Aspergillus nidulans engineering strain AnDAOC-1 and application thereof in fermentation production of DAOC
By co-expressing and overexpressing related genes of Ceptosporidium aristocracy and Streptomyces in Aspergillus nitus, the engineering strain AnDAOC-1 was constructed, solving the problem of achieving efficient DAOC biosynthesis in Aspergillus nitus, and achieving a DAOC yield of 0.593 g/L.
Patent Information
- Application Number
- CN202510325586.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-06-06
AI Technical Summary
There are no reports of heterologous expression of DAOC in China that have obtained high-yield strains, and it is difficult to achieve efficient biosynthesis of deacetoxycephalosporin C in Aspergillus nitritis.
The engineered strain AnDAOC-1 was constructed by co-expressing and overexpressing genes in the biosynthetic gene cluster in Ceptosporidium atrium, except for cefEF, cefG and regulatory gene cefR.
The biosynthesis path of deacetoxycephalosporin C was successfully opened in Aspergillus nitus, and its heterologous expression was achieved, with a yield of 0.593 g/L. For the first time, the efficient synthesis of DAOC was achieved in Aspergillus nitus.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial synthesis, and in particular to the application of genes in heterologous synthesis of deacetyloxycephalosporin C, plasmids and strains. Background Art
[0002] Cephalosporin C (CPC) is a secondary metabolite produced by the filamentous fungus Acremonium chrysogenum and belongs to the β-lactam antibiotics. CPC is not easily destroyed by penicillinase and has the advantages of strong stability, broad antibacterial spectrum and low toxicity. After hydrolysis of CPC to remove the aminoadipoyl group on the side chain, 7-aminocephalosporanic acid (7-ACA) is obtained. 7-ACA is an intermediate of cephalosporin antibiotics widely used in clinical practice.
[0003] 7-ADCA (7-aminodeacetoxycephalosporanic acid) is also an important intermediate in the production of cephalosporin antibiotics. It can produce a variety of cephalosporin drugs with different antibacterial spectra and pharmacokinetic properties, such as cephalexin, cephradine, cefixime, etc. 7-ADCA can simplify the production process steps and reduce production costs in the process of synthesizing some cephalosporin antibiotics.
[0004] At present, the enzymatic production of 7-ADCA has been basically realized in China. The production process route is to use DAOC as a direct precursor, and to generate 7-ADCA through the deacetoxy step by using deacetoxycephalosporin C (DAOC) as a precursor through biosynthesis. The DAOC obtained by bacterial fermentation and separation and purification can be directly used to produce 7-ADCA through enzymatic process. At present, there is no report on high-yield strains obtained by heterologous expression of DAOC in China. Summary of the invention
[0005] In view of this, the present invention provides an engineered strain of Aspergillus nidulans AnDAOC-1, which is obtained by transferring plasmids pDAOC-1, pDAOC-2 and pDAOC-3 into Aspergillus nidulans A1145.
[0006] The present invention aims at an engineered strain of Aspergillus nidulans that heterologously synthesizes deacetyloxycephalosporin C. The biosynthetic genes pcbAB, pcbC, cefD1 and cefD2, transporter genes cefM, cefP and cefT, and SccefE gene from Streptomyces clavuligerus in the cephalosporin C biosynthetic gene cluster in the genome of Cephalosporium acremonium are simultaneously co-expressed and over-expressed in Aspergillus nidulans to obtain strain AnDAOC-1. The biosynthetic pathway of deacetyloxycephalosporin C is successfully opened in Aspergillus nidulans, and the heterologous expression of deacetyloxycephalosporin C is achieved. The yield reaches 0.593 g / L at the shake flask level. The present invention realizes the synthesis of deacetyloxycephalosporin C in Aspergillus nidulans for the first time. The strain constructed by the invention provides a good expression system for heterologous synthesis of deacetyloxycephalosporin C and exploration of the synthesis and regulation mechanism of deacetyloxycephalosporin C in Cephalosporium acremonium. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art are briefly introduced below.
[0008] Figure 1 is a schematic diagram of plasmid pDAOC-1;
[0009] Figure 2 is a schematic diagram of plasmid pDAOC-2;
[0010] Figure 3 is a schematic diagram of plasmid pDAOC-3;
[0011] Figure 4 This is the LC-MS mass spectrum of the fermentation extract of strain AnDAOC-1.
[0012] Figure 5 This is the LC-MS mass spectrum of the fermentation extract of strain AnDAOC-1.
[0013] Figure 6 This is the HPLC spectrum of DAOC standard.
[0014] Figure 7 This is the HPLC spectrum of AnDAOC-1 fermentation products. DETAILED DESCRIPTION
[0015] The present invention discloses the application of genes in heterologous synthesis of deacetyloxycephalosporin C, plasmids and strains. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0016] The present invention aims at an Aspergillus nidulans engineered strain capable of heterologously synthesizing deacetyloxycephalosporin C, co-expresses and over-expresses biosynthetic genes derived from Cephalosporin acremonium in Aspergillus nidulans, obtains strain AnDAOC-1, successfully realizes the reconstruction of the deacetyloxycephalosporin C biosynthetic pathway, realizes the heterologous expression of deacetyloxycephalosporin C, and achieves a yield of 0.593 g / L at the shake flask level.
[0017] Table 1. Primers used in constructing heterologous expression plasmids of the present invention
[0018]
[0019] Note: Underlined sequences represent sequences that are homologous to the vector upstream or downstream of the fragment.
[0020] pcbAB F1 / R1, pcbAB F2 / R2, pcbC F / R, cefD1 F / R, cefD2 F / R, SccefE F1 / R1, SccefE F2 / R2, cefM F / R, cefP F / R and cefT F / R were used as primer combinations, respectively.
[0021] Table 2. Plasmids used in the present invention
[0022]
[0023] Table 3. Strains involved in the present invention
[0024]
[0025] The invention provides the application of the gene in the heterologous synthesis of deacetyloxycephalosporin C, the plasmid and the strain, and the raw materials and reagents used can all be purchased from the market.
[0026] The present invention will be further described below in conjunction with embodiments:
[0027] Embodiment 1,
[0028] Plasmids pDAOC-1, pDAOC-2 and pDAOC-3 required for constructing a strain producing deacetyloxycephalosporin C
[0029] 1. Extraction of Cephalosporium acremonium genomic DNA
[0030] The CGMCC 3.3795 strain of Cephalosporium acremonium was purchased from the China General Microbiological Culture Collection Administration Center (deposit number: CGMCC 3.3795), hereinafter referred to as strain CGMCC 3.3795. A PDA solid plate (potato flour 20 g, glucose 10 g, agar powder 20 g, water 1000 mL) cultured for 3 days of strain CGMCC 3.3795 was placed at -80 ° C for 20 min, and the surface mycelium was scraped off, washed twice with distilled water, freeze-dried and stored at -20 ° C. The freeze-dried mycelium was ground into fine powder using a mortar pre-cooled with liquid nitrogen, resuspended in 500 μL lysis buffer [40 μmol / L Tris-acetate, 20 μmol / L sodium acetate, 1 μmol / L EDTA, 1% (w / v) SDS, pH 7.8], and pipetted until the viscosity of the suspension was significantly reduced and foam was formed. Add RNAaseA and incubate at 37°C for 5 min, then add 165 μL of 5 μmol / L NaCl solution and mix. Centrifuge at 12000 rpm for 20 min, immediately transfer the supernatant to a new EP tube, and add 400 μL of chloroform and 400 μL of phenol. Gently invert until the solution becomes milky. After centrifugation for 20 min, remove the aqueous phase and extract with an equal volume of chloroform. Precipitate the DNA in the supernatant with 2 volumes of 95% ethanol. Wash the precipitated DNA three times with 70% pre-cooled ethanol, dry it, and dissolve it in 50 μL TE buffer (10 μmol / L Tris-HCl, 0.1 μmol / L EDTA, pH 7.8), and store it at -20°C.
[0031] 2. Extraction of Streptomyces genomic DNA
[0032] The strain CGMCC 4.5336 of Streptomyces clavuligerus was purchased from the China General Microbiological Culture Collection (CGMCC 4.5336), hereinafter referred to as strain CGMCC 4.5336. Strain CGMCC 4.5336 was inoculated into TSB liquid medium (tryptone 17 g / L, soy peptone 3 g / L, glucose 2.5 g / L, NaCl 5 g / L, K 2 HPO 42.5 g / L, pH 7.3), shake culture at 28℃ and 200 rpm for 48 hours until the logarithmic growth phase. Take 1.5mL of bacterial solution, centrifuge at 12000rpm for 2 minutes to collect the bacteria, wash twice with sterile distilled water, freeze in liquid nitrogen, and freeze-dry at -80℃ for 24 hours. Transfer the freeze-dried bacteria to a mortar pre-cooled with liquid nitrogen and grind into fine powder (particle size ≤100 μm), resuspend in 500 μL lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA, 1% SDS, containing 1 mg / mL lysozyme), vortex mix, and water bath at 37℃ for 30 minutes. Add 50 μL proteinase K (final concentration 100 μg / mL) and incubate at 55℃ for 1 hour; then add 100 μL CTAB solution (5% CTAB, 0.7 M NaCl) and lyse at 65℃ for 20 minutes to remove lipopolysaccharide. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the lysate, gently invert and mix for 10 minutes, centrifuge at 12,000 × g for 10 minutes, transfer the supernatant to a new centrifuge tube, and repeat the chloroform extraction once. Add 0.6 times the volume of isopropanol to the supernatant, let it stand at -20℃ for 30 minutes to precipitate DNA, and collect the precipitate by centrifugation at 12,000 × g for 5 minutes. Wash twice with 70% pre-cooled ethanol, dry at room temperature, and dissolve in 50 μL TE buffer (containing 20 μg / mL RNase A, treated at 37℃ for 30 minutes to remove RNA), and store at -20℃ for later use.
[0033] 3. Amplification of target fragment
[0034] Primers for amplifying pcbAB, pcbC, cefD1, cefD2, cefM, cefP and cefT sequences were designed based on the DAOC gene sequence of the strain, and primers for amplifying cefE sequences of the Streptomyces clavuligerus strain were designed for the subsequent construction of pDAOC-1, pDAOC-2 and pDAOC-3 plasmids. These eight gene sequences can be retrieved from the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ). The NCBI accession numbers are: pcbAB (X74601.1), pcbC (M33522.1), cefD1 (AJ507632.2), cefD2 (AJ507632.2), cefT (AJ487683.1), cefM (AM231815.1), cefP (AM231816.1), and two copies of SccefE (M32324.1).
[0035] The corresponding primers were designed to amplify the fragments and used to construct pDAOC-1, pDAOC-2 and pDAOC-3 plasmids. The primer sequences are shown in Table 1 (underline indicates the repeating sequence at the junction of the gene and the vector or adjacent fragments):
[0036] The genomic DNA of strain CGMCC 3.3795 was used as a template, and pCbAB-F1 / R1 and pcbAB-F2 / R2 were used as primers to amplify the upper and lower fragments of the pcbAB gene. The demarcation point between the upper and lower parts was the sequence of pcbAB-1F2, that is, the upstream of this sequence was the upper part of pCbAB (pCbAB-up), and the downstream part of this sequence was the lower part of pCbAB (pCbAB-dn).
[0037] The pcbC, cefD1, cefD2, cefT, cefM and cefP gene fragments were amplified from the genome of strain CGMCC 3.3795 using primer pairs: pcbC F / R, cefD1 F / R, cefD2 F / R, cefT F / R, cefM F / R and cefP F / R; meanwhile, the SccefE gene and its second copy DNA sequence were amplified from the genome of strain CGMCC 4.5336 using primer pairs: SccefE F1 / R1 and SccefE F2 / R2. After cloning the above amplified products, the cefD1, cefD2, cefM, and cefT genes were constructed into the pYTR vector through molecular recombination technology to form the recombinant plasmid pYTR-cefD1 / D2 / M / T. At the same time, the pcbC, SccefE and its second copy, and cefP genes were cloned into the pYTU vector in sequence to finally obtain the recombinant plasmid pYTU-pcbC-SccefE / cefP.
[0038] The PCR amplification system is as follows:
[0039]
[0040] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C at 2 kb / min, 33 cycles, extension at 72°C for 5 min, and storage at 4°C.
[0041] 4. Extraction of pYTP, pYTR and pYTU vector plasmids
[0042] Use the Trans EasyPure Plasmid MiniPrep Kit to extract pYTP, pYTR and pYTU vector plasmids. The specific steps are as follows:
[0043] a. Grow E. coli containing pYTP, pYTR and pYTU vectors overnight, take 2-4 mL of bacterial culture, centrifuge at 10,000 rpm for 1 min, and pour off the supernatant.
[0044] b. Add 250 μL of RB solution (containing RNase A) to the collected bacteria and vortex until the bacteria are completely resuspended.
[0045] c. Add another 250 μL of LB solution to the centrifuge tube and gently invert the centrifuge tube 5-6 times.
[0046] d. Add 350 μL of NB solution to the centrifuge tube, gently invert the centrifuge tube 4-6 times, and let it stand at room temperature for 2 minutes.
[0047] e. Centrifuge at maximum speed for 10 minutes, carefully aspirate the supernatant and add it to the centrifugal collection column, centrifuge at 10,000×g for 1 minute, and discard the waste liquid in the collection tube.
[0048] f. Add 650 μL of WB solution to the centrifuge column (80 mL of anhydrous ethanol must be added before use), centrifuge at 12,000 × g for 1 min, discard the waste liquid in the collection tube, and repeat once.
[0049] g. Place the centrifuge column in a clean 1.5 mL centrifuge tube, add 50 μL of sterile water (preheated to 60-70°C) to the centrifuge column, and let it stand at room temperature for 2 minutes.
[0050] h. Centrifuge at 12000×g for 1 min and store the plasmid DNA at -20℃.
[0051] 5. Enzyme digestion of vector and recovery of vector and target fragment
[0052] pYTP and pYTR vectors were digested with SwaI and BamHI endonucleases, and the digestion buffer was NEBuffer 3.1.
[0053] The pYTU vector was double-digested with SwaI and NotI, and the digestion buffer was NEBuffer 3.1.
[0054] The SwaI digestion temperature is 25°C, the BamHI and NotI digestion temperatures are 37°C, and the digestion time is 6 h. The digestion system is as follows:
[0055]
[0056] The vector and target fragment after enzyme digestion were recovered using the OMEGA Gel Extraction Kit. The specific steps are as follows:
[0057] a. Place the cut gel pieces into a 2 mL centrifuge tube, add an equal volume of Binding buffer, and incubate at 60°C until the gel is completely dissolved.
[0058] b. Transfer the solution to a DNA binding column, centrifuge at 12,000 × g for 1 min, and discard the waste liquid in the collection tube.
[0059] c. Add 700 μL SPW Wash Buffer (100 mL ethanol should be added to SPW Wash Buffer before use) to the DNA binding column, centrifuge at 12,000 × g for 1 min, discard the waste liquid in the collection tube, and repeat once.
[0060] d. Place the empty DNA binding column into a collection tube and centrifuge at 12,000 × g for 2 min.
[0061] f. Place the DNA binding column in a clean 1.5 mL centrifuge tube, add 30-40 μL of ultrapure water (preheated at 65°C), and let stand at room temperature for 2 min.
[0062] Centrifuge at 12000×g for 1 min and store the DNA at -20℃.
[0063] 6. Yeast assembly to obtain yeast transformants containing plasmids pDAOC-1, pDAOC-2 and pDAOC-3
[0064] 6.1 Preparation of competent yeast
[0065] Prepare the competent state of yeast strain BJ5464-NpgA (purchased from BioVector NTCC). The competent state preparation refers to the kit: Zymo research-Yeast Transformation II Kit, Catalog No.: T2001. The detailed steps are as follows:
[0066] (1) Inoculate 15 μL of yeast cells in 10 mL of YPD medium and culture the yeast for 18-22 h at 30°C and 220 rpm until the logarithmic phase (OD 600 is between 0.8-1.0);
[0067] (2) Centrifuge at 500 g for 4 min at room temperature to collect yeast;
[0068] (3) At room temperature, wash the yeast precipitate with 5 mL of EZ1 solution and collect the yeast by centrifugation at 500 g for 4 min.
[0069] (4) At room temperature, wash the yeast pellet again with 5 mL of EZ1 solution and collect the yeast by centrifugation at 500 g for 4 min.
[0070] (5) At room temperature, resuspend the yeast pellet with 1 mL of EZ2 solution, dispense 25 μL into each EP tube to obtain competent yeast, and freeze at -80°C for future use.
[0071] 6.2 Yeast homologous recombination transformants
[0072] (1) Yeast competent state after thawing at room temperature;
[0073] (2) Add 0.5 μg of the pYTP vector, pcbAB-up, and pcbAB-dn fragments after restriction digestion to 25 μL of yeast competent medium, add 0.5 μg of the pYTR vector and cefD1 / cefD2 / cefM / cefT fragments after restriction digestion to another 25 μL of yeast competent medium, and add 0.5 μg of the pYTU vector and pcbC / SccefE / SccefE / cefP fragments after restriction digestion to another 25 μL of yeast competent medium.
[0074] (3) Incubate at 30°C for 1 h, vortexing and mixing every 15 min. The incubation time can be extended to 2 h.
[0075] (4) Spread the entire transformation system onto UDMS medium (0.5 g Bacto technical grade casamino acids; 2 g glucose; 2 g agar powder; add 10 mL 10x nitrogen base stock, 1 mL tryptophan stock solution, 1 mL adenine stock solution, and 100 mL deionized water) and culture at 30°C for 2-4 days.
[0076] 6.3 Identification of transformants
[0077] Pick 4-5 transformants and identify them by colony PCR. 2 Transfer to the plate, and after 1 day, perform PCR colony verification on the expanded yeast monoclonal transformants.
[0078] Yeast colony PCR bacterial pretreatment steps:
[0079] Scrape yeast monoclones from the plate into a 1.5 mL EP tube, suspend the cells in 100 μL of 0.2 mM lithium acetate solution containing 1% SDS, and incubate at 70°C for 5 min; then add 300 μL of 96-100% ethanol to the 1.5 mL EP tube and vortex to mix; place the 1.5 mL EP tube in a centrifuge and centrifuge at 15,000 g for 3 min; pour out the supernatant, add 200 μL of 70% ethanol to the 1.5 mL EP tube to wash the residue, centrifuge at 15,000 g for 2-3 min, discard the supernatant and place the precipitate in a 60°C oven for 5 min to evaporate the ethanol; finally, resuspend the residue with 15 μL of ultrapure water, shake it thoroughly on a vortex mixer, and centrifuge it at 15,000 g for 15 s.
[0080] Take 1 μL of supernatant as PCR template for plasmid assembly verification. Plasmid pDAOC-1 was sequenced using pYTP-SF / SR primers; plasmid pDAOC-2 was sequenced using pYTR-SF / SR primers; plasmid pDAOC-3 was sequenced using pYTU-SF / SR primers. The primer sequences are as follows:
[0081] Table 4. Sequencing primer sequences
[0082]
[0083] The sequence obtained by sequencing is compared with the target sequence, and the consistency of the sequences indicates that the constructed plasmid is correct.
[0084] 6.4 Yeast plasmid extraction
[0085] Scrape 1x1 cm pieces of yeast cells containing the correctly identified plasmids pDAOC-1, pDAOC-2 and pDAOC-3 from the plate. 2 The extraction process refers to the kit: Zymo research Zymo prep II Yeast Plasmid Mini prepII Kit, Catalog No.: D2004.
[0086] 6.5 Introducing yeast plasmids into E. coli Top10 for plasmid amplification
[0087] Commercial E. coli Top10 was used for plasmid amplification, and the transformation method was as follows:
[0088] Take out Top10 competent cells from the -80℃ freezer and thaw on ice; add 10μL assembly solution, mix gently, and place on ice for 30 min; place in a 42℃ water bath for 60-90 s, take out and place on ice for 2 min; add 200 μL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl), and incubate in a shaker at 37℃ for 1h; spread the culture medium on an LB solid plate containing 100 μg / mL ampicillin resistance and culture overnight; pick out the grown single clones and culture them with LB liquid medium containing 100 μg / mL ampicillin resistance for 12h, and extract plasmids pDAOC-1, pDAOC-2 and pDAOC-3.
[0089] 6.6 Verification of plasmids pDAOC-1, pDAOC-2, and pDAOC-3
[0090] The extracted plasmids were sent to a sequencing company for sequencing. Plasmid pDAOC-1 was sequenced using pYTP-SF / SR primers; plasmid pDAOC-2 was sequenced using pYTR-SF / SR primers; plasmid pDAOC-3 was sequenced using pYTU-SF / SR primers. The primer sequences are the same as in Table 4. The sequence obtained by sequencing was compared with the target sequence. If the sequence was consistent, it means that the constructed plasmid was correct.
[0091] Embodiment 2,
[0092] Plasmids pDAOC-1, pDAOC-2 and pDAOC-3 were transformed into Aspergillus nidulans A1145 strain
[0093] 1. The preparation method of Aspergillus nidulans protoplasts is as follows:
[0094] Aspergillus nidulans A1145 strain (purchased from BioVector NTCC) was spread or streaked on a solid CD medium containing 0.5 μg / mL vitamin B (pyridoxine HCl), 0.125 μg / mL riboflavin and 10 mM uridine, and cultured at 37°C for about 4 days. After the plate was full of spores, 2 mL of 0.1% Tween-80 was added, and spores from about half of the plate were scraped with a cotton swab and filtered using a spore filter. The solid CD medium formula is: 20 g / L tryptone; 50 mL / L 20x nitrate solution (120 g NaNO 3 ; 10.4 g KCl; 10.4 g MgSO 4 7H 2 O; 30.4 g KH 2 PO 4 dissolved in 1 L of water); 1 mL / L trace elements [2.20 g ZnSO 47H 2 O; 1.10 g H 3 BO 3 ; 0.50 g MnCl 2 ·4H 2 O; 0.16 g FeSO 4 7H 2 O; 0.16 g CoCl 2 6H 2 O; 0.16 g CuSO 4 ·5H 2 O; 0.11 g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O; 5.00 g Na 4 EDTA]; 20 g agar; dissolved in 1 L water.
[0095] The spores were inoculated into 100 mL of liquid CD medium containing 0.5 μg / mL vitamin B (pyridoxine HCl), 0.125 μg / mL riboflavin, and 10 mM uridine. The spore concentration under microscopic examination was about 10 7 / mL. The formula of liquid CD medium is 20g / L tryptone; 50mL / L 20xNitrate salts medium; 1mL / L trace elements. The pH value is adjusted to 6.5 with 1M KOH, and the shaking culture conditions are 37℃, 250rpm. The spores germinate for 6.5h. The best state of spore germination is that the mycelium grows to 3 times the size of the expanded spores, and the spores gather first and then germinate.
[0096] Centrifuge the mycelium in the culture medium at 8000 rpm for 5 min, add 15 mL of Osmotic Medium (1.2 M MgSO 4 , 10 mM sodium phosphate buffer, pH 5.8), and repeated washing three times.
[0097] Add 10 mL of mixed enzymatic solution (30 mg Lysing Enzyme, 20 mg Yatalase dissolved in 10 mL Osmotic Medium Buffer), culture in a shaker at 30°C, 80 rpm, and observe the enzymatic hydrolysis of mycelium under a microscope. The protoplasts after enzymatic hydrolysis are about twice the volume of spores, with uniform morphology and thin walls. The enzymatic hydrolysis time is generally about 2.5 hours.
[0098] Add 3 volumes of STC buffer (1.2 M sorbitol; 10 mM CaCl 2; 10 mM Tris-HCl, pH 7.5), centrifuge at 5000 rpm for 10 min, and remove the liquid.
[0099] Add a small amount of STC buffer to resuspend the protoplasts to a concentration of about 10 8 -10 9 pcs / mL, and dispense into 1.5mL centrifuge tubes, 100μL per tube. All the above operations were performed on ice or at 4℃.
[0100] 2. Transform Aspergillus nidulans protoplasts with plasmids pDAOC-1, pDAOC-2 and pDAOC-3
[0101] Here are the steps:
[0102] Take 5 μg of plasmids pDAOC-1, pDAOC-2 and pDAOC-3, add them to 100 μL protoplasts, mix gently, and place on ice for 60 min.
[0103] Add 1.25 mL of 60% PEG solution (60% PEG4000; 50 mM CaCl 2 ; 50 mM Tris-HCl, pH 7.5), mix gently with a pipette and leave at room temperature for 20 min.
[0104] Gently spread on a solid CD-Sorbitol medium (10 g / L glucose; 50 mL / L 20x nitrate; 1 mL / L trace elements; 1.2 M sorbitol; 20 g / L agar) plate, and be sure to be gentle when mixing and spreading. Incubate upright at 37℃ for 1 day, then invert for 1-2 days, and wait for clones to grow to obtain the engineered strain of Aspergillus nidulans.
[0105] 3. Identification of Aspergillus nidulans products
[0106] Take a single clone transformant and transfer it to solid CD Medium, and culture the transformant strain at 37℃ for seed preservation;
[0107] The bacterial cells obtained in the above step were inoculated into liquid CD-ST Medium and cultured at 37°C and 250rpm for 3 days. The fermentation broth was extracted and the fermentation product was detected, which was determined to be DAOC. The strain was named Aspergillus nidulans engineered strain AnDAOC-1.
[0108] Example 3
[0109] Detection of DAOC production by engineered strain AnDAOC-1
[0110] The correctly engineered strain AnDAOC-1 was inoculated (10 8100 μL of a spore suspension of 100 μL / mL was inoculated into 50 mL of liquid CD-ST medium (20 g / L starch; 20 g / L tryptone; 50 mL / L 20x nitrate; 1 mL / L trace elements; pH adjusted to 6.5) for shake flask fermentation and samples were taken after culturing at 37°C and 250 rpm for 2-3 days.
[0111] Take 50 mL of fermentation broth, extract the mycelium and supernatant with ethyl acetate three times, combine the organic phases, evaporate at 30°C until completely dry, add 300 μL of methanol to dissolve, filter with 0.22 μm filter membrane, and detect DAOC using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).
[0112] LC-MS detection conditions of DAOC: LC-MS analysis was performed on an Agilent-1200HPLC / 6520QTOFMS (USA) system, with acetonitrile (v / v, 0.1% formic acid) and water (v / v, 0.1% formic acid) as the mobile phase, a flow rate of 0.8 mL / min, and an injection volume of 5 μL. 0-15 min, 5%-95% acetonitrile; 15-20 min, 95% acetonitrile. The Q-TOF used dual ESI as the ion source interface, and the ESI source was operated in positive ionization mode. The scan range was m / z 100-1000. The obtained sample mass spectrum ( Figure 4 ), which is consistent with the molecular weight of DAOC standard 357.38.
[0113] HPLC analysis conditions were as follows: 10% methanol was used to balance the column before injection, the mobile phase was 98.7% (v / v) 0.01 M sodium acetate (adjusted to pH 4.75 with acetic acid), 1.3% (v / v) acetonitrile, flow rate 1.0 mL / min, injection volume 10 μL, 0-25 min isocratic analysis. The absorption peak of DAOC in the fermentation product was detected at 254 nm, which was consistent with the retention time of the DAOC standard ( Figure 5 ).
[0114] The DAOC specific absorption peak area of the fermentation extract samples and standards was analyzed by HPLC, and the DAOC yield data in the fermentation extract products were calculated. Three replicates were set for each sample.
[0115] like Figure 4 , 5 As shown in Figure 2, the LC-MS detection results of the fermentation product showed that the Aspergillus nidulans strain AnDAOC-1 produced DAOC. The corresponding HPLC spectrum is shown in Figure 2. Figure 6 , 7As shown, based on the quantitative analysis of DAOC standard (concentration 0.253 g / L, purity 86.20%, characteristic peak area 2,225,868), by comparing the average characteristic peak area of 6,051,581 (n=3) of AnDAOC-1 fermentation product, it was calculated that the yield of DAOC produced by the strain heterologously reached 0.593 g / L.
[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. An engineered strain of Aspergillus nidulans AnDAOC-1, characterized in that: Plasmid pDAOC-1 containing pcbAB genes, plasmid pDAOC-2 containing cefD1 / cefD2 / cefM / cefT genes, and plasmid pDAOC-3 containing pcbC / SccefE / cefP genes were co-transformed into Aspergillus nidulans A1145 strain to obtain the obtained plasmid.
2. The engineered Aspergillus nidulans strain AnDAOC-1 according to claim 1, characterized in that: The plasmid pDAOC-1 contains the pcbAB gene whose expression is regulated by the PamyB promoter, and its plasmid structure is shown in FIG1 .
3. The engineered Aspergillus nidulans strain AnDAOC-1 according to claim 1, characterized in that: The plasmid pDAOC-2 contains the cefD1 gene regulated by the PgpdA promoter, the cefD2 gene regulated by the PamyB promoter, the cefM gene regulated by the PglaA promoter, and the cefT gene regulated by the Ptef1 promoter, and its plasmid structure is shown in FIG2 .
4. The engineered Aspergillus nidulans strain AnDAOC-1 according to claim 1, characterized in that: The plasmid pDAOC-3 contains the pcbC gene regulated by the PglaA promoter, two copies of the SccefE gene regulated by the PgpdA and PamyB dual promoters, and the cefP gene regulated by the Ptef1 promoter, and its plasmid structure is shown in FIG3 .
5. The engineered Aspergillus nidulans strain AnDAOC-1 according to claim 1, characterized in that: The pcbAB, pcbC, cefD1, cefD2, cefM, cefT and cefP genes are derived from Acremonium chrysogenum, and the SccefE gene is derived from Streptomyces clavuligerus.
6. Use of the engineered Aspergillus nidulans strain AnDAOC-1 according to any one of claims 1 to 5 in producing DAOC by fermentation.