| Lignocellulosic biomass is the richest renewable resource in nature, wihch is also seen as the main materials for biofuels and biorefining. Thus, effective degradation of the biomass to polysaccharides (cellulose or starch) is the key step to realize the utilization of these renewable resources. Glycoside hydrolases secreted by microorganisms play important roles in degradation of lignocellulosic biomass. Penicillium. sp is widespread and saprophytic filamentous fungi in nature. Penicillium oxalicum 114-2 (formerly named Penicillium decumbens), isolated from soil, grow faster than Trichoderma reesei. The cellulases system secreted by P. oxalicum contains more reasonable enzymes components. Its high-yield mutant strians have been used for industrial cellulases production for many years. With the sequencing and bioinformatics analysis of its genome, some mechanisms involved in high-yield cellulase production strains have been revealed. However, the regulation of cellulases expression is a multi-level and complex process. More researches were performed on the regulation of cellulases at transcription levels of in recent years. Nowadays, researchers have paid more attentions to pre-process of cellulase expression. Thus, deep investigation on the functions of cellulase signal transduction in sensing and utilization of cellulose or searching of key proteins involved in cellulase signal transduction would be very important for better understanding of regulation mechanisms for lignocellulolytic enzymes production in fungi.In addition to further promote the enzyme production by rational transformation, researchers are trying to explore new enzymes or optimal cocktail enzymes for the degradation of lignocellulosic biomass materials. Although filamentous fungi such as P. oxalicum and T. reesei, are able to secrete a large amount of glycoside hydrolases, however, a considerable number of glycoside hydrolase genes cannot be expressed. Those unexpressed or low-expressed genes are likely to contain new or more excellent single enzyme components. Furthermore, in order to will understand these unknown genes, firstly, they must be effectively expressed outside the cell. However, the proteins of filamentous fungi sometimes cannot be expressed with activity in classic expression system such as E. coli and yeast expression systems due to incorrect glycosylation or protein folding. Thus, constructing a suitable expression system will be necessary for expressing active fungi proteins.The main contents and results of this paper are as follows:1. G protein signaling signal transduction pathway in regulating the synthesis of glycoside hydrolases.Firstly, we found eight genes that likely participate in the cellulase signal transduction pathway through bioinformatics analysis of P. oxalicum. These genes contain four G protein coupled receptors and four intracellular kinases. Knocking out of theses genes in wild-type strain showed no significant impacts on the expression of glycoside hydrolases.We studied the roles of heterotrimeric G proteins in regulating the synthesis of glycoside hydrolases. We found that G protein a subunit PGA3 plays different roles in amylase and cellulase enzyme expression process. Deletion of pga3 resulted in impaired amylase and β-glucosidase while cellobiohydrolase CBHI was not significantly affected. However, when PGA3 were dominantly-activated, the amylase activity was significantly increased while CBHI activity and BGL1 activity decreased slightly. To further assess the changes in extracellular enzyme production, transcriptional levels of the genes encoding these enzymes were determined.In starch or cellulose medium, transcription of amyl5A (the major amylase gene) in deletion strain decreased to very low levels and increased in dominantly-activated strain compared with those in wild-type strain. Up-regulation of cel7A-2 (the major cellobiohydrolase gene) was observed at early time points in the deletion strain. No clear conclusion about transcription levels of the major P-glucosidase gene bgl1 could be drawn in the comparison with wild-type strain and the two mutant strains grown on different carbon sources at different time periods. Supplementation of exogenous cAMP or its analog dibutyryl-cAMP restored amylase production in Apga3 strain, which suggested an essential role of PGA3 in amylase synthesis via controlling cAMP level. The above regulation of PGA3 to amylase synthesis is carbon source-independent, and is achieved, at least, by cAMP-mediated regulation of the expression level of transcription factor AmyR.2. The construction and application of constitutive and cellulase-free background protein expression system.We developed a protein production and evaluation system in cellulase-producing fungus P. oxalicum. First, by deleting the major amylase gene amy15A, a deletion strain Δ15A that produced few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, XynlOB and Cel12A) with original low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. The activities of the main proteins and enzymes activities of corresponding overexpression strains were detected in the supernatant when cultured on starch. When the overexpressed strains were transfered to wheat bran-cellulose medium, a new recombinant cellulases system containing the overexpressed enzyme were formed. Among the three recombinant cellulase systems, overexpression of BGL4 and Cel12A resulted in an increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By changing the culture media in which the overexpression strains were cultured, the expession platform is convenient to produce originally low-expressed lignocellulolytic enzymes with relatively high purities on starch and to obtain more effective complex cellulase systems containing overexpressed cellulase components on cellulose.3. The promotion of protein expression for low-background protein expression system by using the mechanism of G-protein signal transduction pathway.To further enhance the protein expression for low-background protein expression system when cultured on starch, this study performed some optimization work for the expression system. First, we chose the promoter of gene amy15A for protein expression because it could be induced under starch culture. Then, the amylase transcription activator AmyR was overexpressed and another amylase Amy 13 A was knocked out in the dominantly-activated strain PGA3QL. As a result, the transcription levels of amyR and amy15A were increased and SDS-PAGE analysis showed that the band of Amy 13A was disappeared. The protein concentration of the AmyR overexpression strain culture supernatant (mainly Amy 15A) was 3 folds than that in wild type strain 114-2. Finally, a mutant strain with clean background was achieved after deleting the amylase Amy15A. A protein expression plasmid by using Pamy15A promoter and TtrpC terminator was also constructed. In order to detect the efficiency of expression system. P. oxalicum-derived cellobiohydrolase CBHI and T. reesei-derived endoglucanase EGI were successfully overexpressed and their expression levels were significantly improved when compared to host strains Δ15A and PGA3QLΔ15A. |
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