"以重組大腸桿菌進行 1,12 - 十二碳二元醇之生產"

Abstract

中長碳鏈末端二醇是許多重要聚合物之前驅物，可以用於合成保養品和潤滑劑等。工業上生產中長碳鏈末端二醇主要透過石油裂解之烯烴類醛化還原，然而所需高溫高壓反應條件及金屬催化劑除了成本昂貴，也較不環保。另一方面，以微生物催化法是進行末端氧化則是新興具有開發潛力之替代方法；除了微生物酵素具有高度立體選擇性能大幅提升產率，所需反應條件亦較溫和。主要以生物催化進行中長碳鏈的末端氧化反應系統有 AlkB 系統、CYP52 系統和 CYP153A 系統等，但在產程開發上都各有不同難處：其中 AlkB 及 CYP52 系統具有過氧化活性，會將醇進一步氧化成醛和酸。CYP153A 系統過度氧化活性較輕微，因此我們實驗室選用 CYP153A 系統進行後續研究。本研究室先前之研究結果指出海洋桿菌 VT8 之 CYP153AM. aq 操縱子兼具低過氧化特性及高立體選擇性兩種優點，然而，其操縱組卻無法在大腸桿菌中成功表現，本實驗室謝善棋於 2015 年辨認出插入操縱子當中之轉位子，並且成功修復其功能。本研究顯示，使用謝所建構的大腸桿菌 JM109 搭載帶有可受中長碳鏈烷類誘導之 CYP153AM. aq 操縱子的 pBBR1-CYPLR 和鼠李糖誘導的烷類運輸子 psb3C5-rha-alkL 雙質體系統；以 TB 為主培養基，葡萄糖為唯一碳源於 5 升醱酵槽進行批式培養，可成功將十二碳烷及 1 - 十二碳一元醇轉換為 1,12 - 十二碳二元醇，其濃度為 1.2 g/L。以 TB 為主培養基，葡萄糖為唯一碳源，並以葡萄糖和酵母抽出物作為饋料培養基，以碳氮比 5:1 進行添加，於 5 升醱酵槽進行批式饋料培養，則可獲得濃度 1.7 g/L 之二醇，並且將細胞量提升將近兩倍。另一方面，以 TB 為主培養基，甘油為唯一碳源，並以甘油和酵母抽出物作為饋料培養基，以碳氮比 5:1 進行添加，於 5 升醱酵槽進行批式饋料培養，可以觀察到培養中後期質體流失及新生細胞不受基質誘導的現象。在進行抗生素補充和二次誘導之後，在 60 小時的轉化時間內，於醱酵槽中獲得 3.76 g/L 之二醇，轉化效率為 6.267 mg/L/h，轉化率（額外添加之酸鹼液、消泡劑等之體積忽略不計）約為 12.5 %。此外，也利用氣相層析質譜儀進一步確認產物結構，判斷微生物轉化之產物確實為 1,12 - 十二碳二元醇。

Medium-long-chain diterminal alcohols are precursors to many important polymers and can be used in the synthesis of personal care products and lubricants. Industrial approach to medium-long-chain diterminal alcohols production is mainly from olefins using hydroxyfromylation method. However, the high temperature, pressure and the metal catalyst required in this reaction is neither economically nor environmentally friendly. On the other hand, alkane terminal oxidation through microbial catalysis is an emerging alternative with great market potential. The intrinsic nature of high regio-selectivity enables microbial enzymes to achieve high product yield. Furthermore, microbial enzymes demand a less stringent reaction condition. The AlkB system, CYP52 system and the CYP153A system are the most extensively researched field in alkane terminal bio-oxidation, yet they have all encountered different obstacles. AlkB and CYP52 system tends to over-oxidize products and turn alcohols into aldehydes and acids, meanwhile our lab embarks on detailed studies into CYP153A system on the basis of its activity which would not cause over-oxidation. It has been reported that the CYP153AM. aq operon of Marinobacter aquaeoli VT8 has property of both low over-oxidation activity and high regio-selectivity, but it could not be expressed successfully in E.coli. Previously, we have identified the transposon inserted in the CYP153AM. aq and restored its activity by removing the inserted sequence. In this study, we utilized this E. coli harboring two plasmids: pBBR1-CYPLR which contains medium-long-chain-alkane-inducible CYP153AM. aq operon, and the rhamnose inducible psb3C5-rha-alkL. With TB broth as our main medium, and glucose as sole carbon source, we started out a batch culture and successfully transformed dodecane and 1 - dodecanol into 1,12 - dodecanediol at a concentration of 1.2 g/L in a 5 L jar fermentor. We achieved higher diol concentration at 1.7 g/L and nearly doubled bacterial biomass than batch culture in a fed-batch course using TB broth as starting medium, glucose as sole carbon source and feedstock composed of glucose and yeast extract. On the other hand, with TB broth as our main medium, glycerin as sole carbon source, feedstock comprising glycerin and yeast extract supplementing at a C : N ratio of 5 : 1, in a 5 L jar fermentor experiment, we discovered that E. coli culture underwent plasmid loss during the mid-late course of fermentation. Moreover, E.coli cells that were newly reproduced in mid course seemed unaffected by the inducers added. After the treatment of additional antibiotics and inducers to the culture, we ultimately obtained 1,12 - dodecanediol at concentration of 3.76 g/L within 60 hours of transformation session. The productivity was 6.267 mg/L/h and the conversion rate (With the volume of extra acid/base & antifoam added left negligible) was approximately 12.5%. Further- more, we identified the chemical structure of the product being transformed by gas chromatography / mass spectrometry, and verified that it was 1,12 - dodecanediol.