Proteomics Investigation on the Methanol Utilization Metabolism in Escherichia coli
| Autor: | Yi-Wen Fang, 方意雯 |
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| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 Biofuel, a low-cost alternative fuel converted from sugar, starch, and cellulose, has become a potential and renewable biomass in recent years. Methanol is a non-food substitute resource and a sustainable product from natural gas. The biological conversion of methanol to higher energy content alcohols, such as ethanol and butanol, using biological engineering methylotrophic bacteria has been successfully reported in the past decades. An auxotrophic strain of Escherichia coli (E. coli) that can take methanol as growth nutrition and produce ethanol and butanol has been established. However, the mechanism on how microbial host platforms respond or adapt to the exogenous stress of multiple carbon stress remains unknown. In this study, mass spectrometry-based quantitative proteomic analysis was applied to identify the contributing factors and mechanism for biofuel production in E. coli. In the first study, we applied a TMT isobaric labeling strategy to analyze the differential protein expression during the time course of butanol production in the engineered E. coli strain. Among a total of identified 2,268 proteins and 2,200 quantified proteins, about 20% of proteins showed up-regulated expression, which are enriched in functional categories related to export and transport. The result suggests that the mechanism of exporting toxic compounds was activated to tolerate the toxicity from exogenous biofuel. Besides, more than 50% of proteins were down-regulated and they are related to protein translation, tricarboxylic acid cycle, biosynthetic process, which may be due to cell death or severe loss of protein production machinery. Extensive protein modifications were observed and modification degree increased with time, including formylation (44 proteins), methylglyoxalation (69 proteins), ribulosamine-5-phosphate (Ru5P, 26 proteins), erythrulosamine-4-phosphate (E4P, 36 proteins), and sedoheptulosamine-7-phosphate (S7P, 18 proteins), which metabolic modification may likely affect protein structure and enzyme activity. To further study the effect of butanol production to cause extensive modification, we analyzed the E. coli strain without butanol production; a total of 2,358 proteins were identified and 2,224 proteins are quantified. About 20% of proteins were down-regulated and involved in sulfate related metabolism pathway, such as cysteine/methionine and selenoamino acid metabolism. Especially, methionine is the universal message for a ribosome that signals the initiation of protein translation.These suggested either the translation initiation was blocked or newly translated proteins were degraded. In addition, only 9% of proteins showed up-regulation and annotated as cell membrane transporter. The result showed that the mechanism of toxins exporting was activated in absence of butanol. In butanol production defected system, our result show that E4P and erythrulosamine were the most abundant modification, which may be due to accumulation of E4P metabolites in RuMP pathway when methanol was supplied as nutrient resource. Therefore, high concentration of butanol is toxic to microbe and formaldehyde can lead to damage for enzyme structure and activity, following decreased the cell viability and production efficiency. In summary, the low tolerance of formaldehyde and butanol may limit the engineered metabolic pathways for biofuel production in E. coli. Efficient control of toxin compound through cellular export systems can enhance resistance of solvent toxicity and improve cell viability. Here, the proteomics and bioinformatics reveal the molecular mechanism on biofuel tolerance problem and may provide new insight to reduce solvent/chemical toxicity for next-generation biofuel manufacture. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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