Regulation Of The Biosynthesis Of Secondary Metabolites Via Cofactor Metabolic Engineering Strategies In Monascus Purpureus

Monascus pigment is widely used in food and medicine as a natural pigment.As a safe and nutritious natural pigment,it has good economic benefits.There are many types of monascus pigments,which are divided into monascus orange pigments,monascus yellow pigments and monascus red pigments according to their hue.At present,the known chemical structures of monascus pigments are mainly concentrated in 6 kinds of alcohol solubility and 4 kinds of water solubility.Studies have found that monascus pigment biosynthesis is carried out through the FAS and PKS polyketide pathways,requiring a large number of primary metabolites,such as acetyl-CoA,malonyl-CoA,and NAD(P)H.There is more oxidation in the entire metabolic pathway.The reduction reaction involves the participation of NAD(P)H;Monascus is an oxygen-consuming filamentous fungus,disturbing the electron transport chain will definitely change the cofactor metabolic pathway and change the intracellular reducing power balance.Based on this,this project first uses liquid fermentation technology,that is,adding exogenous factors to the fermentation broth to regulate the synthesis of monascus pigments,to study the influence of exogenous factors on the production of pigments;secondly,to mine the nuo on the electron transfer chain complex I through Blast gene analysis I gene,directed to perturb the regulatory pathway of nuo ? on the complex I pathway to increase the pigment production of Monascus,combined with genomics and RNA-seq analysis to analyze the regulation mechanism of cofactors on Monascus pigment synthesis.This experiment aims to clarify the relationship between cofactor metabolism and pigment synthesis,provide a theoretical basis for the study of the regulation mechanism of pigment synthesis,and possibly develop new methods and engineered strains to improve pigment production.The main research contents of the thesis are as follows:(1)Exogenous factors regulate the synthesis of monascus pigmentIn this experiment,purple red yeast rice LQ-6(Monascus purpureus LQ-6)isolated from red yeast rice was used as the parent strain(deposited in the Chinese Type Culture Collection in Wuhan,China,and its deposit number is CCTCC M 2018600).Methyl viologen(MV),rotenone and synthetic cofactors were added to the liquid batch fermentation medium for fermentation regulation.The results showed that after exogenous addition of 1 mg/L methylviologen,1 mg/L rotenone and 50 mg/L synthetic cofactors,the total pigment output reached 170.51 U/mL,172.87 U/mL and 171.82 U/mL,respectively.Compared with the parent strain purple monascus LQ-6,it increased by 39%,40.92%and 40.06%,and the addition of 1 mg/L methylviologen and rotenone significantly increased the yield of yellow monascus pigment,the yield was 77.53 U,respectively./mL,97.74 U/mL,compared with the parent strain purple monascus LQ-6,an increase of 69.76 and%114.01%respectively;while the addition of 50 mg/L synthetic cofactors from external sources significantly increased the production of red monascus pigment.The yield is 67.60 U/mL,which is an increase of 90.74%compared to the parent strain.(2)Directed disturbance of the Electron Transport Chain regulates the synthesis of Monascus PigmentBased on the whole genome sequencing of the parent strain LQ-6,this project uses Blast gene analysis to mine the nuo ? gene on the electron transport chain complex I,to perturb the regulation pathway of nuo ? on the complex I pathway,and perturb the cofactor metabolism to regulate monascus pigment.Synthesis,analysis of the regulation of cofactor synthesis on monascus pigment anabolism.Through the determination of the starting strain M.purpureus LQ-6 and the engineered strain M.purpureus ?4971 intracellular pigment,extracellular pigment,NADH(NADPH),biomass,and sugar consumption,etc.,the incomplete knockout of nuo ? gene was investigated.The effect on the growth of strains and the condition of pigment changes.After perturbing the nuo ? gene encoding NADH-quinone oxidoreductase by genetic engineering,the total pigment production in the fermentation medium of the engineered strain M.purpureus ?4971 was 166.21 U/mL,compared with 122.70 U of the original strain M purpureus LQ-6/mL increased by 35.46%,of which the yellow pigment production was 70.72 U/mL,the orange pigment production was 56.81 U/mL and the red pigment production was 38.66 U/mL,which were 54.86%,36.68%and 9.1%respectively compared with the parent strain.The yield of M.purpureus LQ-6 citrinin was 1.70 mg/L,and the yield of citrinin from the engineered strain M.purpureus ?4971 was increased to 1.80 mg/L,an increase of 5.88%.And the genetically engineered strain M.purpureus ?4971 during the logarithmic growth phase,its NADH and NADPH were significantly increased by 1.29 and 1.61 times,respectively;in addition,its growth rate,biomass and sugar consumption rate were slightly lower than the parent strain in the early stage,and began to slow after 84 h Slowly showing an increasing trend.(3)The regulatory mechanism of cofactors regulating monascus pigment synthesisThe transcriptome comparison results showed that after weakening the nuo ? gene,genes with FDR<0.05 and |log2FC|>1 were selected as significantly different genes(DEG).A total of 1109 genes had significant differences in mRNA expression levels.Compared with the parent strain M.purpureus LQ-6,the expression of 193 genes in the engineered strain M purpureus ?4971 was up-regulated,and the expression of 916 genes was down-regulated.The results showed that the disturbance of cofactor metabolism was compared with that of the parent M.purpureus LQ-6.The glycolysis pathway,fatty acid synthesis pathway,citrinin and pigment synthesis pathway of the engineered strain M.purpureus?4971 were significantly up-regulated,while the expression levels of the TCA cycle,fatty acid degradation,PKS pathway,and electron transport chain pathway were significantly down-regulated.The expression of NADH dehydrogenase,ETC complex I subunit conserved region-domain-containing protein,NADH:ubiquinone oxidoreductase,gNADH dehydrogenase,and NADH-ubiquinone oxidoreductase related to the electron transport chain was significantly reduced.In the citrinin pathway,genes such as ctnC,mrl5,ctnD,ctnB,ctnA,ctnS(pksCT),ctnF,and ctnH were significantly up-regulated.The major up-regulated genes of pigment are MPsGeF,MPsGeC,MPsGeA,MPsGeM,MPsGeK,MPsGeJ.The interfered 4971 gene is named gene-MPDQ₀06632 in transcriptomics,and its log2 Ratio(?4971/WT)=-0.41,which proves that the expression of this gene is reduced after interference.The qRT-PCR verification results are consistent with transcription analysis.Combined with the pigment synthesis,kinetic analysis and physiological characteristics analysis of the incompletely knocked-out nuo ? genetically engineered strain,it provides a theoretical basis for the research on improving the regulation of pigment synthesis.