Construction of γ-PGA-producing strain by chromosomal integration in Bacillus subtilis and optimization of fermentative production

Poly-γ-glutamic acid (γ-PGA) is a versatile high molecular biopolymer material, which has been applied in food, medical, cosmetic, animal feed,and wastewater industry. Enhanced production of γ-PGA is highly recommended. In order to enhance the screening of high γ-PGA yielding strain and quantitation of γ-PGA. A rapid quantification of γ-PGA was established in the beginning of this study. CTAB binds specifically to γ-PGA and form a water-insoluble, highly dispersed micelle-like complex, resulting in an increase in turbidity. The turbidity-based calibration curve of γ-PGA was established as y = 0.0055x – 0.0349 (x and y represent the concentration of γ-PGA and the mixtures turbidity at 400 nm) with a good linearity. The turbidimetric method has advantages of convenience , simplicity and good repeatability and can be used for γ-PGA concentration detecting in the fermentation broth. The host Bacillus subtilis WB800, which possesses the γ-PGA synthesizing genes, pgsBCAE, on its chromosome cannot produce γ-PGA. The efficient constitutive or inducible synthetic expression control sequence (SECS) was introduced into the upstream region of the pgsBCAE genes, resulted in γ-PGA-producing B. subtilis transformants. The transformant strain B. subtilis Dc8006 stably produced high levels of γ-PGA in medium A without extra glutamate supplement. To evaluate the effect of different culture parameters on production of γ-PGA, Plackett–Burman factorial design was preceded. Twelve varients were examined for their significance on γ-PGA production. Based on statistical pre-optimized medium analysis, optimized medium PBD were subjected for fermentation, and achieved 35.2 g/l γ-PGA yield, which is 1.5 times than the original medium A.γ-PGA 是一種高分子的生物材料，已被運用於食品、醫藥、化妝品、飼料及廢水處理工業上，提高 γ-PGA 的生產有其必要性。 為了提升篩選 γ-PGA 高產轉形株及定量 γ-PGA 之便利性，本研究首先建立一個 γ-PGA 快速定量法，利用 γ-PGA 與溴化十六烷基三甲銨 (CTAB)溶液反應可形成不溶於水、懸浮膠狀混合物，依其濁度制定標準 曲線。標準曲線公式為 y = 0.0055x – 0.0349( x 和 y 表示混合物在 400nm波長下 γ-PGA 的濃度及濁度) ，具有良好的線性。應用比濁法測定 γ-PGA的含量具有快速、簡潔、重現性好等優點，可用於發酵液中 γ-PGA 濃度的檢測。 宿主 Bacillus subtilis WB800 其 γ-PGA 合成相關基因 pgsBCAE 是存在的，但此菌株卻非 γ-PGA 生產株。利用持續型及誘導型高效率的人工合成表現元件(SECS)導入宿主中並使其嵌於 pgsBCAE 基因的上游，使得 Bacillus subtilis WB800 轉形為 γ-PGA 生產株。轉形株中 Bacillus subtilis Dc8006 可穩定的生產高量 γ-PGA，且無須額外補充麩胺酸於培養基 medium A。 為了評估不同培養條件對生產 γ-PGA 的效果，進行 Plackett-Burman 因子設計實驗。檢查十二變量對 Bacillus subtilis Dc8006 其 γ-PGA 生產影響程度。基於統計分析得到預優化培養基，在優化培養基 medium PBD中發酵 γ-PGA 產量可達 35.2 克/升，高於原 medium A 培養基之 γ-PGA產量 1.5 倍。中文摘要......................................................................................................................... i Abstract ......................................................................................................................... ii 壹、前言........................................................................................................................ 1 一、Bacillus subtilis 之簡介..................................................................................... 1 二、Bacillus subtilis 相關運用................................................................................. 2 三、Bacillus subtilis 遺傳研究................................................................................. 3 四、Bacillus subtilis 表現系統之研究..................................................................... 4 (一) 質體的選擇.................................................................................................... 4 (二) 表現元件之選擇............................................................................................ 5 (三) 轉形方法之選擇............................................................................................ 6 (四) 菌株之選擇.................................................................................................... 6 五、聚胺基酸(Poly amino acids) ............................................................................. 7 六、聚麩胺酸(Poly-γ-glutamic acid; γ-PGA)介紹 .................................................. 8 (一) 聚麩胺酸(γ-PGA)合成原理.......................................................................... 8 (二) 聚麩胺酸(γ-PGA)生產菌株及生合成途徑.................................................. 9 (三) 聚麩胺酸(γ-PGA)合成及相關降解酶基因................................................ 10 (四) 聚麩胺酸(γ-PGA)合成酶複體(Multienzyme)............................................ 13 七、發酵微生物生產 γ-PGA ................................................................................. 14 (一) 培養基成分的影響...................................................................................... 15 (二) 曝氣和攪拌的影響...................................................................................... 16 (三) 提高細胞膜的通透性.................................................................................. 17 (四) 菌血紅素的應用研究.................................................................................. 17 八、Plackett–Burman 實驗設計............................................................................. 18 九、γ-PGA 定量 ..................................................................................................... 19 十、γ-PGA 商業運用及展望 ................................................................................. 20 貳、實驗緣起與目的.................................................................................................. 23 實驗策略...................................................................................................................... 24 參、材料與方法.......................................................................................................... 25 一、菌種與質體...................................................................................................... 25 (一) 菌種.............................................................................................................. 25 (二) 質體.............................................................................................................. 25 二、藥品與試劑...................................................................................................... 28 三、實驗中所使用引子.......................................................................................... 28 四、質體 DNA 之抽取 ......................................................................................... 28 (一) 大腸桿菌質體之抽取.................................................................................. 28 (二) 枯草桿菌質體之抽取.................................................................................. 29 五、染色體 DNA 之抽取 ..................................................................................... 29 六、DNA 分子相關的操作技術 ........................................................................... 30 (一) 聚合酶連鎖反應(Polymerase Chain Reaction, PCR)................................. 30 (二) DNA 分子電泳 ........................................................................................... 30 (三) DNA 分子剪切 ........................................................................................... 31 (四) DNA 分子之膠體回收 ................................................................................ 31 (五) PCR 產物之回收 ........................................................................................ 31 (六) DNA 分子黏合 ........................................................................................... 32 七、大腸桿菌之電轉形法...................................................................................... 32 八、枯草桿菌之轉形法.......................................................................................... 33 (一) 電轉形法...................................................................................................... 33 (二) 自然轉形法.................................................................................................. 34 九、嵌入質體之構築.............................................................................................. 35 (一) pORI-177-6AFP........................................................................................... 35 (二) pORI-177-7AFP........................................................................................... 35 (三) pORI-177-7lacOGFP ................................................................................... 35 (四) pORI-177-6lacOWSY-1.............................................................................. 35 (五) pPGA6......................................................................................................... 36 (六) pPGA7......................................................................................................... 36 (七) pPGA7o....................................................................................................... 36 (八) pPGA6o....................................................................................................... 37 (九) pBL1 誘導型系統 ...................................................................................... 37 (十) pDcPGA6 ..................................................................................................... 37 (十一) pDcPGA7 ................................................................................................. 38 十、Colony PCR 篩選轉形株 ............................................................................... 38 十一、重組質體的序列確認.................................................................................. 38 十二、γ-PGA 分離、分析及快速定量法 ............................................................. 39 (一) γ-PGA 分離、沉澱與純化 ......................................................................... 39 (二) SDS-PAGE 電泳分析.................................................................................. 39 (三) Agarose gel 電泳分析 ................................................................................. 40 (四) γ-PGA 快速定量法建立 ............................................................................. 40 十三、γ-PGA 生產轉形株篩選、馴化 ................................................................. 41 十四、染色體分析.................................................................................................. 41 (一) Single-crossover 分析.................................................................................. 41 (二) Double-crossover 分析 ................................................................................ 42 十五、Plackett-Burman 實驗設計 ......................................................................... 42 (一) 菌種.............................................................................................................. 42 (二) 菌種活化與前培.......................................................................................... 42 (三) 發酵環境...................................................................................................... 43 (四) 分析因子設計.............................................................................................. 43 (五) 資料分析...................................................................................................... 43 (六) 試驗結果評估.............................................................................................. 44 肆、結果與討論.......................................................................................................... 45 一、γ-PGA 快速定量法 ......................................................................................... 45 (一) 波長與 γ-PGA 標準液濃度範圍之確立 .................................................... 45 (二) CTAB 溶液濃度的選擇 .............................................................................. 45 (三) 反應時間的影響.......................................................................................... 46 (四) γ-PGA 快速定量法確立 ............................................................................. 46 二、嵌入型載體之構築.......................................................................................... 47 (一) 持續型嵌入載體之構築.............................................................................. 47 (二) 誘導型嵌入載體之構築.............................................................................. 48 三、染色體嵌入策略改造 B. subtilis WB800 生產 γ-PGA .................................. 48 (一) Single-crossover 嵌入持續型及誘導型表現元件...................................... 48 1. 挑選與馴化.................................................................................................. 49 2. 生產能力確認.............................................................................................. 49 3. 誘導型表現系統.......................................................................................... 50 4. 培養基改造.................................................................................................. 51 5. 染色體分析及嵌入機制.............................................................................. 52 6. Single-crossover 嵌入探討.......................................................................... 53 (二) Double-crossover 嵌入持續型表現元件 .................................................... 53 1. 挑選與馴化.................................................................................................. 54 2. 生產能力確認.............................................................................................. 54 3. 染色體分析及嵌入機制.............................................................................. 55 4. Double-crossover 嵌入探討 ........................................................................ 55 四、Plackett–Burman design(PBD) ........................................................................ 55 (一) 轉形株的選擇.............................................................................................. 56 (二) 顯著因子篩選.............................................................................................. 56 (三) 因子效應評估.............................................................................................. 58 (四) B. subtilis Dc8006 生產 γ-PGA................................................................... 59 五、未來展望.......................................................................................................... 60 伍、結論...................................................................................................................... 61 陸、參考文獻............................................................................................................ 112 柒、附錄.................................................................................................................... 12