Regulation Mechanism Of Energy Metabolism For Monascus To Enhance Pigment Production Response To Ammonium Chloride Stress

As a traditional medicinal and edible filamentous fungus that has been used for thousands of years in China and other Southeast Asian countries,Monascus sp.can produce many beneficial secondary metabolites.Due to the difficulty in obtaining industrial Monascus strains with high yield target metabolites,only Monascus red pigment has been fully industrialized.Other beneficial polyketides such as Monacolin K are mostly at the stage of laboratory research,the fundamental reason is the lack of theoretical basis for breeding target Monascus strains in industrial environments.Under ammonium chloride stress,the synthesis and secretion of Monascus pigment were promoted,especially yellow pigment.The study on the regulation mechanism of Monascus pigment synthesis under ammonium chloride stress will help to screen out industrial Monascus strains with high stress resistance and secondary metabolite accumulation ability,and provide theoretical basis for the industrial production of Monascus pigment.Currently,most studies on Monascus are the effects of stress on its material metabolism,and we rarely explain the regulation of stress on Monascus pigment biosynthesis from the perspective of energy metabolism.Therefore,in this paper,the energy metabolic pathways such as lipid metabolism,substrate-level phosphorylation and oxidative phosphorylation were combined with lipidomics analysis and proteomics analysis to investigate the energy metabolic regulation mechanism of Monascus pigment synthesis under ammonium chloride stress,hoping to provide a new research direction for increasing the production of Monascus pigment.The main contents of this paper are as follows:(1)Ammonium chloride culture condition at 15 g/L promoted the synthesis of Monascus pigment,with a total color value of 46.23 AU/mL,which was 3.67 times higher than the 0 g/L ammonium chloride culture condition.At the same time,it also significantly up-regulated the expression of MpigA(Polyketide synthase),MpigJ(Fatty acid synthase subunit alpha)and MpigK(Fatty acid synthase subunit beta).Under stress condition,the cell membrane integrity decreased,the intracellular Ca2+concentration,pH gradient and transmembrane potential increased,which resulted in changes in the membrane homeostasis of Monascus.In response to the imbalance of membrane homeostasis,the expression of key enzyme proteins and genes in the metabolic pathways of triglyceride,glycerophospholipid and sphingolipid were upregulated,which in turn resulted in promoted synthesis and accumulation of glyceride(TG)and glycerophospholipid(PG,PS,CL).At the same time,by improving the expression levels of glucose transporter,hexose transporter and their genes,the utilization of carbohydrates was promoted to produce ATP,which provided energy for the proliferation and pigment synthesis of Monascus.In addition,the increased content of CL,a specific lipid component of mitochondrial membrane,might be the regulation mechanism for maintaining the stability of mitochondrial membrane structure and mitochondrial functions such as energy conversion under stress condition.(2)Analysis based on substrate-level phosphorylation pathway(including glycolysis and TCA cycle).Ammonium chloride stress promoted full utilization of carbohydrates such as glucose by Monascus through increasing the activities of hexokinase,phosphofructokinase and pyruvate kinase,the key rate-limiting enzymes of glycolysis,which promoted the synthesis of acetyl-CoA.At the same time,the activities of key rate-limiting enzymes pyruvate dehydrogenase,citrate synthase and malate dehydrogenase in the TCA cycle were increased,and the expression levels of key enzyme proteins and genes in glycolysis and TCA cycle were also up-regulated,indicated that the substrate-level phosphorylation pathways of Monascus were enhanced in ammonium chloride stress.In addition,under ammonium chloride stress,the activity of phosphoenolpyruvate carboxykinase in Monascus gluconeogenesis was increased,which promoted the formation of phosphoenolpyruvate from oxaloacetic acid in the TCA cycle.The phosphoenolpyruvate entered glycolysis for the next cycle and resupplied oxaloacetic acid via oxaloacetate anaplerotic reaction,thereby increased the metabolic flux of glycolysis and TCA cycle.(3)Analysis based on oxidative phosphorylation pathway.Ammonium chloride stress increased the activities of complex Ⅰ,complex Ⅱ,complex Ⅲ and complex Ⅳ in mitochondrial respiratory chain,and the expression levels of related proteins and genes were also significantly up-regulated.In addition,the increase of ATPase activity,F1F0ATP synthase activity-related proteins and genes expression levels indicated that ammonium chloride stress promoted the electron transport and ATP synthesis in oxidative phosphorylation.The promotion of oxidative phosphorylation coupled with the ROS generation resulted in oxidative stress,which led to the increase in the activity of peroxidase,glutathione peroxidase and superoxide dismutase,and the increase in the content of reduced glutathione and mannitol,indicated that non-energy-dependent antioxidase system and non-enzymatic antioxidants played a positive role in resisting the oxidative damage caused by ammonium chloride stress.In summary,ammonium chloride stress regulated the energy metabolism of Monascus from multiple perspectives,including lipid metabolism pathway,substratelevel phosphorylation pathway and oxidative phosphorylation pathway,which in turn maintained the cell growth of Monascus and enhanced the synthesis and secretion of Monascus pigment.