Synthetic biology approach to cellulose degradation
Lakhundi, Sahreena Saleem
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Cellulose, the most abundant biopolymer on earth, is composed of β – 1,4 – linked glucose units, which in turn form a highly ordered crystalline structure that is insoluble and recalcitrant to degradation. It is the world’s most attractive, abundant and renewable energy resource, representing the bioconversion of carbon dioxide into green plants. Cellulosic biomass, such as agricultural and forestry residues, waste paper and industrial waste can therefore be used as an inexpensive and abundantly available source of sugar for fermentation into fuel ethanol. The combustion of biofuels releases carbon dioxide which is thus recycled and hence the use of these fuels in transportation provides an alternative to fossil fuels, solving many environmental problems. The ability to degrade crystalline cellulose seems to be restricted to a specialized group of microorganisms which includes for example Clostridium, Cellulomonas, Cytophaga, Trichoderma etc. Hence the aim of this project was to create BioBricks using different cellulases from cellulose degraders and express them in different expression hosts like Escherichia coli, Bacillus subtilis, Citrobacter freundii etc., using two different promoters, spac and lac. It was observed that the expression of Cytophaga hutchinsonii cellulases (CHU_2103 and CHU_2802) and dehydrogenases (CHU_1944 and CHU_2315) was toxic to the E. coli host for some unknown reason. Therefore it was decided to use cellulases from Cellulomonas fimi, which are well characterized. BioBricks of cellulases (cenA and cex) from C. fimi were introduced into different expression hosts. It was observed that under our experimental conditions Citrobacter freundii SBS197 gave the best results. Both Pspac and Plac were functional in this organism with expression being higher when Pspac was used. When E. coli JM109 was used as an expression host, activity was only detected when the lac promoter was used to control the expression. Although the expression was higher when E. coli JM109 (containing Plac) was used as an expression host, almost all of this activity was residing within the cells, whereas when C. freundii SBS197 was used as an expression host, considerable activity was detected in the surrounding medium, which is essential for cellulose degradation. Growth curve studies were done to see if heterologous cellulases enable the host to use cellulosic substrates as a source of carbon. It was observed that C. freundii SBS197 expressing cenA and cex was able to use filter paper and Avicel as a source of carbon with maximum growth of up to 8.8×108 cfu/ml and 1.2×109 cfu/ml respectively. This was about 2 – 5 fold higher when compared to the control (vector and/or negative) strains. Filter paper completely disappeared within 3 – 4 days when C. freundii SBS197 was used. Slight degradation was observed when E. coli JM109 was used but there was no physical degradation seen when B. subtilis 168 was used as an expression host. Hence it was concluded that heterologous cellulases impart to C. freundii SBS197 with the ability to use cellulosic substrates as a source of carbon. The maximum growth obtained using these cultures is to our knowledge higher than what has been reported so far for recombinant organisms expressing heterologous cellulases using cellulosic substrates as a source of carbon.