以大腸桿菌 K12 MG1655 菌株之誘導型轉運體表現系統生產 1, 12-十二烷二醇之研究

Abstract

中長鏈 α, ω-烷二醇在工業上已被應用為聚氨酯或聚酯之前驅物。但是，此化合物並不存在於自然界，所以人工合成為唯一獲得之方法。至今，最主要合成α, ω-烷二醇之化學催化方法為氫醛化反應，但是此方法具有區域選擇性與立體選擇性之劣勢，影響合成產物的純度。在過去研究中發現，細胞色素P450單氧化酶之 CYP153A具有ω-羥基化之專一性，經由全細胞催化法，可解決化學催化法的缺點。另外，在 α, ω-烷二醇合成中使用的基質為疏水性化合物，不利於穿入細胞膜內中。為了加速基質穿膜率，親疏水性化合物之轉運體 (Transporter) 必要導入此系統。本研究使用長鏈自由脂肪酸轉運體 FadL， 以及可能為轉運體 Maqu0434。接著，以上述系統與 AraC/XylS 家族轉錄調節因子之鼠李糖誘導系統建構於重組大腸桿菌 K12 MG1655。本研究使用1.5 L之饋料批式發酵生物反應器培養過表現上述 FadL運轉體之菌株，以 1.5 g/L/h 之甘油與 0.3 g/L/h 之酵母萃取物的饋料速率可達到 17.69 g/L之1, 12-十二烷二醇的產量。並以動態檢測各基因之表現來分析菌體生理狀況。

Medium to long-chain length α, ω-alkanediols are applied as precursors of polyurethanes and polyesters. However, the absence of these compounds in nature made it ineluctable to synthesis artificially. Hydroformylation, one of a major chemical α, ω-alkanediols synthetic methods, is adverse for its regio-selectivity and stereo-selectivity caused by chemical catalysts. Previously, a cytochrome P450 monooxygenase, CYP153A, was unveiled its capability of ω-hydroxylation, in order that whole-cell bioproduction of α, ω-alkanediols was suggested. However, there was still a bottleneck about substrates uptake rate, since the substrates utilized was hydrophobic. To boost the uptake rate of substrate, long-chain fatty acid transporter FadL and a novel potential transporter Maqu0434 were applied, respectively. The transporters were expressed by rhamnose inducible system with AraC/XylS family of transcriptional regulators. By transfecting Escherichia coli K12 MG1655 with plasmids containing genes of FadL, we recorded 17.69 g/L of 1, 12-dodecanediol in fed-batch bioreactor with 1.5 g/L/h of glycerol and 0.3 g/L/h of yeast extract as feeding strategy. Further, the gene expression level was monitored dynamically to improve the fermentation condition.