Comparative And Functional Genomics Studies Of Lignocellulolytic Fungus Penicillium Decumbens

The use of lignocellulosic biomass for the production of liquid fuels and chemicals has long been considered to be an important alternative for sustaining the human economy and society. In one of the most widely accepted biorefinery schemes, lignocellulosic materials are first hydrolyzed to fermentable sugars by fungal enzymes and then converted to various products. Currently, both the production level and the performance of lignocellulolytic enzymes still need to be improved to make the process economically competitive. The filamentous fungus Penicillium decumbens has been used for industrial-scale cellulase production in China for16years. Cellulase productivities of P. decumbens mutants are comparable with those produced by the widely used cellulase producer Trichoderma reesei. Furthermore, the enzyme system shows a more balanced protein composition than that from T. reseei. Thus, deep investigation of P. decumbens would be important for reducing the cost of lignocellulolytic enzymes production, as well as the understanding of regulation mechanisms for lignocellulolytic enzymes production in fungi.The main results of the research are as follows.1. Genome sequencing and analysis of P. decumbens wild-type strain114-2Lignocellulolytic enzymes and proteins involved in several other biological processes were annotated based on the genome sequence of P. decumbens wild-type strain114-2. Although more related to Penicillium chrysogenum phylogenetically, P. decumbens contains less proteins involved in cellular metabolism and regulation, while the numbers are similar with those of T. reesei. Compared with T. reesei, P. decumbens has genes encoding a set of plant cell wall-degrading enzymes with more diverse components, particularly for cellulose binding domain-containing proteins and several key enzymes to decompose hemicellulose. P. decumbens shares some regulation mechanisms for lignocellulolytic enzymes synthesis with T. reesei, but some different mechanisms between the two species were also indicated by genomic analysis. In addition, genomic regions rich in plant cell wall-degrading enzyme genes were found in P. decumbens, and some genetic events including horizontal gene transfer and gene expansion were observed for lignocellulolytic enzyme-encoding genes.2. Comparative genomic and transcriptome analysis of wild strain and lignocellulolytic enzyme hyper-producing mutants of P. decumbensNumerous genetic variations were found between the wild strain114-2and two lignocellulolytic enzyme hyper-producing mutants JU-A10-S and JU-A10-T through comparative genomic analysis. On the other hand, much less genetic variations were detected between the two mutants. The result indicated that the parent strain114was likely a heterokaryon, and current strains have been purified by subsequent isolation processes. Proteins with sequence differences between strains114-2and JU-A10-T were significantly enriched for those involved in transcriptional regulation, highlighting the important roles of these proteins in the phenotypic differences between the strains. A frame-shift mutation possibly accounting for the abolishment of conidial pigment synthesis in mutants were also found. The result of previous secretome analysis was confirmed, and differential expression of several intracellular biological processes was found, by comparative transcriptome analysis between strains. In addition, the mating-types of wild strain, mutants and another P. decumbens strain Peni-1were established, which providing the possibility for sexual cross in P. decumbens.3. Heterologous expression and characteriztion of endo-p-1,4-glucanases Ce145A and Cel5C from P. decumbensTwo endo-P-1,4-glucanases Ce145A and Ce15C from P. decumbens were recombinantly expressed and characterized for enzymatic properties. Both enzymes produced cellodextrins from cellulose. Compared with reported endo-p-1,4-glucanases from fungi, rCe145A and rCe15C showed relatively lower activities on CMC-Na, which might be related to the mechanisms of enzyme-substrate interactions. Similar to other GH family45proteins, rCe145A had higher temperature optimum and thermal stability, thus indicating the potential for improvement of cellulolytic enzyme systems. In addition, amino acids from position447to655were found to play an important role in the hydrolytic activity of Ce15C, and a protein stability-related mechanism was proposed.4. Preliminary study of the biological function of serine protease PrtB in P. decumbensOrthologs of three hydrolytic enzymes involved in autolysis in Aspergillus nidulans, including chitinase ChiB, metal protease PepJ and endo-β-1,3-glucanase EngA, were found in P. decumbens. However, another autolysis-involved protease PrtA of A. nidulans had no ortholog in P. decumbens. Characterization of gene deletion strains of ChiB, PepJ or EngA revealed that they might not play important roles in carbon starvation response and cellulase production in P. decumbens. Serine protease PrtB of P. decumbens showed the highest sequence identity with A. nidulans PrtA. When prtB was deleted, conidiation was blocked and extracellular acid protease activity was elevated under carbon starvation condition. In addition, deletion of prtB resulted in reduced extracellular protein yields under cellulase inducing condition, while extracellular acid protease activity was similar with that of the parent strain.