Graduate Thesis Or Dissertation

The Path Forward : Using High Throughput Data and Dynamic Flux Balance Modeling to Identify Bottlenecks in the Carbon Metabolism of Industrial Microbes and Suggest Solutions to Improve Product Yield

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  • The economically viable production of value added fuels and chemicals from lignocellulosic feedstocks hinges on our ability to quickly and efficiently transform structural carbon molecules to end products. The sugars in lignocellulosic biomass are held primarily within cellulose and hemicellulose. Cellulose is composed primarily of glucose monomers which are quickly and efficiently consumed by a variety of organisms. Hemicellulose is comprised mainly of xylose. Xylose is less well utilized by industrial yeasts and completely unusable to wild-type Saccharomyces cerevisiae, the most commonly used industrial yeast.Strains of S. cerevisiae capable of fermenting xylose have been developed, but the specific rate of xylose utilization in these strains is slow and product yield is low. Developing a strain capable of fermenting xylose both efficiently and with high product yield would be a massive step towards realizing the sustainable production of lignocellulosic bioethanol.Significant work has gone into identifying the bottlenecks within S. cerevisiae that may be contributing to slow xylose utilization. Research groups have suggested the pentose phosphate pathway, the xylose reductase xylitol dehydrogenase (XR XDH) pathway, xylulokinase deficiency, xylose transport, and cofactor redox imbalance as possible bottlenecks. These groups have further shown that expanding the capacity of any of these pathways can increase xylose utilization under certain conditions. Unfortunately, without a thorough understanding of the interplay between these bottlenecks and the conditions under which they increase the rate of xylose utilization or ethanol production it is nearly impossible to rank their importance and direct the course of future strain development.This dissertation works to identify the most important bottlenecks and strategies to alleviate them. Specifically, this describes the development of a series of S. cerevisiae strains harboring the XR XDH xylose utilization pathway and expressing STB5 (a transcription factor responsible for pentose phosphate pathway regulation) and PGI1 (a required glycolytic enzyme normally down-regulated by STB5p) under the control of a novel xylose-inducible promoter (TEF-X2-2). This novel use of transcription factors to regulate the entire pentose phosphate pathway was an attempt to optimally regulate the pathway using evolutionarily optimal enzyme expression ratios. These strains were then characterized using batch fermentations and high throughput transcriptomic tools to understand pentose phosphate pathway regulation and xylose utilization bottlenecks. These characterizations identified a correlation between cell density and the maximum specific rate of xylose utilization suggesting that oxygen was limiting both respiration and fermentation. Since fermentation is normally performed anaerobically, it was hypothesized that fermentation was actually limited in this case by NADH availability, the production of which would decrease under anaerobic conditions because of the stoppage of the TCA cycle. The high throughput data collected was further used to constrain a regulatory model framework around the previously developed genome scale reconstruction of S. cerevisiae known as iMM904. This model considered pentose phosphate pathway regulation linked to STB5 expression, xylose transport mediated by the HXT family of glucose specific transporters, and the kinetics of oxygen uptake and mass transfer. The novel use of high throughput data and regulatory modeling techniques allowed for a more thorough understanding of the bottlenecks in xylose utilization, supported the hypothesis that NADH availability was limiting fermentation, and suggested that the glyoxylate pathway might allow for anaerobic replenishment of NADH allowing an increased rate of xylose fermentation. Although regulation of the glyoxylate pathway has been considered before, the suggestion that it could increase the rate of xylose utilization and ethanol production is novel.
  • KEYWORDS: Glyoxylate Cycle, Flux Balance Analysis
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