Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method and application of an Aspergillus niger strain for efficiently synthesizing Monacolin J in a heterologous manner.
The aim of the invention is achieved by the following technical scheme:
An aspergillus niger strain (Aspergillus niger) for the efficient heterologous synthesis of Monacolin J, which is obtained by heterologous reconstitution of the polyketide synthase gene lovB, the enoyl reductase gene lovC, the thioesterase gene lovG and the cytochrome P450 monooxygenase gene lovA on the lovastatin synthesis pathway of the natural secondary metabolite of aspergillus terreus (Aspergillus Terreus) regulated by a strong promoter and a strong terminator of a filamentous fungus on the genome of aspergillus niger.
The NCBI database sequence of the DNA sequence of the gene lovB is accession number AF151722, the sequence of the NCBI database is SEQ ID NO. 2, the NCBI database sequence of the DNA sequence of the gene lovC is accession number AAD34554, the sequence of the NCBI database is SEQ ID NO. 4, the FungiDB database sequence of the DNA sequence of the gene lovG is accession number ATEG _09960, the sequence of the NCBI database is accession number AAD34552, and the sequence of the NCBI database is SEQ ID NO. 6.
The NCBI database sequence registration number of the amino acid sequence coded by the gene lovB is AAD39830, the sequence is SEQ ID NO. 7, the NCBI database sequence registration number of the amino acid sequence coded by the gene lovC is AAD34554, the sequence is SEQ ID NO. 8, the NCBI database sequence registration number of the amino acid sequence coded by the gene lovG is XP_001209262, the sequence is SEQ ID NO. 9, the NCBI database sequence registration number of the amino acid sequence coded by the gene lovA is AAD34552, and the sequence is SEQ ID NO. 10.
The promoter for regulating the transcription of the gene lovB is an Aspergillus niger saccharifying enzyme gene promoter PglaA, the terminator is an Aspergillus nidulans tryptophan synthase gene terminator TtrpC, the promoter for regulating the transcription of the gene lovC is an Aspergillus niger 3-glyceraldehyde phosphate dehydrogenase gene promoter PgpdA, the terminator is an Aspergillus nidulans alpha-glucosidase gene terminator TagdA, the promoter for regulating the transcription of the gene lovG is a plasmid pFC330 promoter Ptef, the terminator is an Aspergillus fumigatus 1, 4-beta-xylanase gene terminator TxlnA, the promoter for regulating the transcription of the gene lovA is a plasmid pMT1802 composite promoter PnaII/TPI, and the terminator is a plasmid pFC330 terminator Ttef.
The Aspergillus niger original strain is a delta kusA delta pyrG mutant strain, and is disclosed in a literature Dong Hongzhi, a research [ D ] of a regulating mechanism of endoplasmic reticulum phospholipid, established by a CRISPR gene editing technology of Aspergillus niger, university of North China.
The knocked-out glucoamylase gene glaA utilizes the upstream and downstream 1500bp homologous sequences of the glaA gene, and replaces the Aspergillus niger glaA gene expression cassette with a lovB-lovC-lovG-lovA gene cluster in a homologous recombination mode.
The screening marker used was ANpyrG.
The construction method of the invention specifically comprises the following steps:
(1) And connecting corresponding promoters, terminators and genes regulated by the promoters and terminators on a pMD-18T vector by using a Saccharomyces cerevisiae homologous recombination technology or an In-Fusion seamless cloning technology, and carrying out PCR amplification to obtain an expression frame of a single heterologous secondary metabolic gene.
(2) In Saccharomyces cerevisiae BY4741, a shuttle plasmid pYEP352 is taken as a framework BY utilizing a yeast Transformation coupling recombination technology (Transformation-associated recombination, TAR), and a single gene expression frame of lovB, lovC, lovG, lovA is connected with a homologous sequence of 1500bp on the upstream and downstream of the glaA gene and a pyrG screening marker to obtain a shuttle vector of a Monacolin J synthetic gene cluster;
(3) Preparing protoplast isotonic enzymolysis liquid to obtain fresh aspergillus niger protoplast;
(4) Integrating lovB-lovC-lovG-lovA gene cluster on glaA locus of the genome of Aspergillus niger by using the shuttle vector, and supplementing pyrG screening marker, and screening to obtain Aspergillus niger strain producing Monacolin J;
(5) And (5) fermenting and detecting Monacolin J yield.
The method for producing Monacolin J by fermenting the Aspergillus niger strain comprises the following specific steps:
(1) Purifying on a solid culture medium to obtain single spore-free Aspergillus niger colony, picking a small amount of mycelia, standing and culturing in a liquid culture medium at 30 ℃ until obvious white mycelium pellets are suspended in a clear culture medium;
(2) Taking 7ml-10ml from the stationary liquid culture solution, fermenting at 30deg.C and 220rpm for 7-10days to obtain fermentation supernatant, and detecting Monacolin J by HPLC technique.
The liquid culture medium for stationary culture of the aspergillus niger is glucose 2～3％,NaNO3 0.3～0.6％,KCl0.1～0.3％,KH2PO4 0.1～0.2％,MgSO4·7H2O 0.05～0.2％,FeSO4·7H2O 0.001～0.005％,pH 5.5, solvent which is water.
Preferably, the glucose 2％,NaNO3 0.3％,KCl 0.2％,KH2PO4 0.1％,MgSO4·7H2O 0.05％,FeSO4·7H2O0.001％,pH 5.5, solvent is water.
The fermentation medium comprises 3-6% of corn starch, 2-4% of corn steep liquor, 1.5-3% of soybean meal and water as a solvent. Preferably, the corn starch is 5%, the corn steep liquor is 3%, the soybean meal is 2%, and the solvent is water.
The percentages are mass percentages.
The exploration of the optimal carbon source in the fermentation medium comprises the steps of selecting one of corn starch, glucose, sucrose and maltose as the carbon source, and fermenting and detecting the yield of Monacolin J.
The research of the external additive in the fermentation medium comprises the steps of adding one of glutamic acid, methionine, linoleic acid and sodium citrate, and fermenting and detecting the yield of Monacolin J.
The invention has the advantages and the obtained effects that:
(1) The invention adopts a yeast transformation coupling recombination (TAR) technology to assemble the multi-fragment, rapidly constructs a macromolecular carrier (26.83 kbp) with gene expression frames connected in series, ensures the continuity and the integrity of Monacolin J synthetic gene clusters, and has reference significance on the heterologous reconstruction secondary metabolite gene clusters.
(2) The invention realizes the integration of 4 target genes and 1 screening mark on the genome of Aspergillus niger at one time, and provides a reliable method for simplifying the construction steps of the engineering fungus of the filamentous fungus.
(3) According to the invention, when corn starch is used as a carbon source, monacolin J yield is superior to that of glucose, sucrose and maltose, and after 10mM glutamic acid, 0.18% (m/v) methionine and 520 mu mol/L linoleic acid are respectively added, monacolin J yield can be improved by 2.94-3.49 times, and the highest Monacolin J yield can reach 1605.6mg/L when 100mL fermentation medium is subjected to shake flask fermentation, so that the Monacolin J yield has reference significance for improving the fermentation condition and yield of lovastatin.
Detailed Description
The invention is described in further detail below in connection with examples, but embodiments of the invention are not limited thereto.
The materials used in the present invention are commercially available products unless otherwise specified. The method used in the present invention is carried out according to the conventional laboratory procedures unless otherwise specified.
An aspergillus niger strain (Aspergillus niger) for the efficient heterologous synthesis of Monacolin J, said strain being obtained by heterologous reconstitution of the polyketide synthase gene lovB, the enoyl reductase gene lovC, the thioesterase gene lovG and the cytochrome P450 monooxygenase gene lovA on the natural secondary metabolite lovastatin synthesis pathway of aspergillus terreus (Aspergillus Terreus) regulated by a strong promoter and a strong terminator of a filamentous fungus on the glaA locus of the saccharifying enzyme gene of the genome of aspergillus niger. The knockout saccharifying enzyme gene glaA utilizes the upstream and downstream 1500bp homologous sequences of the glaA gene, and the sequences are SEQ NO.1 and SEQ NO.2.
The NCBI database of the DNA sequence of the gene lovB is provided with the sequence registration number of AF151722, the sequence of SEQ NO.3 and the DNA sequence with more than 70 percent of similarity, the NCBI database of the DNA sequence of the gene lovC is provided with the sequence registration number of AAD34554, the sequence of SEQ NO.4 and the DNA sequence with more than 70 percent of similarity, the FungiDB database of the DNA sequence of the gene lovG is provided with the sequence registration number of ATEG _09960, the sequence of SEQ NO.5 and the DNA sequence with more than 70 percent of similarity, and the NCBI database of the DNA sequence of the gene lovA is provided with the sequence registration number of AAD34552, and the sequence of SEQ NO.6 and the DNA sequence with more than 70 percent of similarity.
The NCBI database sequence of the amino acid sequence coded by the gene lovB is provided with a registration number of AAD39830, the sequence is SEQ NO.7 and the amino acid sequence with the similarity of more than 80%, the NCBI database sequence of the amino acid sequence coded by the gene lovC is provided with a registration number of AAD34554, the sequence is SEQ NO.8 and the amino acid sequence with the similarity of more than 80%, the NCBI database sequence of the amino acid sequence coded by the gene lovG is provided with a registration number of XP_001209262, the sequence is SEQ NO.9 and the amino acid sequence with the similarity of more than 80%, the NCBI database sequence of the amino acid sequence coded by the gene lovA is provided with a registration number of AAD34552, and the sequence is SEQ NO.10 and the amino acid sequence with the similarity of more than 80%.
The starting strain of Aspergillus niger (Aspergillus niger SH. DELTA. kusA. DELTA.pyrG) for producing Monacolin J in the present invention is disclosed in the literature "Dong Hongzhi. Research on the mechanism of endoplasmic reticulum phospholipid regulation established by the Aspergillus niger CRISPR gene editing technique [ D ]. University of North America. Chemical reagents and medicines of the invention ：Takara Premix Primer STAR HSDNA Polymerase、Takara pMD18-T vector、Takara In-Fusion HD Cloning kits、ThermoFisher Scientific Restriction EnzymesPCR product recovery kit (Guangzhou Mei-based), yeared nucleic acid dye (next holy).
Example 1-construction of lovB expression plasmid pYEP-LZ-B method:
The plasmid map was constructed BY designing primer pairs 352-glaA-up-F, 352-glaA-up-R and 352-glaA-dn-F, 352-glaA-dn-R, PCR amplification recovery to obtain a homologous arm of 1500bp upstream of the glaA gene, PCR amplification recovery to obtain a selection marker of lovB-up (4563 bp) and lovB-dn (5000 bp), PCR amplification recovery to obtain a selection marker of ANpyrG, PCR amplification to obtain a linear plasmid conversion map of 352-Ttef-F, 352-Ttef-R, and LbZ-35 BY using the genome of Aspergillus niger CBS 513.88 (purchased from Barceichco) (standard strain), designing primer pairs 352-glaA-up-F, 352-glaA-up-R and 352-glaA-dn-F, 352-glaA-dn-R, PCR amplification recovery to obtain a homologous arm of 1500bp upstream of the glaA gene, PCR amplification recovery to obtain a selection marker of ANpyrG, PCR amplification to obtain a linear plasmid conversion map of 352-Ttef-F, 352-Ttef-R, and PCR amplification to obtain a linear plasmid conversion map of LbZ-35 (BY 35-35) using the plasmid). Yeast transformants can ligate fragments with linearized Ura 3-tagged plasmids into circular plasmids, so that transformants carrying the correctly ligated plasmids can be grown on SD-Ura deficient medium. Plasmid restriction enzyme digestion verification is carried out, three XhoI restriction enzyme sites are arranged on the plasmid, three bands are respectively cut, the sizes of the restriction enzyme sites are 12458bp, 5851bp and 1304bp, and the result of enzyme digestion electrophoresis is shown in figure 2.
Examples 2-lovC construction method of expression plasmid pMD-LC:
The method comprises the steps of taking the genome of aspergillus nidulans FGSC A4 (purchased from Barceichhorns) as a template, designing a primer pair of 18T-TtrpC-F and 18T-TtrpC-R, carrying out PCR amplification and recovery to obtain a terminator TtrpC, taking the genome of aspergillus terreus ATCC 20542 as the template, designing a primer pair of 18T-lovC-F and 18T-lovC-R, carrying out PCR amplification and recovery to obtain a lovC fragment, taking the genome of aspergillus niger CBS 513.88 (purchased from Barceichhorns) as the template, designing a primer pair of 18T-PgpdA-F and 18T-PgpdA-R, carrying out PCR amplification and recovery to obtain a promoter PgpdA, sequentially connecting TtrpC, pgpdA, lovC to a vector pMD-18T by using an In-Fusion seamless cloning technology, converting a connection product into a competent E.coli Match T1, coating the competent product In LB solid medium containing 100 mu g/mL ampicillin, and picking up a monoclonal clone, and constructing to obtain a plasmid D-pMLC (FIG. 3).
Examples 3-lovG construction method of expression plasmid pMD-LG:
A primer pair 18T-TagdA-F and 18T-TagdA-R are designed by taking an aspergillus nidulans FGSC A4 genome as a template, a terminator TagdA is obtained through PCR amplification and recovery, an aspergillus terreus ATCC 20542 genome as a template, a lovG fragment is obtained through PCR amplification and recovery by designing the primer pair 18T-lovG-F and 18T-lovG-R, a plasmid pFC330 as a template, a promoter Ptef is obtained through PCR amplification and recovery by designing the primer pair 18T-Ptef-F and 18T-Ptef-R, tagdA, ptef, lovG is sequentially connected to a vector pMD-18T by using an In-Fusion seamless cloning technology, a connection product is transformed into escherichia coli Match T1 competence, and the obtained product is coated In an LB solid culture medium containing 100 mu g/mL ampicillin, and a single clone is selected to construct a plasmid pMD-LG (figure 4).
Example 4-construction method of lovA expression plasmid pMD-LA:
A primer pair 18T-TxlnA-F and 18T-TxlnA-R is designed by taking an Aspergillus fumigatus Af293 (purchased from Barceichhorns) genome as a template, a terminator TxlnA is obtained by PCR amplification recovery, a Aspergillus terreus ATCC 20542 genome is taken as a template, a primer pair 18T-lovA-F and 18T-lovA-R is designed by PCR amplification recovery to obtain a lovA fragment, a plasmid pMT1802 is taken as a template, a primer pair 18T-PII-F and 18T-PII-R is designed by taking a PCR amplification recovery to obtain a promoter PnaII/TPI, a plasmid pFC330 is taken as a template, a primer pair 18T-Ttef-F and 18T-Ttef-R is designed by taking a PCR amplification recovery to obtain a terminator Ttef, txlnA and Pna II/TPI, lovA, ttef are sequentially connected to a vector pMD-18T by using an In-Fusion seamless cloning technology, a connection product is transformed into E.coli T1 competence, and the connection product is coated on a solid culture medium containing 100 mu g/mL of ampicillin (pMD is obtained by taking a solid map).
Examples 5-lovBCGA construction method of expression plasmid pYEP-L-BCGA:
The plasmid pMD-LC is used as a template, a primer pair 352-LC-F and 352-LC-R is designed, a TtrpC-PgpdA-lovC fragment (2818 bp) is obtained BY PCR amplification and recovery, the plasmid pMD-LG is used as a template, a primer pair 352-LG-F and 352-LG-R is designed, a TagdA-Ptef-lovG (2037 bp) fragment is obtained BY PCR amplification and recovery, the plasmid pMD-LA is used as a template, the primer pair 352-LA-F and 352-LA-R is designed, a TxlnA-PnaII/TPI-gpdA-lovA-Ttef fragment (2474 bp) is obtained BY PCR amplification and recovery (FIG. 6), and the 3 fragments are sequentially transformed with a linearized pYEP-LZ-B into a yeast BY4741 protoplast to construct a plasmid pYEP-L-BCGA (FIG. 7). The cleavage was performed on pYEP-L-BCGA (FIG. 8).
Example 6-heterologous Synthesis of Monacolin J Aspergillus niger Strain construction method:
(1) The medium was removed by suction filtration of Aspergillus niger which was grown in a shaking flask for 48h in DPY medium using a Buchner funnel containing two layers of filter paper. And respectively using sterile water and sterile 0.8M NaCl solution to soak the thalli twice, and scraping 0.6-0.8 g of mycelium into 20mL of enzymolysis liquid by using a medicine spoon after suction filtration and drying, and carrying out enzymolysis for 3 hours in a 30 ℃ water bath shaking table.
(2) The enzymatic hydrolysate after the enzymatic hydrolysis was filtered using four layers of ultrafiltration cloth, the filter cloth was rinsed with 20mL of 0.8M NaCl solution, and the filtrates were all collected in a 100mL centrifuge tube and centrifuged at 600 Xg for 10min at 4 ℃. The supernatant was discarded and placed on ice by adding 20mL of STC solution, gently resuspending with a tip-removing gun head, centrifuging at 4℃900 Xg for 10min, discarding the supernatant, repeating the above step, discarding the supernatant, gently blowing the pellet with 600. Mu.L of STC solution on ice, and observing the protoplast mass with a microscope (FIG. 9).
(3) 160. Mu.L of protoplast was placed in 2mL EP tubes, one tube each for the transformation group, the positive control group and the negative control group, 100. Mu.g of donor fragment for transformation integration was added to the transformation group, an equal volume of STC solution was added to the control group, and the mixture was mixed with 60. Mu. LPEG solution added to each group, and the mixture was left on ice for 30min. 1.5mL of PEG solution was added and the mixture was left at room temperature for 25min.
(4) The liquid in the 2mL EP tube was mixed with 3mL STC solution, 6mL hypertonic CD medium (containing 0.7-0.8% agar) and poured onto the pre-prepared hypertonic CD medium. The positive control group had 10mM uridine added to hypertonic CD medium. Incubated at 30℃for 5 days (FIG. 10).
(5) Single colonies grown on Aspergillus niger transformation plates were picked up onto CD solid medium, after growth was observed, a small amount of mycelia was scraped off into 24 well plates, DPY liquid medium was added, and shaking culture was performed at 30℃for 2 days.
(6) Grinding mycelium pellet of transformant, extracting genome, designing primer pair JD-up-F, JD-up-R and JD-dn-F, JD-dn-R, PCR verifying and screening positive transformant with successfully integrated lovBCGA gene and pyrG screening mark. The principle of PCR verification is shown in FIG. 11.
DPY liquid culture medium for Aspergillus niger shake flask culture comprises glucose 2%, yeast extract 1%, peptone 1%, KH2PO4 0.5％,MgSO4.7H2O 0.5%. The solvent is water. The percentages are mass percentages.
STC solution, 1.2mM sorbitol, 10mM Tris-HCl,50mM anhydrous calcium chloride, pH 7.5.
PEG4000 60% (w/v), 10mM Tris-HCl,50mM anhydrous calcium chloride, pH 7.5.
The hypertonic CD medium contains more than 35％,NaNO3 0.3％,KCl 0.2％,KH2PO4 0.1％,MgSO4·7H2O 0.05％,FeSO4·7H2O 0.001％,pH 5.5. percent of sucrose by mass.
The percentage of the CD culture medium is more than 2％,NaNO3 0.3％,KCl 0.2％,KH2PO4 0.1％,MgSO4·7H2O 0.05％,FeSO4·7H2O 0.001％,pH 5.5. percent of glucose by mass.
Example 7-method for fermentative production of Monacolin J using the above Aspergillus niger strains:
(1) Purifying on a solid culture medium to obtain single spore-free Aspergillus niger colony, picking a small amount of mycelia, standing and culturing in a CD liquid culture medium at 30 ℃ until obvious white mycelium pellets are suspended in a clear culture medium;
(2) 7ml-10ml of the liquid culture solution is taken and fermented in a fermentation medium at 30 ℃ and 220rpm for 7-10days, so that fermentation supernatant can be taken, and Monacolin J (figure 12) is detected by using an HPLC technology.
HPLC detection conditions are adoptedLC Column (250×4.6mm 5 μm), sample injection amount 10 μL, mobile phase methanol: water (0.3% phosphoric acid) =75:25, isocratic elution, ultraviolet detection wavelength 237nm, flow rate 1mL/min, detection time 15min.
The fermentation medium comprises 5% of corn starch, 3% of corn steep liquor, 2% of soybean meal and water as a solvent;
example 8-optimization method of Aspergillus niger Monacolin J fermentation Medium:
(1) The best carbon source in the fermentation medium is studied by selecting one of corn starch, glucose, sucrose and maltose as the carbon source, wherein the concentration is 5% (w/v), and the yield change of Monacolin J is detected through fermentation (figure 13).
(2) Investigation of external additives in the fermentation Medium 10mM glutamic acid, 0.18% (w/v) methionine, 520. Mu. Mol/L linoleic acid, 0.1% sodium citrate and 0.1% sodium acetate were added to the fermentation medium, respectively, and the Monacolin J yield change was detected by fermentation (FIG. 14).
In summary, it is concluded that:
(1) The macromolecular carrier with serially connected gene expression frames can be quickly constructed by adopting a yeast transformation coupling recombination (TAR) technology to assemble multiple fragments, so that the continuity and the integrity of a secondary metabolite synthetic gene cluster are ensured, the synthesis of the product is facilitated, and the Aspergillus niger transformation step and the use of screening markers are saved.
(2) The hydrolyzed starch, the bean pulp and the corn steep liquor are used as fermentation culture media, and the Monacolin J with the Monacolin J expression frame integrated therein is fermented for 10 days in a 500mL shaking bottle to obtain Monacolin J with the concentration of 459.7mg/L, which is 2.0 times that of glucose (236.0 mg/L) when the glucose is used as a carbon source, and 2.5 times that of sucrose (183.9 mg/L) when the sucrose is used as a carbon source, and the Monacolin J has the same effect as that of maltose when the maltose is used as a carbon source (453.8 mg/L). The mass percentage of the carbon source is 5 percent. 10mM glutamic acid, 0.18% (w/v) methionine, 520 mu mol/L linoleic acid, 0.1% sodium citrate and 0.1% sodium acetate are respectively added into the hydrolyzed starch fermentation medium and the maltose fermentation medium, and the effects of improving the yield of Monacolin J in the hydrolyzed starch fermentation medium are more remarkable when only glutamic acid, methionine and linoleic acid are added, and the yield of Monacolin J after improvement in the hydrolyzed starch fermentation medium is about 2 times of the yield of Monacolin J after improvement in the maltose fermentation medium and is up to 1605.6mg/L. And the addition of sodium citrate and sodium acetate would instead decrease the yield of Monacolin J. Even with the simultaneous addition of sodium citrate, sodium acetate, methionine, monacolin J production was still reduced in the hydrolyzed starch medium. This demonstrates that the use of a slow acting carbon source is more suitable for the basic fermentation medium and its subsequent fermentation optimization, and that the addition of sodium citrate, sodium acetate, etc. may enhance the TCA cycle of Aspergillus niger, resulting in growth inhibition production.
The embodiments of the present invention are not limited by the disclosure of the above examples, but are equivalent to the changes, substitutions and modifications without departing from the spirit and essence of the present invention, and are within the scope of the present invention.
Sequence listing of primers used
The above examples of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.