New insights of rotenone targeting cordycepin synthetase 1-cordycepin synthetase 2-glyceraldehyde-3-phosphate dehydrogenase (Cns1-Cns2-GAPDH) triple complex to enhance cordycepin synthesis in Cordyceps militaris.

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Citation abstract

Cordycepin isolated from Cordyceps militaris is an adenosine analogue with various biological activities, and its synthesis is dependent on cordycepin synthetase 1 (Cns1) and cordycepin synthetase 2 (Cns2). Our previous study showed that rotenone induction promotes cordycepin synthesis by regulating energy metabolism pathways such as Embden-Meyerhof-Parnas pathway (EMP). However, the exact molecular mechanism remains unclear. Here, we constructed a yeast hybrid library of Cordyceps militaris CM-001 strain and identified the Cns1/Cns2 interaction protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by yeast two-hybrid (Y2H) screening. Subsequently, the interaction between GAPDH and Cns1/2 was verified by Y2H validation, glutathione S-transferase (GST) pull-down, and Co-Immunoprecipitation (Co-IP). The Co-IP results showed that rotenone promoted the interaction of Cns1-Cns2-GAPDH complex. However, rotenone treatment did not affect the mRNA level of GAPDH but up-regulated the protein level and enzyme activity. In addition, rotenone treatment significantly increased the relative levels of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), but a GAPDH inhibitor partially offsets the promoting effect of rotenone. Our study shows that the Cns1-Cns2-GAPDH triple complex regulates cordycepin synthesis in Cordyceps militaris. Rotenone enhances the metabolic flux of cordycepin biosynthesis pathway by enhancing the Cns1-Cns2-GAPDH interaction and its induced aerobic glycolysis.