重組大腸桿菌生產 1,12-十二烷二醇之烷烴誘導系統與代謝剔除策略之建立

Abstract

1,12-十二烷二醇（1,12-diol）為具高化學穩定性與親水性的中長鏈二醇，可廣泛應用於潤滑劑、聚合物前驅物與界面活性劑等工業用途。目前其製程主要仰賴化學催化法，存在環境污染與製程成本高等問題。為實現綠色永續化生產，本研究以基因工程改造大腸桿菌為宿主，建立1,12-diol之生物製程。本實驗以細胞色素P450 monooxygenase CYP153A 為關鍵氧化酵素，催化1-dodecanol末端碳氧化為1,12-diol，並搭配烷烴轉運蛋白AlkL提升細胞內基質濃度。本研究室先前使用之轉運蛋白alkL表現受rhaBAD啟動子控制，須添加外源性誘導劑 L-rhamnose 調控表現，雖可提高轉換效率，惟操作複雜且增加成本。因此，本研究建構一套以烷烴感應轉錄調控蛋白AlkR為核心之烷烴誘導系統（Alkane Induction System, AIS），同時利用基質1-dodecanol作為誘導劑，自主驅動下游啟動子Pcyp，啟動cyp153A及alkL基因表現，實現誘導與產物轉換同步化之目的。根據qPCR數據顯示AIS菌株於無添加額外誘導劑 L-rhamnose 下，alkR與alkL mRNA表現量顯著提高，且在5公升發酵槽當中1,12-diol生產量達到11.56 g/L。為進一步提升生產量，針對內源性1-dodecanol代謝路徑進行基因剔除。發現在搖瓶實驗中，剔除其中負責啟動β-氧化的fadD，能顯著提升1,12-diol 的累積量。；相反地，當大腸桿菌中之酒精去氫酶adhE與yiaY分別被剔除後卻導致目標產量下降，推測因該基因剔除導致NADH再生能力受損，CYP153A反應受限而產量下降。此策略亦應用於發酵槽試驗，然而fadD剔除株產率不如預期，可能與培養基碳氮比與培養條件相關代謝壓力變動有關。整體而言，本研究成功建立以AIS為基礎，結合代謝剔除策略，展現轉換基質與氧化酶誘導劑合一下，生產1,12-diol之潛力，對未來長鏈脂肪族二醇或其氧化衍生物之綠色製程應用具高度發展潛力。

1,12-Dodecanediol (1,12-diol), a medium- to long-chain diol with high chemical stability and hydrophilicity, is widely used in industrial applications such as lubricants, polymer precursors, and surfactants. Currently, its production primarily relies on chemical catalysis, which poses environmental concerns and high process costs. To enable sustainable and environmentally friendly production, this study engineered Escherichia coli as a microbial host for the biosynthesis of 1,12-diol. The key oxidative enzyme used in this process is cytochrome P450 monooxygenase CYP153A, which catalyzes the terminal oxidation of 1-dodecanol to 1,12-diol. To enhance uptake of substrate, the alkane transporter AlkL was co-expressed. In previous systems, AlkL expression was regulated by the rhaBAD promoter, requiring the addition of the external inducer L-rhamnose. While this improved conversion efficiency, it added operational complexity and increased costs. Therefore, this study established an Alkane Induction System (AIS). In this system, the alkane-responsive regulator AlkR senses the substrate 1-dodecanol and activates the Pcyp promoter, thereby driving cyp153A and alkL expression and achieving synchronized induction and product formation. qPCR data revealed significantly elevated alkR and alkL mRNA levels in AIS strains even without additional inducers, and 1,12-diol production reached 11.56 g/L in a 5-L bioreactor with 1.5 L medium. To further increase yield, endogenous genes involved in 1-dodecanol catabolism were disrupted. Knockout of fadD in β-oxidation pathway effectively enhanced 1,12-diol production, whereas individual knockouts of either adhE or yiaY led to decreased production, likely due to impaired NADH regeneration that limited CYP153A catalytic activity. This knockout strategy was also applied in bioreactor trials; however, the ΔfadD strain did not perform as expected, potentially due to changes in medium carbon-to-nitrogen ratio and scale-dependent metabolic stress. In summary, this study successfully developed a platform that integrates substrate-inducible expression via the AIS with metabolic engineering strategies. It demonstrated the potential for efficient 1,12-diol by utilizing the substrate as an inducer, eliminating the need for additional inducers and offering promising applications for the green biosynthesis of long-chain aliphatic diols and their oxidized derivatives.