以 Marinobacter aquaeolei VT8 之細胞色素氧化酶 CYP153A 操縱組進行中長碳鏈烷類之末端氧化

Whole-cell bio-oxidation of medium-long chain alkanes by Marinobacter aquaeolei VT8 alkane-inducible CYP153A operon

謝善棋(Shan-Chi Hsieh)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11268

摘要

以生物催化進行中長碳烷類的末端氧化面臨諸多困難。常用的 AlkB 系統具有過度氧化活性，會將醇類產物進一步變成醛和酸。CYP153A 系統過度氧化活性較輕微，但其烷類調控蛋白仍未被發現，且廣為使用的AlkS烷類誘導表現系統無法被較長碳鏈（> C12）的烷類誘導。因此以 CYP153A 進行中長碳鏈烷類的生物轉化仍需外加誘導物。本研究分析 Marinobacter aquaeolei VT8 之 CYP153A 操縱組上游的 AraC 家族蛋白，發現其為烷類調控蛋白（AlkRM. aq），可受中長碳鏈（C8-C16）烷類以及月桂酸甲酯（methyl-laurate）誘導，且不會受到烷醇產物的抑制。在發現並移除 CYP153A 操縱組內的轉位子後，包含此調控蛋白的 CYP153A 系統可以讓戀臭假單孢菌 （Pseudomonas putida mt-2）以辛烷為唯一碳源生長，並且在大腸桿菌中可以表現 CYP153A 操縱組將辛烷轉化為辛醇。這證實了 AlkRM. aq 為 CYP153AM. aq 系統的一部份，而且CYP153AM. aq 系統在異源宿主仍保有功能。此烷類誘導調控蛋白亦可以用來評估烷類運輸蛋白 AlkL 的功能和最佳表現強度。藉著 AlkL 以及 AlkRM. aq，我們得以利用 E. coli JM109 進行中長碳鏈烷類的全細胞生物轉化。以小型發酵槽進行生物轉化可以產生約 1.5 g/Ltotal 的十二烷醇和十四烷醇。經 GC/MS 分析發現過氧化現象很輕微。本研究證實並分析了過去未被研究過的 AraC/XylS 家族的烷類誘導轉錄調控蛋白，和過去常用的 AlkB 系統相比，此 CYP153A 系統不但可以受較長碳鏈的烷類誘導，也比較不會有產物過度氧化的問題，是烷醇生產上更好的選擇。

關鍵字

CYP153A ； 烷類調控蛋白 ； 十二烷 ； 烷醇 ； 生物轉化

並列摘要

Bio-oxidation of medium-long chain alkanes has many obstacles. Intensively studied AlkB system tends to overoxidize alcohol products. CYP153A has less overoxidation activity. But the regulator of CYP153A was not characterized and widely used AlkS expression system cannot be induced by alkanes longer than C12, thus the biotransformation of medium-long chain alkanes with CYP153A has to rely on external inducers. This study demonstrated that the AraC family protein upstream of the CYP153A operon from Marinobacter aquaeolei VT8 is medium-long chain (C8-C16) alkane response regulator (AlkRM. aq). The regulator can also be induced by FAME and is not subject to product inhibition in E. coli. After identifying and removing the transposon inside CYP153AM. aq operon, the regulator can drive the CYP153A expression upon induction. With the regulator, the CYP153AM. aq system enabled P. putida mt-2 to use octane as sole carbon source and can bio-oxidized octane in E. coli JM109 without external inducers. These results proved that the AlkRM. aq is part of CYP153AM. aq system, and the CYP153AM. aq system is functional in heterologous hosts. The regulator was also used as biosensor to evaluate the function and optimal expression level of the alkane facilitator AlkL. With AlkL and AlkRM. aq, it is feasible to whole-cell bio-oxidize medium-long chain alkanes in E. coli JM109. The production of 1-dodecanol and 1-tetradecanol reached about 1.5 g/Ltotal in bioreactor with limited overoxidation.

並列關鍵字

CYP153A ； alkane response regulator ； dodecane ； alkanol ； biotransformation

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