PhD Thesis Proposal: Bishal Dev Sharma

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"Understanding factors that limit microbial growth and fermentation at elevated substrate concentrations"

Abstract

Clostridium thermocellum, a thermophilic anaerobe, holds promise for consolidated bioprocessing. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes producing secondary fermentation products like H₂, formate, acetate, and lactate, and the expression of heterologous genes to channel carbon flux toward ethanol production. Despite these efforts, success has been limited, and achieving economically viable ethanol titers remains challenging.

We hypothesized that the study of intracellular metabolites at elevated substrate concentrations might reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at elevated substrate concentrations (≥ 50 g/L) is limited due to a viscous substance in culture media that interferes with filtration and quenching step during metabolite extraction.

In this work, we developed a method for rapid filtration and quenching at elevated substrate concentrations to circumvent the viscosity issue of C. thermocellum cultures during fermentation. Initial studies of C. thermocellum intracellular metabolites showed a metabolic bottleneck at the phosphofructokinase (PFK) reaction, where C. thermocellum uses PPi instead of ATP, which is thermodynamically less favorable. Our study demonstrated that replacing PPi-linked pfk with ATP-linked pfk in engineered strains of C. thermocellum increased the thermodynamic driving force and ethanol titers by an average of 38%. Further analysis of C. thermocellum intracellular metabolites suggested substantial leakage of intracellular metabolites, pointing to a potential area for future research. We also showed that ethanol production in engineered Escherichia coli is limited due to inhibition of pyruvate decarboxylase (PDC) reaction by ethanol, resulting in a subsequent increase in pyruvate concentration followed by the accumulation of upper glycolysis metabolites.

Our results suggest that future metabolic engineering strategies in C. thermocellum should focus on the pyruvate-to-ethanol pathway and/or addressing the issue of metabolite leakage. We also hypothesize that the study of engineered strains of Thermoanaerobacterium saccharolyticum, which can produce up to 70 g/L ethanol, might provide further insights into metabolic engineering strategies in C. thermocellum by uncovering the similarities and differences in metabolism between these microbes.

Thesis Committee

• Daniel G. Olson (Chair)
• Lee R. Lynd
• Jiwon Lee
• Daniel Amador-Noguez (U Wisconsin-Madison)

Contact

For more information, contact Thayer Registrar at thayer.registrar@dartmouth.edu.