Activation Of Two Bgcs From Marine Derived Trichoderma Harzianum By Heterologous Expression Strategy

The secondary metabolites of fungi are important sources of new drugs and lead compounds.However,with the discovery of more and more new drugs,the discovery of natural products derived from fungi hit a plateau after striving for decades.In contrast to the genes required for the primary metabolites,which scattered throughout the fungal genome,genes encoding active enzymes to produce secondary metabolites are usually arranged in a continuous manner to form a biosynthetic gene cluster(BGC).With the development of genomics and bioinformatics analysis,researchers found that much more BGCs existed in fungi.However,due to the limitations of culture conditions in laboratory,even more than 70%of biosynthetic gene clusters are silent or untapped.Therefore,the fungal natural products are still not fully discovered treasure.How to activate these silent gene clusters becomes the key problem to study the fungal natural products.In this paper,the marine derived filamentous fungus Trichoderma harzianum LZDX-32-08 was selected as the research object,and Aspergillus nidulans A1145 and L08030 were used as heterologous expression hosts to investigate the silent or low expression gene clusters in T.harzianum LZDX-32-08.Firstly,the heterologous expression system was built in A.nidulans A1145 and L08030.It was found that the transformation of a commonly used expression vector pQFl(containing the AMA1 sequence)into A.nidulans made the average yield of the bioactive compound cordycepin(COR)higher than the original strain by 1.72-and 1.84-fold,and the yield of A1145 AMA1 T1 in CD-ST medium reached 277 mg/L.Based on bioinformatics analysis and RT-PCR results,it was found that two interesting biosynthetic gene clusters:polyketide synthase(PKS)cluster8(C8)and terpene cluster26(C26)are silent or barely expressed in T.harzianum LZDX-32-08.For the target biosynthetic gene clusters,the recombinant plasmids pQF4(55 kb)containing C8 total genes and pQF3(26 kb)containing C26 total genes were constructed using yeast assembly technology,respectively.Meanwhile,in order to verify the function of two PKSs in C8 and the core structure of the PKS compound,a recombinant plasmid pQF2(42 kb)which only contained the two PKSs core genes were constructed.Then,pQF2-4 were successfully transformed to the expression hosts A.nidulans A1145 and LO8030,respectively.RT-PCR results confirmed the expression of key genes in the two gene clusters.The PKS C8 gene cluster is highly homologous to the tln cluster.By analyzing the LC-MS results of PKS transformants,it was found that there is a compound with molecular weight of 220,presumably tricholignan A.In addition,UPLC results showed that new product peaks appeared in terpene transformants in CD-ST medium.However,it is difficult to isolate the products because of the low absorption.At the same time,due to the impact of the COVID-19,the target compound has not been isolated.