Cloning And Analysis Of The Key Genes In Cordycepin Biosythesis Pathway

Cordycepin?3?-deoxyadenosine?,isolated from Cordyceps militaris as the first nucleoside antibiotic obtained from the fungi.Cordycepin as the key bioactive compound with multi-activities including anti-bacterial,anti-inflammatory,anti-oxidation,anti-viral and anti-tumor,which attracting much attention in the field of pharmaceutical chemistry research.At present,cordycepin can be prepared through chemical or biological synthesis.The application of chemical synthesis has been confined for the high cost in raw material,complicated synthesis process,low product yield,and too many byproducts.While biological synthesis has more advantages considering its low raw material costs,mild conditions during all the production steps,liquid fermentation technology with high space utilization,easy control of the fermentation process,and less byproducts.It is gradually becoming the main trend in the industry of cordycepin production.However,the fermentation period for cordycepin adopted by Cordyceps militaris was so long with low efficiency.That is important to enhance the productivity in codycepin fermentation with higher yield and realizing its large-scale application in economics and practical value.This study aimed to heterologously express the key genes in both Saccharomyces cerevisiae and Escherichia coli,which involved in cordycepin synthesis in Cordyceps militaris to shorten the production period of cordycepin and increase the fermentation intensity.The results are as followed:?1?Two key genes cm1 and cm2 were cloned via nest-PCR technique from Cordyceps militaris 5111.Cm2 was expressed in E.coli separated afterwards via its fused His₆ tag.The SDS-PAGE results proved the soluble expression of CM2,and the polyclonal antibody of CM2 was also successfully prepared.However,in this research cm1 was not expressed in E.coli.Besides,bioinformatical analysis was taken and it showed that CM1 belonged to oxidoreductases and CM2 belonged to the ribose synthase.?2?The chromosomal integration and episomal expression vectors carrying cm1 and cm2 were constructed respectively and successfully introduced into Saccharomyces cerevisiae 4126,obtaining two strains which constitutively produce cordycepin based on chromosomal integration and six strains based on the episomal expression system.The characteristic peak in HPLC and the combination of HPLC-MS analysis confirmed the component of cordycepin in the fermentation broth of those constructed yeasts,and which S.cerevisiae 4126-P6 had the best fermentation performance.The output of cordycepin for P6reached its highest at 28.48 mg/L at 60 h and the final production intensity was 0.47mg/L/h,which was higher than that of C.militaris 5111 by 276%.The analysis of fermentation parameters showed that cordycepin was a secondary metabolite in the metabolic pathways of yeast,for it was synthesized only in the late fermentation stage after glucose was consumed up and the accumulation of biomass had been completed.This research provided a theoretical basis for improving the activity of key enzymes in cordycepin synthesis and uncovered the intact cordycepin biosynthesis pathways,also laying a foundation for the industrial production of cordycepin.