Heterologous Expression Of PKS₅ Gene Cluster Of Stereum Hirsutum

Fungal natural products(NPs)possess diverse chemical structures and biological activities and play an important role in human,animal and plant medicine.However,studies have found that most microbial biosynthetic gene clusters are not expressed under normal laboratory conditions.In our previous work,we found that the five PKS biosynthetic gene clusters in Stereum hirsutum may be in a low expression or silent state.In this dissertation,a PKS₅ cluster was selected and expressed in Aspergillus nidulans(an uracil and uridine auxotroph strains).Firstly,considering the recombinant and heterologous expression of the large DNA fragments,the p SMART BAC vector with high-stability and high-bearing capacity was optimized and rebuilded into p SMART FAC and named pYUZ299,by integrating a constitutive promoter gal A,gene pry G(orotidine-5'-phosphate decarboxylase)and gene Hyg R and an Aspergillus autonomously replicating sequence(AMA1).The shuttle pYUZ299 vector was used to carry the complete gene cluster,and not only for the construction of expression vector in E.coli,but heterologous expression in filamentous fungi.Secondly,from the genome BAC library a complete PKS₅ gene cluster was screened out and then the expression vector was obtained by using Red/ET homologous recombination system to mediate the linear plus circular recombination between the linear shuttle vector pYUZ299 and the circular BAC library vector and named pYUZ301.Finally,spores and mycelium of A.nidulans were deconstructed by enzymes to obtain protoplasts,and PEG-Ca Cl₂ mediate mehod was applied to transformation.The target transformant with the expression plasmid pYUZ301 was obtained,and the transformant with the empty vector pYUZ299 was a control group.We used positive transformants for subsequent product detection,and detected three new molecular ion peaks that did not appear in the control group by using liquid YMG culture.