Functional Study Of Polysaccharide Monooxygenases From Penicillium Decumbens

Producing bioethanol from lignocellulose is an excellent way to alleviate energy crisis, protect environment and promote sustainable development of global economy. The filamentous fungus Penicillium decumbens produce an extracellular cellulase system whose composition is well balanced. The cellulase productivity of P. decumbens mutants obtained by mutagenesis matches that of Trichoderma reesei, the widely used cellulase producer. However, high cost and low efficiency of cellulase is still the bottleneck in industrial application. Thus, studying the synergic proteins enhancing traditional cellulase actvity is important to improve cellulose hydrolysis.P. decumbens was isolated from soil and was able to decompose lignocellulosic biomass rapidly. Current research has already made some important progress on composition of cellulase system and regulation of cellulase expression by analysis of their genome, transcriptome and proteome. In this thesis, the only high expressed polysaccharide monooxygenase gene was deleted and then complemented in order to study its biological function. Meanwhile three polysaccharide monooxygenases were successfully expressed in Pichia pastoris and their synergic action was analyzed. These studies are not only helpful to deepen the understanding of P. decumbens extracellular protein composition, but also can be guidelines for improving P. decumbens strain at the genetic level.The main results of the thesis are as follows:1. Deletion and complementation of PMO5633gene in P. decumbens and characterization of PMO5633genePMO5633gene deletion and complementation cassettes were constructed by fusion PCR and were successfully transformed into P. decumbens. Deletion of the gene made the strain grow better, increased extracellular protein concentration and cellulase activity. Meanwhile, after deleting the gene, hydrolyzing ring on avicel plate amplified and biomass aggrandized. However, the broth from the wild type could hydrolyze microcrystalline cellulose better than the deletion strain when equal FPA activity was added.2. Transcription analysis of PMO genes, CDH gene and cellulolytic enzymes genes of P. decumbens cultured on different carbon sources and its mutant strain cultured on microcrystalline celluloseTranscriptional level of four PMO genes, CDH gene, and other three cellulostic enzymes genes were detected on different carbon source (glucose and microcrystalline cellulose) and in different strains (wild and mutant strain) by means of real-time quantitative polymerase chain reaction (RT-qPCR). For the wild strain, eight genes were coordinately regulated at transcriptional level and are all induced by cellulose. Transcription of PMO genes was about12hours earlier than that of ordinary cellulase genes. PMO1261gene could be transcribed as many as30,000times when growing on cellulose than on glucose, while PMO5633transcribed80,000times more. In the deletion strain, the expression of cdh and cel7A increased6and12times, respectively, when comparing with the wild strain cultured on microcellulose.3. Cloning and recombinant expression of four PMO genes from P. decumbens in Pichia pastoris, and characterization of three successfully-expressed recombinant PMOsFour PMO genes (pmo698, pmo1261, pmo5633, pmo6768) from P. decumbens were cloned using RT-PCR. All PMO genes were expressed in Pichia pastoris. However, the pmo6768could not be expressed in P. pastoris. And only the first three recombinant proteins were obtained and purified successfully. Three recombinant PMOs were glycosylated by different degrees, among which only PMO1261were O-glycosylated. Recombinant PMOs lacked measurable hydrolytic activity by themselves, but in the presence of divalent metal ions and reductants can significantly enhance traditional cellulase activity. PMO5633could even double the hydrolysis rate.