| Cellulose is one of the most abundant carbohydrates and it is also the cheapest renewable resource on earth.The filamentous fungus Trichoderma reesei can efficiently degrade cellulose,and it can rapidly produce a large amount of cellulases in the presence of inducing carbon sources.T.reesei has been a model strain in industry due to its high capacity of cellulase production,but the high cost of cellulase production is still a bottleneck to produce biofuels and bio-based bulk products.Trichoderma spp.are capable of producing many secondary metabolites,whereas the yellow pigment is only produced by T.reesei.The yellow pigment is synthesized by a polyketide synthase(PKS)gene cluster in T.reesei,and its production is strictly regulated.Studies have shown that the the yellow pigment may belong to sorbicillinoids.So far the specific role of the yellow pigment in the physiological growth process of T.reesei is still unclear.Our study will provide a theoretical foundation for improving the production of cellulases and facilitating the cellulase isolation process.In order to study the effect of the yellow pigment on physiological growth and the expression of cellulases in T.reesei,we constructed a series of mutants about two transcription factors ypr1,ypr2 and a PKS-encoding gene sor1 to obtain yellow pigment high-producing and yellow pigment-defective strains respectively,and we also analyzed their phenotypes including the vegetative growth,conidiation,cell wall integrity,stress resistance,and cellulase production.The main results of this study are summarized as followed:1.The transcription factor ypr1-overexpressing strain OEyprl and the ypr2 knockout strain ?ypr2 were constructed respectively.We found that the mutants produced high-yield yellow pigment,and their physiological growth and cellulase expression were seriously affected.OEyprl and ?ypr2 were constructed as two yellow pigment high-producing strains using the wild-type QM9414-?pyr4 as the parent strain.The mutant strains showed defectiveability to utilize glucose in liquid medim;The myceliums of the mutants were larger,and the mycelial branches were thicker and shorter than the wild type strain;The conidia amounts of the mutants were remarkably reduced;Cell wall integrity and stress resistance of the mutants were severely impaired.Moreover,the expression of the main cellulase in the mutants almost disappeared,and this was caused by the decreased cellulase gene expression at the transcriptional level.2.We constructed the ypr1 knockout strain ?ypr1 and the ypr2-overexpressing strain OEypr2,and we also found that whereas these two mutants failed to produce yellow pigmet,their physiological growth and cellulase expression were enhanced.?ypr1 and OEypr2 were obtained by deleting ypr1 and overexpressing ypr2 in the wild-type T.reesei,respectively,and both of them were defective in production of yellow pigment Compared with the wild type QM9414,the biomass and conidia production of the ?ypr1 and OEypr2 mutants were increased;The cell wall integrity and stress resistance were improved;The expression of the main cellulase of the mutants were increased by 15%under induced conditions.3.Analysis of the phenotypes demonstrated by the knockout of sor1 in the wild-type and high-yield yellow pigment strain of OEypr1 revealed that the excessive yellow pigment was the major reason that caused the defects in the physiological growth and cellulase production observed in the OEypr1 strain.The ?sor1 and OEyprl-?sor1 strains were obtained by knocking out sor1 in the wild-type and high-yield yellow pigment strain OEypr1,respectively,and both of the mutants failed to produce yellow pigment,and their abilities in glucose utilization,sporulation,cell wall integrity,stress resistance,and cellulase expression were all improved compared to the wild type.These phenotypes were similar to the ?ypr1 and OEypr2 strains that did not produce yellow pigment,either.The damaged phenotype of the high-yield yellow pigment strain OEypr1 could be completely restored when yellow pigment was eliminated by deleting sor1.Therefore,we concluded that it is the yellow pigment rather than Ypr1 or Ypr2 that plays and important role in T.reesei vegetative growth and cellulase production.HPLC-MS analyses showed that the yellow pigment are mainly dimers and monomers of the sorbicillinoids.We presented that the yellow pigment plays important roles in the physiological growth and cellulase expression in T.reesei,we also proposed that the non-yellow pigment mutants of T.reesei can be used as superior industrial strains to produce cellulases.These results not only enrich the theoretical research on the correlation between primary metabolism and secondary metabolism,but also helps engineer a more efficient and lower-cost model strain for the production of proteins in industrial by T.reesei. |
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