Studies On The Function Of Vacuolar Protein Sorting Receptor In Penicillium Decumbens

Penicillium decumbens is known to secrete a large number of cellulases and hemicellulases, which can make renewable biomass degrade into fermentable sugars. These sugars are used for producting bio ethanol. At present, the relatively low production and hydrolysis efficiency of cellulases is the major bottlenecks in the conversion of biomass into bio ethanol. In order to increase the cellulases production of Penicillium decumbens and reinforce its application in lignocellulose degradation engineering, we conducted a series of experiments.1. Screening P. decumbens Avps::ptrA mutants. VPS10protein sequence of P. decumbens was found through homologous protein sequence comparison.△vpsv::ptrA deletion cassette was constructed by double-joint PCR.△vps::ptrA mutants with the disruption of vps10gene were obtained through homologous double crossover recombination. PCR and enzyme digestion analysis showed that vps10gene was disrupted in P. decumbens. It is of great significance for studying the function of VPS10in P. decumbens.2. Screening P. decumbens△vps::pyrG mutants.△vps::pyrG deletion cassette was constructed by double-joint PCR, restriction enzymes digested and T4ligase. The difference of Avps::ptrA and△vps::pyrG deletion cassette is that△vps::pyrG deletion cassette has a part repeating segments and forms self-cleavage pattern.△vps::pyrG mutants with the disruption of vps10gene were obtained through△pyrG:ptrA protoplast transformation. PCR and enzyme digestion analysis showed that△vps::pyrG mutants were successfully established. It plays an important part in genetic research of P. decumbens.3. Constructing expression vector pbgl-hph of β-glucosidase in Trichoderma reesei. Expression vector pbgl-hph was constructed by double-joint PCR, restriction enzymes digested and T4ligase. β-glucosidase from Trichoderma reesei was expressed in△vps::ptrA mutant. Enzyme digestion analysis showed that the expression vector pbgl-hph was successfully established. It is of great significance for studying the function of VPS10and increasing the production of β-glucosidase in P. decumbens.4. Effects of a novel transcription factor BglR from Penicillium decumbens on cellulase activity. We identified a transcription regulator BglR (PDE-01706) from P. decumbens114-2 by comparing gene expression profiles under different carbon sources condition. This transcription regulator shares a59%sequence identity with the zinc finger protein Pc20g04780from Penicillium chrysogenum. P. decumbens△bglR-1knockout strain was constructed with homologous double crossover recombination. The phenotype of this strain was investigated, including its hyphal growth, protein secretion level, cellulase secretion level and extracellular pH level. It was shown that the deletion of the transcription factor BglR in P. decumbens△bglR-1leads to an increase of extracellular β-glucosidase activity by40%, and a lowered filter paper activity, endoglucanases and xylanase level. From these results it is proposed that BglR plays a major role in the regulation of cellulase production of P. decumbens.