| Monascus pigments(MPs)are a kind of important secondary metabolites that they have azaphilone structure and are produced by Monascus spp.,and they have been utilized for nearly two thousands years in China and Southeast Asia.In 2017,our group has derived a more detailed MPs biosynthesis pathway by gene-knockout strategy and so on,and it was that the enzymes,which coded by the different genes(MpigA,MpigC,MpigD,MpigE,MpigF,MpigG,MpigJ,MpigK,MpigM and MpigON)in MPs gene cluster,participate in the synthesis and modification one after another to produce the final MPs molecule.However,all of the products produced by this pathway are azaphilone structure or its derivatives,and some genes are not directly involved in MPs synthesis.There are more than one hundred types of MPs molecules whose structures are known and multifarious.Most MPs have one azaphilone mother ring structure,while both our group and other researchers have found that the MPs gene cluster can also synthesize a series of naphthoquinones(NQs)molecules,which are different in structure and function with azaphilone pigments.So we inferred that some genes in MPs gene cluster may participate in the synthesis of both NQs and MPs at the same time,and the formation process of two types of compounds was coupling.In order to verify this hypothesis,according to the NQs structure,the genes which may participate in the synthesis were inferred,and the Aspergillus oryzae(AO)heterologous expression strategy was adopted to combinationally express these genes in AO M-2-3.According to detecting the metabolites types and structures of different AO transformants,the synthesis pathway of NQs was studied,and the coupling synthesis relation with MPs was analyzed.1.Construction of the AO M-2-3 heterologous expression transformants containing different combination genes of MPs gene clusterOn the basis of the preliminary study on MPs synthesis pathway of our group,it is showed that polyketide synthase,which is coded by MpigA,is the initial enzyme for the synthesis of MPs,and the products of MpigC gene and other genes are the modification enzymes of MPs synthesis.According to the structure of NQs and the known MPs synthesis pathways,it was inferred polyketide synthase,which was coded by MpigA(A),was the initial enzyme for the synthesis of NQs,however,the products of the four genes MpigC(C),MpigD(D),MpigH(H)?MpigON(ON)were possible the modification enzymes for NQs synthesis.The c DNA,which was obtained from Monascus ruber M7 total RNA by Reverse Transcription PCR,was taken as the template,and the five genes fragments were obtained by PCR.The expression plasmids with different genes combination were constructed by molecular biology technology,and they were transferred into AO M-2-3.After screening and vertification,the corresponding AO transformants were obtained,and their names were AO-A,AO-ON,AO-A-ON,AO-A-C,AO-A-D,AO-A-H and AO-A-C-D,respectively.Among them,transformants AO-A,AO-ON and AO-A-ON have been constructed in the preliminary experiment by our group.2.Discovery and analysis of the coupling synthesis pathway of NQs antibiotics and MPsThe metabolites in the rice culture of the various of AO heterologous expression transformants in part 1 were analyzed by HPLC,the possible target products were analyzed by LC-MS,purification and NMR,and the products structure of the various of gene combination was determined.The results showed that the substance 1(derivative of benzaldehyde)of the product of the transformant AO-A-C was identified,which could be modified by other enzymes to form MPs.The substance 2 of the product of the transformant AO-A-ON was naphthol structure and it could be further catalyzed to form NQs.According to the gene combination type and the molecule structure difference of the corresponding products substance 1 and 2 of this two transformants,we inferred that,on one hand,the products of MpigA were catalyzed by MpigC to produce the derivative of benzaldehyde(the substance 1),which was the pigments precursor substance;on the other hand,the products of MpigA were catalyzed by MpigON to produce the naphthol substance(the substance 2),which was the the NQs antibiotics precusor substance.The two pathways were coupling together and may be competitive.However,under the present experiment conditions,the transformants AO-A-D,AO-A-H and AO-A-C-D did not identify new target metabolism products.In order to construct the AO transformants containing the four genes MpigA,MpigC,MpigD and MpigON,a lot of experiments have been done,but never succeed.We planed to verify whether the four genes were able to catalyze the synthesis of MPs or its precursor substances or not,then the cocultivation experiment of transformants AO-A-C-D and AO-ON which have been constructed was performed,and the substance 3 was identified.According to HPLC,spectrum,the result of reaction with NH3 and literature reports,we inferred that the substance 3 was a kind of atypical orange pigment molecule of MPs(Monasfluol A,Monasfluol B).3.Functional differences of tandem ACPs in polyketide synthase of MPsIt was found that there was a unique tandem ACPs structure domain in MPs polyketide synthase MpigA in preliminary analysis,so the function of tandem ACPs structure domains(ACP1and ACP2)in MPs polyketide synthase coded by MpigA was also studied.The expression plasmids were constructed,which contained MpigA in which the two ACPs were inactivated by site-specific mutagenesis,respectively.Then the corresponding AO transformants AO-?ACP1 and AO-?ACP2were constructed.According to the analysis of fermentation products of the transformants,it was found that AO-?ACP1 was non-capable of producing the corresponding products of transformants AO-A,while AO-?ACP2 was capable of producing the corresponding products of AO-A,but the concentration of the production was only about 10%of AO-A.This result showed that ACP1 may control the activity of the polyketide synthase,while ACP2 could only affect the synthesis efficiency of the polyketide synthase. |
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