Leucine Zipper-Mediated Targeting of Multi-Enzyme Cascade Reactions to Inclusion Bodies in Escherichia coli for Enhanced Production of 1-Butanol 

The utilization of non-edible lignocellulosic biomass assumes importance due to the soaring crude price and depleting reserves of fossil fuels coupled with the rising concern on global warming. Developing countries like India, fourth largest fossil fuel consuming country in the world, produces only 22% of its requirements and rest is imported from several other countries. Fuel derived from renewable lignocellulosic biomass of has been identified globally as most acceptable and sustainable alternative of fossil fuels and future solution for meeting the energy demand. India has huge potential in production of lignocellulosic fuel due to the availability of feedstock in the form of agricultural residues like wheat straw, rice straw and bagasse.

Our current research work is focused on efficient utilization of rice straw as a feed stock for lignocellulosic ethanol production. Lignocellulosic ethanol is immature and commercially non-viable technology because of inefficient and expensive biomass hydrolysing enzymes including several other reasons. To address this bottleneck we have explored gut microbial community of rice stem borer insect which feeds the rice plant and derive nutrition from it. It is envisaged that gut microbial community of these insects must have ability to produce cellulases/hemicellulases that can be utilised in biomass hydrolysis step in lignocellulosic ethanol production. We dissected the insect aseptically and cultured the gut microbial community in the minimal medium (0.1% YE, 0.5 % NaCl, 0.25% K₂HPO₄) having 1% rice straw as main carbon source. Microbial community responsible for the biomass degradation were enriched by passaging at interval of 10 days over a year. We found almost 75% biomass weight reduction in the rice straw by the microbial community. Moreover, protein secreted by the consortium have exocellulase, endocellulase and xylanase activity.

Identification of protein(s) and gene(s) in the consortium were performed by meta-proteomic and meta-transcriptomic studies respectively. We identified almost all classes of biomass hydrolysing enzymes of CAZy family from the consortium. One of the identified enzyme that belongs to GH48 family was cloned and expressed in E. coli is functionally active and releases cellobiose as a result of phosphoric acid swollen cellulose deconstruction.