Study On The Function Of Biosynthetic Gene G7779 Of The Novel Anti-tumor Skeleton Compounds Lithocarpins

Polyketides are important secondary metabolites of marine fungi,and the complexity of their structure makes them have diversified biological activities.So far,more than 10000 kinds of natural polyketides have been found in nature,which has a very broad application prospect.In the early stage,our team isolated four new skeleton compounds,lithocarpins A-D,from the metabolites of marine fungus Phomopsis lithocarpus FS508.Some of them showed good cytotoxicity against human cancer cell lines SF-268,MCF-7 and Hep G-2 and have great development potential.In order to investigate the biosynthesis related genes of lithocarpins,based on the previous research results of transcriptome sequencing and whole genome sequencing of the fungus,the candidate biosynthesis gene cluster 41 was obtained by using anti SMASH,and gene g7779 with completely unknown function was found in this gene cluster.For the purpose of elucidating the biological function of g7779,in this study,we cloned and expressed the gene,analyzed its enzymatic properties,and elucidated the function of g7779 by overexpression and gene knockout.(1)Cloning,expression and characterization of gene g7779.After gene g7779 was expressed in E.coli,the purified protein was analyzed by mass spectrometry,bioinformatics and enzyme activity detection.The results showed that the protein g7779 was a new acyltransferase with glutamic pyruvic transaminase activity;acyltransferase activity analysis showed that the optimum reaction temperature for acetyl Co A was 35?,the highest enzyme activity was at p H7.0,and the thermostability was good at 40?,the values of K_m and V_max of the protein towards acetyl Co A were 761.57?mol/L and 29370?mol/(mg·min),respectively;the protein consisted of 326 amino acid residues with a molecular weight of 36 k D.Its secondary structure consisted of 55.52%?-helix,29.45%random coil,9.82%extended chain and 5.21%?-turn.(2)Overexpression analysis of gene g7779 gene.The overexpression vector of gene g7779was successfully constructed and introduced into the protoplast of P.lithocarpus.The overexpression strain of gene g7779 was obtained by screening.The results of real-time PCR showed that the expression level of gene g7779 was 23.74 times higher than that of wild-type strain.The results of HPLC analysis of fermentation products showed that compared with wild-type strain,the production of lithocarpin A and its derivatives was significantly increased,the abundance of secondary metabolites and the production of related compounds were significantly enhanced.It was preliminarily speculated that gene g7779 was related to the production of lithocarpin A.(3)Knockout analysis of gene g7779.The CRISPR/Cas9 knockout system suitable for P.lithocarpus was constructed,and gene g7779 was knocked out by this system.The HPLC analysis of the fermentation products of gene g7779 deletion strain and wild-type strain showed that the corresponding chromatographic peak of compound lithocarpin A disappeared,and two new chromatographic peaks were produced,which might be the biosynthetic precursor of the former.The results confirmed that the gene g7779 was related to the production of lithocarpin A.In this thesis,the function of gene g7779,the novel anti-tumor skeleton compounds biosynthesis gene of marine fungus P.lithocarpus,was studied.Through in vitro cloning,heterologous expression,enzymatic analysis,overexpression and gene knockout,the function of gene g7779 was elucidated,and its relationship with the biosynthesis of lithocarpins was confirmed.The results of this study can provide necessary gene function information and gene editing tools for further elucidating the biosynthetic pathway of lithocarpins,and lay a foundation for exploring more functional genes and new bioactive secondary metabolites in P.lithocarpus.