Screening And Identification Of Key Cytophaga Hutchinsonii Genes For Cellulose Degradation And Its High-efficiency Promoter Sequences

As the main component of plant biomass,cellulose is the most valuable and inexhaustible renewable resources in the world.However,due to its unique compact structure,the degradation and utilization efficiency of cellulose is quite low.There are a variety of microorganisms that are capable of degrading cellulose in nature,by applying either the extracellular free cellulase system or the cell surface-anchored multiprotein cellulosome to achieve the efficient degradation of cellulose.Among others,the aerobic gram-negative bacterium Cytophaga hutchinsonii is postulated to adopt a third strategy to digest cellulose,which is still a mystery so far.The present work aiming to investigate the novel cellulase-degrading mechanism by C.hutchinsonii can be divided into two parts:firstly,we constructed several mutants wherein several genes putatively involved in cellulase degradation based upon the bioinformatics and transposon mutagenesis analyses were individually inactivated,measured and compared their cellulase-degrading capabilities with that of wild type strain,and successfully identified one gene chu₀089 involved in cellulase degradation;secondly,we picked several promoters from C.hutchinsonii,evaluated their abilities to drive the expression of the reporter gene gfp,and identified an effective promoter that can be used for expression of endo-and exogenous genes.Taken together,our results will not only improve the genetic manipulation system of C.hutchinsonii,but also provide a conceptual framework to understand the unique strategy deployed by C.hutchinsoii to assimilate cellulose.The main contents are presented as follows:1.The gene chu₀089 encoding a putative TonB dependent receptor was found to play an essential role in C.hutchinsonii cellulose degradation.By analyzing the transcriptional profiles of both WT and the mutant T127 wherein the targeted inactivation of chu₁2 76 resulted in a defect in cellulose degradation,we found that the transcriptional level of chu₀089 was apparently down-regulated in T127 compared with that in WT.Further analysis showed that chu₀089 encodes a putative outer membrane TonB dependent receptor.To test whether chu₀089 is involved in cellulose degradation,we constructed the mutant strain chu₀089in wherein chu₀089 was targeted inactivated,and found that,whereas the mutant displayed similar growth to that of WT on glucose,its growth on cellobiose and cellulose was significantly compromised.The complementation of the mutant strain with a CHU 0089-expressing plasmid can restore its ability in cellulose degradation,confirming that the cellulose utilization defect displayed by the mutant is specifically caused by the inactivation of chu₀089,and thus indicating that chu₀089 played an important role in C.hutchinsonii cellulose degradation.2.By using transposon mutagenesis screening,the genes chu₀694 and chu₁812 involved in methionine synthesis were found to be important for C.hutchinsonii cellulose degradation.By using random transposon mutagenesis with the mariner-based pHimarEMl,we successfully obtained two mutants T950 and T964 that were defective in cellulose utilization.The transposon insertion sites were determined to be within the gene loci chu₀694 and chu₁812,respectively,by using plasmid rescue and DNA sequencing.Southern blot analysis indicated that the transposon insertion locus was specific in both mutants.Chu₀694 and chu₁812 encode a putative 2-keto-4-methylthiobutyrate aminotransferase and a O-acetylhomoserine sulfhydrylase,respectively,which are involved in the cyclic regeneration and denovo synthesis of methionine.We also performed targeted insertional inactivation of chu₀694 and chu₁812,and found that,both of them exhibited similar cellulose utilization defect to their corresponding transposon-inserted mutants,indicating that these two genes are important in cellulose degradation,whose detailed action mechanisms underlying methionine synthesis and cellulose utilization await further discovery.3.By testing the expression level of reporter genes under the control of different promoters isolated from C.hutchinsonii,we found that chu₂750 promoter can effectively drive gene expression in C.hutchinsonii.By analyzing the transcriptional profile of C.hutchinsonii and the information from literatures,we picked 15 putative promoter sequences of genes with highly expression levels in C.hutchinsonii,and tested their abilities to drive the expression of reporter gene gfp?green fluorescence protein-encoding gene?.The results showed that,the chu₂750 promoter can successfully drive GFP expression,whereas other 14 promoters failed.When lacZ??-galactosidase-encoding gene?was used as the reporter gene,whereas chu₂750 promoter as well as other three selected promoters can drive LacZ expression,LacZ expression under the control of chu₂750 promoter was highest.The expression of CHU₀089 under the control of chu₂750 promoter can successfully complemented the mutant strain chu₀089in,though the other two commonly used promoters including PompA and Pchu₁284 failed.This study lay the foundation of constitutive expression of both endo-and exogenous genes in C.hutchinsonii.