Research On Function Of Crystal Cellulose Degradation Related Gene In Cytophaga Hutchinsonii

The development of current economy depends on the exploitation of fossil resources such as oil, coal, natural gas, etc. But they are non-renewable and facing shortage. In this situation, the exploitation of renewable energy is paid more attention to by the government. Biomass resources which are important components of renewable resources have irreplaceable advantages. Cellulose is the most abundance biomass resource on the earth. Biofuel production from cellulose is thought to be one of the solutions of the shortage of energy. However, biofuel is not very competitive compared with oil for its low conversion efficiency and the high cost.Cytophaga hutchinsonii is an aerobic bacterium which can degrade crystalline cellulose thoroughly. It had been listed in the listed in the microbial genomic project by the Department of Energy of the USA for its unusual cellulose degradation mechanism. Its complete genome was published in 2005. Based on the the genomic information, the function of the cellulase related genes in C. hutchinsonii was investigated in this paper.Many bioinformatics tools such as TMHMM, SignalP, SMART were used to analysis the sequences of cellulase related genes from the GenBank. The catalytic domains of eleven endoglucanases belong to glycosyl hydrolase family GH9 or GH5, andβ-glucosidases belong to GH3 mostly. Among them, gene CHU₁335 has a special structure that composes of a GH9 catalytic domain and eleven transmembrane regions. These transmembrane regions were thought to form a bate jelly roll. Based on its structure, CHU₁335 was speculated to a channel in the cell membrane, which would be important in substrate transferation during cellulose degradation. Besides this, most of endoglucanases were lack of a carbohydrates binding domain (CBD), which indicate that its binding pattern was different from other well studied cellulases.In this paper, Real-time fluorescent quantitative PCR (RQ-PCR) was used to detect the difference of cellulase related gene transcription under the different culture conditions. In organic medium, when different carbon source (glucose and microcrystalline cellulose) were used, the transcription of the selected genes was nearly the same except gene CHU₁336. But in inorganic medium, the transcription of the selected genes was a bit different. It showed that the difference of transcription level of the selected genes was not very obvious when different carbon source were used.The suicide plasmid pLYL03 was used as the vector to interrupt the target gene. The homologue of target genes was ligated to the plasmid by enzyme digestion and ligation. Then the recombined plasmid was transformed to the E. coli S17-1(λpir) which can transfer DNA to C. hutchinsonii by conjugation. Then the recombinated plasmid was inserted into the target gene by homologue recombination to interrupt the target gene. Several knock-out plasmids were constructed through this protocol. But it's difficult to get gene-interrupted mutant due to the low rate of homologue recombination and conjugation. Considering of this, other methods was used to improve the transformation rate, i.e. electroporation. At the same time, different methods were used to construct the knock-out plasmid, such as LFH-PCR in order to increase the rate of conjugation.In this paper, the growth curve and cellulase production under different culture conditions with different carbon sources were studied. Four kinds of substances were used as the carbon sources: glucose, cellobiose, microcrystalline cellulose, phosphate swollen cellulose. It showed that, extracellular CMCase could be barely detected when glucose or cellobiose was used as the carbon source. And as the running out of carbon source, the optical density decreased immediately, which was thought to be the result cell autolysis. High CMCase could be both detected when using microcrystalline cellulose and phosphate swollen cellulose as the carbon source. And the extracellular CMCase was two times higher when phosphate swollen cellulose was used as the carbon sources than when microcrystalline cellulose was used. At the same time, reducing sugar was detected when using the phosphate swollen cellulose, which has never been found before. Our study showed that cellulose could be induced by cellulose in C. hutchinsonii.More and more details of degradation of cellulose by Cytophaga hutchinsonii were understood through the primary researches in microbiology and biochemistry. These researches would be of great help for future study.