CN109402086B - 2-methylbutyrate side chain hydrolase, expression strain and application thereof - Google Patents

Abstract

The invention belongs to the field of protein engineering and medicine production, and provides 2-methylbutyric acid side chain hydrolase, which is obtained by site-specific mutagenesis of wild type PcEST, wherein the mutagenesis site is 140 th site. The invention also provides biological materials related to the enzyme, application of the enzyme, and an aspergillus strain for coding the enzyme and a construction method. The 2-methylbutyrate side chain hydrolase provided by the invention has high enzyme activity, improves the stability to temperature, and is more beneficial to industrial application.

Description

A kind of 2-methylbutyric acid side chain hydrolase and its expression strain and application

technical field

The invention belongs to the fields of protein engineering and pharmaceutical production, and relates to a 2-methylbutyric acid side chain hydrolase and a strain capable of producing monacolin J.

Background technique

Cardiovascular and cerebrovascular diseases are a serious threat to human health, and their morbidity and mortality ranks first in many countries and regions. Hyperlipidemia (hypercholesterolemia) is an important cause of cardiovascular and cerebrovascular diseases. Therefore, preventing hyperlipidemia, regulating blood lipids and reducing blood lipids have become the keys to preventing and treating cardiovascular and cerebrovascular diseases. Based on the above reasons, cholesterol-lowering drugs have great market prospects and have been at the forefront of the world's best-selling drugs for many years.

Simvastatin (Zocor) is an important hypolipidemic drug developed by Merck, which can effectively inhibit the synthesis of cholesterol in the body and is one of the first-choice drugs for the treatment of hyperlipidemia. The synthesis of simvastatin is chemically synthesized from the fermentation product of Aspergillus terreus, lovastatin. In the synthesis process of simvastatin, it is necessary to remove the 2-methylbutyric acid side chain at the C8 position of lovastatin first to obtain Monacolin J (Monacolin J), and then the 2,2-dimethylbutyric acid is converted into 2,2-dimethyl by subsequent processes. A side chain of butyric acid is added to the C8 position of monacolin J to obtain simvastatin. It can be seen that monacolin J is an important precursor for the synthesis of simvastatin. At present, in the synthetic route of simvastatin, the chemical method is used to hydrolyze lovastatin to prepare monacolin J. A large number of acids, bases and organic reagents are used in the whole preparation process. The process is complicated, time-consuming, low yield, Pollution pressure is high.

2-Methylbutyric acid side chain hydrolase can hydrolyze the ester bond of lovastatin side chain to generate monacolin J and 2-methylbutyric acid. In 1986, Komagata et al. purified a 2-methylbutyric acid side-chain hydrolase from the mycelium of the fungus Emericella unguis with a conversion efficiency of about 86%. In the following decades, researchers successively discovered a number of 2-methylbutyrate side chain hydrolases. However, these enzymes have low catalytic efficiency, obvious substrate inhibition, and the protein Neither the sequence nor the related coding gene information has been identified, so it cannot be directly utilized and modified. These problems greatly limit the application of this enzyme in large-scale industrial production.

The inventor of the present application reported a 2-methylbutyric acid side chain hydrolase PcEST (SEQ ID NO: 1) in 2017, which can effectively hydrolyze the 2-methylbutyryl side chain of lovastatin to generate monac Lin J and 2-methylbutyric acid, with significantly higher catalytic efficiency than previously reported lovastatin side-chain hydrolysis esterase (WO2005040107A2). In addition, by overexpressing PcEST in a high-yielding lovastatin Aspergillus terreus strain, lovastatin is hydrolyzed into monacolin J after it is synthesized in vivo, thereby obtaining an Aspergillus engineered strain capable of directly fermenting and producing monacolin J , a new process for the direct production of monacolin J by one-step fermentation can be established (Huang XN, Liang YJ, Yang Y, Lu XF. Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus. Metab.Eng. , 2017, 42, 109-114). However, the hydrolysis rate of lovastatin in the engineered strain is about 95%, and about 5% of lovastatin remains, which will increase the cost of subsequent separation and purification. Therefore, it is very necessary to construct an engineered strain that can efficiently synthesize monacolin J without lovastatin residue, which can further simplify the production process of simvastatin and reduce the production cost.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new 2-methylbutyric acid side chain hydrolase, which improves the soluble expression effect and thermal stability of PcEST, and further improves its catalytic activity, so that it can be better used in industry production to solve the above-mentioned prior art problems.

In a first aspect, the present invention provides a 2-methylbutyric acid side chain hydrolase, which is a mutant obtained by site-directed mutation of wild-type PcEST (SEQ ID NO: 1), the mutation site is the 140th position, and the mutation The format is Q140L, ie, the sequence of the 2-methylbutyrate side chain hydrolase is shown in SEQ ID NO:2.

In this application document, unless otherwise specified, PcEST/Q140L is abbreviated as Q140L, which means protein mutant. Those skilled in the art can clearly distinguish whether Q140L represents a mutant form or a mutant according to the context.

In this application document, the abbreviations of amino acids and nucleotides have the meanings commonly used in the art.

A second aspect provides biological materials relevant to the 2-methylbutyric acid side chain hydrolase of the present invention, which are:

(1) a nucleic acid molecule, the nucleic acid molecule encodes the 2-methylbutyric acid side chain hydrolase of the present invention; preferably, the nucleic acid molecule is pcest/Q140L, the sequence of which is shown in SEQ ID NO: 3; or

(2) an expression cassette, recombinant vector, recombinant cell or recombinant microorganism comprising the nucleic acid molecule of (1);

Preferably, the microorganism is an Aspergillus strain capable of producing monacolin J.

Regarding the above-mentioned biological materials, those skilled in the art can know the sequence of the nucleic acid molecule encoding the enzyme based on the amino acid sequence of the 2-methylbutyrate side chain hydrolase provided, and can easily select, prepare and use the nucleic acid molecule containing the enzyme. Thereby expressing protein expression cassettes, recombinant vectors, recombinant cells and recombinant microorganisms.

A third aspect provides the application of the 2-methylbutyric acid side chain hydrolase of the present invention in the hydrolysis of statins containing 2-methylbutyric acid side chains; preferably, the 2-methylbutyric acid-containing The statin in the side chain is lovastatin, mevastatin or pravastatin; further preferably, the statin is in acid form, lactone form or salt form.

A fourth aspect provides a method for constructing the above-mentioned Aspergillus strain producing monacolin J, comprising co-coagulating the Aspergillus strain protoplast with a DNA fragment comprising a nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase of the present invention transformation, obtained after screening. Preferably, the Aspergillus sp. strain is Aspergillus sp. HZ01, whose deposit number is CGMCC NO.12970.

Aspergillus sp. HZ01 used in the present invention was deposited in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection Management Committee on October 17, 2016, and the deposit number is CGMCC NO.12970, and the address of the deposit center is Beijing No. 3, Yard 1, Beichen West Road, Chaoyang District.

Preparation of protoplasts of Aspergillus strains, co-culture of protoplasts and DNA fragments, screening of positive Aspergillus species, etc. are all routine techniques in the art.

Preferably, the sequence of the nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase of the present invention is shown in SEQ ID NO:3.

The fifth aspect provides the use of the nucleic acid molecule of the present invention or an expression cassette comprising the nucleic acid molecule, a recombinant vector, a recombinant cell or a recombinant microorganism in the preparation of a monacolin J-producing Aspergillus strain.

A sixth aspect provides a method for preparing monacolin J, comprising fermenting and culturing the Aspergillus strain provided by the present invention, or hydrolyzing lovastatin with the 2-methylbutyric acid side chain hydrolase provided by the present invention.

The seventh aspect provides the application of the above-mentioned biological material or Aspergillus strain or bacterial suspension or fermentation broth or metabolite thereof of the present invention in the preparation of monacolin J.

In an eighth aspect, there is provided the use of the above-mentioned biological material or Aspergillus strain or bacterial suspension or fermentation broth or metabolite thereof of the present invention in the production of a hypolipidemic drug, wherein the hypolipidemic drug is simvastatin.

Compared with the wild-type enzyme, the 2-methylbutyric acid side chain hydrolase provided by the invention has improved enzyme activity, about 2 times higher, improved temperature stability, simplified the production process of simvastatin, and reduced production costs , more conducive to industrial applications.

Description of drawings

Figure 1: A is the electrophoresis analysis of the purification results of wild-type and mutant proteins; B is the result of enzyme activity assay. The unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of monacolin J per minute. Vitality unit (U). The ordinate in the figure represents the number of enzyme activity units per milligram of enzyme protein. WT: wild-type PcEST; Q140L: PcEST/Q140L mutant.

Figure 2: Electrophoretic analysis of soluble expression of wild-type and mutant proteins, WT: wild-type PcEST; Q140L: PcEST/Q140L mutant. S: soluble protein fraction; I: insoluble protein fraction.

Figure 3: Thermal stability analysis plot of wild type and mutant proteins, WT: wild type PcEST; Q140L: PcEST/Q140L mutant.

Figure 4: Schematic representation of the plasmid map of pG3H.

Figure 5: Genome PCR verification results of Aspergillus transformants overexpressing PcEST/Q140L; wherein, 1#, 2#, 7#, 8#, 9#, 10#, 11#, 13# are positive transformants, and C is Plasmid pG3H-pcest/Q140L, wt is the control strain HZ01 strain.

Figure 6: Genome PCR verification results of Aspergillus transformants overexpressing wild-type PcEST; among them, 1#, 4#, and 5# are positive transformants, C is plasmid pG3H-pcest, and wt is control strain HZ01 strain.

Figure 7: HPLC analysis of the 8th day samples of each strain; wherein, HZ01 is a wild-type strain, HZ-PcEST is a control strain overexpressing wild-type PcEST, and HZ-PcEST/Q140L is an Aspergillus project overexpressing a PcEST/Q140L mutant strains, wherein panel B is a partially enlarged schematic view of panel A.

Figure 8: Contents of lovastatin and monacolin J in fermentation samples of each strain on the 8th day; among them, HZ01 is a wild-type strain, HZ-PcEST series is a control strain overexpressing wild-type PcEST, and Q140L series is an overexpressing strain Aspergillus engineered strains of the PcEST/Q140L mutant.

Detailed ways

The content of the present invention will be described below with reference to specific embodiments. Unless otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments in the art, and can be obtained through commercial channels; the methods used are also conventional methods, and those skilled in the art can clearly know from the contents of the description. How to complete the experiment and obtain the corresponding results.

In the present invention, plasmid extraction adopts OMEGA company Plasmid Mini Kit I kit (D6942-01), DNA fragment recovery adopts OMEGA company Cycle-Pure Kit kit (D6492-01), and gel recovery adopts OMEGA company Gel Extraction Kit reagent Box (D2500-01).

The composition of CD medium is: 3g/L NaNO ₃ , 2g/L KCl, 1g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ ·7H ₂ O, 0.02g/L FeSO ₄ ·7H ₂ O, 10g/L glucose.

The composition of CD plate medium is: 3g/L NaNO ₃ , 2g/L KCl, 1g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ ·7H ₂ O, 0.02g/L FeSO ₄ ·7H ₂ O, 10g/L L glucose, 1.5g/L agar.

IPM liquid medium: 60 g L ^-1 glucose, 2 g L ^-1 NH ₄ NO ₃ , 20 mg L ^-1 (NH ₄ ) ₂ HPO ₄ , 20 mg L ^-1 FeSO ₄ , 0.4 g L ^-1 MgSO ₄ , 4.4 mg L ^-1 ZnSO ₄ , 0.5 g/L corn steep liquor, pH 3.5.

Lovastatin Fermentation Seed Medium: 9g L ^-1 glucose, 10g L ^-1 sucrose, 1g L ^-1 yeast extract, 1g L ^-1 peptone, 1g L ^-1 sodium acetate, 0.04g L ^-1 KH ₂ PO ₄ , 0.1 g L ^-1 MgSO ₄ , 5 g L ^-1 soybean meal, 1.5 g L ^-1 calcium carbonate, pH 6.8.

Lovastatin fermentation medium: 70g L ^-1 glucose, 20g L ^-1 sucrose, 1.5g L ^-1 yeast extract, 20g L ^-1 peptone, 7g L ^-1 sodium acetate, 0.5g L ^-1 KH ₂ PO ₄ , 0.5g L ^-1 MgSO ₄ , 5g L ^-1 soybean meal, 5g L ^-1 calcium carbonate, foam Di (Yantai Hengxin Chemical Technology Co., Ltd., model THIX-298) 0.1mL L ^-1 , pH 6.5.

Example 1: Obtainment of 2-methylbutyrate side chain hydrolase mutants

1. Construction of 2-methylbutyrate side chain hydrolase mutant Q140L

(1) Mutation PCR: site-directed mutagenesis was performed using the QuikChange method. Take pEASY-E2-pcest plasmid (HuangXN, Liang YJ, Yang Y, Lu XF. (2017). Single-step production of the simvastatinprecursor monacolin J by engineering of an industrial strain of Aspergillusterreus. Metab.Eng., 42, 109-114) As a template, primers were designed and synthesized according to the mutation site, and PCR amplification was performed to obtain a gene encoding a 2-methylbutyrate side chain hydrolase mutant.

The PCR reaction system was: sterile water 32.5 μL, template DNA 1 μL, 5× PrimeSTAR HS buffer (Mg ²⁺ plus) 10 μL, dNTP mixture (2.5 mmol/L each) 4 μL, mutation primer pair Q140L-F (5′ -CAAAATGGCGCGCACTATACGCGAATCCGG-3') and Q140L-R (5'-CCGGATTCGCGTATAGTGCGCGCCATTTTG-3') 1 μL each (20 pmol/μL), TaKaRa PrimeSTAR HS DNA polymerase 0.5 μL. The reaction conditions were: pre-denaturation at 95 °C for 5 min; 18 cycles of 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 7.5 min; and a final extension at 72 °C for 10 min.

(2) Enzyme digestion: Take out the PCR product after the reaction, add Dpn I enzyme at a ratio of 1:50 (Dpn I enzyme:PCR product, v:v), and digest at 37°C for 1 hour.

(3) Take out, transform into Escherichia coli BL21 (DE3) competent cells, spread on LB plates containing kanamycin (final concentration 50 μg/ml), and cultivate overnight at 37°C.

After the transformants were grown, single clones were picked and sent for sequencing, and the correct mutant strain and the corresponding vector pEASY-E2-pcest/Q140L contained therein were screened according to the mutant form shown in SEQ ID NO: 3 of the mutant nucleotide sequence.

2. Construction of wild-type 2-methylbutyric acid side chain hydrolase expression vector:

The wild-type PcEST genes pcest and PcEST/Q140L were amplified by conventional PCR with primer 1: 5'GGAATTCCATATGGATACCACCTTTCAGGCG 3' and primer 2: 5'CCGCTCGAGTCACTGCTGACCTTTCCAGGC 3' from plasmids pEASY-E2-pcest and pEASY-E2-pcest/Q140L, respectively The mutant gene pcest/Q140L, the PCR product was purified and digested with restriction enzymes Nde I and Xho I, and then ligated with the pET28a-smt3 vector digested by the same enzyme, and the obtained vectors were named pET-pcest and pET-pcest respectively /Q140L, the plasmids were transformed into E. coli BL21 (DE3) competent cells, and the transformants were picked and sequenced to identify their gene sequences. The pcest/Q140L mutant sequence is shown in SEQ ID NO:3, and the wild-type pcest is shown in SEQ ID NO:4.

3. Protein purification

E. coli BL21 (DE3) expressing wild-type enzyme and mutant enzyme was cultured at 37°C to logarithmic growth phase, and the inducer isopropyl-β-D-thiogalactoside (IPTG) was added, the final concentration was 0.2mM ), after inducing expression overnight at 16°C, the cells were collected, resuspended in 40 ml of Tris-HCl buffer (20 mM, pH 8.0), and the cells were sonicated to obtain crude enzyme solution, which was purified by Ni-NTA. The protein was collected, digested with Ulp1 (Ulp1:PcEST mass ratio of 1:50) overnight, and continued to purify the protein by inverting the Ni column. Tight, high concentrations (250 mM) of imidazole are required to elute, so pure protein can be obtained (Figure 1A).

After sequencing, the sequence of the mutant enzyme was as expected.

Example 2: Determination of enzymatic activity

The protein prepared in Example 1 was used for enzyme activity assay. The concentration of the enzyme protein was 0.05 μM, the concentration of the substrate lovastatin was 400 μM, and the reaction was carried out at 37° C. for 10 minutes, and the produced monacolin J was measured by HPLC. The column used for the HPLC reaction was Agilent ZORBAX XDB-C18 (4.6×150 mm, 5 μm), the mobile phase was acetonitrile:0.1% phosphoric acid (50:50, v:v), the flow rate was 1 ml/min, and the injection was 10 μl. The results are shown in Figure 1B, the enzyme activity of the 2-methylbutyrate side chain hydrolase of the mutant Q140L was 2.51 times that of the wild enzyme.

Example 3: Determination of enzyme properties of 2-methylbutyrate side chain hydrolase mutants

1. 2-Methylbutyrate side chain hydrolase wild-type and mutant protein soluble expression assay

Cultivate 100ml of Escherichia coli BL21(DE3) encoding the wild-type and mutant 2-methylbutyrate side chain hydrolase, cultivate to logarithmic growth phase at 37℃, add the inducer isopropyl-β-D-galactothioate Glycoside (IPTG, final concentration is 0.2mM), protein expression was induced at 25°C, the bacterial solution was collected overnight, sonicated and centrifuged, the supernatant and the pellet were separated, and the same volume of Tris-HCl buffer was added to the pellet. solution (50mM, pH 8.0) for suspension. Take the same amount of supernatant and precipitated protein solution for SDS-PAGE protein electrophoresis analysis, the results are shown in Figure 2. Most of the wild-type protein is in the precipitate part, and there is only a small amount of PcEST protein in the supernatant part, while the amount of PcEST protein in the supernatant part of the mutant Q140L is significantly increased, indicating that the soluble expression of the Q140L mutant protein is relatively high. increased in wild type.

2. Determination of temperature stability of 2-methylbutyrate side chain hydrolase wild-type and mutant proteins

Set the temperature conditions of the reaction tank of the PCR instrument, take out equal amounts of wild-type and mutant proteins, and place them at different temperatures (20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, 45°C and 50°C), incubate for 10 minutes, immediately place on ice to cool for 10 minutes, and then remove the protein from it for enzyme activity assay. The assay conditions are: the concentration of the enzyme is 0.05 μM, the concentration of the substrate lovastatin was 400 μM, the reaction was carried out at 37° C. for 10 minutes, and the generated monacolin J was measured by HPLC. Taking the activity of the protein without temperature treatment as 100%, calculate the relative value of the enzyme activity of the protein after different temperature treatments, and draw a curve. As shown in Figure 3, the T50 value of the mutant _Q140L was 3°C higher than that of the wild type.

Example 4: Construction of Monacolin J-producing Aspergillus strains

The two plasmids pEASY-E2-pcest and pEASY-E2-pcest/Q140L constructed in Example 1 were used as templates, and the primers pcest-F1 (5'-gatccatggataccacctttcaggc-3') and pcest-R1 (5'-gatgcggccgcgttagcagccggatctcagtg- 3') Amplify the wild-type pcest and mutant pcest/Q140L gene fragments, digest with restriction enzymes Nco I (Thermo, FD0573) and Not I (Thermo, FD0593) respectively, and recover the target fragment (1.2kb) . The vector pG3H (Figure 4, Xuenian Huang, Xuefeng Lv, Jianjun Li*, Cloning and functional characterization of anative glyceraldehyde-3-phosphate dehydrogenase promoter for Aspergillusterreus, Journal of Industrial Microbiology and Biotechnology, 2014, 41(3): 585-592) for enzyme digestion, and a fragment with a size of about 7.3 kb was recovered. The recovered pcest and pcest/Q140L fragments were ligated with pG3H vector using T4 ligase, respectively, and transformed into E. coli DH5a. The correct plasmids verified by PCR and sequencing were recorded as pG3H-pcest and pG3H-pcest/Q140L, respectively. Using pG3H-pcest and pG3H-pcest/Q140L as templates, PCR amplification was performed with primers PgpdAt-F630 (5'-catcatcgcattctccctctcg-3') and TtrpC-R2 (5'-ttactattgtatacccatcttag-3') to obtain the expression of PcEST Elements PgpdAt-pcest-TtrpC and PgpdAt-pcest/Q140L-TtrpC, the expression element includes promoter PgpdAt, PcEST (or PcEST/Q140L) coding sequence pcest (or pcest/Q140L), terminator TtrpC, the size is 2490bp, by PCR Product recovery kit for recovery and purification. Using plasmid pPTR II (TAKARA, Catalog No.: 3621) as a template, ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primers, the obtained size is about The 2.0kb screenable marker pyridinethiamine resistance gene ptrA fragment was recovered and purified by a PCR product recovery kit.

The Aspergillus strain HZ01 (preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC), the preservation number is CGMCC No. 12970, and the address of the preservation center is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing). It was inoculated on a PDA solid plate (BD, Difco ^™ Potato Dextrose Agar, 213400) and cultured at 30°C for 7 days to obtain spores. Spores of HZ01 strain were inoculated into 50 mL liquid medium IPM (60 g L ^-1 glucose, 2 g L ^-1 NH ₄ NO ₃ , 20 mg L ^-1 (NH ₄ ) ₂ HPO ₄ , 20 mg L ^-1 FeSO ₄ , 0.4 g L ^-1 MgSO ₄ , 4.4 mg L ^-1 ZnSO ₄ , 0.5 g/L corn steep liquor (Sigma, C8160-500G), pH 3.5), the spore concentration was about 10 ⁷ /mL, and cultured at 200 rmp and 32°C 12-18h. Use sterile single-layer 500-mesh nylon cloth to filter and collect the grown mycelium, rinse with sterilized 0.6M _MgSO4 solution three times, press dry and place in a sterile 50ml conical flask; weigh 1g of mycelium, add 10ml The enzymatic hydrolyzate was treated at 30°C and 60rpm for 1-3h. The components of the enzymatic hydrolysis solution are: 0.8% (mass volume ratio) cellulase (Sigma, Catalog No.: C1184), 0.8% (mass volume ratio) lyase (Sigma, Catalog No.: L1412), 0.4% (mass volume ratio) than) helicase (Shanghai Shenggong, Catalog No.: SB0870), 0.6M MgSO ₄ , and sterilized by filtration through a 0.22 μm sterile filter. The mixed solution after the enzymolysis was first filtered with 8 layers of lens-wiping paper, and the filtrate was collected. Protoplasts were collected by centrifugation at 4°C, washed once with pre-cooled 1.0M sorbitol solution, and then once with pre-cooled STC (STC was 1.0M sorbitol, 50mM Tris·HCl-pH 8.0, 50mM CaCl ₂ ), and finally the protoplasts were washed once. The body weight was resuspended in pre-cooled STC, and the protoplast concentration was adjusted to 2×10 ⁸ /mL with STC to obtain a protoplast suspension.

The DNA fragments PgpdAt-pcest/Q140L-TtrpC (or PgpdAt-pcest/Q140L-TtrpC) (about 3 μg) and ptrA (about 0.5 μg) were simultaneously added to 150 μl of the above protoplast suspension, the total volume was 15 μl, the expression element and ptrA The molar ratio is about 1:5. Then add 50 μl of PSTC in ice bath (PSTC is 40% PEG4000, 1.2 M sorbitol, 50 mM Tris-HCl-pH8.0, 50 mM CaCl ₂ ), mix gently, and ice bath for 30 min. Add 1 mL of PSTC at room temperature, mix well and place at room temperature for 20 min; then mix with 30 mL of upper agar (CD+1.2M sorbitol+4g/L agarose, sterilized at 48°C) and pour into 10 pieces of CD-SP for regeneration screening Culture medium plates (CD plates + 1.2M sorbitol + 0.1 mg/L pyrithione (Sigma, P0256)), and cultured at 30°C under dark conditions for 5-7 days.

The transformants with pyrithione resistance were picked from the transformation screening plate and transferred to the screening plate CD-P (CD plate + 0.1 mg/L pyrithione), cultured at 32°C for 5 days for passage purification, and serially passaged for 4 Second-rate. 4 transformants (2#, 3#, 4#, 5#) of stable passage were selected for single spore separation and purification, and the obtained single colony spores were inoculated in IPM liquid medium, and cultivated for 48 hours at 30° C. and 200 rpm. Collect the hyphae to extract the genome. PCR was performed with primers PgpdAt-F630/TtrpC-R2 to verify that a band with a size of about 2.6kb (PcEST/Q140L or wild-type PcEST expression element) could be amplified as positive. It can be seen from Figure 5 that 1#, 2#, 7#, 8#, 9#, 10#, 11#, 13# transformants are positive, and the PcEST/Q140L expression element PgpdAt-pcest/Q140L-TtrpC is integrated on the genome. The eight HZ-PcEST/Q140L engineering strains were recorded as Q140L-1, Q140L-2, Q140L-7, Q140L-8, Q140L-9, Q140L-10, Q140L-11, and Q140L-13, respectively. It can be seen from Figure 6 that the transformants 1#, 4# and 5# are positive transformants integrated with the PcEST expression element PgpdAt-pcest-TtrpC, which are respectively recorded as HZ-PcEST-1, HZ-PcEST-4, HZ-PcEST-5 .

Example 5: Fermentation verification of Aspergillus strains producing monacolin J

The Aspergillus strains that need to be fermented were inoculated on a PDA solid plate, and cultured at 30°C for 7 days to obtain spores, including: 8 engineering strains Q140L-1, Q140L-2, Q140L-7, Q140L-overexpressing PcEST/Q140L mutants 8. Q140L-9, Q140L-10, Q140L-11, Q140L-13, 3 engineered strains HZ-PcEST-1, HZ-PcEST-4, HZ-PcEST-5 overexpressing wild-type PcEST, and the starting strain HZ01 . The spores were inoculated in 35 ml of lovastatin fermented seed medium (250 mL conical flask), and cultured with shaking at 28° C. and 220 rpm for 48 hours. The control strain HZ01 strain was inoculated into 4 flasks. 3.5 mL of seed liquid was inoculated into 30 mL of lovastatin fermentation medium (250 mL Erlenmeyer flask), and cultured with shaking at 28° C. and 220 rpm for 8 days. Take 0.8mL of fermentation broth (with a cut tip) into a 15mL centrifuge tube, add 0.8mL of 0.2M NaOH solution, then add 8mL of anhydrous methanol, and place it on a mixer for 4 hours. The samples were filtered through a 0.22 μm organic filter and sent to HPLC for analysis. The HPLC analysis method is: the liquid chromatography column is Agilent ZORBAX SB-C18 liquid chromatography column 883975-902 (4.6×150 mm, 5 μm), the mobile phase is acetonitrile/0.1% H ₃ PO ₄ =0.7/0.3, and the flow rate is 0.5 ml /min, UV detector (237 nm), 25°C. The results are shown in Figures 7 and 8. In the starting strain HZ01, the product is mainly lovastatin, and the content of monacolin J is very small. The main product in the engineered strain overexpressing wild-type PcEST is monacolin J, and the residual lovastatin is very small, about 5%-8%. In the engineering strain of Aspergillus overexpressing the PcEST/Q140L mutant, the main product contained a large amount of monacolin J, while lovastatin could not be detected in some strains, and the content in some strains was very low, less than 0.5%, Subsequent separation and purification are no longer required, which further simplifies the simvastatin production process and reduces production costs. This shows that the PcEST mutant with improved enzyme performance can improve the intracellular hydrolysis efficiency of lovastatin, thereby increasing the hydrolysis rate of lovastatin, reducing the residue of lovastatin, and obtaining monacolin-producing Aspergillus with better characters. Engineered strains.

sequence listing

<110> Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences

Zhejiang Hisun Pharmaceutical Co., Ltd.

<120> A kind of 2-methylbutyric acid side chain hydrolase and its expression strain and application

<130> DP1F171503ZX

<160> 4

<170> SIPOSequenceListing 1.0

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<211> 399

<212> PRT

<213> Penicillium chrysogenum

<400> 1

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Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 2

<211> 399

<212> PRT

<213> Artificial Sequence

<400> 2

Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Asp Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Leu Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 3

<211> 1200

<212> DNA

<213> Artificial Sequence

<400> 3

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacta 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 4

<211> 1200

<212> DNA

<213> Artificial Sequence

<400> 4

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacaa 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

Claims (14)

1. The 2-methylbutyrate side chain hydrolase has the sequence shown in SEQ ID NO 2.

2. A biomaterial, being:

(1) a nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase according to claim 1; or

(2) An expression cassette, a recombinant vector, a recombinant cell or a recombinant microorganism comprising the nucleic acid molecule of (1).

3. The biomaterial according to claim 2, wherein the sequence of the nucleic acid molecule is as shown in SEQ ID NO 3.

4. The biomaterial according to claim 2, wherein the recombinant microorganism is an aspergillus strain capable of producing monacolin J.

5. The use of 2-methylbutyrate side chain hydrolase according to claim 1 for hydrolyzing statins containing a 2-methylbutyrate side chain.

6. The use according to claim 5, wherein the statin-like substance having a side chain of 2-methylbutyric acid is lovastatin, mevastatin or pravastatin.

7. The use according to claim 6, wherein the statin is in the acid, lactone or salt form.

8. A method for constructing the biomaterial according to claim 4, comprising co-transforming protoplasts of an Aspergillus oryzae strain with a DNA fragment comprising a nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase according to claim 1, followed by screening.

9. The method according to claim 8, wherein the Aspergillus starter strain is Aspergillus (Aspergillus sp.) HZ01 with the accession number CGMCC NO. 12970.

10. The method according to claim 8 or 9, wherein the nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase according to claim 1 has the sequence shown in SEQ ID NO 3.

11. Use of the biomaterial of claim 2 or 3 in the preparation of monacolin J producing aspergillus strain.

12. A method of preparing monacolin J comprising fermenting the biomaterial of claim 4 or hydrolyzing lovastatin with the 2-methylbutyrate side chain hydrolase of claim 1.

13. Use of the biological material of claim 4 or a bacterial suspension thereof or a fermentation broth thereof or a metabolite thereof in the preparation of monacolin J.

14. Use of a biological material or a bacterial suspension thereof or a fermentation broth thereof or a metabolite thereof according to claim 4 in the manufacture of a hypolipidemic agent, wherein the hypolipidemic agent is simvastatin.

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