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Construction of novel fatty acid ester library and its application

新型脂肪酸酯库的构建及其应用

基本信息

批准号：
17380051
负责人：
HOSHINO Tskayuki
金额：
$ 10.16万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

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.

本研究的目的是建立包括胆固醇和植物甾醇在内的多种甾醇的新型脂肪酸酯文库。为了使甾醇与脂肪酸酯化，我们使用了一种胆固醇酯酶，该酶是我们在Streptomyces sp. X9的培养滤液中发现的。对该酶进行了纯化和基因克隆，发现它是分布在放线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，产生真菌细胞色素P450foxy (P450foxy)，该系统能羟基化脂肪酸的亚末端碳。该系统将提供各种羟基脂肪酸与甾醇酯化。在对P450foxy的研究过程中，我们发现尽管反应速率低于脂肪酸，但它也能羟基化脂肪族醇生成烷基二醇。本研究的另一个目的是揭示嗜热热菌独特的类胡萝卜素化合物的生理和物理特性。主要结果如下：(1)利用大肠杆菌基因组合研究细菌β -胡萝卜素3,3′-羟化酶、CrtZ和P450在虾青素生物合成途径和adonirubin生产中的作用；(2)发现玉米黄质、玉米黄质糖苷和热玉米黄质等类胡萝卜素对脂质体膜的稳定作用，这些类胡萝卜素都是嗜热t菌的特异性产物。最后，我们成功地利用放线菌胆固醇酯酶合成了多种脂肪酸的甾醇酯。所产生的酯是与棕榈酸、肉豆蔻酸、亚油酸和硬脂酸结合的脂酰部分和胆固醇、麦角甾醇和豆甾醇结合的脂酰部分。我们体内的羊毛甾醇没有活性。