Preliminary Study On Nitrogen Metabolism And Regulation Of Penicillium Oxalicum

Cellulose is a rich and renewable resource in nature,with advantages such as wide distribution and large reserves.Biofuels obtained by conversion of cellulose can effectively alleviate environmental pollution and promote economic sustainable development.Many filamentous fungi are capable of synthesizing and secreting cellulolytic enzymes that degrade cellulose.Among them,Penicillium oxalicum has a relatively complete lignocellulose degrading enzyme system.The high-yield mutant strain of Penicillium oxalicum has been applied to industrial production and has certain potential in the development of biofuel industry.Protein synthesis is closely related to carbon and nitrogen metabolism.At present,researchers have gained a deeper understanding of the relationship between cellulase synthesis and carbon metabolism in filamentous fungi,and related regulatory mechanisms have been extensively studied.However,although it is known that nitrogen sources are important for the production of extracellular proteins such as cellulases,studies on related molecular mechanisms are rather weak.In this thesis,the relationship between nitrogen source and cellulase production,the biological role of nitrogen metabolism-related transcription factors and its relationship with cellulase synthesis were studied in P.oxalicum.The research results can provide new ideas for optimization of fermentation medium and strain modification.The main research progress of this thesis is as follows:1.Effects of nitrogen sources and proteases on the yield of cellulaseFermentation medium with corn steep liquior,dried distiller grain with solubles,bean cake powder,ammonium sulfate and sodium nitrate as sole nitrogen source was designed respectively.The cellulase-producing strain RE-8 was subjected to a 9-day shake flask fermentation culture.The results showed that P.oxalicum had the strongest ability to produce cellulase on the complex nitrogen source medium,and the enzyme production ability was the lowest on ammonium sulfate as the sole nitrogen source.In the complex nitrogen source medium,the ammonium concentration in the fermentation broth first decreased and then increased,while the activities of acid protease and neutral protease gradually accumulated.After adding protease inhibitor to the culture medium,the activity of extracellular protease was significantly decreased,the concentration of extracellular protein increased,and the activity of cellulase was not changed remarkably,which still showed a downward trend in the late stage of fermentation.2.Biological functions of nitrogen catabolism transcription factor AreAAreA is a conserved nitrogen catabolic metabolic transcription factor in fungi and is generally thought to be responsible for the activation of secondary nitrogen source metabolic gene expression.This paper constructed a DNA binding domain deletion mutant strain and a replenishing strain of areA.The effects of nitrogen source species on the expression of cellulase genes and the role of transcriptional regulator AreA in the utilization of nitrogen source and cellulase gene expressionwere studied.The results showed that the effect of nitrogen sources on the expression of cellulase genes in wild strain was related to the type of carbon source.AreA is not only necessary for the use of secondary nitrogen sources for P.oxalicum,but also for the utilization of readily available nitrogen sources such as ammonium sulfate,which is different from the results reported in Aspergillus species.AreA has a regulatory effect on the expression of ammonium metabolism related genes and also cellulase genes.Knockout of the areA gene resulted in a significantly lower cellulase gene expression under cellulose-induced condition.3.The role of amino acid synthesis regulator CpcA in the growth and extracellular enzyme productionCpcA is a conserved cross-pathway amino acid anabolic transcriptional activator in fungi.In this paper,the cpcA-knockout strain AcpcA,cpcA replenishing strain RcpcA and the cpcA overexpression strain were constructed.In the medium with or without the addition of the amino acid synthesis inhibitor 3-amino-1,2,4-triazole,the cpcA knockout strain showed a very significant disadvantage compared to the wild strain and the replenished strain,and this phenotype can be compensated in the amino acid-rich medium.The cellulase and amylase activities of the cpcA knockout strain were significantly lower than those of the wild strain,and the addition of corn steep liquior could reduce the difference between the strains.By tracking cell growth and cellulase production during fermentation and the determination of cellulase gene transcription levels,it was confirmed that the decrease in cellulase production after cpcA knockout was not only caused by cell growth disadvantage.In addition,it was found that overexpression of cpcA can significantly increase the production of amylase,which provides a new idea for the improvement of amylase producing strains.