Studies On Cellodextrin Transporters In The Cellulolytic Fungus Penicillium Oxalicum

With the concerns of increasing energy demands, environment pollution, and intensification of fossil fuels shortage, the development of alternatives to fossil fuels as an energy source is an urgent global priority. Liquid biofuels produced from lignocellulosic biomass are an environmentally clean and renewable source of energy that could displace a significant fraction of the current demand for petroleum, protect environment and maintain sustainable economic development. Filamentous fungi are among the most efficient degraders of plant biomass, which could hydrolyze insoluble plant cell wall polysaccharides to fermentable sugars. Then the fermentable sugars can be converted to bioethanol and other value-added products. Currently, both the enzymatic hydrolysis and fermentation processes need to be improved to make the process economically competitive. Oligosaccharides such as cellodextins and its modifiers are thought to function as the actual molecules that trigger enzyme induction and play important roles in sugar utilization. It is possible that organisms sense cellulose through recognition of these oligosaccharides by a transporter in the membrane. Thus, deep investigation of the functions of cellodextrin transporters in sensing and utilization of cellulose would be very important for understanding of regulation mechanisms for lignocellulolytic enzymes production in fungi.Penicillium oxalicum114-2, formerly classified as Penicillium decumbens, is a fast-growing filamentous fungus that secretes various lignocellulolytic enzymes. The genome of strain114-2was recently sequenced and annotated and there have been several researches about the regulation mechanisms for lignocellulolytic enzymes production in P. oxalicum. However, the researches on cellodextrin uptake, the regulation mechanism for sugar transport, and the relationship between cellodextrin transporters and cellulase induction remains unclear. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum were identified, and their crucial roles in cellulose utilization and cellulase induction were analyzed. The main results of the research are as follows: 1. Three cellodextrin transporters in the cellulolytic fungus Penicillium oxalicum were identified using Saccharomyces cerevisiae as a host.A total of11putative cellodextrin transporters (named from CdtA to CdtK) were predicted in P. oxalicum. All these transporters are members of the major facilitator superfamily, and were annotated as lactose permeases in P. oxalicum. Because S. cerevisiae doesnot contain cellodextrin transporters and intracellular β-glucosidase, it cannot ferment the cellodextrins naturally released by cellulose, which allow us to probe the transport properties of these transporters. The genes encoding nine of the candidate cellodextrin transporters were separately transformed into a recombinant S. cerevisiae host YGH1-1expressing intracellular P-glucosidase (named from CdtB to CdtK). Of the nine strains evaluated, three exhibited significant growth phenotypes, which indicate that the three expressed transporters CdtC, CdtD, and CdtG could transport cellobiose. Furthermore we discovered that the three recombinant yeasts could also use cellodextrins with a degree of polymerization higher than two. The whole phylogenetic tree of all the predicted candidate cellodextrin transporters in P. oxalicum, N. crassa and T. reesei poorly recapitulates the species tree, indicating fast evolution of this group of membrane transporters.2. Three cellodextrin transporters have different expression pattern in P. oxalicum.To examine the effects of different carbon sources on cdt gene expression in P. oxalicum, the transcript abundances of cdts were monitored and we found the expression pattern of cdtC, cdtD, and cdtG is different. The transcription level of cdtD is the highest among the three genes, and cdtG shows very low expression level in P. oxalicum. cdtC was induced by wider range of substrates, which can be induced to cellulose, cellobiose and lactose, and be repressed by glucose and xylose. The expression level of cdtD was induced by cellulose and repressed by glucose. However, cellulose cannot trigger the expression of cdtG. 3. Cellodextrin transporters exhibited critical functions cellulose utilization and lignocellulolytic enzymes inductionTo study the function of cellodextrin transporters during cellulase induction, cdt single deletants were first constructed. The phenotypes of cdt single deletants, including colony radial, growth rate, conidia formation or conidia color, were not affected in glucose, cellulose, cellobiose and xylose. The mutants showed a slight decrease in cellobiose consumption than the parental strain114-2. In addition, no significant difference in cellulase production on cellulose was detected between the mutants and114-2. The transcription of cdtC was significantly up-regulated in ΔcdtD and ΔcdtG than that in114-2. We assumed that cellodextrin transporters have overlapping activities, so we constructed cdt double deletion strains, including ΔcdtDC, ΔcdtGC, and ΔcdtGD. The colony radial and growth rate of ΔcdtDC was decreased in glucose, cellulose and cellobiose than that in114-2and the cellulose hydrolysis circle of ΔcdtDC was not detected. ΔcdtDC could hardly grow on cellulose as the sole carbon source, and showed a decrease of about50%in cellobiose consumption than the wild-type strain114-2which means the lack of both cdtC and cdtD results in a poor capacity to transport cellobiose. The mutant lost half of the extracellular proteins production, most of the hemicellulose activity and nearly all of the extracellular cellulase activity. The transcription levels of the major cellulase gene cbhl (cel7A-2) and the major xylanase gene xynl (xynlOA) in mutant ΔcdtDC decreased significantly compared with those in the wild-type strain114-2. The results suggested that cellodextrin transporters exhibited critical functions in cellulase induction by cellulose. CdtC and CdtD, but not CdtG, could compensate for the loss of other cellodextrin transporters. The cdt Triple deletion strains show more obvious growth defects on cellulose, cellobiose and lactose. 4. Three cellodextrin transporters are regulated differently in Penicillium oxalicumThe previous transcriptome analysis showed that the expression levels of cdtC and cdtD were up-regulated in the mutant lacking transcription factor AmyR. After deletion of creA, the expression levels of cdtC and cdtD were similar with the wildtype strain. Under cellulose conditions, cdtC expression was undetectable without the cellulose degradation regulator gene clrB, but cdtD expression was up-regulated in the same condition. In the mutant lacking creA and overexpression clrB, both cdtC and cdtD expression were up-regulated. However, cdtG expression was down-regulated in all mutants. These results suggest that ClrB is the major regulator for cdtC, whereas AmyR and CreA do not directly regulate these transporters. In summary, the three cellodextrin transporters are regulated differently in Penicillium oxalicum.5. Over-expression of cellodextrin transporters affect cellulase regulation in P. oxalicumThe three cellodextrin transporter genes were separately over-expressed under the control of the cbhl promoter in P. oxalicum114-2. The expression levels of cdtC, cdtD and cdtG increased in the corresponding over-expressing mutants, especially for cdtG with low-expression in114-2. The expression levels of major (hemi-)cellulase genes cbhl, egll (cel7B) and xynl significantly increased in the three mutants compared with those in114-2, with the highest fold changes in cdtG over-expressing mutant. Consistently, the extracellular cellobiohydrolase activities of the mutants were elevated compared with those in114-2.To avoid possible hydrolysis of intracellular cellodextrins, strain Δbg12lacking the major intracellular β-glucosidase gene bgl2was used as the starting strain. However, over-expression of cdtC or cdtD led to neither improved cellobiose transport nor improved cellulase production, although the expression levels of cdtC and cdtD did increased in the cdt over-expression mutants. We suggest that cellodextrin transport might not be a rate-limiting process for cellulase induction in this strain and the cbhl promoter placed in cellodextrin transporter might affect the expression of cbhl.6. Cellodextrin transporters may have the ability to transprot lactoseBoth cellodextrin transporter single and double deletants showed growth defect on lactose, especially for AcdtDC. Thus we assume that CdtC, CdtD and CdtG might have the ability to transport lactose. The single deletion of cdts improved the extracellular cellulase activity. However, the cdt double deletants decreased the extracellular cellulase activity. SDS-PAGE analysis suggested that the secretion level of amylase was improved. The extracellular amylase activity was enhanced in cdt mutants except AcdtG.