8 research outputs found
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A modeling and experimental approach to understanding the bottlenecks in xylulose utilization in S. cerevisiae
The production of fuel ethanol from lignocellulosic biomass has the potential to replace a significant portion of non-renewable transport fuels. Woody feedstocks are composed of cellulose, hemicellulose, and lignin. Glucose, the monomer of cellulose, is readily utilized by wild-type S. cerevisiae, but xylose, which comprises 60% of the sugar in hemicellulose, is not. To make the process economically competitive with conventional fossil fuels, both five and six carbon sugars must be utilized efficiently.
One approach to improving xylose utilization is to convert it to the more readily usable xylulose using an extracellular enzyme. Xylulose is taken up by wild-type S. cerevisiae and incorporated into the pentose phosphate pathway. To our knowledge there are no reports that elucidate the kinetics of this pathway, an important hurdle to overcome for strain development.
This thesis documents the work carried out to gain a better understanding of the xylulose utilization pathway in S. cerevisiae. The work was comprised of a series of batch fermentations that identified xylulokinase as a limiting enzyme in wild-type strains and transport through the HXT family of hexose transporters as a possible limiting step in xylulokinase enhanced strains. Batch experiments with HXT knockout strains suggest that alternative modes of xylulose transport are possible and may be up regulated in the knockout. An existing genome scale model for S. cerevisiae (iMM904) was used as the basis to develop a dynamic flux balance model. This model was used to verify the batch fermentation findings. The model has strong predictive capacity for xylulose and glucose consumption under anaerobic conditions and sugar levels sufficient to support growth
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A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae
A combination of batch fermentations and genome scale flux balance analysis were used to identify and quantify the rate limiting reactions in the xylulose transport and utilization pathway. Xylulose phosphorylation by xylulokinase was identified as limiting in wild type Saccharomyces cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through the HXT family of non-specific glucose transporters. A genome scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain xylulose utilization limitation in S. cerevisiae
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Economic and cradle-to-gate life cycle assessment of poly-3-hydroxybutyrate production from plastic producing, genetically modified hybrid poplar leaves
Poly-3-hydroxybutyrate (PHB) is a renewable, biodegradable biopolymer that has
shown great promise to offset the use of hydrocarbon-derived plastics. The genes
encoding the bacterial PHB production pathway have been engineered into several
higher order plant species providing an opportunity to produce PHB as a
co-product on an industrial, agricultural scale. This study investigates the economic
feasibility and estimates the life-cycle greenhouse gas (GHG) emissions produced
during the PHB production from hybrid poplar leaves. An established, bench scale
extraction procedure was extrapolated upon using SuperPro designer to estimate
the product cost, raw materials and energy used during extraction of PHB
from poplar leaves on an industrial scale. Assuming an economically feasible
concentration of PHB could be produced in the leaf material, a cradle-to-gate life
cycle assessment was performed under two of the most likely poplar production
scenarios for the Northwest United States where poplar is commonly grown for
biomass applications. The cost of PHB production was found to vary greatly with
the PHB content in the leaves; from 1.72 per kg at
20% PHB content. Poplar production scenarios varied greatly in their emission of
GHGs. An irrigated poplar production scenario is estimated to produce 248.8%
more GHGs than production of the displaced polypropylene. An un-irrigated poplar
production scenario produced 76.1% less GHGs. Both production scenarios
produced significant amounts of volatile organic compounds (VOCs) associated
with normal poplar growth that could prove detrimental to local air quality. PHB
content of 15% in the poplar was required to bring the PHB production price to
$2.26 per kg and make production competitive with petroleum-derived plastics
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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
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 Analysi
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Hohenschuh WilliamBiologicalEcologicalEnginDynamicFluxBalance.pdf
A combination of batch fermentations and genome scale flux balance analysis were used to identify and quantify the rate limiting reactions in the xylulose transport and utilization pathway. Xylulose phosphorylation by xylulokinase was identified as limiting in wild type Saccharomyces cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through the HXT family of non-specific glucose transporters. A genome scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain xylulose utilization limitation in S. cerevisiae.Keywords: Xylose, Fermentation, Pentose, Flux balance analysis, Cellulosic ethano
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Hohenschuh WilliamBiologicalEcologicalEnginDynamicFluxBalanceSupplementaryMaterialAppendicesA-B.pdf
A combination of batch fermentations and genome scale flux balance analysis were used to identify and quantify the rate limiting reactions in the xylulose transport and utilization pathway. Xylulose phosphorylation by xylulokinase was identified as limiting in wild type Saccharomyces cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through the HXT family of non-specific glucose transporters. A genome scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain xylulose utilization limitation in S. cerevisiae.Keywords: Fermentation, Pentose, Cellulosic ethanol, Xylose, Flux balance analysi