| Lignocellulosic biomass is the largest reserves of renewable resource on the earth. With the intensification of fossil fuels shortage and environment pollution, effective use of these low-cost resource to produce biofuel and biochemical is an efficient way to relieve the energy crisis, protect environment and maintain sustainable economic development. There are many lignocellulose degradation systems in nature, in which conversion by enzyme systems from microorganism was one of the most efficient pathways. Fungi play an important role in the environmental lignocellulose degradation. The researches on the composition of their lignocellulolytic enzyme system and the regulation of enzyme expression are very important for improvement of cellulase production and enzyme system composition.The filamentous fungus Penicillium decumbens114-2can produce a balanced extracellular lignocellulolytic enzyme system with more β-glucosidase, which is very efficient for lignocellulose degradation. However, the plenty of β-glucosidases can hydrolyze the oligosaccharides to glucose immediately, which restricts the accumulation of inducers and the induction of cellulase genes. It shows that, as an important component of lignocellulolytic enzyme system, β-glucosidase can also participate in the regulation of cellulase gene expression by regulating the concentration of inducer.There have been several researches about the regulation mechanisms for lignocellulolytic enzymes production in P. decumbens. However, traditional methods studying on single-gene is hard to unravel the holistic regulation mechanisms for lignocellulolytic enzymes production due to the complexity of cellulase enzyme system and multifarious of regulatory levels and pathways. Transcriptomics, which can reveal the genome-wide differences in gene transcription of different strains under different culture conditions, is very useful in perfecting the holistic network of regulation mechanisms for lignocellulolytic enzymes production.The main results of the thesis are as follows: 1. Characterization of the major extracellular β-glucosidase BGL1and its role in regulation of lignocellulolytic enzymesA β-glucosidase was purified from extracellular enzymes of P. decumbens114-2, which was identified the major extracellular β-glucosidase BGL1by MS/MS. The properties of BGL1, including its pH and temperature optima, the high affinity to substrates and high specific activity, make it has great potential to be utilized as supplementation in conversion of corncob residue and other lignocellulosic biomass into simple sugars. And it showed similar ability in boosting enzymatic degradation of substrate with the P-glucosidase from commercial enzymes NS-50010.After deletion of bgll, the phenotypes, including colony radial, growth rate, conidia formation or conidia color, were not affected in Abgll. When incubated in CW (cellulose+wheat bran) medium, the production of extracellular proteins (except for BGL1) was almost the same as the wild type strain. In accordance with this, expression levels of the major extracellular lignocellulolytic enzyme genes egl, cbhl and xyn10were similar with the parent strain on1%MCC in24h, although the extracellular oligosaccharides were accumulated during the incubation. Meanwhile, addition of oligosaccharides in the medium could not induce the higher expression of cellulase genes. When the intracellular cellobiose was determined on MCC (microcrystalline cellulose) medium, no more cellobiose was accumulated in Abgll, which might be due to the existence of intracellular β-glucosidase. The expressions of detected genes were all up-regulated after cultivation with0.02%cellobiose for4h, while the intracellular cellobiose concentration was in synchronous with gene repressions. We assumed that the oligosaccharides should be transported into the cell to trigger the induction, and the intracellular cellobiose concentration was important for the induction degree.2. Characterization of the major intracellular β-glucosidase BGL1and its role in regulation of lignocellulolytic enzymesThe six intracellular p-glucosidases were deleted in P. decumbens114-2 respectively. As the major intracellular β-glucosidase, the absence of BGL2improved the extracellular FPA, EG, CBH and xylanase activities dramatically on CW medium compare with the wild type strain. While deletion of other β-glucosidases barely make any difference. SDS-PAGE analysis suggested that the secretion improvement of the extracellular proteins was not general and only some of proteins increased dramatically in Abgl2, which were probably lignocellulolytic enzymes. Meanwhile the calculated specific EG and xylanase activities of Abgl2broth also confirmed the conclusion. As a constitutive enzyme, the production of extracellular β-glucosidase was not promoted synchronously with those of cellulose-induced enzymes. In accordance with the enzymes production, the transcription levels of egl, cbhl, xynlOA were improved dramatically on0.02%cellobiose and1%MCC in Abgl2. When the intracellular cellodextrins of P. decumbens114-2and Abgl2were analyzed, greater amounts of cellobiose was determined in the cytoplasm of Abgl2than that of114-2. To explore the mechanism of the improved production of lignocellulolytic enzymes by deficiency of BGL2, bgl2was expressed in E. coli BL21(DE3) and the characters of purified rBGL2were analyzed. rBGL2showed efficient hydrolytic activities toward cellodextrins with degree of polymerization from2to4. Besides cellodextrins with the glycosidic bond type of β-1,4, rBGL2could also hydrolyze sophorose (β-1,2bond) and gentiobiose (β-1,6bond). The results strongly supported that the improved induction of lignocellulolytic enzymes expression in Abgl2by cellobiose and cellulose might be mediated by the intracellular accumulation of cellobiose.3. Preliminary study of the cellulase induction mechanism of P. decumbens in double deletion strain of P-glucosidase genesThe mutant lacking both bglland bgl2was constructed. The mutant AbgllΔbgl2lost nearly all of the extra-β-glucosidase and most of intracellular β-glucosidase activities. However, AbgllΔbgl2could survive on the medium with cellobiose as the sole carbon source normally. The production of extracellular lignocellulolytic enzymes was not further enhanced compared with that of Abgl2as expected when incubated in CW medium. Transcriptional analysis of egl, cbhl and xyn10also revealed that simultaneously deletion of bgll and bgl2did not cause the further improvement of lignocellulolytic genes expression when incubated either on cellobiose or MCC. In accord with this, there was less cellobiose determined in the cytoplasm of AbgllΔbgl2compared with that of Abgl2, which probably be the reason resulting in the less efficient expression of lignocellulolytic genes. The results suggested that there seemed to be some other pathways activated to utilize cellobiose in the absence of main β-glucosidases, which caused the less accumulation of intracellular cellobiose and lower production of lignocellulolytic enzymes in AbgllAbgl2. The transcription levels of egl and cbhl were analyzed when induced by two potential inducers sophorose and gentiobiose in mutant AbgllAbgl2and no inductive effect was observed. The results suggested that despite acting as cellulase inducers in some other fungi, the two disaccharides did not participate in the induction of cellulase expression in P. decumbens. In other words, the induction mediated by cellobiose and cellulose was unrelated to the formation, if any, of sophorose or gentiobiose from cellobiose in P. decumbens.4. Transcriptional analysis of P. decumbens wild type strain114-2and mutant JU-A10-TTranscriptome of P. decumbens114-2and JU-A10-T on different carbon sources were analyzed using digital gene expression tag profiling (DGE) technology. The differential expression genes between114-2and JU-A10-T, as well as the differential expression genes on different carbon sources, were analyzed by Gene Ontology functional enrichment. The genes co-regulated with lignocellulolytic enzyme genes were worth studying, which might be related to the up-regulation of lignocellulolytic enzyme genes. The down-regulated genes in JU-A10-T relative to114-2were involved in organics transport, secondary metabolism and synthesizing amylases and proteases, which might contribute to the up-regulation of lignocellulolytic enzyme genes by alleviating burdens of protein synthesis and energy consumption. |
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