טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZeltzer Tal
SubjectStudying Gene Expression in the Cellulolytic Bacterium
Clostridium Thermocellum in Batch and
Continuous Cultures
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham
Full Thesis textFull thesis text - English Version


Abstract

    Motivated by potential sustainability, security, and rural economic benefits, ethanol produced from cellulosic biomass is a leading candidate for alternatives to petroleum-derived transportation fuels. Clostridium thermocellum is an anaerobic, thermophilic, soil bacterium that utilizes crystalline cellulose and ferments it to ethanol, allowing a relatively low-cost conversion of biomass to biofuel. The hallmark of the cellulose degradation system is the cellulosome, an extracellular, multi-enzyme complex. The composition of the cellulosome was found to be regulated by a set of seven ECF alternative RNA polymerase σI-factors, released upon binding to extracellular polysaccharides. In addition, several cellulosomal genes are up-regulated during slow growth rates in continuous cultures under carbon limitation. Yet, it is possible that this phenomenon is derived from the carbon limitation itself and not caused by the growth rate.   In the framework of this study, we investigated C. thermocellum physiology using three different approaches: (1) DNA-pull down assays were used to identify regulatory proteins; (2) gene knockout was used for studying the role of regulatory genes; and (3) transcriptomes generated by next generation sequencing were used for studying the physiology of the bacterium under carbon or nitrogen limitations.


    The in vitro DNA-pull-down approach did not yield putative cellulosomal genes regulators due to high background of non-related proteins, demonstrating the significance of applying gene manipulations in vivo. Therefore, a genetic system was assimilated and C. thermocellum null-mutants (ΔsigI2 and ΔrsgI2) were generated. Comparing cellulosomal gene expression of the parental strain with the ΔsigI2 strain (both grown with cellulose as carbon source) detected down-regulation in several key celluosomal cellulases including cbh9A, cel9V and cel5L.


    Nitrogen-limited continuous cultures elucidated the nitrogen metabolism in
C. thermocellum. Ammonium enters the cell though a specialized transporter fused to a response regulator (designed as AtrP) and is assimilated by three glutamine synthetases.  Urea can enter the cell not only by diffusion, but also by active transport and further metabolize by an intracellular urease. The regulation of nitrogen assimilation is at least partially conducted by several transcriptional regulators from the CopY, AraC and PodR families. In addition, nitrogen starvation up-regulated the sporulation alternative sigma factors SigE, SigG and SigF, which known to promote transcription of sporulation genes. Carbon limitation influenced the expression of key cellulosomal genes, notably the major cellulosomal components cel48S, cbh9A, xyn10A, cipA and olpB. Interestingly, an oligopeptide permease system (OPP) was also up-regulated during carbon limitation. Up-regulation of an OPP system could hint it is aimed for bacterial communication by pheromone transport. The growth rate per se also influenced transcription of many systems including carbon utilization, chemotaxis and signal transduction. Yet, the regulatory mechanism of growth rate impacts gene regulation was not identified.

    Understanding the physiology of C. thermocellum in continuous cultures together with the ability to generate mutants can assist in designing strains and fermentation processes for biomass hydrolysis and its fermentation to ethanol aimed for transportation fuel.