Heterologous Expression Of Str Gene Cluster In Endophytic Fungus Stachybotrys Sp.g12 From Paris Polyphylla Var.yunnanensis And Its Product Mining

Secondary metabolites are often called natural products,and play a very important role in the discovery of drug precursors for the treatment of human diseases.For example,more than half of clinically approved drugs are natural products or their derivatives.Although significant progress has been made in natural resource research,traditional natural product discovery methods can not meet the growing demand for new bioactive molecules in drug research and development,for one thing,the discovery of"duplicative"natural products leads to a serious imbalance in the input-output ratio of research and development,and for another many natural products have low or no production under the conventional laboratory culture conditions.Therefore,activating the"silent"natural product biosynthesis genes/gene clusters in fungi by heterologous expression is an effective way to find new natural products.Generally,the natural product biosynthesis gene cluster of fungi contains 2 to10genes,and the heterologous expression of 10 or more natural product biosynthesis genes is rarely studied.In this study,we discovered an orphan biosynthetic gene cluster of salicylaldehyde derivatives in the fungus Stachybotrys sp.g12,which has 18 genes.We used bioinformatics tools to calibrate and annotate the genes in this gene cluster.However,we focused on the str A,str B,str C and str D genes,because previous comparisons between this gene cluster and similar gene clusters found that these gene clusters all contained these four homologous genes,so we guessed that the formation of salicylaldehyde derivatives might be related to these four genes.Then the target genes were constructed into corresponding plasmids by using relevant operations of molecular biology.The constructed plasmid was detected and verified,and then the constructed plasmids p TAex3-str A,p Ade A-str B,p Ade A-str B+str D and p USA-str C were introduced into Aspergillus oryzae protoplasts for heterologous expression using high osmotic pressure solution PEG/Ca Cl₂.Firstly,the PKS gene str A was expressed separately in Aspergillus oryzae to produce metabolites 1-5.Compared with vir A products in previous literature^[111],the carbon atom number of 1 was two less,and conjugated dienes replaced trienes,but the overall structure of the two products was similar,indicating that str A was also involved in the biosynthesis of salicylaldehyde derivatives.Then,the SDR genes str A and str B were expressed in Aspergillus oryzae together to obtain 6 as the main product.Then another SDR gene str D was introduced,and a complex metabolic profile was obtained,including six new isolated products 7-12,among which compound 9-12,which we named stachysalicyloids A-D,contained salicylaldehyde or salicylol parts with different unsaturation in the alkyl chain.Thus,it was found that PKS str A and the two SDR str B and str D could produce salicylaldehyde derivatives,albeit in very low yields.Therefore,we added cupin domain protein gene str C on the basis of positive strain,and found that the yield of compound 9 increased significantly.We successfully activated the"silent"genes（str A gene,str B gene,str C and str D gene）in the str gene cluster by using the filamentous fungus Aspergillus oryzae as the heterologous expression host.Finally,12 compounds were identified and the function of these genes were determined.The structures of these compounds were determined by NMR,and the stereoconfiguration of compound 1 was determined by X-ray single crystal diffractometer,thus the stereoconfiguration of compound 2-8 was deduced.The function of these genes was also clarified by heterologous expression.The str A gene produced compounds 1,2,3,4 and 5 in the heterologous host of Aspergillus oryzae,the str A and str B genes produced compound 6,and the str A,str B and str D genes produced compounds 7,8,9,10,11 and 12,and the yield of compound 9 was significantly increased after adding the str C gene to these three genes.Using Aspergillus oryzae NSAR1 as the host for heterologous expression can not only activate the"silent"gene/gene cluster and clarify the function of the silent gene cluster,but also can get some intermediates and products,which is of great help to the study of fungal natural products and their biosynthetic pathways.Reconstructing the biosynthesis mechanism in the heterologous host Aspergillus oryzae can not only determine each step of the enzyme catalysis in the synthesis process,but also determine the intermediates and final natural products.