טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAdi Rolider
SubjectThe Expression of the Cellulosomal Genes from Clostridium
thermocellum in a Continuous Culture
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Shoham Yuval


Abstract

Cellulose is the main component of the plant cell wall and is the most abundant polymer in terrestrial environments. Cellulose is made of glucose chains linked by b-1,4 bonds that form inter and intrachain hydrogen bonding to produce a highly stable, crystalline, polymer.  The natural degradation of cellulose is carried out mainly by microorganisms that secrete a battery of cellulolytic enzymes. In some cases, these enzymes are organized into large enzymatic complexes called, “cellulosomes”. The first cellulosome to be studied was that of the anaerobic, thermophilic, bacteria, Clostridium thermocellum.  The purpose of this research was to study the regulation of expression of the cellulosomal genes from C. thermocellum in continuous cultures. The expression of genes was determined by overall enzymatic activity on various substrates and by the mRNA transcript levels of selected cellulosomal genes. Continuous culture was performed at dilution rates ranging from 0.04hr-1- 0.235hr-1 under carbon and nitrogen limitation. Overall cellulolytic activity towards, carboxymethyl cellulose (CMC), crystalline cellulose (Avicel), and  p-nitrophenyl cellobioside (PNPC), was not affected by the dilution rate (growth rate). The activity towards PNPC under carbon limitation was significantly higher than the activity obtained in batch cultures and under nitrogen limitation suggesting the cellulosomal genes are repressed by cellobiose and by nitrogen limitation. The mRNA levels of the cellulosomal genes, cbhA, celK (both cellobiohydrolases) and xynA, (xylanase) was also determined by RNase Protection Assay (RPA). The transcript levels of these genes was not affected by growth rate under carbon or nitrogen limitation. The number of the cbhA mRNA transcripts under carbon limitation was higher than under nitrogen limitation suggesting that this gene is either repressed by cellobiose or its expression is inhibited under nitrogen limitation. The transcript levels of the xynA and celK genes was not affected by carbon or nitrogen limitation.  Overall, the expression of the cellulases of C. themocellum is not affected by growth rate at the range of 0.04 hr-1- 0.235 hr-1 and evidence for catabolite repression by cellobiose was demonstrated.