The Regulation Of The Cell Growth And Biosynthesis Of Secondary Metabolites By Disturbing The Chitin Synthesis Pathway In Monascus Purpureus

It was reported that the production level of secondary metabolites in the submerged fermentation process is closely related to the growth and morphology of the cell.The chitin synthase genes,as morphological regulatory genes,can affect the morphological development of filamentous fungi during the submerged fermentation process.However,the functional annotation of chitin synthesis genes and their regulation on morphological growth,secondary metabolism,and molecular mechanisms are still insufficient in Monascus.Therefore,in this work,Monascus purpureus LQ-6 was used as the parental strain,and the chitin synthase genes family was mined through whole genome sequencing and NCBI comparison analysis.The gene chsⅥ was knocked out by genetic engineering technology,thereby perturbing the chitin synthesis pathway,changing the morphological development of the hyphae,and regulating the synthesis of secondary metabolites.Combined with the analysis of image technology and transcriptomic,the regulation and molecular mechanisms of growth and secondary metabolism in Monascus was preliminarily elucidated.The main contents of this paper are as follows:1.Construction of chsⅥ recombinant strainsTaking M.purpureus LQ-6 as the parental strain,seven chitin synthase genes were mined through whole genome sequencing and NCBI comparison analysis.The gene Monascus₅162 was confirmed as chsⅥ chitin synthase with bioinformatics analysis.The gene knockout vector pXS-5162 and the complement expression vector pBA-5162 were obtained by one step cloning technology,and the gene Monascus₅162 was knocked out by PEG mediated protoplast transformation to obtain 4 transformants.Finally,the deletion strain M.purpureus Δ5162 was obtained by PCR and sequencing verification.Similarly,the complementation expression strain M.purpureus cp:5162 was gained.2.The regulation effect of chsⅥ on cell growth,cell morphology and secondary metabolitesThe effect of chsⅥ on colony morphology,mycelial morphological development,cell growth,and cell wall structure were investigated.After knocking out the gene chsVI,there were almost no significant changes in the morphology of mycelium（except the aerial hyphae on PDA medium were short and sparse）and the grow rate of colony diameter between the chsⅥ-deleted mutation and LQ-6 strains.And the sensitivity to environmental factors in the strain Δ5162 was improved.During submerged fermentation,the number of tip balloons further increased,but the number of cleistothecia was significantly reduced at day 6 in M.purpureus Δ5162 compared with that in the parent strain.The results of TEM revealed that the cell wall thickness showed significant changes,and the cell wall was thinner in the mutant strain than in the wild type.The analysis of the physiological characteristics of cell wall and cell membrane showed that the content of chitin first decreased and then increased,and the structure of cell wall and cell membrane was destroyed after knocking out the chsⅥ gene.In the process of submerged submerged fermentation,the biosynthesis of Monascus pigment in strain A5162 was significantly inhibited,and almost no citrinin was produced.On account of the increasing of permeability,the proportion of extracellular pigment increased by 22%（YMPs-370 nm）and 20%（YMPs-410 nm）and 40%（RMPs-510 nm）,respectively.The effect of chitin synthase inhibitor polyoxomycin B on the cell growth and secondary metabolites in M.purpureus LQ-6 was studied.It was found that The growth of Monascus was inhibited in the presence of inhibitors at low concentrations（50 mg/L）,but the total pigment yields were 55.45 U/mL（YMPs-370 nm）,63.60 U/mL（YMPs-410 nm）and 61.90 U/mL（RMPs510 nm）.Compared with the parental strain LQ-6,the production of extracellular pigments increased by 45.07%,64.60%,and 56.75%and respectively.3.Transcriptomic analysisTranscriptomic sequencing results showed that M.purpureus Δ5162 had 2664（996 genes up-regulated,1698 down-regulated）and 2495 genes（1894 up-regulated,601 down-regulated）in the mRNA from day 4 and day 7,respectively.There was a significant difference in expression level（p<0.05）.Comparative transcriptome analysis revealed that the pathways related to spore development and growth,including MAPK signaling pathway,Ca2+-dependent signaling pathway,chitin biosynthetic pathway,regulatory factor laeA gene and WetA gene,were significantly downregulated at the early phase of fermentation.Deletion of gene chsVI also could disturb the carbon metabolism,amino acid metabolism and lipid metabolism during the fermentation.The mRNA expression levels of genes in the cluster of Monascus pigments and citrinin biosynthesis were significantly downregulated,especially those related to citrinin biosynthesis.