Construction of novel fatty acid ester library and its application

本研究的目的是构建一个由多种甾醇(如胆固醇和植物甾醇)的新型脂肪酰酯组成的文库。为了将甾醇和脂肪酸酯化，我们使用了一种胆固醇酯酶，这是我们在本研究中在链霉菌培养滤液中发现的。X9.对该酶进行了纯化和基因克隆，发现该酶是分布在放线杆菌中的一个新的酯酶家族。我们还构建了一个基于重组大肠杆菌系统的高效生物转化系统，该系统产生真菌细胞色素P450foxy(P450foxy)，该真菌细胞色素P450foxy可使脂肪酸亚端碳羟化。该系统将提供各种羟基脂肪酸与甾醇进行酯化反应。在对P450foxy的研究过程中，我们发现它可以将脂肪醇羟化，生成烷基二醇，尽管反应速度低于脂肪酸。本研究的另一个目的是揭示嗜热嗜热菌独特的类胡萝卜素化合物的生理和物理性质。主要研究结果如下。(1)细菌β-胡萝卜素3，3‘-羟基酶、CrtZ和P450在虾青素生物合成途径中的特性和在大肠杆菌中通过基因组合产生腺红素；(2)发现玉米黄质、玉米黄质葡萄糖苷和热玉米黄质等类胡萝卜素对脂膜的稳定作用，这三种类胡萝卜素都是嗜热木霉的特异性产物。最后，我们成功地利用放线细菌胆固醇酯酶合成了各种甾醇酯。所产生的酯是那些与棕榈酸、肉豆蔻酸、亚油酸和硬脂酸相结合的脂肪酰基部分，以及胆固醇、麦角甾醇和豆甾醇相结合的甾醇部分。羊毛甾醇在我们的体内是不活跃的。

The aim of this study was to construct a library consisted of novel fatty acyl esters of various sterols such as cholesterol and phytosterols. To esterify sterols with fatty acids, we used a cholesterol esterase, which we found in this study in culture filtrate of the Streptomyces sp. X9. The enzyme was purified and its gene was cloned, and revealed to constitute a novel family of esterases distributed in actinobacteria. We also constructed an efficient bioconversion system based on recombinant Escherichia coli system producing the fungal cytochrome P450foxy (P450foxy) that hydroxylates subterminal carbons of fatty acids. This system will provide variety of hydroxyl fatty acids to be esterified with sterols. During course of studies on P450foxy, we revealed that it hydroxylated aliphatic alcohols to produce alkyl diols as well although reaction rates were lower than for fatty acids. Another objective of this study was to reveal physiological and physical properties of unique carotenoid compounds of the thermophilic Thermus thermophilus. Major results were as follows. (1) Characterization of bacterial beta-carotene 3, 3'-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli and (2) finding of stabilizing effect of liposomal membranes by carotenoids such as zeaxanthin, zeaxanthin glucoside and thermozeaxanthin both of which are specific products of T. thermophilus. Finally, we succeeded in synthesizing various sterol esters of fatty acids by the actinobacterial cholesterol esterase. The produced esters were those combined with palmitic, myristic, linoleic, and stearic acids as fatty acyl moieties and cholesterol, ergosterol, and stigmasterol as sterol moieties. Lanosterol was inactive in our system.