Mechanism Of Manganese Ion And N,N-Dimethylformamide Inducing Cellulase Production Of Trichoderma Reesei

Trichoderma reesei has excellent ability of protein secretion and expression,and is one of the important industrial strains for cellulase production.The cellulase produced by T.reesei is widely used in textile,paper,feed and bioenergy industries.Therefore,modern bioengineering technology is required to further increase cellulase production and reduce costs of cellulase to meet the needs of commercial applications.Additionally,with the completion of the whole genome sequencing of T.reesei,the establishment and improvement of efficient molecular biology and genetic operation system,T.reesei has become a potential model host for studying the mechanism of high-yield cellulase production,signal transduction and regulation,and for the expression of heterologous proteins,attracting more and more attention.In order to research the mechanism of cellulase production and the regulation mechanism of signal transduction,the following three aspects of research were carried out in this paper.1)Mechanism of manganese ion regulating cellulase gene expression.Our study found that 10 mM Mn2+markedly stimulated cellulase production and total protein secretion of T.reesei.Furthermore,by determining the content of intracellular and extracellular manganese ions by ICP-MS,we identified two Mn2+ transport proteins,designated as TPH084-1 and TPH084-2,which were responsible for transporting extracellular Mn2+into the cell,indicated that they are involved in Mn2+-induced cellulase production.By using calcium ion fluorescence probe Fluo-3 AM,we also found that Mn2+ induced a significant increase in cytosolic Ca2+ concentration,which may be the key reason for Mn2+-mediated regulation of cellulase gene transcription and production.The utilization of LaCl3 to block plasma membrane Ca2+ channels,and deletion of crz1(calcineurin-responsive zinc finger transcription factor 1)to interrupt calcium signaling,showed that Mn2+ exerts the induction of cellulase genes via calcium channels and calcium signaling.To substantiate this,we identified a Ca2+/Mn2+P-type ATPase,TPMR1,which was responsible for the Ca2+/Mn2+balance in T.reesei.Under the condition of 10 mM Mn2+,TPMR1 functions as Ca2+/Mn2+ exchanger,pumps Ca2+ into the cells,which leads to a increase of intracellular Ca2+concentration,and then stimulates calcium signaling pathway to induce cellulase production.This study first discovered and revealed the mechanism of Mn2+-induced cellulose production;this study also provides a theoretical basis for the further study of mechanism of cellulase production and calcium signaling pathway.2)The mechanism of DMF regulating cellulase expression and secretion.DMF is a common organic solvent,widely used in industry,especially as a solvent for various water-insoluble substrates in biological experiments.Our study found for the first time that biologically relevant concentrations of extracellular DMF induced cellulase production in T.reesei.In order to study the mechanism of DMF-induced cellulase production,our study,through transcriptomics analysis,found that the plc-e gene associated with the regulation of intracellular Ca2+ was activated by DMF,indicating that intracellular Ca2+ was induced by DMF in T.reesei.The utilization of EGTA(a putative extracellular Ca2+chelator),LaCl3(a plasma membrane Ca2+ chalnel blocker),and deletion of crz1(interrupt calcium signaling),we demonstrated that DMF induced a cytosolic Ca2+ burst via extracellular Ca2+ and Ca2+channels in T.reesei,and that the cytosolic Ca2+ burst induced by DMF mediated overexpression of cellulase through calcium signaling.Additionally,0.5-2%DMF increases the permeability of T.reesei mycelia for cellulase releasing.These results indicate that DMF induces cellulase expression and secretion through calcium signaling and permeability.Simultaneous supplementation with 1%DMF and 10 mM Mn2+ to T.reesei Rut-C30 increased cellulase activity approximately four-fold compared to that without treatment and was also more than that observed in response to either treatment alone.This study found for the first time that DMF induced cellulase production and revealed its induction mechanism,which provided guidance f-or the further study of the role of calcium signaling in cellulase production.3)DMF in wastewater was degraded by engineered T.reesei.Since DMF is widely used in many chemical industries,DMF is usually discharged at a high concentration together with industrial wastewater.DMF is both hepatotoxic and carcinogenic and can be harmful to human health.Thus,the removal of DMF from industrial wastewater is essential.However,there are few studies on the degradation of DMF in biological methods.Our study found that T.reesei can grow under high concentrations of DMF(20 g/L).At the same time,we found that the addition of DMF wastewater can still induce the cellulase production of T.reesei.In addition,T.reesei cannot degrade DMF.Therefore,based on synthetic biology,our study used T.reesei as the original strain,introduced the degradation pathway of exogenous DMF into T,reesei cells,and constructed an engineering strain that utilizes and treats DMF in wastewater effectively.Firstly,a method for detecting DMF is established based on HPLC methods.Secondly,N,N-dimethylformamidase(DMFase),a key enzyme in the DMF degradation pathway,was obtained through GenBank,the heterologous expression vector CA21T was constructed,and the T.reesei CA21T strain which can degrade DMF was obtained.Finally,DMF wastewater was added during the fermentation of T.reesei CA21T strain to degrade DMF at the same time of producing high-yield cellulase.Combination of production of high-yield cellulase induced by DMF with degradation and utilization of DMF wastewater achieve"killing two fish with one stone".T.reesei CA21T strain can be widely used as a chassis for the utilization and treatment of DMF wastewater effectively.