Screening And Characterization Of Genes Involved In Cellulose Utilization By Cytophaga Hutchinsonii

The issue of resource as well as environment has always been focused by human society.It is also an important problem to be solved during the development of our social economy.With the gradual depletion of non-renewable resource of oil and coal,it becomes urgent to explore new renewable sources.As the most abundant renewable resources,the plant biomass with cellulose as the main component is promising biomass material.But the low efficiency of cellulose degradation becomes the main obstacle for its industrialization.The research on cellulose degradation ability and the mechanism of microorganisms will help us understand the cellulose utilization mechanism as well as establish the theoretical foundation for the cheap and efficient exploitation of biomass resources.Cellulose degradation strategy in nature present of high diversity.As an aerobic gram-negative bacteria,Cytophaga hutchinsonii is capable to degrade crystalline cellulose effectively.But its cellulose degradation mechanism is different from the known free Cellulase system model,nor a composite cellulosome model.In order to systematically elaborate the unique cellulose degradation mechanism of C.hutchinsonii,we obtained genes that involved in cellulose degradation by mutagenesis screening,analyzed the related metabolic pathways by bioinformatics methods and transcriptome data.Moreover,we also confirmed possible functions of identified genes via gene knockout.The main contents and results obtained are as follows:1.The beta-glucosidase CHU₃811 was identified to play a significant role in cellulose utilization of C.hutchinsonii.The transcriptome data showed that genes encoding cellulases were induced differently by various carbon sources.For instance,as an endoglucanase,CHU₁655 was specifically up-regulated,while the beta-glycosidase CHU₃811 was down-regulated when cultivated with cellulose as the sole carbon source.The results demonstrated that the inactivation of CHU₁655 led to the slightly weaker growth compared to the wild type strain when cultured on glucose or cellobiose as the sole carbon source,and a slightly longer log phase on the cellulose.The inactivation of beta glycosidase CHU₃811 caused the mutant strain completely deficient in cellulose degradation as well as a quite long log phase on cellobiose or low concentration of glucose as the sole carbon source.Previous studies on other cellulose genes also inactivated separately indicated that the lack of a single cellulase may not affect the cellulose utilization.Therefore,our investigation of CHU₃811 proved that it may be essential for the expression of other cellulase components in cellulose degradation.2.Inactivation analysis of glucose dehydrogenase CHU1221 and CHU₂315,proved that the glucose metabolic pathway of C.hutchinsonii was connected with its cellulose utilization.Through the analysis of growth phenotypes of mutants deficient in cellulose utilization,a long time delay on low concentration of glucose was displayed by most strains.We therefore predicted that the metabolism of low concentration of glucose may be related to the cellulose utilization.The whole genome sequence analysis of C.hutchinsonii and other species from Bacteroidetes indicated that C.hutchinsonii contained the complete metabolic enzyme system thansforming alpha-D-glucose to gluconate as well as proteins participating in gluconate transport and degradation.In addition,it encoded unique non-phosphorylation glucose degradation pathway.The inactivation of CHU₁221 or CHU 2315 led to the deficiency of cellulose degradation and the growth delay on low concentration glucose or cellobiose at different levels.The phenotypes of mutant strains ?1221 and ?2315 demonstrated that CHU 2315 played more vital role in glucose metabolism compared to CHU₁221.The glucose dehydrogenase activity assay of outer membrane proved that the glucose dehydrogenase may influence cellulose metabolism by affecting the glucose degradation.3.By inactivation of chu₀547 and chu₀554 we proved that the proteins annotated as SusD-like in C.hutchinsonii were not necessary for the cellulose degradation.By random transposon mutagenesis screening we identified a gene cluster that may be associated with oligosaccharides transport.Genes encoding SusC-like and SusD-like proteins which were important components of starch utilization system?Sus?in Bacteroidesthetaiotaomicron are essential for the starch degradation.SusC and SusD are involved in binding and transporting oligosaccharide,respectively,and thus contribute to the effecient starch degradation.Inactivation of two SusC-like genes of C.hutchinsonii previously did no effect to cellulose degradation.In this paper we concluded that two genes,chu₀547 and chu₀554,encoding SusD-like proteins were not essential for cellulose utilization as well as other carbon sources by insertional inactivation of these two genes respectively.A library containing strains deficient in cellulose utilization was constructed via the method of transposon mutagenesis mediated by pHimarEM1.The research on a mutant strain of which the inserted gene locus was chu₃508,encoding a hypothetical protein indicated that the gene cluster chu₃508-chu₃509 may be related to oligosaccharide transport.The phenotypes that the mutant strain could recover the ability to use cellulose after being pre-cultured in low concentration of glucose or cellobiose showed that low concentration of glucose or cellobiose may induce the cellulose utilization of the mutant strain.4.We identified a gene cluster coding sigma factor regulators which were important for cellulose degradation in C.hutchinsonii.We obtained two deficient cellulose utilization mutants MT2007 and MT2013 whose inserted locus were both chu₀195 annotated sigB regulation protein RsbU.The complemented strain confirmed that it was the inactivation of chu₀195 leading to the specific phenotypes of MT2007.chu₀193 and chu₀194 were the downstream genes of chu₀195,annotated as aF antagonist and anti-? factor,respectively.It was predicted by bioinformatics analysis that they may function as Spo?E,Spo?AA and Spo?AB that participate in ?F regulation of B.subtilis.And they were named as ChSR,ChAAS and ChAS respectively.According to the genetic and biochemical analysis,we speculated that although ChAAS and ChAS can interact with each other,the interactions between these three proteins and the way they involving in cellulose utilization may be different from that of B.subtilis,and the specific pattern need more exploration.