Identification And Functional Analysis Of Genes Involved In Cellulose Utilization In Cytophaga Hutchinsonii

With the development of modern society, the nonrenewable resources such as oil and coal are gradually depleted leading to the more serious problems of energy crisis and environmental pollution. Therefore, exploration and utilization of clean and environmentally friendly energy has become urgent. Cellulosic biomass from plants is the most abundant renewable resource on the earth, thus more and more attention has been paid to the utilization of cellulosic biomass. However, due to its compact structure and the presence of crystalline region, utilization of cellulose is still ineffective. Clarification of the mechanism underlying efficient cellulose degradation in nature would contribute to improve cellulose degradation efficiency, which is essential for cellulosic biomass utilization.There are many kinds of cellulolytic microorganisms in the natural environments using different strategies and mechanisms to degrade crystalline cellulose. An aerobic bacterium Cytophaga hutchinsonii, which is ubiquitously distributed in natural environment, can degrade cellulose efficiently, rapidly and thoroughly. C. hutchinsonii probably adopts an unique strategy to degrade cellulose, neither that in cellulolytic fungi by synergetic cooperation of a set of dissociative cellulase, nor that in anaerobic cellulolytic bacteria by cellulosome. The investigation and clarification on the novel mechanism of crystalline cellulose degradation by C. hutchinsonii would not only improve and enrich our understandings of the microbial cellulose utilization strategies, but also promote to exploit and take advantage of cellulosic biomass.This research aims to investigate the cellulose utilization mechanism in C. hutchinsonii by identification and functional analysis of genes involved in cellulose degradation. By screening a mutant library generated by random transposon mutagenesis, we successfully obtained a mutant defective in cellulose degradation, which is termed MT1-5. Identification and functional analysis of the inactivated gene in MT1-5was also performed. Moreover, two genes of cbpl （chu₃654） and OppA （chu₃498） in C. hutchinsonii that encodes proteins possibly involved in cellulose utilization were interrupted respectively and the influences of their absence on cellulose utilization were also studied. Specific works and results are described as follows:1. Identification of Mutant MT1-5with Defects in Cellulose DegradationBy screening the mutant library generated by random transposon mutagenesis, we successfully obtained a mutant named MT1-5defective in cellulose utilization. The growth of MT1-5on cellulose or filter paper but not glucose was abolished. We confirmed the specific inactivated gene locus in MT1-5is chu₂981, which has an open reading frame of393bp that encodes a putative protein with130amino acids. We named the putative protein encoded by chu₂981as Spp1, which is a conserved hypothetical protein by analysis of Blast algorithm.2. Functional study of the gene sppl involved in cellulose utilizationAlthough mutant MT1-5did not show remarkable growth difference with wild types strain on medium with glucose or cellobiose as the sole carbon source, its growth was completely abolished on cellulose. SDS-PAGE analysis showed that, in comparison with that in wide type strain, the outer membrane proteins from MT1-5decreased remarkably in both types and amounts. In addition, MT1-5also showed a compromised ability in spreading on agar surface and bacterial motion on glass surface. Recomplementation of sppl to MT1-5neither restored the growth on cellulose, nor restored spreading on agar surface and motion on glass. The above results indicated that, Spp1, which is absent in MT1-5, plays an essential role in cellulose degradation, colony spreading and bacterial motion. Western blot analysis showed Sppl is located in periplasmic space and its function awaits further investigation.3. Heterologous expression, purification and functional study of periplasmic protein SpplWe obtained recombinant protein GST-Sppl with an N-terminal GST tag by heterologous expression and purification of Sppl in E.coli. Addition of recombinant GST-Sppl into the medium can not restore the growth of MT1-5. Attempts to isolate the proteins from C. hutchinsonii that interacted with GST-Sppl by using "GST pull-down" assay also failed. Molecular sieve analysis indicated the recombinant GST-Spp1exist in the form of monomer but not polymer in Na2HPO3-NaH2PO3buffer.4. Insertional inactivation and functional study of genes cbpl （chu₃654） and oppA （chu₃498） putatively related to cellulose degradation in C. hutchinsoniiWe targetedly inactivated the gene cbp1（chu₃654） that encodes a putative cellulose banding protein and the gene oppA （chu₃498） that encodes a putative cellobiose-binding protein respectively in C. hutchinsonii. Analysis of the two mutants suggested that, while the inactivation of Cbp1results in a decrease in the amount of outer membrane proteins exhibiting binding affinity to cellulose, it does not significantly influenced the ability to bind and utilize cellulose. In the case of OppA, although its absence results in a compromised utilization of cellobiose, it does not remarkably affect cellulose utilization. These results suggested the absence of Cbpl and OppA does not compromised the strain to utilize cellulose, thus identification of essential genes involved in cellulose utilization required further investigation in future.