Regulation Of Cordycepin Metabolism Via Redox Balance In Cordyceps Militaris

Cordyceps militaris is a traditional Chinese medicinal fungus that contains a variety of biologically active ingredients.Cordycepin is one of its main active ingredients and has various physiological functions such as anti-tumor,anti-virus and immune regulation.At present,the synthetic methods of cordycepin include chemical synthesis and biosynthesis.The biosynthesis includes fruit body extraction and mycelial liquid fermentation.Liquid fermentation is an important production route for cordycepin.However,the fermentation yield of cordycepin at this stage is low and cannot meet the needs of industrial production.Therefore,increasing the yield of cordycepin has important economic and application value.A large number of studies have shown that oxidative stress is involved in the regulation of the secondary metabolism of filamentous fungi.This study found that the addition of reduced glutathione to the liquid fermentation system can significantly enhance the synthesis of cordycepin.Based on this,this study used fermentation process optimization,genetic engineering,transcriptomics analysis and other strategies to explore the relationship between GSH and cordycepin metabolism.The main research conclusions of this paper are as follows:(1)Taking the Cordyceps militaris submerged liquid fermentation system as the research object,adding GSH to regulate the redox state of the cell,and investigating its influence on the metabolism of cordycepin.When 3.0 g/L GSH was added,the cordycepin yield reached439.69±12.43 mg/L after 20 days of fermentation,which was an increase of 471.24% compared with the control without addition,and the activity of glutathione peroxidase increased by414.82%.Gene expression analysis showed that after adding GSH,the expression of cordycepin biosynthesis key genes Cns1 and Cns2 were significantly up-regulated,and the expression levels in 15 days of fermentation were 540.67 times and 25.81 times that of the control group,respectively.(2)Overexpression of glutathione peroxidase-encoding gene GpxA,construct a genetic engineering strain of Cordyceps militaris,and explore the effect of GpxA on cordycepin synthesis.The results showed that after overexpression of GpxA,the yield of cordycepin in 20 days fermentation with Cordyceps militaris genetically engineered bacteria reached868.53±50.11 mg/L,which was 4.30 times that of the control group;gene expression analysis showed that the key gene Cns1 for cordycepin biosynthesis after overexpression of GpxA.The expression of GpxA and Cns2 were significantly up-regulated.In 20 days,the expression levels were 5.92 times and 28.95 times that of the control group,and the expression levels of GpxA were 1.26 times that of the control group.The intracellular NADPH content of Cordyceps militaris decreased with the down-regulation of GpxA expression,which was 1.49～1.64 times that of the control group.At the same time,the level of ROS was significantly lower than that of the control group.(3)To add 3.0 g/L GSH and GSH was not added liquid fermentation system for the study carried out transcriptomics analysis showed that after addition of GSH,significant enrichment upregulated genes redox process,indicating that GSH involved in regulation of intracellular redox state of Cordyceps militaris.Gene expression analysis showed that the expression level of cordycepin synthesis key gene Cns1、Cns2 and Cns3 were significantly up-regulated after adding GSH,which was 4.29、6.81 and 3.95 times as much as that of the control group in 15 days,and 3.50、4.24 and 1.48 times as much as the control group in 20 days;in addition,pentose phosphate pathway and purine metabolism such as 6-phosphogluconate dehydrogenase,ribosephosphate pyrophosphokinase and amidophosphoribosyltransferase and other expressions are significantly up-regulated,indicating that the metabolic flux of GSH is enhancedafter adding GSH,which helps to accumulate cordycepin synthesis precursor substances.By studying the regulation analysis of GSH on cordycepin metabolism in Cordyceps militaris,it is preliminarily confirmed that redox is involved in the regulation of cordycepin metabolism in Cordyceps militaris,laying the foundation for further gene redox regulation strategies to develop high-efficiency cordycepin production strategies,and also for in-depth exploration of cordycepin redox regulation provides a theoretical basis and methods.