Study On The Breeding Of Monascus Purpureus With High Production Of Monacolin K By X-Ray Mutation And Optimization Of Fermentation Process

Monascus is one of the main sources of microbial fermentation to produce Monacolin K.It is widely recognized by various countries that Monacolin K in Monascus is the most effective drug for reducing cholesterol and regulating blood lipids,with characteristics such as high efficiency,low toxicity,and safety.It has been widely concerned in the food and pharmaceutical fields.In order to improve the yield of Monacolin K produced by dliquid submerged fermentation of Monascus,this project mainly focuses on two aspects:the selection of Monacolin K producing strains from Monascus and the optimization of fermentation process;L-citrulline can significantly improve the synthesis of Monacolin K,and further analyze the mechanism of L-citrulline promoting the synthesis of Monacolin K using Transcriptome.The experimental details are as follows:1.Breeding of a high-yield Monacolin K Monascus strain.Using X-ray mutation breeding technology,the effects of different treatment doses(250,300,350,400,450,500 Gy)on bacterial survival rate were investigated.The optimal irradiation dose was determined to be 400 Gy,and the spore survival rate was 9.4%.A high-yield Monacolin K Monascus strain M400-6 was obtained under this irradiation condition.After ITS sequence identification,the mutant strain belongs to Monascus purpureus.After 5 consecutive generations of cultivation,the average yield of Monacolin K produced by the liquid fermentation process of M.purpureus M400-6 was 181.97 mg/L.Compared with its parent strain M.purpureus M1,the yield of Monacolin K increased by 54.20%,and it was demonstrated that the strain has good passability characteristics.2.Response surface optimization of fermentation media significantly improves the production level of Monacolin K during liquid fermentation.Single factor experiments and BoxBehnken response surface design were conducted to optimize the liquid fermentation medium.Through a single factor experiment,three substances that are most effective for the synthesis of Monacolin K were selected from carbon sources,nitrogen sources,metal ions,and amino acids,namely glycerol,peptone,and L-citrulline.The addition amounts were 91.65 g/L,14.68 g/L,and 4.01 g/L,respectively.It was verified that the yield of Monacolin K at this addition amount was 266.63 mg/L,which was 98.51%of the predicted value.The optimized fermentation medium composition was determined as follows:rice flour 20 g/L,glycerol 91.65 g/L,peptone 14.68 g/L,MgSO4 1 g/L,ZnSO4 2 g/L,KH2PO4 2.5 g/L,NaNO3 5 g/L,L-citrulline 4.01 g/L,with natural pH.To verify the effect of the optimized and non optimized fermentation media,(M400-6 was fermented in the optimized medium;parent strains M1 and M400-6 were fermented in the non optimized fermentation medium),the yield of Monacolin K in the optimized group M400-6 reached a peak of 262.71 mg/L in 20 days,which was 128.20%and 54.78%higher than that of the non optimized parent strains M1 and M400-6(111.34 mg/L and 178.19 mg/L),respectively.3.To analysis the mechanism of citrulline promoting the synthesis of Monacolin K based on tra1scriptomics.According to the differential gene screening criteria FDR<0.05 and |log2FC|>1,the analysis showed that there were 2215 significantly different genes in the sample,584 genes were up regulated,and 1631 genes were down regulated.According to GO analysis,the differential genes mainly focus on annotation items such as Cell,Cell part,Organelle,Catalytic activity,Binding ability,and Transporter activity.KEGG analysis showed that the metabolic pathway was mainly concentrated on items such as Carbohydrate metabolism,Amino acid metabolism,and Energy metabolism.The results showed that the pathways of galactose metabolism,starch and sucrose metabolism,and glycolysis metabolism were significantly up-regulated,and glycolysis provided sufficient energy and acetyl-CoA for the synthesis of Monacolin K.The differentially expressed genes in serine,glycine,threonine degradation and alanine,aspartic acid and glutamate metabolism were significantly changed.Amino acids were decomposed into acetyl-CoA,energy and precursors conducive to the synthesis of Monacolin K.Monascus pigment and Monacolin K belong to polyketids.Combined with qRT-PCR validation analysis,Monacolin K synthesis genes mokA,mok B,mok C,mok D,mokF,mok H and mok I were up-regulated,while Monascus pigment genes were down-regulated.Energy from metabolism and acetyl-CoA are presumed to act on Monacolin K synthesis.