Construction Of Highly Efficient Gene Knock-Out System For Monascus Ruber M7 And Analysis Of Citrinin Biosynthetic Pathway

The filamentous fungus Monascus spp.is the traditional microorganism used to make red fermented rice(RFR),which can produce several beneficial secondary metabolites.RFR has been used as fermentation starters,food colorants and medicinal agents in China and East Asian countries for centuries,and it's also Chinese traditional export product.Since 1995 the first time to discover some Monascus strains can produce a nephrotoxic citrinin(CIT),the popularization and development of Monascus-related products have been challenged.Thus,elimination of citrinin production in Monascus spp.is essential to increase the safety of Monascus-related products and extend their application.Researches revealed that CIT belong to polyketide(PK),which was synthetized by gene cluster including polyketide synthase(PKS)gene.However,many of these efforts are pale as little is known about the function of genes in CIT gene cluster and CIT biosynthetic pathways and its regulation mechanisms.Gene knock-out and heterologous expression have been extensively used as important molecular research methods to analyse gene function.In gene knock-out,how to increase the gene knock-out efficiency is very important.Researches revealed that gene knock-out efficiency can be dramatically increased by inactivating the ku70,ku80 or ligase IV participating in non-homologous end-joining pathway.Gene knock-out can block biosynthetic pathway and make the mutant accumulating substrate of targeted gene,while heterologous expression can make the transformant synthesizing product of targeted gene.Through analyzing the structure of metabolites produced by gene knock-out and heterologous expression strains with mass spectrum and nuclear magnetic resonance,we can uncover the substrate,product and function of targeted gene,then explore the biosynthetic pathways of secondary metabolites.This research use Monascus ruber M7 as research object,firstly we constructed the highly efficient gene knock-out system for Monascus ruber M7 by inactivating the ku70,ku80 or ligase IV genes.Based on this,CIT biosynthetic gene cluster was obtained through analyzing the genomic sequence of M.ruber M7.The functions of genes within CIT gene cluster were predicted using bioinformatic technology and some key genes were given a further identification by gene knock-out.Besides,we uncovered the CIT biosynthetic pathway by gene heterologous expression.These results will be elaborated as follows.1 Construction of highly efficient gene knock-out system for M.ruberThe ku70,ku80 and ligase ? genes were cloned and individually knocked out from M.ruber M-7.Colonial morphology,conidial production,growth rate and Monascus pigment and CIT production of these mutants(MrAku70,MrAku80 and MrAlig4)were analyzed and the results were as follows.The knock-out of these three genes did not affect growth,morphology,differentiation,development,breeding and the production of Monascus pigments and CIT.But gene knock-out efficiencies in ku70,ku80 and ligase IV null mutants were two,three and four times compared with that in wild-type strain,respectively,and the highest gene knock-out efficiency reached 85%.Thus we successfully constructed the highly efficient gene knock-out system for M.ruber which can be used for functional genomics.2 Analysis of CIT biosynthetic gene clusterThrough analysis of M ruber M7 genomic sequences,a 44 kb CIT gene cluster including polyketide synthase gene(pksCT)was obtained.There are 16 genes in CIT gene cluster,including pksCT and 7 genes upstream and 8 genes downstream,encoding polyketide synthase(pksCT),serine hydrolase(MRL1),dioxygenase(MRL2),transcriptional regulator(MRL3),aldehyde dehydrogenase(MRL4),glyoxalase(MRL5),dehydrogenase(MRL6),oxidoreductase(MRL7),transporter(MRR1,MRR8),phosphoglycerate mutase(MRR2),WD protein(MRR4),carbonic anhydrase(MRR5),oxidoreductase(MRR7)and unknown protein(MRR3,MRR6).Domain analysis suggested that CIT PKS belongs to non-reducing PKS with SAT-KS-AT-PT-ACP-CMeT-R domain structure.Sequences comparison revealed that first nine genes(MRL1-MRL7,pks CT and MRRl)in the cluster were highly conserved in different Monascus strains and Penicillium expansum.Among these 9 genes,MRL3 is regulator gene,MRR1 encodes transporter,the protein encoded by MRL5 is too short,pksCT,MRL1,MRL2,MRL4,MRL6 and MRL7 are structural genes,maybe directly participate CIT biosynthesis.Thus we will detailed analyze these 6 genes in following gene knock-out and heterologous expression experiments.3 Construction and analysis of gene knock-out mutantsUsing the constructed highly efficient gene knock-out mutant(MrAku80)as original strain,the pksCT,MRL1,MRL2,MRL4,MRL6 and MRL7 were knocked out from M.ruber M7 based on homologous recombination to generate Mr?ku80?pksCT,Mr?ku80?L1,Mr?ku80?L2,Mr?ku80?L4,Mr?ku80?L6 and Mr?ku80?L7,respectively.Analysis of CIT-related metabolites of these mutants in PDB medium indicated that,compared with MrAku80 strain,Mr?ku80?pksCT can't produce CIT,Mr?ku80?L1 still produced CIT but with 95%reduction in amount,knock-out of MRL2,MRL4,MRL6 and MRL7 both abolished CIT production and accumulated metabolite 2,the production of 2 reached 8.6 mg/L in Mr?ku80?L2,while Mr?ku80?L4,Mr?ku80?L6 and Mr?ku80?L7 only accumulated very less amount of 2.After structure identification,2 has the molecular formula of C13H16O4[2,4-dihydroxy-3,5-dimethyl-6-(3-oxobutan-2-yl)benzaldehyde],according domain analysis of pksCT,2 may be the first intermediate in CIT biosynthetic pathway.4 Construction and analysis of heterologous expression transformants and CIT biosynthetic pathway hypothesisThe cloned pksCT,MRL1,MRL2,MRL4,MRL6 and MRL 7 genes were used to construct 10 different Aspergillus oryzae heterologous expression transformants(A.oryzae-pksCT,A.oryzae-pksCT-L1,A.oryzae-pksCT-L1-L2,A.oryzae-pksCT-L1-L2-L4,A.oryzae-pksCT-L1-L2-L6,A.oryzae-pksCT-L1-L2-L4-L6,A.oryzae-pksCT-L1-L2-L7,A.oryzae-pksCT-L1-L2-L4-L7,A.oryzae-pksCT-L1-L2-L6-L7 and A.oryzae-pksCT-Ll-L2-L4-L6-L7).The metabolites produced by these transformants in MPM medium were analyzed by HPLC-MS and then purified and structurally identified(compounds 1-9).According to the structure differences of the metabolites produced under different gene expression,the CIT biosynthetic pathway was proposed,one molecular acyl-CoA and three moleculars malonyl-CoA were catalyzed by the PKS encoded by pksCT to synthetize compound 2,then the dioxygenase encoded by MRL2 catalyze the oxidation of C-12 methyl in compound 2 to yield the alcoholic hydroxyl group in compound 4,this alcoholic hydroxyl group subsequently was oxidized to a aldehyde group by the oxidoreductase encoded by MRL7,and finally oxidized to a carboxyl group by the aldehyde dehydrogenase encoded by MRL4 to obtain compound 15,the last step in CIT biosynthesis was the reduction of the carbonyl at C-3 into hydroxyl and to get CIT after cyclization and dehydration.5 Analysis of pre-mRNA alternative splicing in pksCTThe sequencing of mRNA transcribed by pksCT demonstrated the existence of pre-mRNA alternative splicing in pksCT.pksCT can transcribe into two different mRNA,the low abundant one indicated pksCT has only one 56-bp intron located at 640-695 base,while the other high abundant one indicated pksCT has another 62-bp intron located at 6549-6610 base.The PKSs translated by two different mRNA has domain structure of SAT-KS-AT-PT-ACP-CMeT-R and SAT-KS-AT-PT-ACP-CMeT,respectively.Heterologous expression vectors of pksCT with different intron-removed were constructed and transformed into Aspergillus oryzae M-2-3,results revealed PKS can produce 2 only with complete SAT-KS-AT-PT-ACP-CMeT-R domains,while the PKS lacking R domain can't produce 2 also other metabolites.