Identification Of Ilicicolin H Biosynthetic Gene Cluster In Neonectria Sp.DH2

Fungal secondary metabolites are complex and diverse in structure and species.Many of them have various biological activities such as antibacterial,anti-tumor,antiviral and immunosuppressive,which are important sources of drug development.However,the separation of secondary metabolites directly from the fermentation of the production strain is difficult and inefficient,because the yield of fungal secondary metabolites is quite low generally.Synthetic biology is an emerging field in recent years.Molecular biological techniques are used to manipulate the metabolic pathways of secondary metabolites and the biosynthetic gene clusters are transferred to heterologous hosts for expression for high-yielding of secondary metabolites.Aspergillus nidulans is a model strain with clear genetic background,which is commonly used for heterologous expression of fungal genes.The target gene clusters are transferred to A.nidulans for heterologous expression,which could help identify the biosynthetic gene cluster function,create new secondary metabolites and improve the yield thereof.The subject of this study is Neonectria sp.DH2 isolated from Meconopsis grandis Prain in Tibet.In the previous work,we isolated many active secondary metabolites from Neonectria sp.DH2,including compound ilicicolin H.Ilicicolin H is a hybrid polyketide-nonribosomal peptide natural product,which exhibits potent,broad-spectrum antifungal activity,with sub-μg/mL MICs against Candida spp.,A.fumigatus and Cryptococcus spp.,which is generally superior to comparators.It has a good prospect in clinical application.There are no reports on the biosynthetic gene clusters of ilicicolin H until now.Therefore,we carried out the identification of the biosynthetic gene cluster of ilicicolin H from DH2 by heterologous expression in A.nidulans and predicted the biosynthetic way of ilicicolin H.Then we added promoter adgA and xylP upstream of the biosynthetic genes to achieve higher-level production in the heterologous expression strain.The details are given as follows.Firstly,the wild-type strain Neonectria sp.DH2,which produces ilicicolin H was subjected to genome sequencing.The whole genome sequence was obtained and analyzed.The potential biosynthetic gene clusters were predicted by using the software antiSMASH.According to gene annotation and sequence alignment with homologs,Cluster3 was proposed to be ilicicolinH biosynthetic gene cluster.Then the core genes orf7-11(named as iliA-E)of Cluster 3 were amplified from the genomic DNA and inserted in the vector pRG-AMA1 to give plasmid pDH01.HPLC-MS analysis revealed A.nidulans expressing pDH01 accumulates ilicicolin H and its homolog compound ilicicolin J,in contrast to the original strain and the strain expressing empty vector.The structure of the compound was further determined by NMR.Moreover,three more plasmids(pDH02-04)were constructed and expressed in A.lividans as well.Ilicicolin H was found only in the strain expressing pDH04(iliB-E),but not in the strains expressing pDH02(iliB-C)or pDH03(iliB-D).Therefore,Cluster3 in DH2 can be identified as ilicicolin H biosynthesis gene cluster.While iliA is not directly involved in the biosynthesis of ilicicolin H,iliE is indispensable for the synthesis.Bioinformatics analysis suggests iliD might encode an S-adenosyl-L-methionine(SAM)-dependent Diels-Alderase,which is one of a few enzymes of this kind found in fungal secondary metabolism reported to date.The expression of exogenous proteins in filamentous fungi is usually very low and strong promoter can be used to drive the efficient production of heterologous proteins as demonstrated in many literatures.In order to further efficiently express the target product and increase the yield of ilicicolin H,agdA-promoter of glucosidase and xylP-axylose inducible promoter were jointly used to express genes iliB-E.After adding the strong promoters,the yield of ilicicolin H was increased by 3.23 times,up to 2.33mg/L.In order to find out the components of more suitable fermentation medium,carbon sources in the medium were optimized.The results showed that the yield of ilicicolin H generated in the medium containing 1%xylose was the highest,reaching 3.87mg/L.In this study,the biosynthetic gene cluster of ilicicolin H of DH2 was successfully identified by hetero-expression in A.nidulans,which lays a foundation for comprehensive understanding of the biosynthetic pathway of ilicicolin H and engineering transformation to obtain new structural derivatives.The discovery of more novel active ilicicolin compounds may contribute to the research and development of innovative drugs.