| Lycopene is an important carotenoid and has been widely used in food,medicine,cosmetics and other production fields.Lycopene synthesis by microbial method has attracted more and more attention in recent years.At present,the biosynthesis of lycopene by microbial method mainly uses Escherichia coli and Saccharomyces cerevisiae as hosts,but it is far from meeting the industrial needs.In order to promote the industrialization of lycopene,it is of great significance to develop efficient lycopene synthetic chassis cells.Pichia pastoris has been widely used in the industry for heterologous expression of proteins,and P pastoris has been shown to be a potential chassis cell for the production of high value-added chemicals.Effective synthetic biology tools are important for industrial and biotechnology studies of microbial strains.However,the development of P pastoris gene editing tools is scarce.In addition,the lack of clear metabolic network studies also makes the studies on compound synthesis of P pastoris far inferior to model strain S.cerevisiae.In this study,lycopene was synthesized and optimized using P.pastoris as the chassis cell.A novel,simple and efficient CRISPR-Cpf1 system was developed in this yeast and applied to the reconstruction of lycopene synthesis branch of recombinant strain to promote lycopene synthesis.On this basis,the lipid synthesis was optimized to improve lycopene storage,further increase the yield of lycopene,and obtain lycopene high-yield strain.Through transcriptome analysis,metabolomics analysis and correlation analysis,the specific regulatory effect of sterol regulatory element SREBP(Sre)in the process of optimizing lipid synthesis of the recombinant strain was analyzed.The specific research contents and results are as follows:(1)Construction and optimization of lycopene synthesis pathway in P.pastorisLycopene was synthesized in P.pastoris GS115,and the lycopene synthesis pathway was optimized and reconstructed by screening lycopene synthesis pathway gene,optimizing lycopene synthesis pathway gene dose,and optimizing NADPH supply of lycopene synthesis pathway.The lycopene yield of recombinant engineering strain P2 OZPP was 33.19 mg/g DCW.(2)Development of CRISPR-Cpf1 system and its application in lycopene synthesis bypass reconstructionA novel efficient genome editing method mediated by CRISPR-Cpf1 system was established.The gene editing system was optimized by screening Cpf1 protein sources and optimizing the combination of different component.The efficiency of this optimized system for P.pastoris gene editing was 99±0.8% for single gene editing,65±2.5-80±3% for double genome editing and 30±2.5% for triple gene editing,respectively.In addition,the deletion of large DNA fragments(20 kb)and one-step integration of multiple genes in P.pastoris were achieved for the first time.It provides a new,simple and efficient gene editing tool for P.pastoris.Based on the established CRISPR-Cpf1 gene editing method,the FPP branch pathway of lycopene synthesis pathway of recombinant strain was blocked,and the established CRISPRdCpf11 method weakened the squalene pathway,which is another branch pathway of lycopene synthesis pathway.The yield of lycopene was increased to 38.99 mg/g DCW.The CRISPR-Cpf1 gene editing method and CRISPR-dCpf1 gene inhibition method were established for the first time in P.pastoris and effectively applied in metabolic engineering.(3)Lipid synthesis optimization to improve lycopene production and specific regulation analysisLycopene production was further increased by optimizing lipid synthesis,and lycopene high yield strain was obtained.It was found for the first time that SREBP(Sre)could promote lycopene synthesis in P.pastoris,and the lycopene yield was 43.23 mg/g DCW.Under high density fermentation in 5 L fermentor,the yield of this strain was 7.5 g/L,which was higher than the model strain S.cerevisiae,and the highest level in P.pastoris.Transcriptome,metabolome and correlation analysis were used to analyze the specific regulation of Sre on lycopene synthesis of high lycopene strain.Omics data showed that Sre not only regulated lipid synthesis but also up-regulated MVA pathway related genes and promoted lycopene synthesis of the recombinant strain.Moreover,the lycopene synthesis precursors of recombinant strains were insufficient.These results provide a method reference for the synthesis of other high value-added compounds and optimization of related metabolic pathway in P.pastoris and provide guidance for further synthesis and optimization of lycopene... |
PDF Full Text Request