Modulating The Terpene Biosynthesis Pathway In Yeast To Improve The Yield Of α-Amyrin By Using CRISPR/Cas9 Techniques

ObjectiveTerpenoids are naturally occurring compounds found primarily in the roots,stems,leaves,flowers and fruits of plants.Triterpenoid saponins consist of 6 isoprenoid moieties.They have complex chemical structures and diverse pharmacological activities.Ursane-type triterpenoid saponins are derived from α-amyrin.A variety of ursane-type triterpenoids like ursolic acid and 18-dehydroursolic acid have biological activities such as antibacterial,antiinflammatory,anti-thrombotic,hepatoprotective,etc.,and demonstrate great value in drug development.However,low titer of the triterpene saponins in the plants and difficulties in isolation and extraction have limited the wide application of triterpene saponins.The development of synthetic biology provides a powerful means for obtaining large amount of medicinal ingredients through heterologous biosynthesis.Therefore,according to the principles of synthetic biology,the yeast strain Saccharomyces cerevisiae WAT11 was used as the host,combined with the regulation of the key enzyme genes related to the endogenous anthraquinone synthesis pathway in yeast,to construct a yeast strain with high production of α-amyrin,which was later constructed.A complete triterpene saponin synthesis pathway is established,and a foundation is established for the continuous and mass production of ursolic triterpene compounds such as ursolic acid and 18-dehydrouric acid in Saccharomyces cerevisiae.Methods 1.Analysis of target sites in WAT11.By consulting the literature,it was learned that the Saccharomyces cerevisiae strain WAT11 preserved in the laboratory is an auxotrophic strain.Search NCBI for the distribution of the defective genes in the S288 c chromosome of Saccharomyces cerevisiae,extract the genome,PCR amplification fragments,sequencing the actual sequence of the fragment,acquire the inactivation of the WAT11 auxotrophic genes and the upstream and downstream sequences of each gene coding region.The auxotrophic gene locus was selected as a gene editing target.2.Gene editing in WAT11.Based on the actual sequence of the target site,use the online software CHOPCHOP(http://chopchop.cbu.uib.no)to design the 20 bp RNA sequence using the pRS42H-trp plasmid with the trp1-gRNA sequence as a template,and use the inverse PCR(Inverse PCR,iPCR)method amplifies a linearized fragment containing the gRNA sequence of the target target site and cyclizes with T4 ligase to construct a specific gRNA plasmid for each target site.The genes of the MVA pathway were amplified using the genomes of Saccharomyces cerevisiae WAT11,E.coli and Bacillus subtilis as templates and inserted into the p426-GPD vector.Primers with corresponding target site homology arms were designed to amplify the donor fragments containing the expression cassette of the target gene,and genomic integration of the target gene or mutation of the target gene was achieved using the CRISPR/Cas9 technique to obtain the WAT11-derived strains.3.Metabolites anlysis of WAT11 derivative strains.The WAT11-derived strains were cultured in the same conditions,and the production efficiency of the derivative strain was evaluated using squalene as a labeled compound.GC/MS was used to determine the content of squalene(α-amyrin after oxidation and cyclization)of common triterpenoids.Using WAT11 as a reference,the most productive derivative strain of squalene was selected.Further,the expression plasmid pEXPR-IaAS1 of the amyrin synthesis enzyme IaAS1 gene,which was constructed in the earlier stage of the research group,was transferred to the derivative strain to obtain a yeast strain with a free plasmid,and at the same time,the IaAS1 gene expression was performed using a CRISPR/Cas9 technique.The yeast strains integrated into the genome of the cassette were compared for the yield of α-amyrin with the episomal plasmid strains and the integrated genome strains,and the target strain with higher yield was screened.Results 1.Screening the targeting sites in WAT11.It can be known from the literature that WAT11 is an auxotrophic strain of trp1,ade2,can1,ura3,his3,and leu2,and the gene sequence is continuous and does not have introns.The BTS1 gene encoding GGPPS of the diterpene branching pathway is intact and continuous,so the TRP1,CAN1,URA3,ADE2,HIS3,LEU2 and BTS1 targets were selected as genetic editing sites for follow-up experiments.2.10 WAT11 derivative strains have been constructed.WAT11t,WAT11 f,WAT11s,WAT11i1,WAT11i2 WAT11 ts,WAT11ti1,WAT11 tf and WAT11 tfA these 9 knock-in strains were successfully built based on the enzymes tHMGR(labeled as t),FPPS(f),SS(s),IPPI1(i1),IPPI2(i2)and ACS(A)in the terpenoid synthesis pathway.And a BTS1(g)mutation strain for the diterpene pathway disruption was also constructed,named WAT11Δg.Under the same conditions for 72 hours,the squalene yields of the 10 S.cerevisiae strains were compared.It was found that the squalene production of the WAT11 tfA strain was increased by nearly 20 times compared to WAT11.3.WAT11 tfA with high-yield α-amyin have been constructed.Treat the WAT11 tfA strain obtained above as the host,the pEXPR-IaAS1 expression plasmid was transformed into it by the lithium acetate transformation to construct the WAT11tfA/IaAS1 strain.Meanwhile,the IaAS1 gene expression cassette was integrated into the HIS3 site of the WAT11 tfA genome using CRISPR/Cas9 technology to construct the WAT11 tfAX strain.Using the corresponding medium cultured for 3 days,the accumulation of α-amyrin by two strains was compared.The results showed that compared with WAT11/IaAS1,the α-amyrin yields of WAT11tfA/IaAS1 and WAT11 tfAX strains were significantly increased,and the α-amyrin yield of WAT11tfA/IaAS1 strain was 4.67±0.21 mg/L,about 21 times that of WAT11,WAT11 tfAX The α-amyrin yield of the strain was 2.18±0.14 mg/L,about 10 times that of WAT11,and the yield of WAT11tfA/IaAS1 was slightly higher than that of WAT11 tfAX.The WAT11tfA/IaAS1 and WAT11 tfAX strains were cultured in the same culture conditions for 7 days.The yield of α-amyrin of WAT11tfA/IaAS1 strain was 13.26 mg/L,and the accumulation of squalene was 105.47 mg/L.The α-amyrin of WAT11 tfAX strain was obtained.The yield was 10.39 mg/L and the accumulation of squalene was 210.20 mg/L.ConclusionIn this dissertation,we focused on the MVA pathway of terpenoids and regulated the key enzymes of terpenoids through CRISPR/Cas9 technology to construct a WAT11-derived strain WAT11 tfA and two strains of WAT11tfA/IaAS1 with high production of α-amyrin.WAT11 tfAX.The construction of these strains not only enables the mass production of ursolic triterpenoids,such as ursolic acid and 18-dehydrouric acid,but also lays the foundation for the research on triterpenoids of other configurations and shows the synthesis.The great potential of biology in the microbial production of active ingredients of medicinal plants.