Gene Sequence Analysis Of Primary Metabolism In High-and Low-yeild Acremonium Chrysogenum And Functional Study On Ssadh Gene

Cephalosporin C,the important precursor of ?-lactam antibiotics in industrial production,plays an important role in the antibiotic market with its enormous economic value and its producer Acremonium chrysogenum also receives more attention.After several rounds of mutation and selection through conventional breeding methods,CPC production capacity of A.chrysogenum significantly increased,but the molecular mechanism of genetic variation and metabolic differences are not fully known,which restricts the possibility of molecular breeding for further enhancing the production of CPC to a great extent.At present,the elucidation of the biosynthesis pathway of CPC and the application of omics technologies provide us with very convenient conditions for understanding the metabolic network and regulation mechanism of CPC.As wild type strain ATCC11550 genome sequencing completing,we planed to conduct comparative genome study of A.chrysogenum,combined with the transcriptomic and proteomic data in the previous research,to conduct a comprehensive in-depth research of the metabolic regulation in the mutation and screening process,and find out regulating factors that affect CPC synthesis metabolism.This article aimed at gene sequence analysis of primary metabolism in high-and low-yield A.chrysogenum,investigated whether these genetic mutation could affect their enzyme activity and how they affect CPC metabolism.Firstly,genome of high-yield A.chrysogenum LD-7 served as template,amplified and sequenced 38 key enzymes involved in central carbon metabolism and precursor amino acids synthesis pathways,then aligned with wild type A.chrysogenum ATCC11550,found that amino acids mutations were taken place in only 3 enzymes.They are MDH1121 of TCA cycle,SSADH10578 of GABA pathway and PSAT1046 of serine synthesis pathway.According to the study of transcriptional level,we chose SSADH to conduct the change of enzyme activity research before and after the mutation and found that there was nosignificant differences in the activity between two kinds of SSADH protein.Finally,we proved that ssadh could upregulate CPC yield through the overexpression experiment cause it provided more NADPH to participate in CPC synthesis.In summary,there was very few enzyme of primary metabolic pathway related to CPC synthesis in high-yield strain mutated,while enzyme activity of the transcriptional upregulated protein didn't change due to the mutation.Therefrom,we hypothesized that metabolic regulation changes might be the main reason for the increase of CPC production in the process of the traditional strain breeding.This provides reference for further study on metabolic regulation of CPC biosythesis through the follow-up comparative genome research between high-yield and wild type A.chrysogenum strains,and build a theoretical basis for establishing a better high-yield strain.