Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
Escherichia coli--Testing||Xylose||Alcohol as fuel
Due to its capability to ferment biomass-derived pentose and hexose, Escherichia coli is considered a potential biocatalyst for production of bioethanol. Gene deletions and promoter replacements were previously employed to engineer a non-transgenic homoethanol pathway in E. coli [glucose (or 1.2 xylose) => (glycolysis) => 2 pyruvate + 2 NADH => (pyruvate dehydrogenase, aceEF-lpd operon) => 2 acetyl-CoA + 4 NADH => (alcohol dehydrogenase, adhE) => 2 acetaldehyde + 2 NADH => (alcohol dehydrogenase) => 2 ethanol]. The resulting strain, RM10, is able to produce ethanol from xylose and glucose, with a yield greater than 90% theoretical (one mole of glucose to 2 mole of ethanol). In this study, the transcription of five genes involved in the homoethanol pathway [ aceEF-lpd, adhE, and aldB (encoding for aldehyde dehydrogenase)] were overexpressed in RM10 for improved ethanol fermentation. Acetaldehyde is an intermediate of the homoethanol pathway. In theory, blocking the oxidation of acetaldehyde (catalyzed by aldehyde dehydrogenase) will prevent diverting the carbon skeleton to acetic acid, which should in turn improve ethanol production. Contrary to this hypothesis, however, blocking acetaldehyde oxidation (deletion of aldB) resulted in decreased ethanol production compared to that of the parent strain. Furthermore, overexpression of aldB increased ethanol production over that achieved by the parent strain. The improved ethanol production likely attributes to improved acetaldehyde tolerance because the aldB overexpression showed an improved acetaldehyde tolerance than the parent strain. The anaerobically expressed pyruvate dehydrogenase complex (PDH) is encoded by a three-gene operon (aceEF-lpd) and is essential to ethanol production by RM10. Previous studies suggested that PDH expression might be one of the limiting steps for ethanol fermentation. This hypothesis was tested by overexpression of the PDH via: 1) cloning aceE, aceF, lpd and the complete aceEF-lpd operon into a high copy plasmid; 2) integrating of a second copy of aceEF-lpd into chromosome. The results showed that: 1) overexpression of aceEF-lpd operon significantly improved cell growth and ethanol production; 2) chromosomal integration of a second copy of the aceEF-lpd operon did not show an improved transcription rate nor improved ethanol production; 3) overexpression of individual aceE , aceF, lpd genes decreased cell growth and ethanol fermentation. Alcohol dehydrogenase (adhE) is the final enzyme of the homoethanol pathway in RM10. Prior studies suggested that deletion of RNase G resulted in an increase of adhE mRNA, which in turn improved cell growth and ethanol fermentation. We assessed the impact overexpression of adhE on ethanol production by increasing the adhE copy number and by promoter replacement. The results showed that overexpression of adhE had a beneficial impact on cellular growth and ethanol production. Additionally, replacing the native adhE promoter with a pflBp6 promoter further enhanced ethanol fermentation. Nevertheless, chromosomal integration of a second copy of adhE with either pflBp6 promoter or native promoter did not achieve the improved adhE expression nor improved ethanol fermentation. In summary, the expression of aldB, aceEF-lpd , and adhE appears to be a rate-limiting factor during RM10 xylose fermentation. Successful overexpression of those genes resulted in improvements in cellular growth and/or ethanol production. Increasing chromosomal expression of genes involved in the homoethanol pathway should improve the fermentative capabilities of RM10 and move non-transgenic E. coli forward as an option for industrial bioethanol production.
Manow, Ryan Allan, "Enhancing the ethanol pathway in Escherichia coli RM10 for improved ethanol production from xylose" (2016). Graduate Research Theses & Dissertations. 2885.
ix, 94 pages
Northern Illinois University
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