Characterization Of Thiazole Biosynthetic Enzyme From Acremonium Chrysogenum And The Effect Of Thiamine On CPC Production

Cephalosporin(CPC),one of the major precursors of ?-lactam antibiotics,is produced by Acremonium chrysogenum(A.chrysogenum),which is an important industrial fungus.Over the past years,its fermentation yield has been increased significantly.But it is not very clear about molecular changes between the industrial strain and the wild type strain.In our previous study,our lab used comparative proteomics to study the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer(HY)strain and a wide-type(WT)strain and found an thiazole syntheis enzyme(Acthi S).The cDNA sequence was exacted from the Illumina sequencing in Acremonium chrysogenum and named Acthi gene.Manipulation of Acthi gene indicated that Acthi might relate to the production of thiamine and the transcriptional regulation of CPC biosynthesis.Thus,in this study,the function of ActhiS and its role in thiazole biosynthesis were identified using a series of biochemical methods in vitro for the first time.The size exclusion chromatography and simulated structure results showed that ActhiS might be a homooctamer.The bound substance that consists of the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid(ADT)was characterized by HPLC and 1H NMR analysis.Bound-metabolite-free ActhiS expressed in M9 minimal medium catalyzed the conversion of NAD and glycine to ADT.Furthermore,we identified that C217 provide the sulfur donor to thiazole moiety.These observations confirmed that ActhiS is a thiazole biosynthetic enzyme in A.chrysogenum,and it serves as a sulfur source at the same time.Then we further explore the effect of thiamine on CPC synthesis.Adding 1 mg/mL thiamine to the fermentation solution of A.chrysogenum can increase CPC production,keeping to increase the concentration of thiamine result in the inhibit of CPC production.At the same time,the transcriptional differences of biosynthetic genes involved in CPC were studied.1mg/m L and 100mg/mL thiamine were added to the fermentation medium,and the levels of cefEF and cefG were up-regulated and down-regulated respectively.This shows that thiamine can regulate the synthesis of CPC gene transcription,which in turn affects the yield of CPC.At the same time,the addition of thiamine in the fermentation medium had different effects on mycelial growth and biomass of Acremonium chrysogenum.We confirmed that the ActhiS protein is a thiazole synthase in Acremonium chrysogenum for the first time,which participates in the synthesis of thiazole and further affects the formation of thiamine.Thiamine is a coenzyme for a variety of enzymes,involved in amino acid metabolism and carbohydrates in vivo,and further affect the synthesis of CPC.The result indicated that the thiamine metabolic pathway and Acthi gene could be a potential target for molecular breeding of CPC producer in industry.