Degradation Of Rice Straw By Aspergillus Fumigatus Z5 And Heterologous Expression Of Its Polysaccharide Momooxygenase

Lignocellulose,as the main component of plant cell walls,is the most abundant biomass resources,and it is hard to decompose because of the complex structure.The lignocellulose can be degraded by lignocellulolytic enzymes secreted by various microorganisms,which is important to reduce the environmental pollution,energy crisis,food shortages and help to guarantee the human sustainable development.Degradation of lignocelluloses is the major biochemical reaction during the composting process,which is catalyzed by various lignocellulolytic enzymes secreted by microorganisms,and there is a positive correlation lignocellulase activity between the composting efficiency.In order to reveal the capacity of lignocellulose degradation,A.fumigatus Z5 conidia suspension was inoculated into the Mandels’ salt solution with the dry rice straw,so we could track the degradation process of rice straw by A.fumigatus Z5.The result illustrated cellulose and hemicellulose in rice straw is not so hard to be degraded,lignin was hard to be degraded.Polysaccharide monooxygenase(PMOs)belong to the glycoside hydrolase family member 61(GH61),and they played an important role during the lignocellulose degradation process.In order to reveal the regulation relationship between GH61 genes and carbon metabolic regulation factors,fluorescence quantitative PCR method was used to quantify the transcriptional level of different GH61 genes in A.fumigatus Z5 by use rice straw and glucose as sole carbon source,respectively.The highest expressed GH61 gene of A.fumigatus Z5 with the PMOs function module was expressed in Pichia pastor is X33 under methanol induction condition.The function of PMOs was studied in detail,and the results revealed the mechanism of lignocellulose hydrolysis and synergistic promoting effect on lignocellulose degradation,which would provide a theoretical basis to improve the composting efficiency.As a eukaryote,Pichia pastoris X33 owned a eukaryotic expression systems with many advantages,such as protein processing,folding,and other post-translational modification.So,Pichia pastoris X33 was used as the expression host.The total RNA of A.fumigatus Z5 cultured with rice straw as sole carbon source was extracted,and then the cDNA were synthesized by reverse transcription Kit.The specific primers of different PMOs were designed to amplified different PMOs genes,and then connected to the Pichia expression vector pPICZaA.Finally,the recombinant plasmid were transformed into the Pichia pastoris X33.The results obtained in this study showed that three different polysaccharide PMOs are successfully expressed,and the enzyme activities were determined by use fluorescence method.