Preliminary Study On Biosynthesis Pathway Of Sanxiapeptin In Penicillium Oxalicum

With the increasing difficulty of antibiotics mining,people refocused their attention on the discovery and research of secondary metabolites in filamentous fungi,and are committed to finding new antibiotics.Penicillium oxalicum is an important filamentous fungus with many biological functions.P.oxalicum has a rich variety of secondary metabolites,mainly including polyketides,terpenes,alkaloids,fatty acids and various alkanes and other compounds.Previously,our laboratory isolated a new linear pentapeptide "Sanxiapeptin" from P.oxalicum SG-4,the structure of which was Amoba-N-Me-L-Thr-D-Thr-N-Me-L-Val-L-Ser,in order to study the mechanism of the biosynthesis of Sanxiapeptin,genomics and transcriptomics methods were applied to research the synthesis of Sanxiapeptin gene cluster,which laid the theoretical foundation for the improvement and popularization of Sanxiapeptin production.The main results of the current study are as follows:1.The whole genome sequencing result of P.oxalicum SG-4 showed that the SG-4sequence was 30.1 Mb in size and consists of 9 Scaffolds,including 1 mitochondrial Scaffold.The sequence size was consistent with the sequencing results of P.oxalicum submitted strains.The GC content of P.oxalicum SG-4 was 50.6%,which was similar to the strains of the same genus.2.By analyzing of the genome sequence,it was found that the genome contained a wealth of natural product synthetic gene clusters,a total of 34,of which 22 were NRPS gene clusters,and the rest were terpenoid,polyketone,and indole compound synthetic gene clusters.3.The transcriptomic analysis of different P.oxalicum strains,it was shown that NRPS-9gene cluster was screened and identified as a possible gene cluster for the synthesis of Sanxiapeptin.Combined with the transcriptomic analysis of SG-4 under different culture conditions,it was further verified that the gene cluster was a possible synthetic gene cluster.4.The inability of strain 114-2 to produce Sanxiapeptin may be related to the deletion of the 312 bp in gene cluster and the high expression of McrA transcription factor PDE?01139.5.The NRPS-9 gene cluster was very similar to the reported synthetic gene clusters of oxaline and histidyltryptophanyldiketopiperazine,but both of these synthetic gene clusters were only part of the NRPS-9 gene cluster.6.The homologous recombination vectors of NRPS-9-3,NRPS-9-10,NRPS-9-16 and NRPS-21-6 were successfully constructed with HygR as the marker gene.The NRPS-9-16 homologous recombinant vector was transformed into SG-4 protoplast,and no mutation was found in the monoclone strain.