以蛋白質體學探討在大腸桿菌中甲醇利用代謝途徑

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方意雯(Yi-Wen Fang) 查詢紙本館藏

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論文名稱

以蛋白質體學探討在大腸桿菌中甲醇利用代謝途徑
(Proteomics Investigation on the Methanol Utilization Metabolism in Escherichia coli)

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摘要(中)

『生質燃料』是一種是可以經由糖質、澱粉和纖維素轉換形成的低成本燃料，近年來已成為一具有潛力的再生能源。甲醇可作為其生產原物料之一，由天然氣轉換生成，屬於非食物競爭性的替代原物料，已被廣應用於生質能源的發展。在過去幾十年中，利用基因工程以甲基營養菌為載體，在大腸桿菌中使甲醇透過生物轉換，可以合成較高能量含量的醇類，如:乙醇和丁醇。然而，在生產含碳量較高之生質然料的同時，微生物系統如何應對與適應來自外源性的壓力，其調控機制仍未被清楚的研究與了解。在本研究中，利用質譜技術與定量蛋白質體學，分析大腸桿菌於製造生質燃料時，細胞生長與燃料產能如何受到影響與調控。
在實驗設計I中，我們以串聯質譜標籤(TMT)同位素標記技術分析基因改造大腸桿菌蛋白質體在生產丁醇過程隨著時間的變化，共可鑑定出2,268個蛋白質，在2,200個可定量的蛋白質中，大約20％的蛋白質會隨著時間表現量上升，包括 440個蛋白與蛋白質或小分子運輸相關，推測在大腸桿菌中，將具生物毒性的代謝物(如:甲醛與丁醇)與化合物排出體外，可作為對抗外源性生物燃料毒性策略之一。此外，超過50％的蛋白質表現量會隨時間下降，多與蛋白質轉譯功能、能量生產之三羧酸循環與胺基酸生合成相關，藉此可猜測細胞可能已失去蛋白質製造功能，甚至隨時間趨於死亡。同時，我們也鑑定到多種蛋白質經代謝物的化學修飾，如:甲醛(44個)、甲基乙二醛(69個)、核酮醣-5-磷酸(Ru5P, 26個)、赤鮮醣-4-磷酸(E4P, 36個)、景天庚酮醣-7-磷酸(S7P, 18個)。其中，甲醛和甲基乙二醛所造成的化學修飾隨著時間增加，表示當高濃度的代謝物累積於細菌細胞內，會對蛋白質進行化學修飾，可能影響蛋白質的結構與活性，導致細胞死亡與產能下降。
在實驗設計II中，分析以利用甲醇及木糖代謝卻無丁醇生產能力的大腸桿菌之RuMP路徑中之代謝物化學修飾的變化趨勢，共鑑定出2,358個蛋白質並有2,224個蛋白質可被定量，約20％的蛋白質表現量隨著時間下降，而這些蛋白質多參與硫相關之代謝路徑，而因與硫相關之代謝調控很多胺基酸合成過程像是:蛋胺酸合成，並且在胺基酸合成過程中必須消耗能量，因此這類蛋白質表現量下降顯示細菌RuMP路徑的活化，使蛋白質轉譯上游途徑受到抑制，並同時也降低能量的消耗；另外，表現量上升的蛋白僅佔了9％，且仍與細胞表面汞幫浦相關，推測在無丁醇的存在下，此類蛋白被啟動負責輸出具生物毒性化合物的機制，以調控代謝物累積濃度。此外，實驗結果顯示E4P和Erythrulosamine在無丁醇生產之系統中為主要的修飾代謝物，推測由於RuMP循環在此系統為甲醇主要的代謝路徑，產生代謝物快速累積，進而發生化學修飾。統整上述實驗，可知高濃度丁醇的存在對細菌生長具有毒性，且甲醛修飾對蛋白質的結構與活性可能造成影響，進而造成細胞死亡與產能下降。
總結來說，以大腸桿菌作為生產生質燃料的平台，甲醛和丁醇的濃度耐受程度可限制酵素活性與細胞生長，若可經細胞輸出系統排除這些化合物，進行有效的細胞內濃度控制，應可有效提高生物毒性耐受度和改善細胞生存。透過蛋白質體學與生物資訊分析，解析溶劑與有毒代謝物耐受性影響生產效率之分子機制，提供可能的改良策略應用於新一代的生物燃料發展。

摘要(英)

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.

關鍵字(中)

★ 生質燃料
★ RuMP代謝路徑
★ 溶劑耐受度
★ 定量蛋白質體學

關鍵字(英)

★ Biofuel
★ RuMP pathway
★ solvent tolerance
★ quantitative proteomics

論文目次

Table of Contents
中文摘要 i
Abstract iii
Table of Contents v
List of Figures vii
1. Introduction 1
1.1 Economic and environmental benefits of the biofuel 1
1.2 The generations and dilemma of biofuel production 1
1.3 Utilizing metabolic pathway for bioconversion of methane and methanol 3
1.4 The characteristic and superiority of butanol as biofuels 3
1.5 Challenges of engineering microbial for biofuel production 4
1.6 The proteomics approach characterizes the metabolic responses in Escherichia coli during biofuel production 5
1.7 Hypothesis and Objective 5
2. Materials and Methods 7
2.1 Materials and chemical reagent 7
2.1.2 Apparatus 7
2.2 Sample preparation for LC-MS/MS 8
2.2.1 Total protein extraction of E. coli 8
2.2.2 BCATM protein assay Kit 8
2.2.3 Reduction, alkylation and In-solution digestion 9
2.2.4 Desalting with C18-beads Stage-Tip 9
2.2.5 Tandem Mass Tags (TMTs) isobaric chemical labeling 10
2.2.6 High pH reversed-phase peptide fractionation 10
2.2.7 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis 11
2.3 Data process and analysis 11
2.3.1 Database search and quantification 11
2.3.2 Relative quantitative strategy in an isobaric labeling tandem mass tag (TMT) 12
2.3.3 Statistics and bioinformatics analysis 13
3. Results and Discussion 14
3.1 Butanol production in synthetic methanol auxotrophic E. coli system 14
3.1.1 Engineered E. coli produce butanol through RuMP Pathway 14
3.2 Quantitative analysis of E. coli proteome during butanol production through engineered RuMP pathway 15
3.3 Analysis of chemical modification by metabolites produced in RuMP pathway 18
3.3.2 Identification of formylation and methylglyoxalation for enzymes in RuMP pathway 20
3.3.3 Enrichment of function and pathway of metabolically modified proteins 21
3.4 Investigate the alteration of protein expression and modification in the engineered RuMP pathway without butanol generating ability 22
3.4.1 Bioinformatics analysis of differentially regulated protein in engineered RuMP pathway without butanol production ability 24
3.4.2 Bioinformatics analysis of chemically modified proteins by metabolites in RuMP pathway in butanol absent system 25
3.4.3 Comparison of protein expression and metabolic modifications between butanol synthesis and its absence in RuMP pathway 26
3.4.4 Proteomics result identify protein candidates for improvement of solvent tolerance and biofuel production efficiency 27
4. Conclusions 30
Reference 32
Supplementary Figure 56
Supplementary Table 61
Appendix 64

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指導教授

陳玉如侯敦仁(Yu-Ju Chen Duen-Ren Hou)

審核日期

2018-7-30

推文