| Shiraia bambusicola belongs to Shiraiaceae,and has been used as a traditional Chinese medicine in south of China for several centuries.Hypocrellin A(HA)is a perylenequinone which can be extracted from S.bambusicola,was usually used in photodynamic therapy(PDT).Hypocrellin A(HA)has antibacterial,antiviral and antitumor activities,and has shown great potential in the treatment of diseases such as cardiovascular diseases or skin diseases,and in the development of new photoelectric conversion materials.However,the current producing methods of HA are inefficient and unable to meet the huge market demand.In recent years,more and more attentions have been paid to the study of HA biosynthesis pathway expecting to transform the wild strains through genetic technology and other methods to make them adapt to large-scale industrial production.In order to take further study of the relevant genes involved in HA synthetic pathway in S.bambusicola S4201 strains,this study used the S4201 strains as the material,an efficient genetic transformation system was established and optimized based on the protoplasts made with hypha of the S4201 strains.The benomyl was used as a selection marker,the protoplasts preparing method with hypha has been optimized and a Polyethylene glycol(PEG)-mediated transformation method was established.Meanwhile,the endogenous GAPDH promoter Pgpd1 of the S4201 strains was identified through promoters selecting.We introduced a rigid 2A peptide(EAAAK)3 in the construction of plasmids,which can form three a helixes,ensuring that the two proteins between 2A peptide can not affect each other in co-expression of genes.In the further study,we overexpressed the O-methyltransferase/FAD-dependent monooxygenase gene(mono)and the hydroxylase gene(hyd)in the S4201 strains.By measuring the biomass,HA yield,and the relative expression level of mono and hyd genes or other adjacent genes in the HA synthetic gene cluster such as:multicopper oxidase gene(mco),major facilitator superfamily gene(mfs),polyketide synthase gene(pks),O-methyltransferase gene(omef)and FAD/FMN-dependent oxidoreductase gene(fad)in the transformants,the effects of overexpression in S.bambusicola was determined.The results showed that the expression level of mono and hyd genes in transformants was increased by 6 and 11 times than the controls,and the HA yield in the mono and hyd transformants was increased by 3 and 2 times than that in the control strain,respectively.The relative expression levels of other adjacent genes in the HA synthetic gene cluster also increased.These results indicated that the mono and hyd genes are involved in the HA biosynthesis.Furthermore,high HA-producing strains were obtained by overexpression studies.In this study,we further explored the relative genes involved in HA biosynthetic pathway,determined the important role of mono and hyd in HA biosynthetic pathway and also pointed out the direction of further research in HA biosynthetic genes. |
