Method for rapidly and iteratively acquiring saccharomyces cerevisiae recombinants with high copy integration of long-fragment DNA (deoxyribonucleic acid)

Abstract

The invention belongs to the field of genetic engineering and metabolic engineering, and discloses a method for rapidly iteratively acquiring a saccharomyces cerevisiae recombinant with high copy integration of long-fragment DNA (Deoxyribose Nucleic Acid). By means of a CRISPR-Cas9 gene editing technology, an auxotrophic gene weakening and back-supplementing screening strategy and a delta sequence / rDNA sequence multi-copy integration strategy are combined to construct a method for rapidly iteratively acquiring a long-fragment DNA high-copy integrated saccharomyces cerevisiae recombinant, and the method is applied to construction of saccharomyces cerevisiae engineering bacteria for producing natural products. By applying the method, the yield of two natural products of ergothioneine and cordycepin is greatly improved. The method for obtaining the saccharomyces cerevisiae recombinant with high copy integration of the long fragment DNA through rapid iteration is beneficial for developing saccharomyces cerevisiae engineering bacteria and applying the saccharomyces cerevisiae engineering bacteria to efficient production of natural products.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and metabolic engineering, and particularly relates to a method for rapidly iteratively obtaining a saccharomyces cerevisiae recombinant with high-copy integration of long-fragment DNA. Background Art

[0002] Natural products are small organic molecules from animals, plants and microorganisms in nature, with very important biological activities and physiological functions such as anti-inflammatory, antibacterial and antioxidant. According to statistics, about 40% of modern clinical drugs come directly or indirectly from natural products. The 2015 Nobel Prize in Physiology or Medicine was awarded to Chinese pharmacist Tu Youyou, Irish scientist Campbell and Japanese scientist Satoshi Omura in recognition of their great contributions to the research of plant-derived artemisinin and fungal-derived avermectin. The issuance of this award has increased the research enthusiasm for natural products. At present, the production of natural products is mainly through plant extraction and chemical synthesis, but traditional synthesis methods are often subject to many restrictions. Using modern biotechnology such as synthetic biology to construct microbial cell factories to heterologously synthesize natural products can break through the bottleneck of traditional production methods, provide new strategies for the green, efficient and low-cost synthesis of natural products, and promote the research and application of natural products. In recent years, with the rapid development of synthetic biology, many natural products have made important progress through heterologous synthesis of microorganisms.

[0003] As a model fungus, Saccharomyces cerevisiae has the advantages of high biosafety, easy cultivation, clear genetic background, mature gene editing technology, and suitability for large-scale fermentation production. It is widely used as a host for heterologous synthesis of plant natural products. At present, scientists have used Saccharomyces cerevisiae as a chassis cell to synthesize a variety of complex natural products such as artemisinic acid, ginsenosides, and vinblastine, laying the foundation for their industrial production. In order to efficiently produce natural products in yeast cells, metabolic engineering strategies such as heterologous gene expression and overexpression are often used to enhance the metabolic flux of natural product synthesis pathways. However, traditional free expression systems are greatly affected by culture conditions, genes, products, and the strain itself, and product yields are unstable. Saccharomyces cerevisiae has a high homologous recombination efficiency, so integrated expression has become the preferred strategy for stable gene expression. Studies have shown that the performance of transformants using single-copy gene integration is often not ideal. In general, the gene transcription level and protein expression level will increase with the increase in the number of gene copies, and the yield of the target product will also increase accordingly, so it is necessary to increase the number of gene copies. The integration method using the multi-copy sites of Saccharomyces cerevisiae itself (such as delta sites and rDNA sites) can further increase the copy number of foreign genes, which is suitable for metabolic engineering transformation. The Delta site is located on the flank of the Ty1 retrotransposon of Saccharomyces cerevisiae. In the haploid yeast genome, there are about 300 delta sequences. In addition to the Delta site, rDNA (i.e., ribosomal RNA gene) is highly repeated in all eukaryotic genomes, containing about 150 copies. Due to its highly repeated sequence, it is also one of the commonly used integration sites. Currently, rDNA-mediated multi-copy integration has been applied to various yeasts.

[0004] However, the common multi-copy integration strategies currently have certain limitations. On the one hand, some genomic integration methods that do not use multi-copy sites (such as delta sites and rDNA sites) often obtain limited gene copies and have a long operation cycle; in addition, the integration methods that only use multi-copy sites of rDNA sites or delta sites do not comprehensively utilize the reusability of these sites; secondly, the current more mature integration and transformation technologies are mainly aimed at small fragments of DNA, and the integration efficiency decreases with the increase of the length of the integrated fragment. On the other hand, in the screening process, in order to prevent the omission of excellent transformants, the later transformation verification of the traditional multi-copy integration strategy needs to be verified one by one, and needs to be verified by sequencing, RT-PCR or fermentation detection, which is time-consuming, complicated, and dependent on detection equipment. Therefore, it is of great significance to develop a method for the integration of high-copy integration of long-fragment DNA in Saccharomyces cerevisiae recombinants that is efficient in integration, simple in screening, and not overly dependent on detection instruments.

[0005] CN113564206A is a method for integrating multiple copies of a target gene into rDNA of the chromosome of Saccharomyces cerevisiae. The disclosed method is: the Saccharomyces cerevisiae to be transformed is first pre-cultured on a culture medium containing hydroxyurea, and then the DNA fragments with rDNA homologous sequences at both ends are transferred into Saccharomyces cerevisiae by a conventional transformation method, and finally the transformants are screened on the screening culture medium to achieve. However, this method only takes 6-8d in the hydroxyurea pretreatment stage of the yeast cells in the early stage, which greatly increases the overall experimental time. In addition, the method introduces antibiotic markers, needs to apply antibiotic stress, is costly and not environmentally friendly, and the residues of antibiotic screening marker genes and their expression products will limit the application of related technologies in food and medicine. Summary of the invention

[0006] In view of the problems of the existing heterologous gene integration methods, such as long cycle, low efficiency, difficult screening and dependence on detection instruments, the present invention provides a method for rapidly iterating and obtaining Saccharomyces cerevisiae recombinants with high-copy integration of long-fragment DNA. On the one hand, two multi-copy integration sites (delta site and rDNA site) are simultaneously used to provide sufficient integration sites to obtain high-copy integration; on the other hand, weak promoters are used to achieve weak complementation of nutrient deficiency-related genes, and then high-copy integration recombinants are quickly screened through strain growth conditions.

[0007] The technical solution of the present invention is as follows:

[0008] A method for rapidly and iteratively obtaining a saccharomyces cerevisiae recombinant with high-copy integration of a long-fragment DNA fragment, the method comprising:

[0009] S1 Construction of Cas9-gRNA vector: A free expression plasmid suitable for Saccharomyces cerevisiae and containing Cas9 and sgRNA expression frames was selected as the starting vector, and the starting vector was double-digested with restriction endonucleases to remove the sgRNA expression frame to obtain a linearized vector. Two pairs of sgRNAs were designed according to the sequences of the multi-copy integration sites (delta sites and rDNA sites) of Saccharomyces cerevisiae, and four sgRNA expression frames containing the target sgRNA sequences were constructed by restriction ligation or overlap PCR (the nucleotide sequences of the fragments are shown in SEQ ID NO.1-4), and further connected to the linearized vector by restriction ligation or seamless cloning to construct the Cas9-gRNA vector.

[0010] Amplification of S2 genomic integration fragment: The donor DNA fragment (Donor DNA) is amplified by overlap PCR, including the target gene expression frame, the weak promoter-driven nutritional deficiency marker gene expression frame and the left and right homologous arms.

[0011] S3 Iterative transformation of Saccharomyces cerevisiae: Prepare competent cells of Saccharomyces cerevisiae, transform an appropriate amount of Cas9-gRNA plasmid and the corresponding Donor DNA fragment by electroporation, and then spread them on a nutrient-deficient solid plate.

[0012] S4 Rapid Iterative Screening: After completing one electroporation transformation, place the nutritionally deficient solid plate coated with yeast cells upside down in a 25-30°C incubator for static culture, and observe the growth of yeast colonies on the plate. When 5-15 transformants appear, add an appropriate amount of sterile physiological saline, scrape all colonies with a coating rod, transfer to a nutritionally deficient liquid culture medium for overnight enrichment culture, prepare competent cells in the form of mixed bacteria, and start the next round of yeast transformation.

[0013] Depending on the target gene copy number in the experimental design, the above screening method can be used to perform multiple rounds of transformation iterative screening to obtain the target transformants.

[0014] The single colonies of yeast transformants obtained after multiple rounds of iterative screening were inoculated into multi-well deep plates for liquid culture. 1-5 mL of nutrient-deficient medium was added to each well and cultured overnight at 25-30°C and 150-200 rpm. After the culture was completed, the gene copy number was preliminarily estimated based on the cell growth.

[0015] S5 shake flask liquid fermentation: inoculate the screened target strain into 20-50mL liquid culture medium, culture at 25-30℃, 150-200r / min for 16-24h; then transfer to 50-100mL fermentation medium without nutritional deficiency, control the initial bacterial OD 600 The temperature is 0.2-0.4, and the culture is carried out for 120-168 hours at 25-30℃ and 150-200r / min.

[0016] S6 Fermentation parameter determination: OD of bacterial solution was measured using a spectrophotometer 600 , the copy number of the target gene in the genome was determined by qPCR, and the yield of the target product in the fermentation broth was detected by high-performance liquid chromatography.

[0017] Furthermore, the starting vector suitable for Saccharomyces cerevisiae and containing Cas9 and sgRNA expression frames described in step S1 above is pCas9-URA3-gRNA, and the restriction endonucleases used for linearizing the vector are Nhe I and Hpa I.

[0018] Furthermore, the weak promoter in step S2 above includes P BTS1 , P PDA1 , P ERG1 etc. Nutritional deficiency genes include URA3, LEU2, MET15 / 17 and HIS3.

[0019] Furthermore, the Saccharomyces cerevisiae in step S3 is auxotrophic Saccharomyces cerevisiae, and all yeasts containing auxotrophic defects can be used for this method, including but not limited to BY4741, BY4742, BY4743, INVSc1, EBY100, and AH109.

[0020] Furthermore, the nutritional deficiency medium in the above step S3 is determined according to the complementing genes on the plasmid and the integrated fragment. The nutritional deficiency medium components are (1L): 8g SC-Trp-Leu-His-Ade-Ura-Met medium, 25-30g glucose, 0.1-0.4g tryptophan, 0.05-0.2g histidine, 0.1-0.4g adenine, 0.1-0.4g uracil, 0.05-0.2g methionine, 0.1-0.4g leucine. 2-3% agar powder needs to be added to the solid medium.

[0021] Furthermore, the fermentation medium components in the above step S5 are (1 L): YNB (containing ammonium sulfate) 6.7-8 g, D-glucose 25-30 g, L-arginine 0.2-0.5 g, L-cysteine ​​0.05-0.1 g, L-lysine 0.05-0.1 g, L-threonine 0.05-0.1 g, L-aspartic acid 0.1-0.3 g, L-isoleucine 0.2-0.4 g, L-phenylalanine 0.02-0.05 g, L-proline 0.05-0.1 g, L-serine 0.2-0.4 g, L-tyrosine 0.01-0.05 g, L-valine 0.02-0.05 g, L -Methionine 0.2-0.5g, L-tryptophan 0.2-0.5g, L-histidine 0.2-0.5g, L-leucine 0.1-0.3g, adenine 0.05-0.1g, L-glutamic acid 0.02-0.04g, L-glutamine 0.02-0.04g, L-glycine 0.1-0.2g, L-alanine 0.01-0.05g, L-asparagine 0.05-0.08g, ammonium sulfate 5-10g, copper sulfate 0.1-0.16mg, pyridoxine hydrochloride (vitamin B6) 1.0-1.6mg, biotin 0.25-0.5mg, ferrous sulfate 0.025-0.05mg.

[0022] The objects of the present invention are as follows:

[0023] The present invention uses CRISPR-Cas9 gene editing technology to combine the screening strategy of weakening and complementing auxotrophic gene with the delta sequence / rDNA sequence multi-copy integration strategy to construct a method for rapidly iteratively obtaining cerevisiae recombinants with high-copy integration of long-fragment DNA, which can be applied to the construction of cerevisiae engineered bacteria that produce natural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the operation process of a method for rapidly iteratively obtaining large fragment high copy integrated recombinants of Saccharomyces cerevisiae;

[0025] Figure 2 This is a schematic diagram of the Donor DNA fragment structure that needs to be amplified to construct an engineered strain of Saccharomyces cerevisiae that produces ergothioneine;

[0026] Figure 3 This is a schematic diagram of the Donor DNA fragment structure that needs to be amplified to construct a Saccharomyces cerevisiae engineered strain that produces cordycepin;

[0027] Figure 4 It is the main reaction in which the key genes EGT2 and EGT1 in heterologous synthesis of ergothioneine in Saccharomyces cerevisiae participate;

[0028] Figure 5 It is the main reaction in which the key genes Cns1 and Cns2 in heterologous synthesis of cordycepin in Saccharomyces cerevisiae participate;

[0029] Figure 6 The growth condition of the transformant of ergothioneine producing bacteria constructed by the method provided by the present invention; Figure 6 (a) shows the growth of bacterial colonies on the nutritional deficiency plate after the first transformation; Figure 6 (b) shows the growth of the colonies on the nutritional deficiency plate after the second iterative transformation; Figure 6 (c) The growth of randomly selected strains obtained by two transformations (L1-1, L1-2, L1-3 and L1-4 are strains obtained after one round of transformation, and L2-1, L2-2, L2-3 and L2-4 are strains obtained after two rounds of transformation) in a nutrient-deficient medium containing the corresponding attenuated complementation selection marker;

[0030] Figure 7 The growth of the transformant of cordycepin-producing bacteria constructed by the method provided by the present invention; Figure 7 (a) shows the growth of bacterial colonies on the nutritional deficiency plate after the first transformation; Figure 7 (b) shows the growth of the bacterial colonies on the nutritional deficiency plate after the second iterative transformation; Figure 7 (c) Growth of randomly selected strains obtained by two transformations (CNS01-09 is a strain obtained after one round of transformation, and CNS10-18 is a strain obtained after two rounds of transformation) in a nutrient-deficient medium containing the corresponding attenuated complementation selection marker;

[0031] Figure 8 The key gene copy number of the ergothioneine producing bacteria obtained by the method provided by the present invention and the ergothioneine yield and growth after 144h fermentation, i.e., OD 600 ;

[0032] Fig. 9 The key gene copy number of the cordycepin-producing bacteria obtained by the method provided by the present invention, as well as the cordycepin yield and growth after 144 hours of fermentation, i.e., OD 600 . DETAILED DESCRIPTION

[0033] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0034] Embodiment 1: utilize the present invention to construct the saccharomyces cerevisiae engineering bacteria producing thioneine

[0035] Construction of S1 CRISPR / Cas9 plasmid: First, restriction endonucleases NheI and HpaI were used to double-digest pCas9-URA3-gRNA to obtain vector fragments, and then ScF100 and ScR101, ScR102, ScR103, ScR104, and ScF101, ScF102, ScF103, ScF104 and ScR100 were used as primers, and pCas9-URA3-gRNA was used as a template to obtain four groups of fragments (two fragments in each group). The sgRNA sequence was replaced into the fragments by overlap primers, and then overlap PCR was performed using ScF100 and ScR100 as primers, and the corresponding two fragments in the four groups were used as templates to obtain four fragments. The nucleotide sequences of the four fragments are SEQ ID NO.1-4; The four fragments obtained above were connected with the vector fragment obtained after enzyme digestion by seamless cloning to obtain four plasmids: pCas9-gRNA (Delta1), pCas9-gRNA (Delta2), pCas9-gRNA (rDNA1), and pCas9-gRNA (rDNA2). The plasmid was quickly replicated by E. coli, and the target plasmid was extracted with a plasmid extraction kit for later use.

[0036] S2 Amplification of Donor DNA fragments: The Donor DNA fragments that need to be integrated into the yeast genome include the upstream and downstream homology arms of the multi-copy site, the expression frames of the key synthetic genes EGT1 and EGT2 of ergothioneine, and the expression frames of the weakened complementation nutrient deficiency genes. The Donor DNA structure is as follows: Figure 2The nutritional deficiency gene selected for complementation in this embodiment is the key gene MET15 / 17 for the synthesis of methionine (an important synthetic precursor of ergothioneine), and the weak promoter selected is BTS1p. The primers listed in Table 3 were used to amplify the required target fragments. The S288C genome was used as a template, and ScF201 and ScR201, ScF202 and ScR202, ScF203 and ScR203, ScF204 and ScR204 were used as primers to amplify the upstream and downstream homology arms of Delta, and the upstream and downstream homology arms of rDNA; pYES2-KanMX-EGT1-EGT2 was used as a template, and ScF211 and ScR211, ScF214 and ScR214 were used as primers to amplify the expression frames of the key genes EGT2 and EGT1 for ergothioneine synthesis, respectively; the S288C genome was used as a template, and ScF212 and ScR212 were used as primers to amplify the weak promoter BTS1p, whose nucleotide sequence is SEQ ID NO.5; pYES2-DLPU-8-MET17 was used as a template and ScF213 and ScR213 were used as primers to amplify the MET15 / 17 gene, whose nucleotide sequence has a GeneBank number of 851010; and overlap PCR was performed using the homology arms on the primers to amplify the entire fragment. The entire expression cassette to be integrated is 8.2 Kb in length. Because the DonorDNA fragment is obtained by PCR amplification in the present invention, in order to ensure the fidelity of the amplification, Figure 2 The donor DNA was divided into three segments. The fragments amplified above were used as templates and ScF201 and ScR221, ScF213 and ScR222, ScF223 and ScR202, ScF203 and ScR221, ScF223 and ScR204 were used as primers to amplify P11, P12, P13, P14 and P15, among which P11, P12 and P13 formed a group (Delta site) and fragments P14, P12 and P15 formed a group (rDNA site).

[0037] S3 electroporation transformation of Saccharomyces cerevisiae: Prepare Saccharomyces cerevisiae BY4741 competent cells, transform them with 2000 ng of each of the four Cas9-gRNA plasmids and 40000 ng of the corresponding donor DNA fragments P11, P12 and P13, and P14, P12 and P15, and spread them on SC-URA-MET nutritional deficiency plates.

[0038] S4 Rapid Iterative Screening Method: After completing one electroporation transformation, place the coated nutritional deficiency plate upside down in a 30°C incubator for static culture and observe the growth of the colonies on the plate. Figure 6(a) Add 500 μL of sterile saline to the plate, use a spreader to slowly scratch the colonies on the plate, culture overnight for 14-16 hours, and prepare the competent cells in the form of mixed bacteria and start the next round of transformation. The second round of iterative operations is similar to the first one (such as Figure 6 (b) Then, the transformants with faster growth were selected and placed into 96-well deep plates with 1 mL of SC-MET medium to observe the growth of the strains (e.g. Figure 6 (c)).

[0039] S5 shake flask fermentation: The growth of transformants in S4 in nutrient-deficient medium (such as Figure 6 (c)) is used to preliminarily estimate the copy number based on the size of the bacterial mass accumulated at the bottom of the well plate. In order to determine whether the growth is related to the copy number, four transformants with significantly different bacterial mass sizes were selected and activated in 20 mL SC-MET medium and cultured at 30°C and 200 rpm for 24 h. They were then transferred to 50 mL of fermentation medium without nutritional deficiencies and the initial OD was controlled. 600 The concentration of 0.2 was used and the culture temperature was 30°C and 200 rpm for 144 h.

[0040] S6 Fermentation parameter determination: Use UV spectrophotometer to measure the OD of bacterial solution 600 The copy number of the target gene in the cell was determined by conventional RT-qPCR, and the yield of the target product in the fermentation broth was detected by high performance liquid chromatography. The wavelength of the UV detector was 254 nm, the chromatographic column was Xcharge C18 (5 μL 100A4.6×250 mm Column), the mobile phase was 0.1% formic acid, the injection volume was 10 μL, and the flow rate was 0-20 min, 0.5 mL / min; 21-29 min, 0.8 mL / min; 30-35 min, 0.5 mL / min.

[0041] Ergothioneine (EGT) is a rare natural amino acid derivative with unique cell physiological protection functions such as scavenging free radicals, detoxification, maintaining DNA biosynthesis, normal cell growth, cellular immunity, anti-radiation, whitening, and anti-aging. Ergothioneine has broad application prospects in industries such as food, cosmetics, functional foods, and biomedicine, and was included in the list of novel foods in the European Union in 2018. The methods for obtaining ergothioneine include chemical synthesis and natural extraction, but both of these methods have high costs and low yields. The biosynthesis method using microbial cells has the advantages of high yield, low cost, and environmental friendliness, and has become the main synthesis strategy for ergothioneine. In the eukaryotic biological system, the biosynthesis of ergothioneine mainly involves two key enzymes, Egt1 (methyltransferase & 5-histidylcysteine sulfoxide synthase, which simultaneously catalyzes the methylation of cysteine or histidine to desacetylcysteine and the conversion of desacetylcysteine to desacetylcysteine sulfoxide) and Egt2 (hercynylcysteine S-oxide lyase, which binds to the sulfoxide after the formation of the C-S bond to form ergothioneine). The Egt1 and Egt2 on the ergothioneine synthesis plasmid used in this study to amplify gene fragments refer to the amino acid sequences of Egt1 from Clavieps purpurea and Egt2 from Neurospora crassa (such as Figure 4 ).

[0042] Such as Figure 8 , the highest yield of the strain obtained by the method provided by the present invention reached 67.63 ± 9.27 mg / L, which was 161.62% higher than that of the strain with free expression (25.85 ± 1.2 mg / L) and 86.57% higher than that of the strain that only underwent one round of δ integration (36.25 ± 0.25 mg / L). The highest copy number reached 11, and the copy number could reach 7 after one transformation. By comparing the copy number and the size of the bacterial blocks in its 96-well deep plate, it was found that the sizes of the bacterial blocks of the four selected strains from small to large were L1-3 < L2-2 < L1-1 < L2-3, and the copy numbers of these four strains from low to high were also L1-3 < L2-2 < L1-1 < L2-3. This result proved the feasibility of the method for screening high-copy transformants using a auxotrophic medium. Theoretically, the more copies of the gene integrated into the yeast chromosome, the higher the protein expression level and the higher the yield of the target product. However, through Figure 8It is not difficult to find from the data that the L2-2 (11) with the highest copy number does not have the highest yield. Therefore, it is inferred that the copy number and yield are only positively correlated within a certain range. Exceeding a certain range may also reduce the yield. This is consistent with the results of many studies. Different proteins have different optimal copy numbers. The higher the copy number, the better. Increasing the copy number may result in unchanged or reduced yield. It can also be found that the growth of L2-2 is also inhibited. It is speculated that the introduction of too many heterologous genes will affect the normal metabolism of cells, which can be improved through other metabolic modifications. The method provided by the present invention only takes 3-4 days for one round of integration operation, eliminating the screening work between each round of integration, and greatly reduces the probability of missing high-copy transformants through mixed bacteria treatment. The method provided by the present invention can be operated by a robotic arm, which provides the possibility for the application of high-throughput technology, so the efficiency can be further improved by high-throughput methods. In addition, from Figure 8 OD of the strain 600 It can be found that the integrated strains are better than the free strains in terms of growth, and do not require fermentation conditions to provide screening pressure, and can be more stably synthesized target products. The integrated strains are more tolerant to the culture medium, which provides opportunities for reducing fermentation costs, and saccharomyces cerevisiae is a kind of generally recognized as safe (GRAS) strains identified by the U.S. Food and Drug Administration, which has a very wide range of applications in industries such as food, especially the brewing industry, so the integrated strains do not require culture conditions to provide screening pressure and can synthesize the ability of natural products with excellent biological activity, which can provide an effective way for the development of the food industry and the application of rare natural products. Therefore, the results of the present embodiment further prove that the method for obtaining high-copy transformants provided by the present invention is more efficient and has stronger universality.

[0043] Example 2: Construction of an engineered yeast strain for producing cordycepin using the present invention

[0044] Construction of S1 CRISPR / Cas9 plasmid: First, restriction endonucleases NheI and HpaI were used to double-digest pCas9-URA3-gRNA to obtain vector fragments, and then ScF100 and ScR101, ScR102, ScR103, ScR104, and ScF101, ScF102, ScF103, ScF104 and ScR100 were used as primers, and pCas9-URA3-gRNA was used as a template to obtain four groups of fragments (two fragments in each group). The sgRNA sequence was replaced into the fragments by overlap primers, and then overlap PCR was performed using ScF100 and ScR100 as primers, and the corresponding two fragments in the four groups were used as templates to obtain four fragments. The nucleotide sequences of the four fragments are SEQ ID NO.1-4; The four fragments obtained above were connected with the vector fragment obtained after enzyme digestion by seamless cloning to obtain four plasmids: pCas9-gRNA (Delta1), pCas9-gRNA (Delta2), pCas9-gRNA (rDNA1), and pCas9-gRNA (rDNA2). The plasmid was quickly replicated by E. coli, and the target plasmid was extracted with a plasmid extraction kit for later use.

[0045] S2 Amplification of Donor DNA fragments: The Donor DNA fragments that need to be integrated into the yeast genome include the upstream and downstream homology arms of the multi-copy site, the expression frames of the key synthetic genes Cns1 and Cns2 of cordycepin, and the expression frame of the weakened complementation nutrient deficiency gene. The Donor DNA structure is as follows: Figure 3The nutritional deficiency gene selected for complementation in this embodiment is the key gene for methionine synthesis MET15 / 17, and the weak promoter selected is BTS1p. The primers listed in Table 3 were used to amplify the desired target fragments. The S288C genome was used as a template, and ScF301 and ScR301, ScF302 and ScR302, ScF303 and ScR303, ScF304 and ScR304 were used as primers to amplify the upstream and downstream homology arms of Delta, and the upstream and downstream homology arms of rDNA; pPICZA-Cns1-Cns2 was used as a template, and ScF312 and ScR312, ScF314 and ScR314 were used as primers to amplify the genes and terminators of the key genes Cns1 and Cns2 for cordycepin synthesis, respectively; plasmid pESC-URA3 was used as a template, and ScF313 and ScR313 were used as primers to amplify the promoter GAL1,10p of Cns1 and Cns2, the nucleotide sequence of which is SEQ ID NO.6; using S288C genome as template, ScF316 and ScR316 as primers, amplify weak promoter BTS1p, whose nucleotide sequence is SEQ ID NO.5; using pYES2-DLPU-8-MET17 as template, ScF217 and ScR217 as primers, amplify MET15 / 17 gene, whose nucleotide sequence GeneBank number is 851010; and using the homologous arms on the primers to perform overlap PCR to amplify the entire fragment. The entire expression cassette required for integration is 7.5Kb in length. Because the present invention obtains the DonorDNA fragment by PCR amplification, in order to ensure the fidelity of amplification, Figure 3 The donor DNA was divided into three segments. The fragments amplified above were used as templates and ScF301 and ScR313, ScF313 and ScR221, ScF317 and ScR302, ScF303 and ScR313, ScF213 and ScR304 were used as primers to amplify P21, P22, P23, P24 and P25, among which P21, P22 and P23 formed a group (Delta site) and fragments P24, P22 and P25 formed a group (rDNA site).

[0046] S3 electroporation transformation of Saccharomyces cerevisiae: Prepare Saccharomyces cerevisiae BY4741 competent cells, transform them with 2000 ng of each of the four Cas9-gRNA plasmids and 40000 ng of the corresponding donor DNA fragments P21, P22 and P23, and P24, P22 and P25, and spread them on SC-URA-MET nutritional deficiency plates.

[0047] S4 Rapid Iterative Screening Method: After completing one electroporation transformation, place the coated nutritional deficiency plate upside down in a 30°C incubator for static culture and observe the growth of the colonies on the plate. Figure 7 (a) Add 500 μL of sterile saline to the plate, use a spreader to slowly scratch the colonies on the plate, culture overnight for 14-16 hours, and prepare the competent cells in the form of mixed bacteria and start the next round of transformation. The second round of iterative operations is similar to the first one (such as Figure 7 (b) Then, the transformants with faster growth were selected and placed into 96-well deep plates with 1 mL of SC-MET medium to observe the growth of the strains (e.g. Figure 7 (c)).

[0048] S5 shake flask fermentation: The copy number was preliminarily estimated by the growth of the transformants in S4 in the nutrient-deficient medium, that is, the size of the bacterial mass accumulated at the bottom of the well plate. In order to determine whether the growth was related to the copy number, four transformants with significantly different bacterial mass sizes were selected and activated in 20 mL SC-MET medium, cultured at 30°C and 200 rpm for 24 h; then transferred to 50 mL fermentation medium without nutrient deficiency. Since cordycepin is toxic to Saccharomyces cerevisiae, the synthetic gene of cordycepin is driven by the inducible promoter P GAL1 and P GAL10 Driven, during the fermentation process, when the OD600 of the fermentation liquid reaches 0.6-0.8, it is centrifuged, and the bacteria are washed twice with sterile Wahaha water and then inoculated into the fermentation medium with the carbon source replaced with galactose to continue fermentation, and cultured at 30°C and 200r / min for 144h.

[0049] S6 Fermentation parameter determination: The OD600 of the bacterial solution was determined using an ultraviolet spectrophotometer, the copy number of the target gene in the cells was determined by conventional RT-qPCR, and the yield of the target product in the fermentation broth was detected by high-performance liquid chromatography. The chromatographic conditions were a mobile phase of water: methanol (95:5) gradient, a column temperature of 25°C, a flow rate of 0.8 mL / min, a detection wavelength range of 210-400 nm, and an injection volume of 10 μL.

[0050] Cordycepin is the first nucleoside antibiotic isolated from fungi. It has multiple biological activities such as antioxidant, anti-inflammatory, antiviral, anti-tumor and immunomodulatory, and has broad application prospects in clinical medicine. Traditional methods for obtaining cordycepin mainly include extraction from natural Cordyceps and chemical synthesis. The first two methods have always had problems such as long cycle and high cost, and cannot meet market demand. With the development of biosynthesis and the gradual identification of cordycepin biosynthesis genes, the method of constructing cell factories to produce cordycepin in a green and efficient manner has become a very promising research strategy. In 2011, the key genes for cordycepin synthesis were analyzed, and then the key genes Cns1 and Cns2 for synthesizing cordycepin (such as Figure 5 ).

[0051] like Fig. 9 After two rounds of iterative transformation, the maximum number of copies reached 8, and the yield reached 62.01±2.4 mg / L, which was 222.13% higher than that of the free expression strain (19.25±0.55 mg / L). Comparing the copy number with its growth in nutritional deficiency culture, that is, the size of the bacterial mass accumulated at the bottom of the 96-well plate, it can be found that there is a positive proportional relationship, which also confirms the feasibility of the screening method of the present invention. Fig. 9 It can be found that with the increase of copy number, the cordycepin yield of the strain also increases. Consistent with the result of thioneine production strain, the growth of the integrated expression strain of cordycepin synthesis strain is significantly better than that of free expression strain. This result proves that the present invention is more efficient and has stronger universality.

[0052] All nucleotide sequences in the present invention are shown in Table 1. The genotypes of plasmids and strains are shown in Table 2. All key primer sequences are shown in Table 3.

[0053] Table 1 All nucleotide sequences

[0054]

[0055]

[0056] Table 2 Plasmids and genotypes of strains

[0057]

[0058] Table 3 Key primer sequences

[0059]

[0060]

[0061]

[0062] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment, characterized in that: The method combines the screening strategy of weakening and complementing auxotrophic gene with the strategy of multi-copy integration of delta sequence / rDNA sequence by means of CRISPR-Cas9 gene editing technology, including the construction of Cas9-gRNA vector, amplification of genomic integration fragments, iterative transformation of Saccharomyces cerevisiae and rapid screening.

2. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 1, characterized in that: The method for constructing a Cas9-gRNA vector is as follows: a free expression plasmid suitable for Saccharomyces cerevisiae and containing Cas9 and sgRNA expression frames is selected as a starting vector, the starting vector is double-digested with a restriction endonuclease, the sgRNA expression frame is removed, and a linearized vector is obtained, two pairs of sgRNAs are designed according to the sequences of the multi-copy integration sites of Saccharomyces cerevisiae: the delta site and the rDNA site, and four sgRNA expression frames containing the target sgRNA sequences are constructed by enzyme ligation or overlap PCR, and the nucleotide sequences of the fragments are shown in SEQ ID NO.1-4, and they are further connected to the linearized vector by enzyme ligation or seamless cloning to construct a Cas9-gRNA vector.

3. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 1, characterized in that: The method for amplifying the genome integration fragment is: amplifying the donor DNA fragment by overlap PCR, including the target gene expression frame, the weak promoter-driven nutritional deficiency marker gene expression frame and the left and right homologous arms.

4. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 1, characterized in that: The method for iterative transformation of Saccharomyces cerevisiae is as follows: prepare Saccharomyces cerevisiae competent cells, electroporate an appropriate amount of Cas9-gRNA plasmid and the corresponding Donor DNA fragment, and then spread them on a nutrient-deficient solid plate.

5. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 1, characterized in that: The method for rapid iterative screening is as follows: after completing one electroporation transformation, the nutritionally deficient solid plate coated with yeast cells is inverted and placed in a 25-30°C incubator for static culture, and the growth of the yeast colonies on the plate is observed. When 5-15 transformants appear, an appropriate amount of sterile saline is added, all colonies are scraped with a coating rod, and transferred to a nutritionally deficient liquid culture medium for overnight enrichment culture. Competent cells are prepared in the form of mixed bacteria and the next round of yeast transformation is started until the target strain is obtained.

6. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 2, characterized in that: The starting vector suitable for Saccharomyces cerevisiae and containing Cas9 and sgRNA expression frames is pCas9-URA3-gRNA, and the restriction endonucleases used for linearization of the vector are Nhe I and Hpa I.

7. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment according to claim 3, characterized in that: Promoters include but are not limited to P BTS1 , P PDA1 , P ERG1 .

8. The method for rapidly iteratively obtaining a Saccharomyces cerevisiae recombinant with high-copy integration of a long-fragment DNA fragment according to claim 3, characterized in that: Auxotrophic genes include URA3, LEU2, MET15 / 17, and HIS3.

9. The method for rapidly iteratively obtaining a Saccharomyces cerevisiae recombinant with high-copy integration of a long-fragment DNA fragment according to claim 4, characterized in that: Saccharomyces cerevisiae is a nutritionally deficient strain of Saccharomyces cerevisiae.

10. The method for rapidly iteratively obtaining a cerevisiae recombinant with a high copy number of a long DNA fragment integrated according to claim 9, characterized in that: Auxotrophic Saccharomyces cerevisiae include but are not limited to BY4741, BY4742, BY4743, INVSc1, EBY100, and AH109.

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