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系統識別號 U0026-3007202010050700
論文名稱(中文) 大腸桿菌生產五胺基酮戊酸之蛋白表達模組及代謝調控
論文名稱(英文) Development of 5-Aminolevulinic Acid Production in Escherichia coli by Expression Modular Design and Metabolic Regulation
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 108
學期 2
出版年 109
研究生(中文) 施億泰
研究生(英文) I-Tai Shih
學號 N36074398
學位類別 碩士
語文別 英文
論文頁數 83頁
口試委員 指導教授-吳意珣
口試委員-張嘉修
口試委員-趙雲鵬
口試委員-李思禹
口試委員-陳柏庭
中文關鍵字 大腸桿菌  5-胺基酮戊酸生產  磷酸吡哆醛(PLP)  GroESL 分子伴侶蛋白  代謝工程  整合菌株 
英文關鍵字 5-Aminolevulinic acid  Pyridoxal 5’-phosphate  GroESL chaperones  Metabolic engineering  Plasmid-free  Escherichia coli 
學科別分類
中文摘要 5-胺基酮戊酸(5-ALA)是生物體中必需的代謝中間體,在農業和醫學領域也有極大用途,例如用作可生物降解的除草劑、殺蟲劑、農作物生長的促進因子及腫瘤診斷。5-ALA之酸鹽衍生化學品已被美國食品和藥物管理局(FDA)批准,用作疑似高度惡性神經膠質瘤的手術光學成像劑。
近年來,由於基因和代謝工程學的迅速發展,利用基改及代謝調控的微生物生產5-ALA逐漸成為兼具可行性及永續性之製程。本研究探討亞比多球型紅桿菌之ALA合成酶(RsHemA)、非專一性之ALA輸送蛋白(RhtA)和GroE分子伴侶三者的表達系統模組。磷酸吡哆醛(PLP)為ALA合成酶(ALAS)的輔因子,通過表達基因pdxK和pdxY或直接添加於培養基中皆可提高ALA的產量。添加終濃度為30 µM之PLP時,可將5-ALA濃度由1.35 g/L提升至2.44 g/L。然而,RsHemA的包涵體為5-ALA生產的障礙之一,透過DnaKJ和GroESL分子伴侶共表達以重新折疊不可溶的包涵體,結果顯示僅GroESL分子伴侶可以顯著解決ALAS包涵體的問題。在雙質體表達系統中,5-ALA進一步提高至3.46 g/L;而當groESL基因簇整合至基因組中,即使沒有明顯的GroE蛋白表達,5-ALA產量略提升至4.01 g/L。
基因敲除是將碳代謝流由其他分流導向目標產品之常用策略。在本研究討論了ldhA,pta,sucC和pck基因敲除對於5-ALA生產之影響。ldhA、pck或pta的敲除對於提高ALA產量較為有益,sucC之剔除則導致生長缺陷。另外結合多基因合一的質粒系統在個別pck、pta基因剔除菌株中,5-ALA產量提昇至5.11 g/L和5.23 g/L。
最後,本研究中構築了不含質體之RrGI整合菌株,達成無生物標記且穩定之細胞工廠。此菌株呈現了高穩定性且5-ALA濃度可達初始菌株Rsr之5.53倍。利用相對轉錄水平、代謝物分析及電子顯微鏡分析細胞形態,以期闡明無質體菌株之差異。根據牽涉於糖解路徑、TCA循環和重組表達基因之轉錄水平,發現RrGI整合菌株的轉錄水平相較於質體表達菌株低10倍,然而重組蛋白表達卻相似,意味著RrGI菌株中的能量消耗可能更少,故葡萄糖、甘氨酸及琥珀酸消耗量相似,也能轉化出高產量5-ALA;同時,RrGI整合菌株之細胞更完整。整體而言,整合菌株RrGI可以執行更具效率且穩定的5-ALA轉化,並節省轉錄能耗,從而達到7.47 g/L產量和0.588 g/L/h的生產率。整合菌株也呈現較佳的穩定性,高ALA產量更可達三代;相較之下,RsrG菌株即使加有抗生素仍無法提供穩定ALA生產。
英文摘要 5-Aminolevulinic acid (5-ALA) is an essential intermediate in organisms and has also considerable applications in the agricultural and medical field. Beyond the applications as biodegradable insecticide and promoting factor for crops, 5-ALA has been approved by the US Food and Drug Administration (FDA) as an intraoperative optical imaging agent for suspected high-grade gliomas. Thanks to the advance in genetic and metabolic engineering, biosynthesis of 5-ALA in microorganisms has become a sustainable process for efficient production.
In this study, several modules for expression systems of ALA synthase from Rhodobacter sphaeroides (RsHemA), a non-specific ALA exporter (RhtA) and chaperones were first developed and discussed. As the co-factor of ALA synthase (ALAS), 5-ALA production was enhanced by the key cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes pdxK and pdxY or direct addition. With final concentration of 30 M PLP, the 5-ALA titer could increase from 1.35 g/L to 2.44 g/L. However, inclusion bodies of RshemA served as an obstacle; thus, DnaKJ and GroESL chaperones were introduced to refold the insoluble proteins. However, only GroESL chaperones could significantly solve the issue of inclusion bodies. In dual plasmid system, the 5-ALA titer was further enhanced to 3.46 g/L. While with integrated groESL cluster in chromosme, the 5-ALA production reached 4.01 g/L even with no obvious expression of chaperones.
As gene deactivation is a common strategy for redistributing the carbon fluxes oriented to target product instead of bypass pathways, deactivation of ldhA, pta, sucC and pck genes were discussed. Silencing either pck or pta was more beneficial for improving 5-ALA production. However, sucC-defect caused inpaired cell growth. To adjust the GroESL expressions, an all-in-one plasmid encoding RshemA, rhtA and groESL was further been constructed. Coupling with the effect of all-in-one plasmid system and gene deactivation, the 5-ALA titer reached 5.11 g/L and 5.23 g/L with knock-out of pck and pta respectively. Lastly, plasmid-free strain called RrGI was constructed with expectation of being a robust and marker-free strain. Surprisingly, it was found robust for 5.53-folds enhancement on 5-ALA titer. Thus, the aspect of relative transcription levels, metabolites analysis and cell morphology were discused to clarify the criticle difference of plasmid-free strain. In accordance with the transcription level of genes involved in glycolysis, TCA cycle and recombinant expression, the energy consumption of RrGI strain may be less due to the much less transcription level of recombinant protein but similar protein expression. Therefore, 5-ALA yield was significantly higher than other strains even with similar consumption of glycine, succinate and glycine. In addition, the morphology analysis also showed the poor cell integrity of plasmid-harboring strains. Generally, the plasmid-free strain (i.e., integration of gene cluster to the chromosme), RrGI, could perform higher 5-ALA titer by reduced energy consumption for transcription, thus lead to 7.47 g/L titer and 0.588 g/L/h productivity. Besides, RrGI strain could show more stable ALA production for three generations compared to plasmid-harboring RsrG strain.
論文目次 摘要 I
Abstract III
致謝 V
Table of Contents VI
List of tables IX
List of figures XI
Chapter 1 Introduction 1
1.1 Preface and research backgrounds 1
1.2 Research purpose 2
Chapter 2 Literature review 4
2.1 Properties and application of ALA 4
2.1.1 Agricultural applications of ALA 5
2.1.2 Medical applications of ALA 6
2.2 Developmetnt of ALA biosynthesis 6
2.2.1 The introduction of ALA biosynthesis pathways 7
2.2.2 The development of ALA biosynthesis by microorganisms via C5 pathway 10
2.2.3 The development of ALA biosynthesis by microorganisms via C4 pathway 13
2.2.4 Metabolic regulation and gene deactivation for enhancing ALA production 15
2.3 Inclusion bodies and heat shock proteins (Hsp) 16
2.3.1 Solution of inclusion bodies formation 16
2.3.2 Mechanism of protein re-folding by Hsp 17
2.3.3 The example of chemical production assisted by GroESL chaperones 19
Chapter 3 Materials and methods 20
3.1 Materials and chemicals 20
3.2 Microorganisms, plasmids and primers 23
3.3 Equipments 26
3.4 Strains, media and culture condition 27
3.5 Plasmid construction and genome editing 27
3.5.1 Plasmid DNA extraction 27
3.5.2 Modification of commercial plasmid with multiple cloning sites 28
3.5.3 Construction of expressing plasmid 29
3.5.4 Heat shock transformation method 31
3.5.5 Electroporation method and gene knock-out 32
3.5.6 Integration of expressing cassette containing RshemA, rhtA and groESL genes 34
3.5.7 Preservation of constructed strains 34
3.6 Sample preparation for scanning electron microscope (SEM) 34
3.6 Analytical methods 35
3.6.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 35
3.6.2 Colorimetric analysis by Ehrlich’s reagent assay 37
3.6.3 HPLC analysis of extracellular metabolites 38
3.6.4 Quantitative real time polymerase chain reaction (qRT-PCR) 38
Chapter 4 Result and discussion 43
4.1 Establish the ALA-producing system in E. coli 43
4.1.1 Gene selection for catalyzing the ALA formation 43
4.1.2 Co-expression of rhtA and pdx genes affected ALA production 44
4.2 Co-expression of GroESL chaperones assist the protein folding of ALAS 47
4.3 Blocking bypass pathways increases carbon flux toward ALA 48
4.4 Robust ALA-production by plasmid-free strain 55
4.5 Metabolic flux analysis 61
Chapter 5 Conclusion and prospection 66
5.1 Conclusion 66
5.2 Prospection 67
Reference 68
Appendix 80
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