HZ-DeltalovF 3-lovE-1 and HZ-DeltalovF 3-lovE-2 are two HZ-DeltalovF 3-lovE engineering strains, and HZ-PcEST-58 is monacoliform J oxytetracycline-producing strain with lovastatin residues.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Aspergillus HZ01(Aspergillus sp.HZ01) is deposited in China general microbiological culture Collection center (CGMCC) at 2016, 10 and 17 days (address: Xilu No.1 Hospital, Beijing, Chaoyang, Beijing) with the deposit number of CGMCC No.12970 and is classified and named Aspergillus sp. The strain is aspergillus terreus.

IPM liquid medium: 60g/L glucose, 2g/LNH₄NO₃，20mg/L(NH₄)₂HPO₄，20mg/L FeSO₄，0.4g/L MgSO₄，4.4mg/L ZnSO₄0.5g/L corn steep liquor and the balance of deionized water, the pH value is 3.5, and the mixture is autoclaved at 121 ℃ for 20 minutes.

Enzymolysis liquid: 0.4g of cellulase (Sigma product, catalog No. C1184), 0.4g of lyase (Sigma product, catalog No. L1412), 0.2g of snailase (Bio-engineering, Shanghai, Ltd., catalog No. SB0870) were weighed out and dissolved in 50mL of 0.6M MgSO₄The aqueous solution was sterilized by filtration through a 0.22 μm sterile filter.

In the present invention, Plasmid Extraction was performed using the Plasmid Mini Kit I Kit (D6942-01) from OMEGA, DNA fragment recovery was performed using the Cycle-Pure Kit (D6492-01) from OMEGA, and Gel recovery was performed using the Gel Extraction Kit (D2500-01) from OMEGA.

pXH-106 plasmid sequence is shown in SEQ ID No. 5.

pXH2-1 plasmid sequence is shown in SEQ ID No. 37.

PDB top agar: 24g/L of potato and potato culture medium PDB dry powder (product of BD company, catalog number: 7114771), 1.2M sorbitol, 4g/L of agarose and the balance of deionized water, sterilizing at 121 ℃ for 20 minutes under high pressure, and keeping the temperature at 48 ℃.

PDA flat panel: 39g/L of potato/potato medium PDA dry powder (product of BD company, catalog No. 633840), and the balance deionized water, was autoclaved at 121 ℃ for 20 minutes, and was cooled to about 60 ℃ to prepare a plate.

PDAS plate: 39g/L of potato/potato medium PDA dry powder (product of BD company, catalog No. 633840), 1.2M sorbitol, and the balance of deionized water, autoclaved at 121 ℃ for 20 minutes, and prepared into plates after cooling to about 60 ℃.

CD plate: 3g/L NaNO₃，2g/L KCl，1g/L KH₂PO₄，0.5g/L MgSO₄·7H₂O，0.02g/LFeSO₄·7H₂O, 10g/L glucose, 20g/L agar, and the balance deionized water, autoclaved at 121 ℃ for 20 minutes, and prepared into plates after cooling to about 60 ℃.

CD top agar: 3g/L NaNO₃，2g/L KCl，1g/L KH₂PO₄，0.5g/L MgSO₄·7H₂O，0.02g/LFeSO₄·7H₂O, 10g/L glucose, 1.2M sorbitol, 4g/L agarose, autoclaving at 121 ℃ for 20 minutes and then incubating at 48 ℃.

CD-SFU: CD plate +1.2M sorbitol +1 g/L5-fluoroorotic acid +10mM uridine.

CD-FU: CD plate +1 g/L5-fluoroorotic acid +10mM uridine.

Lovastatin fermentation seed culture medium: 9g/L glucose, 10g/L sucrose, 1g/L yeast extract, 1g/L peptone, 1g/L sodium acetate, 0.04g/L KH₂PO₄，0.1g/L MgSO₄5g/L of soybean meal, 1.5g/L of calcium carbonate, pH6.8 and the balance of deionized water, and autoclaving at 121 ℃ for 20 minutes.

Lovastatin fermentation medium a: 70g/L glucose, 20g/L sucrose, 1.5g/L yeast extract (product of Angel Yeast Co., Ltd., catalog number: FM888), 20g/L peptone, 7g/L sodium acetate, 0.5g/L KH₂PO₄， 0.5g/LMgSO₄5g/L of soybean meal, 5g/L of calcium carbonate, 0.1mL/L of natural enemy (product catalog number: THIX-298 of Nicotai Henxin chemical technology Co., Ltd.), pH6.2 and the balance of deionized water, and is sterilized at 121 ℃ under high pressureAnd (5) sterilizing for 20 minutes.

Lovastatin fermentation medium B: 70g/L glucose, 20g/L sucrose, 1.5g/L yeast extract (OXOID, Cat. ID: LP0021), 5g/L peptone, 7g/L sodium acetate, 0.5g/L KH₂PO₄，0.5g/L MgSO₄20g/L of soybean meal, 5g/L of calcium carbonate, 0.1mL/L of natural enemy (Nicotai Henxin chemical technology Co., Ltd., product catalog number: THIX-298), pH7.2, and the balance of deionized water, and autoclaving at 121 ℃ for 20 minutes.

HZ-PcEST-58 is an engineering strain No. 58 of the invention patent application with the publication number CN108118042A and the title of 2-methylbutyrate side chain hydrolase and Monacolin J producing aspergillus strain, and a construction method and application thereof, and is publicly available from Zhejiang Henyang pharmaceutical industry Co.