Transcriptomic Responses Involved In Enhanced Perylenequinones Production Of Endophytic Fungus Shiraia Sp. Slf14 With Fructose As The Sole Carbon Source

Perylenequinones?PQ?are a class of natural light-sensitive compound,which can be used in anticancer,antiviral and skin diseases treatment.However,due to the limited sources and insufficient production of PQ,which can not meet the needs of large-scale clinical treatment and development,the production of secondary metabolites by liquid fermentation has the advantages of short cycle and high production efficiency,so it has been attached great importance to by people.In this study,a strain of endophytic fungus Shiraia sp.Slf14,which was screened in the early stage of laboratory,was used to study the effect of carbon source on it in liquid fermentation.The effect of PQ production showed that fructose was beneficial to the PQ synthesis of Shiraia sp.Slf14,and the mechanism of fructose promoting PQ synthesis was analyzed by transcriptome technique.This study can not only provide a reference for further exploring the synthetic pathway of PQ,but also lay a foundation for the large-scale production of PQ.?1?The effects of different carbon sources on the synthesis of PQ from Shiraia sp.Slf14 were studied.It was found that fructose was the best carbon source,at the optimum concentration of fructose 60 g/L,the PQ yield of Shiraia sp.Slf14 reached1753.64 mg/L,which was 1.73 times of that of glucose as carbon source.?2?Transcriptome analysis of Shiraia sp.Slf14 with fructose or glucose as carbon source was carried out.After sequencing on the Illumina HiSeq^TM4000platform,a total of 9627310966 nt data were obtained.14596transcripts were assembled,the average length was 1525.37 bp,and the N50 was 2597 bp.Transcripts are annotated in the NR,Swiss-Prot,Pfamn,String,Go and KEGG databases,the respective numbers were 10441,5624,6777,3465,7151 and 3913 espectively.Based on the annotation results in the KEGG database,3913 Unigenes were annotated into327 KEGG pathways.According to the analysis of differentially expressed genes,there are a total of 9183 differentially expressed gene,of which 212 were up-regulated,mainly involved in transport systems,energy metabolism and carbon metabolism pathways.?3?Based on transcriptome analysis,the molecular mechanism of fructose promoting Shiraia sp.Slf14 synthesis of PQ was further discussed.Genes involved in glycolysis,pentose phosphate pathway,and encoding citrate synthase,ATP citrate lyase and acetyl coenzyme A carboxylase were significantly up-regulated.This is beneficial to the synthesis of acetyl coenzyme A and malonyl coenzyme A precursor compounds.At the same time,the gene expression of glyoxylic acid cycle pathway and fatty acid synthesis were significantly down-regulated.This will inevitably lead to more acetyl coenzyme A flowing into the PQ synthesis pathway.Furthermore,qRT-PCR and enzyme activity analysis of related pathways were carried out,and the results were consistent with transcriptome analysis.In addition,we found that when fructose was used as carbon source,hypocrellin A biosynthesis clusters were significantly up-regulated.These results not only help us understand the mechanism of fructose promoting PQ synthesis,but also lay a foundation for revealing the PQ biosynthesis pathway of Shiraia sp.Slf14.?4?The feasibility of producing PQ by Shiraia sp.Slf14 fermentation with fructose based biomass inulin was discussed,and the inulinase was cloned and analyzed.It was found that inulin was suitable for PQ synthesis of Shiraia sp.Slf14.At the same time,the inulinase gene?Inu?of Shiraia sp.Slf14 was cloned successfully,and the open reading frame length of Inu is 1774 bp and it encodes 572amino acids.The Inu gene was ligated with pET-22b?+?vector and transformed into E.coil DH5?,and then expressed heterologous in E.coil BL21.Inactivated inulinase was obtained?inclusion body?,and the activity of inulinase was obtained by renaturation of inclusion body.The enzymology property indicates that the inulinase is an exo-inulinase,meanwhile,bioinformatics analysis of which is carried out,the reason of Shiraia bambousicola using inulin to produce PQ was studied.In this paper,the mechanism of fructose promoting the synthesis of perylenequinones by endophytic fungi Shiraia sp.Slf14 has been preliminarily studied,and through the study of inulinase as a starting point to provide a direction for the subsequent Shiraia sp.Slf14 to use cheap inulin to produce perylenequinones,this will also lay a foundation for metabolic regulation of perylenequinones.