Construction And Fermentation Optimization Of Engineering Strain Related To The Genes Involved In The Production Capacity Improvement Of Chaetoglobosin A

Chaetomium globosum,which is widely distributed in various habitats,is a natural cell factory and an important source of bioactive metabolites,such as azaphilones,terpenes,and cytochalasins.Chaetoglobosin A(che A),which is mainly produced by C.globosum,is a major active substance catalyzed by polyketide synthase and a member of the cytochalasin family.Che A has been comprehensively investigated for its unique inhibitory activity against phytopathogen s,parasitic nematodes,and strong cytotoxicity in tumor cells.Studies on che A are essential in accelerating the development of green agriculture and biomedicine.However,little is known regarding the transcriptional regulation of che A biosynthesis and no study has investigated the associated efflux systems.Based on this research status,this study aims to increasing the production of the bioactive metabolite che A,functions of the CgMfs1 and CgXpp1 genes,which encode the major facilitator superfamily transporter and b HLH-type regulatory factor in C.globosum,respectively,were identified.A more comprehensive and in-depth understanding of the transportation and the biosynthesis regulation of che A could facilitate the construction of genetically engineered strains,enhance target metabolite production,and provide sufficient theoretical basis and superior strain resources for large-scale production.In this study,CgMfs1 was initially used as the target gene.RNAi-mediated gene silencing and strong promoter gpd A-driven gene overexpression strategies were used to determine the function of the CgMfs1 gene in parental cells.The variations in the expression levels of CgMfs1 did not have a significant impact on the diameter of mycelium,size and number of spores,and growth state of the strain,suggesting that the gene was not involved in the growth,development,and morphological differentiation of C.globosum.In addition,inactivation of CgMfs1 caused a notable decrease in che A yield from 58.66 mg/L to 19.95 mg/L(MFS1-3)and 17.13 mg/L(MFS1-4),and the total yield were significantly lower than that of the wild-type strain.The production of target metabolites in each transformant increased significantly following the overexpression of CgMfs1.The extracellular yields of mutants OEX1 and OEX19 were 147.56 and 155.92 mg/L,respectively,during the fermentation equilibrium period.The mutant OEX13 exhibited a significant change in trend and the extracellular yield was 298.77 mg/L,which was4.82-fold higher than that of the parental cells.Furthermore,the efflux ratio of target metabolites was substantially enhanced,suggesting that the predicted efflux gene CgMfs1 could influence che A transportation,and that the immediate efflux of che A facilitates the biosynthesis of target metabolite.To comprehensively elucidate the function of relevant regulatory factor of che A biosynthesis,this study identified the function of CgXpp1 by employing CgXpp1-disruption and CgXpp1-complementation strategies.The results showed that CgXpp1 disruptive gene led to an irreparable reduction in spore production but had no significant effect on hyphal diameter and spore size,which demonstrate the positive regulatory role of CgXpp1 in spore formation.In addition,inactivation of CgXpp1 considerably increased che A production from 60.32 mg/L to 274.61 mg/L(CgXpp1-N14)when compared to the wild-type strain;however,che A production was restored in the CgXpp1-Com strain.Knocking out the target gene considerably increased the transcription levels of key genes associated with che A biosynthesis and regulation,a significant increase in CgMfs1 expression levels also being observed.According to the results of electrophoretic mobility shift assay analysis,clarified that CgXpp1 has an indirect negative regulatory effect on CgPKS,CgP450,CgFMO,and CgER,and a direct negative regulatory effect on the transcription of CgMfs1.Furthermore,metabolome analysis results further verified the debilitation effect of CgXpp1 inactivation on various primary metabolic pathways and its activation impact on secondary metabolic pathways.To increase che A yield,based on the above results,CgMfs1 was overexpressed based on CgXpp1 deletion.Cornstalks,which was the abundant and inexpensive resources,used as the fermentation substrate.Selected a high-yielding strain NM33 of the target metabolite.Che A yield of the NM33 strain was highest and reached at275.30 mg/L,which was 7.54-fold higher than that of C.globosum.Meanwhile,the fermentation culture conditions were optimized by response surface methods.After optimization,cultivated for 12 days,che A yield of 351.16 mg/L was achieved,improved to 27.56% compared with the unoptimized strain.In addition,che A yield under optimized conditions was 9.79-fold higher than the wild-type strain.The optimized NM33 strain could destroy the structure of cornstalk s more efficiently.This study focused on improving the production of the bioactive metabolite che A and elucidated the functions of CgMfs1 and CgXpp1 genes,which are involved in the transportation and regulation of target products in C.globosum.Constructing engineered strains,optimizing the fermentation conditions,and establishing a novel high-efficiency and low-cost production strategy for che A form the basis for future industrial production.