Microbial Engineering

INDUSTRIAL BIOTECHNOLOGY  / Biofuels and Industrial Biotechnology

Research Interests

Second generation biofuels, fungal and bacterial genome engineering, engineered cellulases, C5/C6 fermentation, n- and iso-butanol production, microbial alkane and cetearyl alcohol production.

Description of Research

Discovery and design of novel enzymes and enzyme systems for biofuels
Recent interest towards shifting to non-food-based feedstock for biofuel production, in addition to biomass burning issues, has resulted in the exploitation of agricultural residues as feedstock. However, the recalcitrant nature of this biomass makes it extremely difficult to hydrolyze into fermentable sugar. 

The filamentous fungi are found to be efficient in carbon cycling in nature and thus are treated as potential sources of enzymes for converting recalcitrant lignocellulosic biomass into precursors for industrial purposes. We use an intense mathematical model-based screen to identify fungal isolates whose secretome can degrade biomass more efficiently than commercial cellulase formulation. Several enzymes have been characterized and engineered to increase the efficiency. Prominent among them are cellobiohydrolase 1 (CBH1), Lytic Polysaccharide Monooxygenase (LPMO) and beta-glucosidase.  We have performed genome engineering of the new fungal isolate to disrupt its catabolite repressor and overexpress the cellulase gene activator to enhance the enzyme production by several folds. Many leads have been obtained to understand the role of several unannotated transcription factors involved in cellulase production via transcriptomic and proteomic studies, which are being valorized to construct superior biocatalysts. Furthermore, the Group identified a drug efflux mechanism of hypercellulolytic fungus Penicillium funiculosum and developed strategies to block these efflux pumps for the development of effective transformation tools for genome engineering. The highest enzyme titer-producing engineered fungal strain has been the scale-up to a pre-commercial 15,000 litre scale for use in lignocellulosic biomass hydrolysis. 

Engineering microbes to produce biofuels and biochemicals
Lignocellulosic biomass consists of ~30% of pentose sugars, and therefore, its effective fermentation will certainly have a positive impact on the economy of biofuel production. The traditional yeast, Saccharomyces cerevisiae, is unable to ferment pentose sugars into ethanol. E. coli is a robust host for various genetic manipulations and can readily consume both hexose and pentose sugars. However, the availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity in this microbe. In our lab, we have constructed an E. coli strain to produce a high yield of ethanol from hexose and pentose sugars by modulating endogenous pathways without the need for foreign genes. We also worked on disrupting catabolite repression for simultaneous utilization of C5/C6 sugar as well as on making the engineered E. coli tolerant towards the inhibitors present in the biomass hydrolysate. We further worked towards the formation of more energy-dense fuel molecules, such as butanol, hexanol, pentadecane and hexadecene. Butanol was made in E. coli by integrating Clostridial pathway in its genome via CRISPR/Cas9 technique. On the other hand, long-chain alkanes and alcohols are being made via metabolic model-assisted engineering of E. coli, with the highest titers reported so far. Efforts are also ongoing to produce 1,4-butanediol via novel pathway engineering with industry support.

Recent Publications

Patents