Microbial Chassis Development For The Expression F Silent Genes In The Secondary Metabolites Cluster From Colletotrichum Gloeosporioides ES026

The diversity of fungal species and the diversification of biosynthetic gene clusters underscores a nearly limitless potential for metabolic variation and an untapped resource for drug discovery and synthetic biology.However,they house a multitude of silent biosynthetic gene clusters that are poorly expressed and whose products are unknown or’cryptic’.Stimulating the expression of these clusters and accessing their associated molecules is a major priority,as they are expected to have a veritable cornucopia of bioactivites.Access to dolastanes is of high interest because several oxidized members of these class modulate the effects of other drugs in resistant Staphylococcus aureus strains.Our preliminary study has demonstrated a chemical investigation into the organic extracts from the heterogenous expression of Cgt14 gene from Colletotrichum gloeosporioides ES026 resulted in the separation of one diterpene compound featuring the relatively scarce dolastane frame isolated from ocean sources exclusively.In this study,several bioinformatics tools had been utilized to detect the gene cluster of Cgt14 gene and elucidate the molecular functions of core genes,including SMURF and anti SMASH.Then we constructed the overexpression,heterologous expression systems of core genes(CYP450 genes-Cgt15,2-hydroxy acid dehydrogenase gene-Cgt23)from the same biosynthesis gene cluster using microorganisms(filamentous fungi,Saccharomyces cerevisiae and Escherichia coli)as chassis cells.(1)To elucidate the biosynthesis of dolastane type diterpenoids,the expression of silent genes was strengthened in Filamentous fungi.C.gloeosporioides ES026 was transformed using the 3 plasmids in accordance with the methods for Agrobacteriummediated transformation,and a randomly chosen transformant was verified by PCR.Unfortunately,the new dolastane type analogue was not detected when the CYP450 geneCgt15,2-hydroxy acid dehydrogenase gene-Cgt23 were overexpression in the original strain Colletotrichum gloeosporioides Es026,which may be due to insufficient precursor.(2)Moreover,Saccharomyces cerevisiae was chosen as the chassis strain.The t HMG1,ERG20(FPPS)and Cgt14 genes were integrated into the genome of S.cerevisiae to construct a host strain TCgt14 for efficient synthesis of dolastane diterpenes.Then heterologous reconstitution of CYP450 genes-Cgt15 in S.cerevisiae leads to produce a new dolastane type analogue.(3)In vitro biochemical assays,2-hydroxy acid dehydrogenase-Cgt23 protein was expressed and purified by E.coli,and the result proved that the enzyme could catalyze pyruvate to lactic acid,which had certain catalytic activity.These results not only provide efficient methods to further improve the product yield and generate new dolastane type compounds,but also provide theoretical guidance for the rational design and construction of microbial strains to produce natural products.