Thesis

Analysis of an ethanol inducible promoter in escherichia coli and tobacco for production of cellulase

Cellulase is widely used as an enzyme for saccharification of cellulosic materials. However, commercially available cellulase is an expensive enzyme. Recombinant ethanologenic bacteria have also become an important tool for production of bioethanol from lignocellulosic biomass. Heterogolous gene expression in transgenic microorganisms can be improved by utilization of an efficient promoter system to regulate genes encoding for cellulase or ethanol production pathway. Here, we evaluated the feasibility of bioengineering E. coli cells with an ethanol inducible promoter system driving a cellulase gene. We tested the strength of an ethanol inducible native alcA promoter from Aspergillus nidulans in E. coli and compared this promoter with T5, a strong promoter commonly used for gene expression in bacteria, and cauliflower mosaic virus 35S (CaMV 35S), a strong and widely used promoter in plants. The activity of alcA promoter was also compared to CaMV 35S promoter in tobacco by transiently expressing a GFP gene under the control of 35S and alcA promoters. E. coli cells were transformed with various plasmid constructs and quantitative PCR was performed to analyze promoter activities. The highest transcription level was observed for alcA promoter when it was expressed along with AlcR transcription factor in absence of ethanol in which alcA expression was 11 times higher than T5 promoter. The alcA promoter showed basal level expression in absence of AlcR, although, its expression level was higher in presence of AlcR. All cassettes harboring alcA promoter showed non-significant ethanol dose-dependent activity. Conducting SDS-PAGE and CMCase activity assay displayed no or low protein expression and no enzyme activity. Lack of protein expression might be due to the presence of 14 rare codons in cellulase DNA sequence inhibiting protein translation in E. coli. In contrast to E.coli, the inducible expression of alcA promoter system was tightly regulated in tobacco. We suggest further study for the utilization of ethanol inducible system in ethanol-tolerant ethanologenic E. coli strains which may lead to significant success in production of bioethanols and other biofuels. We hope, our work on inducible promoter system for in vivo cellulase production will open new avenues of research in the fields of developments of biofuel from prokaryotic and eukaryotic systems.

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