Multi-omics Analysis Of The Effects Of X-ray Irradiation On Monascus Pigment Synthesis And Fungal Growth

Hongqu has been recorded in Compendium of Materia Medica as a product of affinal drug and diet.With the help of molecular biology,the study of the metabolic pathway of monascus pigment has gradually developed.Monascus has a wide variety of species and different forms.Even in the same species and genus,they also have great differences in morphology,and the yield of metabolites also changes accordingly.The relation between growth and metabolism is complex.In this article,a high-yield monascus pigment producing strain MT-21 was obtained by X-ray irradiation mutagenesis breeding.Multi-omics analysis was performed at the molecular level of growth and secondary metabolic pathways,and the apparent traits and properties of the strain were investigated.The main research of the paper is as follows:1.Breeding of high yield monascus pigment mutant strains using X-ray irradiation mutagenesis,the results showed as follows:The optimal mutagenic dose was determined to be 300 Gy,and the survival rate of the strain was maintained at 7.8%.High throughput screening fungus obtained a high-yield monascus pigment strain MT-21,which was from mutagenesis breeding of strain M183 at a dose of 300 Gy,and the pigment yield of the strain under deep-hole plate liquid fermentation conditions was increased to 159U/mL,the yield of monascus pigment increase to 130.43%.and the pigment yield of red yeast rice was increased to 4310 U/g.Compared with the parent strain M183,the yield of monascus pigment increase to 312.44%.After passaging in 5 times,the monascus pigment yield keep in 150 U/mL,which shows that the mutant strain MT-21 had good passage stability,ITS identification determined that the mutant strain is Monascus purpureus,we named it Monascus purpureus MT-21.2.Comparative genomics analysis and transcriptomics analysis was carried out on the effects of mutation on secondary metabolism and growth and development by X-Rays irradiation in MT-21.The main results are as follows:The genes of the mutant strain MT-21 were compared with the known M183 whole genome sequence by comparative genomics and a total of 3838 mutation sites were found.Expasy was used to predict the readable frame and protein sequence,uploaded to NCBI for sequence comparison,and screened out 10 genes with greater influence and uploaded to NCBI for protein function prediction,we found that most of the mutant genes were related to growth and development.Transcriptomic analysis of parent strain M183 and mutant strain MT-21 showed that the pathways with significant upregulation including amino acid degradation pathway and nucleic acid repair,and KEGG analysis showed that peroxisome,branched-chain amino acid degradation and base excision repair were significantly upregulated.GO analysis showed that downregulated genes were mainly related to membrane composition and transport,and KEGG analysis showed that starch and sucrose and other related sugar metabolism were inhibited.Comparative genomics and transcriptomics combined analysis showed that MT-21 mutant strains had gene mutations in the starch domain,the disturbance of this domain affected the synthesis of glucan and trehalose,the genes in the metabolic pathway showed significant differences,and the polysaccharide controlled surface mycelium synthesis was significantly inhibited,presenting smooth colonies.In comparative genomics,G1/S-specific cyclins were affected,and transcriptomics showed significant up-regulation of telomere-region genes and prolonged cell cycle.At the same time,the mutation led to the change of MT21 endonuclease RNase HI.From the transcriptional level,it could be found that the related nucleic acid excision repair levels were significantly up-regulated,which also had a certain impact on the cell cycle.3.The molecular mechanism of X-Ray irradiation on monascus pigment synthesis and fungal growth was verified from the perspective of fermentation over time,results are as follows:The growth and development of the mutant strain MT-21 was significantly inhibited,the diameter of the strain was reduced from 4.8 cm to 1.45 cm,there was less aerial hyphae,and the synthesis of spores and sporangia were significantly inhibited under the microscopic observation.The corresponding genes expression levels were also significantly reduced.The peroxisome genes of the mutant strain MT-21 were up-regulated,and the antioxidant repair ability of the mutant strain was measured by the determination of T-SOD,POD and ROS contents.The enhanced antioxidant repair ability of the mutated strain was reconfirmed.Combied with metabolic pathways changes,cofactor levels were selectively measured,the ATP content and pyruvate content of the mutant strain MT-21 increased,the ratio of NADH/NAD+and NADPH/NADP+of the mutant strain MT-21 was higher than that of the parent strain M183,the metabolic level was vigorous,and more energy was accumulated for secondary metabolic synthesis with acetyl-CoA.Fermentation kinetics verified that the monascus pigment yield of the strain increased from 179 U/mL to 225 U/mL,and the synthesis of citrinin was significantly inhibited,which was reduced to 4.6%.