Author ORCID Identifier

http://orcid.org/0000-0002-8326-4444

Date of Award

Spring 4-8-2025

Document Type

Thesis (Ph.D.)

Department or Program

Engineering Sciences

First Advisor

Daniel G. Olson

Abstract

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at the elevated substrate concentrations (≥ 50 g/L) needed to observe cessation of fermentation limited by ethanol are challenging. Filtration, a key step in metabolite quenching and extraction, initially did not work for C. thermocellum cells at elevated substrate concentration. We eventually determined that the filter pore size could be substantially increased without loss of cells, allowing 20-fold increase in the initial substrate concentration that could be used during fermentations to study intracellular metabolites. We used this technique to reveal a metabolic bottleneck at the phosphofructokinase (PFK) reaction, where C. thermocellum uses PPi instead of ATP, making this reaction thermodynamically less favorable. Our study demonstrated that replacing PPi-linked pfk with ATP-linked pfk, substituting pyruvate dikinase with pyruvate kinase, and expressing a soluble pyrophosphatase (PPase) in engineered strains of C. thermocellum increased the thermodynamic driving force which led to an average 38% increase in ethanol titers. Further intracellular metabolite analysis suggested substantial metabolite leakage, indicating a previously unrecognized limitation in C. thermocellum that could restrict high-titer ethanol production. This highlights membrane-associated metabolite loss as a new avenue for investigation to enhance product titers. Additionally, we investigated ethanol production cessation in ethanologen strains of Escherichia coli and Thermoanerobacterium saccharolyticum by profiling glycolysis metabolites during fermentation. Our results showed that ethanol production cessation is characterized by pyruvate accumulation, followed by the accumulation of upper glycolysis metabolites. These findings highlight new targets for strain optimization, emphasizing the need to improve pyruvate-to-ethanol conversion and address metabolite leakage to enhance ethanol titers.

Keywords: Clostridium thermocellum, biofuels, ethanol, extracellular polymeric substance, metabolic bottleneck, metabolic engineering, metabolomics, viscosity

Download

Share

COinS