Study On The Metabolic Regulation Mechanism Of Trisporic Acid On Blakesla Trispora

Lycopene production through microbiology has gained greater attraction with the rapid development of biotechnology.Among various types of lycopene producing strains,B.trispora is considered as important strain at industrial production level.B.trispora belong to Zygomycetes and can be divided into plus(+)and minus(-)mating-type.The co-culture of these two mating-type help to produce large amount of lycopene.Among the two mating type,B.trispora(-)are the main producers of lycopene while B.trispora(+)are considered as the pairing strain greatly promoting the synthesis of lycopene of minus strain.It is generally believed that the deficiency of sex hormone Trisporic acid is the main reason for not synthesizing lycopene alone by B.trispora(-)in large quantities.Though extensive research has been conducted on Trisporic acid but the up or down-regulated genes triggered by Trisporic acid and the differentially expressed genes related to the synthesis of lycopene are unknown.Further,the metabolic regulation mechanism of Trisporic acid on the synthesis of lycopene of B.trispora is still unclear.Therefore,in the current study RNA-Sequencing was used to analyze the differential expression of genes of B.trispora(-)under Trisporic acid stimulation,and some key metabolic pathways and the genes stimulated by Trisporic acid were obtained.The gene redY encoding 5 dehydrogenase was further analyzed as follows:(1)Effects of Trisporic acid on the synthesis of lycopene of B.trispora(-): In this study saponifying agent and organic extraction agent were selected for optimization which are key factors affecting trisporic acid extraction.The effects of different concentrations of Trisporic acid on lycopene production were investigated.The relationship between the time of culture after the addition of Trisporic acid and the expression of key genes in the lycopene synthesis pathway were analyzed.The results showed that sodium carbonate as saponifying agent and chloroform containing 5% isopropanol as organic phase had the highest extraction efficiency.The amount of Trisporic acid obtained by extraction is 24 g/L.At 6 h after the addition of Trisporic acid,the difference in the expression of key genes was the most obvious.(2)The mechanism of high lycopene production of B.trispora(-)stimulated by Trisporic acid was analyzed by RNA-Sequencing.Based on BGISEQ-500 platform RNA-Sequencing was performed on B.trispora(-)cultured with trisporic acid and B.trispora(-)cultured without Trisporic acid.A total of 1026 genes with significant different expression were obtained,including 487 up-regulated genes and 539 down-regulated genes.Gene Ontology and Pathway Analysis of different expression genes showed that Trisporic acid has significant effects on the synthesis pathway of terpenoids,fatty acid degradation pathway,unsaturated fatty acid synthesis pathway,carotenoid synthesis pathway and amino acid degradation pathway of B.trispora(-),Among the annotated differentially expressed genes,sexM,SOX5 and 5 dehydrogenase encoding genes are closely related to the regulation mechanism of Trisporic acid,therefore,These genes are used as candidate genes for further study.(3)Functional investigation of 5 dehydrogenase encoding gene-redY.Among the Candidate genes obtained by transcriptome analysis,The gene encoding 5 dehydrogenase first attracted our attention.Since this gene has not been reported in fungi,it is named redY in this study.In the fermentation process,redY gene product inhibitor-Dutasteride and its end product involving the metabolic pathway-Brassinolide were added,and molecular biological methods were used to overexpress redY gene in B.trispora(-)to explore the function of gene.The results showed that both Dutasteride and Brassinolide could significantly inhibit the stimulation of Trisporic acid on B.trispora and reduce the synthesis of lycopene.In the presence of Trisporic acid,overexpression of redY gene can stimulate sexM,carRA,carB gene expression,while in the absence of Trisporic acid,overexpression of redY gene can only stimulate sexM gene expression,but has no effect on carRA,carB gene expression.