Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51299
標題: Development of constitutive and inducible expression systems for expression of autologous, homologous and heterologous proteins in Bacillus subtilis and establishment of Bacillus probiotics expression systems
開發枯草桿菌持續型及誘導型表現系統以應用於自體、同源及異源蛋白質之表現暨建立芽孢桿菌益生菌表現系統
作者: 王志鵬
Wang, Jyh-Perng
關鍵字: 枯草桿菌表現系統
Bacillus subtilis expression system
芽孢桿菌益生菌表現系統
持續型及誘導型表現元件
果聚醣酶
甘露聚醣酶
阿拉伯糖異構酶
聚麩胺酸合成酶
複合體
第一型抗凍蛋白類似物
苦瓜抗病毒及抗腫瘤蛋白質
甜味蛋白質
靈芝免疫調節蛋白質
Bacillus probiotics expression systems
constitutive and inducible expression control sequences
levanase
mannanase
arabinose isomerase
poly-γ-glutamate synthetase complex
type I antifreeze protein analogue
Momordica anti-viral and anti-tumor protein MAP30
sweet protein brazzein
Ganoderma lucidum immunomodulatory protein Ling Zhi-8
出版社: 食品暨應用生物科技學系
摘要: 枯草桿菌(Bacillus subtilis)為革蘭氏陽性、兼性厭氧之產孢桿菌,廣泛存在於自然界之土壤及水源中。此菌之產業應用性極為廣泛,可運用於發酵食品之製造、酵素及特用化學品之生產、作為益生菌(probiotic)及作為植物病害防治試劑等用途。此外,其亦被開發成為基因表現之宿主,用以生產同源或異源蛋白質。 欲以枯草桿菌表現重組蛋白質時,重組蛋白質之基因上必需帶有枯草桿菌可辨識之表現元件,包括轉錄及轉譯之訊號,方能達到基因表現之目的。表現元件除了可自菌體之染色體中篩選外,亦可利用化學合成法合成或以聚合酶連鎖反應增幅已知基因之表現元件。過往至今,有關枯草桿菌持續型及誘導型表現元件之研究成果陸續的發表,但由於某些表現元件取得不易或受國外專利之束縛,因此實有必要開發一具競爭性之新穎表現元件以供國內學術界及產業界應用。 本論文之主要目的即在開發新穎創新之枯草桿菌持續型及誘導型表現元件,並運用於自體、同源及異源蛋白質之表現。本實驗室先前曾以枯草桿菌veg 表現元件之DNA序列為藍圖,設計及合成一持續型表現元件。有鑒於此表現元件之UP element及核糖體結合部位仍有改進之空間,我們對此表現元件進行改造,建構出7種衍生表現元件。以增強型綠螢光蛋白基因作為報導基因測試這些表現元件之表現效率,結果顯示改造表現元件之UP element或核糖體結合部位可提升表現元件之表現效率,若同時改造兩者則可獲最高表現效率。衍生表現元件之表現效率亦高於文獻中常用之持續型表現元件包括veg、cdd及rpsJ等基因之表現元件及HpaII表現訊號。接著,我們利用聚合酶連鎖反應將lacO、gntO或xylO等操縱子之DNA序列加入高表現效率之持續型表現元件中,以建構誘導型表現元件。結果顯示帶有lacO之誘導型表現元件具有最高之表現效率及誘導效果。相較於T5誘導系統、spac誘導系統、木糖誘導系統、nisin誘導系統及阿拉伯糖誘導系統,本論文中利用人工合成誘導表現元件所建立之新型誘導表現系統具有最高之表現效率。這些結果顯示,本論文中所建構之表現元件具有運用於大量生產重組蛋白質之潛力。 為了測試這些系統之應用性,我們選擇數種具有市場價值之自體、同源及異源基因進行表現。在自體蛋白質表現方面,我們利用持續型表現系統大量分泌表現果聚醣酶及β-甘露聚醣酶;利用誘導型表現系統大量表現阿拉伯糖異構酶。此外,我們利用嵌入性策略將聚麩胺酸合成酶複合體結構基因上游之DNA序列置換為持續型表現元件,成功改造枯草桿菌DB430成為聚麩胺酸生產株。在同源蛋白質表現方面,我們利用持續型表現系統大量分泌表現源自於Bacillus sp.之subtilisin YaB。在異源蛋白質表現方面,我們利用持續型表現系統成功表現第一型抗凍蛋白類似物及苦瓜抗病毒及抗腫瘤蛋白質MAP30。利用誘導型表現系統表現甜味蛋白質brazzein時,卻無法觀察到brazzein之分泌表現。我們推測帶有四個雙硫鍵之brazzein於分泌時會遭遇瓶頸。 最後,我們針對芽孢桿菌益生菌B. subtilis 3A16及B. pumilus 14A1建立基因操作相關技術,包括電轉形條件之建立及評估不同報導基因及表現訊號之適用性等,以期開發芽孢桿菌益生菌成為基因表現系統。結果顯示,質體可藉由電轉形法送入芽孢桿菌宿主中,轉形效率在102 transformants/μg DNA以上;葡萄糖醛酸酶基因(gusA)、subtilisin YaB基因(ale)及增強型綠螢光蛋白基因(gfp+)可運用於這些宿主做為報導基因;人工合成持續型表現元件及其衍生表現元件均可被這些宿主所辨識,可作為表現訊號。在表現實例方面,我們利用芽孢桿菌益生菌系統表現出苦瓜抗病毒及抗腫瘤蛋白質MAP30及靈芝免疫調節蛋白質LZ-8,顯示芽孢桿菌益生菌具有開發成為異源蛋白質表現宿主之潛力,未來可運用於醫藥及保健用蛋白質之生產或作為疫苗攜帶者。此外,重組芽孢桿菌益生菌亦可應用於基礎學術研究,探討菌種於腸道中生長、分布及拓殖之情形。
Bacillus subtilis is a Gram-positive, facultative anaerobic rod-shaped endospore-forming bacterium that occurs naturally in soil and water. It has been used in the production of fermented food, extracellular enzymes as well as specialty chemicals. Some strains of B. subtilis are exploited as probiotics or biological control agents. In the field of genetic engineering, B. subtilis has been developed as a host for the production of homologous and heterologous proteins. Expression control sequence which comprises of a transcriptional signal (promoter) and a translational signal [Shine-Dalgarno (SD) sequence] is indispensable for gene expression. The crucial element is necessary for construction of an efficient B. subtilis expression system. Several strategies have been applied to obtain suitable expression control sequences from B. subtilis and related organisms. One strategy uses promoter-trap plasmid to isolate unknown expression control sequences from bacterial genomic DNA. The second strategy uses polymerase chain reaction (PCR) to amplify well-studied expression control sequences from bacterial genomic DNA. The third strategy uses chemical DNA synthesis method to create novel expression control sequences. To date, many B. subtilis expression systems which were based on various constitutive and inducible expression control sequences have been constructed for high level expression of recombinant proteins. However, most of the high level expression systems have been patented and theirs availabilities are limited. The objective of this study was to develop novel constitutive and inducible expression control sequences for high level expression of autologous, homologous and heterologous proteins in B. subtilis. In previous study, a synthetic expression control sequence was constructed using overlapping extension polymerase chain reaction (OEPCR) in our laboratory and its sequence designed on the basis of the expression control sequence of the B. subtilis veg gene. In this study, the UP element (an A+T-rich sequence located upstream of the -35 hexamer) and SD sequence or both of the synthetic expression control sequence were engineered and the effects of their modification on expression efficiencies were examined. Our results showed that the substitution of the SD sequence or UP element could enhance the expression efficiency of synthetic expression control sequence, while combined substitution of both gave the highest expression. In addition, the expression efficiencies of engineered synthetic expression control sequences were higher than those of expression control sequences of veg, cdd, rpsJ and HpaII expression signal. Furthermore, regulatory elements including lacO, gntO, and xylO were respectively introduced into constitutive expression control sequences for construction of isopropyl-β-D-thiogalactoside (IPTG), gluconate, and xylose inducible expression control sequences. Our results showed that the newly constructed inducible expression system based on the lacO gave the highest expression efficiency and induction ratio. Its expression efficiency was higher than those of commonly used inducible expression systems including T5 exression system, spac expressin system, xylose inducible expression system, nisin inducible expression system, and arabinose inducible expressin system. These novel constitutive and inducible expression systems could potentially be used to express homologous and heterologous proteins in B. subtilis at high level. In order to test the application of these novel expression systems on the expression of recombinant proteins, various autologous, homologous, and heterologous proteins were selected as model proteins. High level expression and secretion of autologous enzymes including levanase and β-mannase were achieved in B. subtilis by using constitutive expression system. Autologous enzyme arabinose isomerase was expressed at high level by using inducible expression system. The DNA sequence uspstrem of ywsCAB encoding poly-γ-glutamate synthetase complex was replaced with constitutive expression control sequence by using integration strategy in this study. Using this approach, B. subtilis DB430 was successfully engineered into a poly-γ-glutamate producer. The constitutive expression system could also be used to expression of subtilisin YaB, type I antifreeze protein analogue, and Momordica anti-viral and anti-tumor protein, MAP30. The sweet protein brazzein was not expressed in B. subtilis. It is suggested that there are bottlenecks for secretory production of brazzein in B. subtilis. Finally, we have developed the probiotic strains B. subtilis 3A16 and B. pumils 14A1 as expression hosts. The electrotransformation procedure, reporter systems, and expression systems have been established. The transformation efficiencies of Bacillus probiotics were above 102 transformants per microgram of plasmid DNA using the established electrotransformation procedure. Reporter genes gusA, ale, and gfp+ could be used to investigate expression efficiencies of various expression control sequences. Novel constitutive expression control sequences could be used to drive gene expression in Bacillus probiotics. These probiotic expression systems have been used to express Ganoderma lucidum immunomodulatory protein, Ling Zhi-8, and Momordica anti-viral and anti-tumor protein, MAP30. We suggested that these Bacillus probiotics could potentially be used to express functional peptides and pharmaceutical proteins or used as oral vaccine vehicles. The recombinant Bacillus probiotics could also be used in study of the growth, distribution and colonization of probiotics in gastrointestinal tract.
URI: http://hdl.handle.net/11455/51299
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