Construction method and application of aspergillus niger strain for efficient heterologous synthesis of Monacolin J

Abstract

The invention discloses a construction method of an aspergillus niger strain for efficient heterologous synthesis of Monacolin J. According to the invention, in saccharomyces cerevisiae BY4741, a transformation coupled recombination (TAR) technology is utilized to construct an expression vector of a lovastatin precursor Monacolin J, wherein the expression vector is formed by serially connecting lovB, lovC, lovG and lovA genes regulated and controlled by a strong promoter and a strong terminator of filamentous fungi. According to the invention, the steps of expression vector construction and aspergillus niger homologous recombination transformation are simplified, the lovastatin is efficiently and heterologously synthesized in the aspergillus niger by optimizing fermentation components, and a research idea is provided for the construction of a macromolecular vector and the research of metabolic engineering and synthetic biology of filamentous fungi.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and fermentation engineering, and specifically to a method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J and its application. The invention provides reliable research ideas for the construction of macromolecular carriers, metabolic engineering of filamentous fungi, and synthetic biology research. Background Art

[0002] Lovastatin, a natural secondary metabolite of the polyketide family, is a gene cluster found in the genome of Aspergillus Terrus. The biosynthesis of the lovastatin precursor, monacolin J, proceeds sequentially through the polyketide synthase gene lovB, the enoyl reductase gene lovC, the thioesterase gene lovG, and the cytochrome P450 monooxygenase gene lovA. Lovastatin is then synthesized by the acyltransferase LovD, which adds a side chain synthesized by LovF.

[0003] Statins inhibit HMG-CoA reductase activity, reducing cholesterol synthesis in the body. Since the 1990s, they have become lipid-lowering drugs that account for over 80% of the total market. In the industrial production of lovastatin, biofermentation offers advantages over chemical synthesis, including lower cost, less pollution, higher efficiency, and higher purity. With the continuous advancement of genetic engineering, fermentation engineering, and metabolic engineering, biofermentation still has significant room for optimization.

[0004] Compared with prokaryotic expression systems such as Saccharomyces cerevisiae and Escherichia coli, filamentous fungi can recognize introns and complete post-transcriptional modifications, and have PPTase, which is more conducive to the heterologous synthesis of polyketide products. Aspergillus niger has active metabolism and can use lower-cost carbon sources for growth and fermentation.

[0005] Low expression levels of secondary metabolite genes themselves can lead to low yields of heterologous secondary metabolites. The coherence and integrity of the secondary metabolite gene cluster are also crucial for secondary metabolite production. In summary, regulating the expression of heterologous secondary metabolite genes through strong promoters and terminators to construct a coherent and complete secondary metabolite gene cluster is beneficial for improving the fermentation of heterologous secondary metabolites. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J and its application.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The invention discloses an Aspergillus niger strain for the efficient heterologous synthesis of Monacolin J. The strain is obtained by heterologously reconstructing the polyketide synthase gene lovB, the enoyl reductase gene lovC, the thioesterase gene lovG, and the cytochrome P450 monooxygenase gene lovA in the synthesis pathway of the natural secondary metabolite lovastatin of Aspergillus Terreus, which are regulated by a strong filamentous fungal promoter and a strong terminator, into the Aspergillus niger genome.

[0009] The NCBI database sequence accession number of the DNA sequence of the gene lovB is AF151722, and its sequence is SEQ ID NO: 2; the NCBI database sequence accession number of the DNA sequence of the gene lovC is AAD34554, and its sequence is SEQ ID NO: 4; the FungiDB database sequence accession number of the DNA sequence of the gene lovG is ATEG_09960, and its sequence is SEQ ID NO: 5; the NCBI database sequence accession number of the DNA sequence of the gene lovA is AAD34552, and its sequence is SEQ ID NO: 6.

[0010] The amino acid sequence encoded by the gene lovB has an NCBI database sequence accession number of AAD39830, and its sequence is SEQ ID NO: 7; the amino acid sequence encoded by the gene lovC has an NCBI database sequence accession number of AAD34554, and its sequence is SEQ ID NO: 8; the amino acid sequence encoded by the gene lovG has an NCBI database sequence accession number of XP_001209262, and its sequence is SEQ ID NO: 9; and the amino acid sequence encoded by the gene lovA has an NCBI database sequence accession number of AAD34552, and its sequence is SEQ ID NO: 10.

[0011] The promoter for regulating the transcription of the gene lovB is the Aspergillus niger saccharifying enzyme gene promoter PglaA, and the terminator is the Aspergillus nidulans tryptophan synthase gene terminator TtrpC; the promoter for regulating the transcription of the gene lovC is the Aspergillus niger 3-phosphoglyceraldehyde dehydrogenase gene promoter PgpdA, and the terminator is the Aspergillus nidulans α-glucosidase gene terminator TagdA; the promoter for regulating the transcription of the gene lovG is the plasmid pFC330 promoter Ptef, and the terminator is the Aspergillus fumigatus 1,4-β-xylan endonuclease gene terminator TxlnA; the promoter for regulating the transcription of the gene lovA is the plasmid pMT1802 composite promoter PnaⅡ / TPI, and the terminator is the plasmid pFC330 terminator Ttef.

[0012] The starting strain of Aspergillus niger is a ΔkusAΔpyrG mutant strain, which is disclosed in the document "Dong Hongzhi. Establishment of Aspergillus niger CRISPR gene editing technology and research on endoplasmic reticulum phospholipid regulation mechanism [D]. South China University of Technology."

[0013] The glucoamylase gene glaA was knocked out by utilizing the 1500bp upstream and downstream homologous sequences of the glaA gene, and the Aspergillus niger glaA gene expression cassette was replaced with the lovB-lovC-lovG-lovA gene cluster through homologous recombination.

[0014] The selection marker used was ANpyrG.

[0015] The construction method of the present invention specifically includes the following steps:

[0016] (1) Using Saccharomyces cerevisiae homologous recombination technology or In-Fusion seamless cloning technology, the corresponding promoter, terminator and the gene regulated by them are connected on the pMD-18T vector, and the expression frame of a single heterologous secondary metabolic gene is obtained by PCR amplification.

[0017] (2) In Saccharomyces cerevisiae BY4741, the shuttle vector for the Monacolin J synthetic gene cluster was obtained by ligating the single gene expression cassettes of lovB, lovC, lovG, and lovA with 1500 bp of homologous sequences upstream and downstream of the glaA gene and the pyrG selection marker using the shuttle plasmid pYEP352 as the backbone using yeast transformation-associated recombination (TAR) technology.

[0018] (3) preparing a protoplast isotonic enzymatic hydrolyzate to obtain fresh Aspergillus niger protoplasts;

[0019] (4) Using the above shuttle vector, the lovB-lovC-lovG-lovA gene cluster was integrated into the glaA locus of the Aspergillus niger genome, and the pyrG selection marker was complemented to screen for Aspergillus niger strains that produced Monacolin J;

[0020] (5) Fermentation detection of Monacolin J production.

[0021] The method for producing Monacolin J by fermentation using the above-mentioned Aspergillus niger strain comprises the following specific steps:

[0022] (1) Purify a single asporous Aspergillus niger colony on solid culture medium, pick a small amount of hyphae and culture them statically in liquid culture medium at 30°C until small white hyphae balls can be seen suspended in the clarified culture medium;

[0023] (2) 7 ml to 10 ml of the static liquid culture was added to the fermentation medium and fermented at 30°C and 220 rpm for 7 to 10 days. The fermentation supernatant was then collected and monacolin J was detected using HPLC.

[0024] The liquid culture medium for static culture of aspergillus niger comprises: 2-3% glucose, 0.3-0.6% NaNO3, 0.1-0.3% KCl, 0.1-0.2% KH2PO4, 0.05-0.2% MgSO4·7H2O, 0.001-0.005% FeSO4·7H2O, pH 5.5, and the solvent is water.

[0025] Preferably, the composition is: glucose 2%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.001%, pH 5.5, and the solvent is water.

[0026] The fermentation medium comprises: 3-6% corn starch, 2-4% corn steep liquor, 1.5-3% soybean meal powder, and water as solvent, preferably: 5% corn starch, 3% corn steep liquor, 2% soybean meal powder, and water as solvent.

[0027] The above percentages are all mass percentages.

[0028] Exploration of the optimal carbon source in the fermentation medium: selecting one of corn starch, glucose, sucrose, and maltose as the carbon source, and fermenting to detect the yield of Monacolin J.

[0029] The exogenous additives in the fermentation medium were investigated: one of glutamic acid, methionine, linoleic acid, and sodium citrate was added, and the Monacolin J production was detected by fermentation.

[0030] The advantages and effects achieved by the present invention are:

[0031] (1) The present invention uses yeast transformation-coupled recombination technology (TAR) to assemble multiple fragments and rapidly construct a macromolecular vector (26.83 kbp) with a series of gene expression cassettes, ensuring the coherence and integrity of the Monacolin J synthetic gene cluster, which has reference significance for heterologous reconstruction of secondary metabolite gene clusters.

[0032] (2) The present invention realizes the integration of four target genes and one screening marker into the Aspergillus niger genome at one time, providing a reliable method for simplifying the construction steps of filamentous fungal engineering bacteria.

[0033] (3) When corn starch is used as a carbon source, the yield of Monacolin J in the Aspergillus niger strain of the present invention is better than that when glucose, sucrose, and maltose are used as carbon sources; after adding 10 mM glutamic acid, 0.18% (m / v) methionine, and 520 μmol / L linoleic acid, respectively, the yield of Monacolin J can be increased by 2.94 to 3.49 times. The maximum yield of Monacolin J in shake flask fermentation with 100 mL fermentation medium can reach 1605.6 mg / L, which has reference significance for the fermentation conditions of lovastatin and the improvement of its yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a map of the lovB expression plasmid pYEP-LZ-B constructed in the present invention.

[0035] Figure 2 This is the enzyme digestion verification of the lovB expression plasmid pYEP-LZ-B in the present invention.

[0036] Figure 3 This is a map of the lovC expression plasmid pMD-LC constructed in the present invention.

[0037] Figure 4 This is a map of the lovG expression plasmid pMD-LG constructed in the present invention.

[0038] Figure 5 This is a map of the lovA expression plasmid pMD-LA constructed in the present invention.

[0039] Figure 6 This is a product map of PCR amplification of the lovC, lovG, and lovA expression cassettes constructed in the present invention.

[0040] Figure 7 This is a map of the lovBCGA expression plasmid pYEP-L-BCGA constructed in the present invention.

[0041] Figure 8 This is the double enzyme digestion verification of the lovBCGA expression plasmid pYEP-L-BCGA in the present invention.

[0042] Figure 9 This is the protoplast of Aspergillus niger under a microscope.

[0043] Figure 10 These are the Aspergillus niger protoplast transformation plate and the control plate.

[0044] Figure 11 Schematic diagram of the integration of the lovBCGA expression cassette and the ANpyrG selection marker by homologous recombination at the glaA site of the Aspergillus niger genome.

[0045] Figure 12HPLC analysis of Monacolin J in Aspergillus niger fermentation broth.

[0046] Figure 13 Effects of different carbon sources on the yield of Monacolin J fermented by Aspergillus niger.

[0047] Figure 14 The effect of different exogenous additives on the production of Monacolin J by Aspergillus niger fermentation. DETAILED DESCRIPTION

[0048] The present invention will be described in further detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0049] Unless otherwise specified, the materials used in the present invention are all commercially available products. Unless otherwise specified, the methods used in the present invention were performed according to conventional laboratory procedures.

[0050] A highly efficient heterologous synthesis of monacolin J in Aspergillus niger is achieved by heterologously reconstructing the polyketide synthase gene lovB, enoyl reductase gene lovC, thioesterase gene lovG, and cytochrome P450 monooxygenase gene lovA, all of which are regulated by a strong filamentous fungal promoter and terminator, at the glaA locus of the Aspergillus niger genome. The glaA gene is knocked out by utilizing 1500 bp of homologous sequences upstream and downstream of the glaA gene, as shown in SEQ NOs. 1 and 2.

[0051] The NCBI database sequence registration number of the DNA sequence of the lovB gene is AF151722, and the sequence is SEQ NO.3 and DNA sequences with a similarity of more than 70% thereto; the NCBI database sequence registration number of the DNA sequence of the lovC gene is AAD34554, and the sequence is SEQ NO.4 and DNA sequences with a similarity of more than 70% thereto; the FungiDB database sequence registration number of the DNA sequence of the lovG gene is ATEG_09960, and the sequence is SEQ NO.5 and DNA sequences with a similarity of more than 70% thereto; the NCBI database sequence registration number of the DNA sequence of the lovA gene is AAD34552, and the sequence is SEQ NO.6 and DNA sequences with a similarity of more than 70% thereto.

[0052] The amino acid sequence encoded by the gene lovB has an NCBI database sequence registration number of AAD39830, and its sequence is SEQ NO.7 and amino acid sequences with a similarity of more than 80% thereto; the amino acid sequence encoded by the gene lovC has an NCBI database sequence registration number of AAD34554, and its sequence is SEQ NO.8 and amino acid sequences with a similarity of more than 80% thereto; the amino acid sequence encoded by the gene lovG has an NCBI database sequence registration number of XP_001209262, and its sequence is SEQ NO.9 and amino acid sequences with a similarity of more than 80% thereto; and the amino acid sequence encoded by the gene lovA has an NCBI database sequence registration number of AAD34552, and its sequence is SEQ NO.10 and amino acid sequences with a similarity of more than 80% thereto.

[0053] The starting strain of Aspergillus niger (Aspergillus niger SH2ΔkusAΔpyrG) used to produce Monacolin J in the present invention is disclosed in the document "Dong Hongzhi. Establishment of CRISPR gene editing technology in Aspergillus niger and study of endoplasmic reticulum phospholipid regulation mechanism [D]. South China University of Technology." Chemical reagents and drugs involved in the present invention: Takara Premix Primer STARHSDNA Polymerase, Takara pMD18-T vector, Takara In-Fusion HD Cloning kits, ThermoFisher Scientific Restriction Enzymes PCR product recovery kit (Guangzhou Meiji), Yeared nucleic acid dye (Yisheng).

[0054] Example 1-Construction of lovB expression plasmid pYEP-LZ-B:

[0055] Using the genome of Aspergillus terreus ATCC 20542 (purchased from Baosai Biological Company) (standard strain) as a template, primer pairs lovB-up-F, lovB-up-R and lovB-dn-F, lovB-dn-R were designed, and PCR amplification and recovery of lovB-up (4563 bp) and lovB-dn (5000 bp) fragments were obtained. Using the genome of Aspergillus niger CBS 513.88 (purchased from Baosai Biological Company) (standard strain) as a template, primer pairs 352-glaA-up-F, 352-glaA-up-R and 352-glaA-dn-F, 352-glaA-dn-R were designed, and PCR amplification and recovery of 1500 bp upstream and downstream homology arms of the glaA gene were obtained. Primer pairs 352-ANpyrG-F, 352-ANpyrG-R were designed, and PCR amplification and recovery of the ANpyrG selection marker were obtained. Plasmid pFC330 (Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9.PLoS One.2017.) was used as a template to design primer pairs 352-Ttef-F and 352-Ttef-R, and the terminator Ttef was recovered by PCR amplification. pYEP352 (Cloning and expression of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field.BioresourTechnol.2009.) was linearized with XbaⅠ and then transformed into yeast BY4741 protoplasts with the above six fragments to construct plasmid pYEP-LZ-B ( Figure 1 Yeast transformants can ligate the fragment to the linearized plasmid with the Ura3 marker to form a circular plasmid. Transformants carrying the correctly linked plasmid can then grow on SD-Ura-deficient medium. Plasmid enzyme digestion verification revealed three XhoⅠ restriction enzyme sites on the plasmid. Enzyme digestion revealed three bands, sized 12458 bp, 5851 bp, and 1304 bp, respectively. The results of enzyme electrophoresis are shown below. Figure 2 shown.

[0056] Example 2-Construction of lovC expression plasmid pMD-LC:

[0057] Using the genome of Aspergillus nidulans FGSC A4 (purchased from Baosai Biotechnology Co., Ltd.) (standard strain) as a template, the primer pair 18T-TtrpC-F and 18T-TtrpC-R were designed, and the terminator TtrpC was recovered by PCR amplification; using the genome of Aspergillus terreus ATCC 20542 as a template, the primer pair 18T-lovC-F and 18T-lovC-R were designed, and the lovC fragment was recovered by PCR amplification; using the genome of Aspergillus niger CBS513.88 (purchased from Baosai Biotechnology Co., Ltd.) as a template, the primer pair 18T-PgpdA-F and 18T-PgpdA-R were designed, and the promoter PgpdA was recovered by PCR amplification. TtrpC, PgpdA, and lovC were sequentially ligated to the vector pMD-18T using the In-Fusion seamless cloning technology, and the ligation products were transformed into Escherichia coli Match. T1 competent cells were plated on LB solid medium containing 100 μg / mL ampicillin, and single clones were picked to construct the plasmid pMD-LC ( Figure 3 ).

[0058] Example 3-Construction of lovG expression plasmid pMD-LG:

[0059] Using the Aspergillus nidulans FGSC A4 genome as a template, the primer pair 18T-TagdA-F and 18T-TagdA-R were designed, and the terminator TagdA was recovered by PCR amplification. Using the Aspergillus terreus ATCC 20542 genome as a template, the primer pair 18T-lovG-F and 18T-lovG-R were designed, and the lovG fragment was recovered by PCR amplification. Using the plasmid pFC330 as a template, the primer pair 18T-Ptef-F and 18T-Ptef-R were designed, and the promoter Ptef was recovered by PCR amplification. TagdA, Ptef, and lovG were sequentially ligated into the vector pMD-18T using the In-Fusion seamless cloning technology. The ligation products were transformed into Escherichia coli Match T1 competent cells and plated on LB solid medium containing 100 μg / mL ampicillin. Single colonies were picked to construct the plasmid pMD-LG ( Figure 4 ).

[0060] Example 4-Construction of lovA expression plasmid pMD-LA:

[0061] The Aspergillus fumigatus Af293 (purchased from Baosai Biotechnology Co., Ltd.) (standard strain) genome was used as a template, and primer pairs 18T-TxlnA-F and 18T-TxlnA-R were designed. The terminator TxlnA was recovered by PCR amplification. The Aspergillus terreus ATCC Using the 20542 genome as a template, the primer pair 18T-lovA-F and 18T-lovA-R were designed, and the lovA fragment was recovered by PCR amplification. Using the plasmid pMT1802 as a template, the primer pair 18T-PⅡ-F and 18T-PⅡ-R were designed, and the promoter PnaⅡ / TPI was recovered by PCR amplification. Using the plasmid pFC330 as a template, the primer pair 18T-Ttef-F and 18T-Ttef-R were designed, and the terminator Ttef was recovered by PCR amplification. TxlnA, PnaⅡ / TPI, lovA, and Ttef were sequentially ligated into the vector pMD-18T using the In-Fusion seamless cloning technology. The ligation products were transformed into Escherichia coli Match T1 competent cells and plated on LB solid medium containing 100 μg / mL ampicillin. Single colonies were picked to construct the plasmid pMD-LA ( Figure 5 ).

[0062] Example 5-Construction method of lovBCGA expression plasmid pYEP-L-BCGA:

[0063] Using plasmid pMD-LC as template, primer pair 352-LC-F and 352-LC-R were designed, and the TtrpC-PgpdA-lovC fragment (2818 bp) was recovered by PCR amplification. Using plasmid pMD-LG as template, primer pair 352-LG-F and 352-LG-R were designed, and the TagdA-Ptef-lovG fragment (2037 bp) was recovered by PCR amplification. Using plasmid pMD-LA as template, primer pair 352-LA-F and 352-LA-R were designed, and the TxlnA-PnaⅡ / TPI-gpdA-lovA-Ttef fragment (2474 bp) was recovered by PCR amplification. Figure 6 ), the above three fragments were sequentially transformed with linearized pYEP-LZ-B into yeast BY4741 protoplasts to construct plasmid pYEP-L-BCGA ( Figure 7 ). Enzyme digestion verification of pYEP-L-BCGA ( Figure 8 ).

[0064] Example 6 - Construction of an Aspergillus niger strain for heterologous synthesis of Monacolin J:

[0065] (1) Filter the Aspergillus niger cells cultured in DPY medium for 48 h using a Büchner funnel containing two layers of filter paper, and remove the medium. Wash the cells twice with sterile water and sterile 0.8 M NaCl solution, filter and dry them, and then scrape 0.6-0.8 g of mycelium with a spatula and add it to 20 mL of enzymatic hydrolysis solution. Hydrolyze in a shaking water bath at 30°C for 3 h.

[0066] (2) The enzymatic hydrolysate was filtered using a four-layer ultrafiltration cloth, and the filter cloth was rinsed with 20 mL of 0.8 M NaCl solution. The filtrate was collected in a 100 mL centrifuge tube and centrifuged at 4°C 600 × g for 10 min. The supernatant was discarded and placed on ice. The following steps were performed: 20 mL of STC solution was added, and the suspension was gently resuspended with a sharpened pipette tip, and centrifuged at 4°C 900 × g for 10 min; the supernatant was discarded and the previous step was repeated; the supernatant was discarded, and the precipitate was gently dispersed with 600 μL of STC solution on ice. The protoplast quality was observed under a microscope ( Figure 9 ).

[0067] (3) Place 160 μL of protoplasts in a 2 mL EP tube. One tube each for the transformation group, positive control group, and negative control group. Add 100 μg of the donor fragment used for transformation and integration to the transformation group and an equal volume of STC solution to the control group. Add 60 μL of PEG solution to each group, mix well, and incubate on ice for 30 min. Add 1.5 mL of PEG solution and incubate at room temperature for 25 min.

[0068] (4) Mix the liquid in the 2mL EP tube with 3mL STC solution and 6mL hypertonic CD medium (containing 0.7-0.8% agar) and pour it on the pre-prepared hypertonic CD medium. 10mM uridine should be added to the hypertonic CD medium of the positive control group. Culture at 30℃ for 5 days ( Figure 10 ).

[0069] (5) Single colonies growing on the Aspergillus niger transformation plate were picked onto CD solid medium. After observing their growth, a small amount of mycelium was scraped into a 24-well plate, DPY liquid medium was added, and cultured in a shaking incubator at 30°C for 2 days.

[0070] (6) Grind the mycelial pellet of the transformant, extract its genome, design primer pairs JD-up-F, JD-up-R and JD-dn-F, JD-dn-R, perform PCR verification, and screen out positive transformants that have successfully integrated the lovBCGA gene and pyrG selection marker. The principle of PCR verification is as follows Figure 11 shown.

[0071] DPY liquid medium for shake flask culture of Aspergillus niger: 2% glucose, 1% yeast extract, 1% peptone, 0.5% KH2PO4, and 0.5% MgSO4·7H2O. The solvent is water. All percentages are by mass.

[0072] STC solution: 1.2 mM sorbitol, 10 mM Tris-HCl, 50 mM anhydrous calcium chloride, pH 7.5.

[0073] PEG solution: PEG4000 60% (w / v), 10 mM Tris-HCl, 50 mM anhydrous calcium chloride, pH 7.5.

[0074] Hypertonic CD medium: sucrose 35%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.001%, pH 5.5. All percentages are by mass.

[0075] CD culture medium: glucose 2%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.001%, pH 5.5. All percentages are by mass.

[0076] Example 7 - Method for producing Monacolin J by fermentation using the above-mentioned Aspergillus niger strain:

[0077] (1) Purify a single asporous Aspergillus niger colony on solid culture medium, pick a small amount of hyphae and culture them statically in CD liquid medium at 30°C until small white hyphae balls can be seen suspended in the clarified medium;

[0078] (2) Take 7ml-10ml of the liquid culture medium and add it to the fermentation medium. Ferment it at 30℃ and 220rpm for 7-10 days. Then take the fermentation supernatant and use HPLC technology to detect Monacolin J ( Figure 12 ).

[0079] HPLC detection conditions: using LC Column (250×4.6 mm 5 μm), injection volume 10 μL, mobile phase methanol: water (containing 0.3% phosphoric acid) = 75:25, isocratic elution, UV detection wavelength 237 nm, flow rate 1 mL / min, detection time 15 min.

[0080] Fermentation medium: corn starch 5%, corn steep liquor 3%, soybean meal 2%, solvent is water;

[0081] Example 8 - Optimization of Aspergillus niger Monacolin J fermentation medium:

[0082] (1) Exploration of the optimal carbon source in the fermentation medium: corn starch, glucose, sucrose, and maltose were selected as the carbon source, with a concentration of 5% (w / v). The yield of Monacolin J was detected by fermentation. Figure 13 ).

[0083] (2) Investigation of exogenous additives in the fermentation medium: 10 mM glutamic acid, 0.18% (w / v) methionine, 520 μmol / L linoleic acid, 0.1% sodium citrate and 0.1% sodium acetate were added to the fermentation medium, and the changes in Monacolin J production were detected by fermentation ( Figure 14 ).

[0084] In summary, we can draw the conclusion:

[0085] (1) The use of yeast transformation-coupled recombination technology (TAR) to assemble multiple fragments can quickly construct a large molecular vector with a series of gene expression cassettes, ensuring the coherence and integrity of the secondary metabolite synthesis gene cluster, which is more conducive to product synthesis and saves the Aspergillus niger transformation step and the use of screening markers.

[0086] (2) Using hydrolyzed starch, soybean meal, and corn steep liquor as fermentation media, an Aspergillus niger strain harboring the Monacolin J expression cassette was fermented in a 500 mL shake flask for 10 days to produce 459.7 mg / L of Monacolin J. This is 2.0 times the concentration when glucose was used as the carbon source (236.0 mg / L), 2.5 times the concentration when sucrose was used as the carbon source (183.9 mg / L), and comparable to the concentration when maltose was used as the carbon source (453.8 mg / L). The mass percentage of the carbon source was 5% in all cases. Addition of 10 mM glutamate, 0.18% (w / v) methionine, 520 μmol / L linoleic acid, 0.1% sodium citrate, and 0.1% sodium acetate to hydrolyzed starch and maltose fermentation media, respectively, revealed that the addition of glutamate, methionine, or linoleic acid alone increased monacolin J production, particularly in the hydrolyzed starch medium. The enhanced monacolin J yield in the hydrolyzed starch medium was approximately twice that of the maltose medium, reaching a maximum of 1605.6 mg / L. However, the addition of sodium citrate and sodium acetate decreased monacolin J production. Even with the simultaneous addition of sodium citrate, sodium acetate, and methionine, monacolin J production in the hydrolyzed starch medium remained low. This suggests that using a slow-release carbon source is more suitable for basic fermentation media and subsequent fermentation optimization; the addition of sodium citrate, sodium acetate, and other sources may enhance the TCA cycle of Aspergillus niger, thereby inhibiting growth and production.

[0087] The embodiments of the present invention are not limited to the contents disclosed in the above embodiments. Any changes, substitutions, and modifications that do not depart from the spirit and essence of the present invention are equivalent replacement methods and are within the scope of protection of the present invention.

[0088] Primer sequence list used

[0089]

[0090]

[0091]

[0092] The above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J, characterized in that: It was obtained by heterologously reconstructing the polyketide synthase gene lovB, enoyl reductase gene lovC, thioesterase gene lovG, and cytochrome P450 monooxygenase gene lovA in the synthesis pathway of the natural secondary metabolite lovastatin of Aspergillus Terreus, which are regulated by a strong promoter and strong terminator of filamentous fungi, on the Aspergillus niger genome.

2. The method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J according to claim 1, characterized in that: The steps include: (1) Using Saccharomyces cerevisiae homologous recombination technology or In-Fusion seamless cloning technology, the corresponding promoter, terminator and the gene regulated by them are connected to the pMD-18T vector, and the expression cassette of a single heterologous secondary metabolic gene is obtained by PCR amplification; (2) In Saccharomyces cerevisiae BY4741, the yeast transformation-associated recombination (TAR) technique was used to connect the single gene expression cassettes of lovB, lovC, lovG, and lovA with 1500 bp of homologous sequences upstream and downstream of the glaA gene and the pyrG selection marker using the shuttle plasmid pYEP352 as the backbone to obtain the shuttle vector for the Monacolin J synthetic gene cluster; (3) preparing a protoplast isotonic enzymatic hydrolyzate to obtain fresh Aspergillus niger protoplasts; (4) using the shuttle vector, integrating the lovB-lovC-lovG-lovA gene cluster at the glaA site of the Aspergillus niger genome, complementing the pyrG selection marker, and screening to obtain a Monacolin J-producing Aspergillus niger strain; (5) Fermentation detection of Monacolin J production.

3. A method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J according to claim 1 or 2, characterized in that: The DNA sequence of lovB is SEQ ID NO: 2; the DNA sequence of lovC is SEQ ID NO: 4; the DNA sequence of lovG is SEQ ID NO: 5; the DNA sequence of lovA is SEQ ID NO: 6; The amino acid sequence encoded by lovB is SEQ ID NO: 7; the amino acid sequence encoded by lovC is SEQ ID NO: 8; the amino acid sequence encoded by lovG is SEQ ID NO: 9; and the amino acid sequence encoded by lovA is SEQ ID NO:

10.

4. The method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J according to claim 2, characterized in that: The promoter used to regulate the transcription of the lovB gene is the Aspergillus niger saccharifying enzyme gene promoter PglaA, and the terminator is the Aspergillus nidulans tryptophan synthase gene terminator TtrpC; the promoter used to regulate the transcription of the lovC gene is the Aspergillus niger 3-phosphoglyceraldehyde dehydrogenase gene promoter PgpdA, and the terminator is the Aspergillus nidulans α-glucosidase gene terminator TagdA; the promoter used to regulate the transcription of the lovG gene is the plasmid pFC330 promoter Ptef, and the terminator is the Aspergillus fumigatus 1,4-β-xylan endonuclease gene terminator TxlnA; the promoter used to regulate the transcription of the lovA gene is the plasmid pMT1802 composite promoter PnaⅡ / TPI, and the terminator is the plasmid pFC330 terminator Ttef.

5. The method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J according to claim 2, characterized in that: The Aspergillus niger starting strain is a ΔkusAΔpyrG mutant strain; the saccharifying enzyme gene glaA is knocked out by utilizing the upstream and downstream 1500bp homologous sequences of the glaA gene, and the Aspergillus niger glaA gene expression cassette is replaced with the lovB-lovC-lovG-lovA gene cluster by homologous recombination.

6. The Aspergillus niger strain obtained by the method for constructing an Aspergillus niger strain for efficient heterologous synthesis of Monacolin J according to claim 1 is used for fermentation production of Monacolin J, characterized in that: The steps include: (1) Purify a single asporous Aspergillus niger colony on solid culture medium, pick a small amount of hyphae and culture them statically in liquid culture medium at 30°C until small white hyphae balls can be seen suspended in the clarified culture medium; (2) 7 ml to 10 ml of the static liquid culture was added to the fermentation medium and fermented at 30°C and 220 rpm for 7 to 10 days. The fermentation supernatant was then collected and monacolin J was detected using HPLC.

7. The use according to claim 6, characterized in that The liquid culture medium for static culture of aspergillus niger comprises, by mass percentage, 2-3% glucose, 0.3-0.6% NaNO3, 0.1-0.3% KCl, 0.1-0.2% KH2PO4, 0.05-0.2% MgSO4·7H2O, 0.001-0.005% FeSO4·7H2O, pH 5.5, and the solvent is water.

8. The use according to claim 6, characterized in that The fermentation medium comprises, by mass percentage, 3-6% corn starch, 2-4% corn steep liquor, 1.5-3% soybean meal powder, and water as the solvent.

9. The use according to claim 6, characterized in that The carbon source in the fermentation medium is selected from one of corn starch, glucose, sucrose and maltose.

10. The use according to claim 6, characterized in that The exogenous additive in the fermentation medium is selected from one of glutamic acid, methionine, linoleic acid and sodium citrate.

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