| Monascus spp.are traditional edible fungi,which fermented rice,has been widely used as food colorants,fermentation starters and folk medicines in China.Monascus spp.could produce diversiform secondary metabolites,and the well-known ones are Monascus pigments(Mps),monacolin K,ergosterol and citrinin.Among them,Mps,as one kind of the main secondary metabolites produced by M.spp,are a azaphilone mixture compound composed by red,orange and yellow colors),providing various functions,such as antihypertension,anti-hyperlipidemia and antidiabetics.Up to now,at least 90 kinds of Mps have been identified.Currently,solid fermentation of red yeast rice,liquid fermentation of Monascus red,Monascus yellow are the main three kinds of the Mps products in China.Although red yeast rice has several strengths such as mature production technology and little pollution,but it also has many shortcoming such as easily contaminated,low automation,labour-intensive in anduneven colouring.So Monascus red and Monascus yellow are the most common used Mps products in China.The Mps in the mycelium are extracted by ethanol after filtration,but extracellular Mps in the fermentation supernatant are discarded as wastewater,which caused great waste and increased the pressure of environmental protection.Hence,how to improve the intracellular Mps production and reduce the extracellular Mps prodution is urgent needs to be solved.Many researchers devoted themselves to the study of improve Mps production by strain screening and fermentation technology optimization,but the results are not very ideal.The study of the transport and secretion mechanisms of Mps from the molecular level may provide theoretical basis for the consturction of Monascus strains which have high intracellular Mps production and little extracellular Mps prodution.Related research has less been reported.Results showed that active transport and vesicle transport are the main pathway for the transport and secretion of secondary metabolites in filamentous fungi.Active transport is the movement of a substance against its concentration gradient(from low to high concentration),which requires the chemical energy or electrochemical gradient and the aid of carrier proteins.ATP-binding cassette(ABC)and major facilitator superfamily(MFS)are the main known carrier proteins.Vesicle transport is the movement of a substance from donor membrane to receptor membrane or cell membrane by vesicles.It includes four processes,namely vesicles budding,transport,binding and fusion.Ras-associated binding GTPases(Rab),ankyrin(Ank),tethering factors and many proteins involved in the regulation of vesicle transport.Some encoding genes of these proteins located in the gene cluster of secondary metabolites,other genes located in the genome.Besides,results showed that the quantity and ratio of vesicles(Diameter<2.5 ?m)and vacuoles(Diameter>2.5 ?m)had a significant effect on the synthesis and transport of metabolites.Since vesicles usually contain many enzymes and intermediates,so the increase of the vesicles quantity and the ratio of vesicles and vacuoles could improve the enzymes and intermediates transport and increase the intracellular metabolites content.Our previous study discovered the encoding gene of MFS and Ank in the Mps gene cluster of M.ruber M7,and the encoding gene of Rab protein in the genome of M.ruber M7,named MpigP,MpigL and Ypt7 respectively.In this study,in order to realize the transport pathway of Mps and improve the intracellular Mps production,the gene deletion(including single gene and double gene deletion),complemation and overexpression strains of MpigP,MpigL and Ypt7 will have been constructed to analyze the effect of MpigP,MpigL and Ypt7 on the intracellular and extracellular production of Mps,and the change of vesicles and vacuoles.The effect of Ypt7 on the transcriptional level of the key genes in the Mps gene cluster and the tranport-related genes including ABC/MFS,Ank and other vesicle transport associate proteins in the genome will be analyze by RNA-Seq.These results will be elaborated as follows.1 Construction and characteristic analysis of MpigP deletion,complementation and overexpression strainsHomologous recombination method was used to construct the MpigP deletion strain(OMpigP)and MpigP overexpression strain(M7::PtrpC-MpigP)driven by constitutive trpC promoter in M.ruber M7.A MpigP-complementatin vector containing an entire MpigP was introduced into AMpigP,forming MpigP complementation strain(?MpigP::MpigP).Colonial morphology,sporulation capacity,the quantity and ratio of vesicles and vacuoles in the mycelium and the intracellular and extracellular production of Mps of these strains were analyzed and the results were listed as follows.After cultivation for 15 days' at 28? on the 4 common medium(Potatodextrose agar medium,PDA;Malt extract agar,MA;Czapek yeast exatract agar,CYA;25%Glycerol nitrate agar,G25N),the colonial morphology,sporulation capacity,the quantity of vesicles and vacuoles of ?MpigP,?MpigP::MpigP,M7::PtrpC-MpigP were similar to M7.After cultivation for 11 days' at 28 on PDA,there were no difference in the extracellular Mps production of ?MpigP,?MpigP::MpigP,M7::PtrpC-MpigP and M7.While for the intracellular Mps production compared with M7,?MpigP had a 2-fold increase in yellow pigments in the entire cultivation process,the orange and red pigments had 70%decrease in 3rd day while was similar to M7 in 5-11d;AMpigP::MpigP had similar prodution in yellow,orange and red pigments;M7::PtrpC-MpigP had similar prodution in yellow pigment,while in orange and red pigments,it had a 1-fold increase in 3 rd day and had similar prodution in 5-11d.All these results indicated that MpigP may involve in the transport of yellow pigments and affect the synthesis of orange and red pigments.2 Construction and characteristic analysis of MpigL deletion,complementation and overexpression strainsHomologous recombination method was used to delete MpigL in M.ruber M7,generating AMpigL;A MpigL-overexpressing vector containing an entire MpigL driven by constitutive trpC promoter was introduced into M7 and ?MpigL,forming MpigL complementation strain ?MpigL::PtrpC-MpigL and overexpression strain M7::PtrpC-MpigL.Colonial morphology,sporulation capacity,the number of vesicles and vacuoles in the mycelium and the intracellular and extracellular production of Mps of these strains were analyzed and the results were list as follows.After cultivation for 15 days' at 28? on PDA,MA,CYA and G25N,the colonial morphology,sporulation capacity,the quantity of vesicles and vacuoles of ?MpigL were similar to M7;While for ?MpigL::PtrpC-MpigL and M7::PtrpC-MpigL,compared with M7,there are no difference in clony size,sporulation capacity and the number of vesicles,but the colour of the clony was lighter,the number of vacuoles(per 100?m mycelium)was decreased about 50%.After cultivation for 11 days' at 28 C on PDA,for intracellular Mps production compared with M7,AMpigL had a 30%decrease in yellow pigments from 3-7 d and had similar production in 9-11 d,and had similar prodution in orange and red pigments in the entire cultivation process;?MpigL::PtrpC-MpigL and M7::PtrpC-MpigL had a 50%decrease in yellow pigments in 3 rd day and had similar production in 5-11 d,had a 90%and 50%decrease in orange and red pigments in 3rd day,50%decrease in orange pigment and similar production in red pigment in 5-11 d.For extracellular Mps production compared with M7,in the entire cultivation process,?MpigL had no difference in yellow pigment,had a 50%decrease in orange pigment,had a 50%decrease in red pigment in 3rd day,nad a 1-fold increase in 5-7 d and had no difference in 9-11 d;In the entire cultivation process,AMpigL::PtrpC-MpigL and M7::PtrpC-MpigL had no difference in yellow pigment,had a 50%and 60%decrease in orange and red pigments.All these results indicated that MpigL may involve in the binding of vesicle membrane(contain yellow pigments)and organelle membrane(orange pigment synthesis site),then affect the intracellular production and secretion of orange and red pigments.Besides,the overexpression of MpigL caused the reduction of intracellulae and extracellular orange and red pigments as a negative control.3 Construction and characteristic analysis of Ypt7 deletion and overexpression strainsHomologous recombination method was used to construct the Ypt7 deletion strain(?Ypt7)and Ypt7 overexpression strain(M7::PtrpC-Ypt7)driven by constitutive trpC promoter in M.ruber M7.Colonial morphology,sporulation capacity,the number of vesicles and vacuoles in the mycelium and the intracellular and extracellular production of Mps of these strains were analyzed and the results were list as follows.After cultivation for 15 days' at 28? on PDA,MA,CYA and G25N,the colonial morphology,sporulation capacity,the number of vesicles and vacuoles of M7::PtrpC-Ypt7 were similar to M7;While for ?Ypt7,the clony was smaller than M7,the clony colour was dark red,the number of conidium and cleistothecium decreased obviously,the number of vesicles was 20-times more than M7,the vacuoles amount decreased raletively and distributed unevenly.After cultivation for 11 days' at 28? on PDA,for intracellular Mps production compared with M7,in the entire cultivation process,?Ypt7 had a 80%,30%and 1.8-fold increase in yellow,orange and red pigments;M7::PtrpC-Ypt7 had a 20%,30%and 10%increase in yellow,orange and red pigments.For extracellular Mps production compared with M7,in the entire cultivation process,?Ypt7 had a 63%,45%and 83%decrease in yellow,orange and red pigments;M7::PtrpC-Ypt7 had a 40%,10%and 20%increase in yellow,orange and red pigments.All these results indicated that Ypt7 may regulat the secretion of Mps.4 Construction and characteristic analysis of MpigP,MpigL and Ypt7 double deletion strainsHomologous recombination method and double screening markers were used to construct the MpigP,MpigL and Ypt7 double deletion strains,forming ?MpigP?Ypt7,?MpigL?Ypt7 and ?MpigP?MpigL.Colonial morphology,sporulation capacity,the number of vesicles and vacuoles in the mycelium and the intracellular and extracellular production of Mps of these strains were analyzed and the results were list as follows.After cultivation for 15 days' at 28? on PDA,MA,CYA and G25N,the colonial morphology,sporulation capacity,the number of vesicles and vacuoles in the mycelium of?MpigP?Ypt7 and ?MpigL?Ypt7 were similar to ?Ypt7.The clony of ?MpigP?MpigL was a little bigger and the clony colour was much lighter than M7,the number of conidium,cleistothecium,vesicles and vacuoles decreased obviously.After cultivation for 11 days' at 28? on PDA,for intracellular Mps production,in the entire cultivation process,the yellow and red pigments production in ?MpigP?Ypt7 were higher and the orange pigments was lower than that in AMpigP and ?Ypt7,while compared with M7,?MpigP?Ypt7 had a 2.5-fold increase in yellow pigment,75%decrease in orange?pigment and 1-fold increase in red pigment;the yellow,orange and red pigments production in AMpigLAYpt7 were both higher than that in ?MpigL and ?Ypt7,while compared with M7,?MpigL?Ypt7 had a 1-fold increase in yellow pigment,1-fold increase in red pigment,while orange pigment production was similar to M7.the yellow,orange and red pigments production in ?MpigP?MpigL were both lower than that in ?MpigP and ?MpigL,while compared with M7,?MpigP?MpigL had a 88%decrease in yellow pigment,97%decrease in red pigment,and no orange pigment was detected.For extracellular Mps production,in the entire cultivation process,the yellow,orange and red pigments production in ?MpigP?Ypt7,?MpigL?Ypt7 and ?MpigP?MpigL were both lower than that in ?MpigP,?MpigL and ?Ypt7,while compared with M7,?MpigP?Ypt7 had a 38%,88%and 50%decrease in yellow,orange and red pigments,AMpigLAYpt7 had a 39%,83%and 50%decrease in yellow,orange and red pigments,?MpigP?MpigL had a 82%decrease in yellow pigment,and no orange and red pigments were detected.All these results indicated that MpigP involved in the transport of intracellular pigments,especially the transport of yellow pigment,and affected the synthesis of orange pigment.The interaction of MpigL and MpigP influenced the Mps synthesis efficiency.Ypt7 mediated the Mps transport and secretion.The transport and secretion of Mps may mainly rely on the vesicle transport pathway mediated by Ypt7.5 Analyze of the interaction of different transport protein-encoding genes by RNA-seqThe results in part 3 and part 4 indicated that Ypt7 affect the Mps transport and secretion obviously,in order to reveal the interaction of different transport protein-encoding genes,and the effect of transport protein-encoding genes on Mps transport and secretion,the differential expression genes and their functions of M7 and ?Ypt7 after 3 days' and 7 days' cultivation at 28?,120r/min in PDB were analyzed by RNA-seq implemented on high-throughput sequencing platform(BGISEQ-500RS),and the results were list as follows.Compared with M7,after 3 days' cultivation,there were 376 genes up-regulated expression,456 genes down-regulated expression in ?Ypt7;after 7 days' cultivation,there were 598 genes up-regulated expression,425 genes down-regulated expression in ?Ypt7.Among them,The results showed that compared with 263 ABC/MFS tranporters,18 Ank,70 vesicle transport related proteins in the ?Ypt7 genome,after 3 days' cultivation,6 ABC/MFS tranporters,1 Ank and 6 vesicle transport related proteins up-regulated expression;8 ABC/MFS tranporters,2 Ank,2 vesicle transport related proteins and 1 Mps-Polyketone synthetase(PKS)down-regulated expression in ?Ypt7.Besides,there were 52 ribosomal protein up-regulated expression in ?Ypt7.After 7 days' cultivation,13 ABC/MFS tranporters,1 Ank,9 vesicle transport related proteins,3 ribosomal protein and 15 PKS/regulatory factors up-regulated expression;6 ABC/MFS tranporters,2 vesicle transport related proteins and the global regulatory factors LaeA down-regulated expression in ?Ypt7.All these results indicated that Ypt7 mediated the Mps transport and secretion by affecting the expression of the encode genes involved in transmembrane transport,vesicle transport and secondary metabolites synthesis.6 The propesed Mps transport and secretion pathwayThe Mps transport and secretion pathway was proposed based on above results.The details were explained as fellow:first,the intermediates and the synthetases of Mps were secreted from golgi apparatus to early endsome(EE)in cytoplasm,then they were transported to yellow pigment endsome(YPE),orange pigment endsome(OPE)and red pigment endsome(RPE).The yellow pigment was fisrt synthesised in YPE.Second,part of the yellow pigments were secreted to extracellular through vesicles under the regulation of Ypt7;Another part of yellow pigments were transported to OPE for orange pigments synthesis,this process was mediated by the interaction of Vpsl(Vesicle protein sorting protein)and MpigP or Vps2 only through binding with different binding sites in Ank(encoded by MpigL).Third,part of orange pigments were secreted to extracellular through vesicles under the regulation of Ypt7;Another part of orange pigments were transpored to RPE for red pigments synthesise under the regulation of Vps3 and SNARE.Similar to orange pigments,part of red pigments were secreted to extracellular through vesicles under the regulation of Ypt7;Another part of red pigments were accumulated in cytoplasm. |
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