Whole Genome Sequencing And Functional Annotation Of A.Westerdijkiae And Prediction Of Important Secondary Metabolite Biosynthesis Gene Clusters

Aspergillus westerdijkiae is a filamentous fungus in Aspergillus section Circumdati.It has caused wide concern due to its significant capability in ochratoxin A(OTA)production.OTA,a polyketide secondary metabolite,is potentially carcinogenic in humans through its induction of oxidative DNA damage and is neurotoxic,with a strong affinity for the brain.In addition,OTA also induce renal adenomas and hepatocellular carcinomas in rodents.A.westerdijkiae has a worldwide distribution and is responsible for the contamination of agricultural crops,fruits,and food commodities,as its secondary metabolite ochratoxin A poses a potential threat to the health of human and animals.As a member of the filamentous fungi family,its capacity for enzymatic catalysis and secondary metabolite production is valuable in industrial production and medicine.To understand the genetic factors underlying its pathogenicity,enzymatic degradation,and secondary metabolism,we sequenced and analyzed the whole genome of A.westerdijkiae and compared it with eight other sequenced Aspergillus species.We sequenced the complete genome of A.westerdijkiae and assembled approximately 36 Mb of its genomic DNA,in which we identified 10,861 putative protein-coding genes.We constructed a phylogenetic tree of A.westerdijkiae and eight other sequenced Aspergillus species and found that the sister group of A.westerdijkiae was the A.oryzae-A.flavus clade.These two close species could provide additional reference sequence for A.westerdijkiae annotation.By searching the associated databases,we identified 716 cytochrome P450 enzymes,633 carbohydrate-active enzymes,and 377 proteases.By combining comparative analysis with Kyoto Encyclopaedia of Genes and Genomes(KEGG),Conserved Domains Database(CDD),and Pfam annotations,we predicted 228 potential carbohydrate-active enzymes related to plant polysaccharide degradation(PPD).We found a large number of secondary biosynthetic gene clusters,which suggested that A.westerdijkiae had a remarkable capacity to produce secondary metabolites.Furthermore,we obtained two more reliable and integrated gene sequences containing the reported portions of ochratoxin A biosynthesis and identified their respective secondary metabolite clusters.We also systematically annotated these two hybrid tlpks-nrps gene clusters involved in ochratoxin A biosynthesis.These two clusters were separate in the genome,and one of them encoded a couple of GH3 and AA3 enzyme genes involved in sucrose and glucose metabolism.The genomic information obtained in this study is valuable for understanding the life cycle and pathogenicity of A.westerdijkiae.We identified numerous enzyme genes that are potentially involved in host invasion and pathogenicity,and we provided a preliminary prediction for each putative secondary metabolite biosynthesis gene cluster.In particular,for the ochratoxin A biosynthesis related secondary metabolite gene clusters,we delivered their components with domain and pathway annotations.This study sets the stage for experimental verification of the biosynthetic and regulatory mechanisms of ochratoxin A and for the discovery of new secondary metabolites.