Wohlert S, Lomovskaya N, Kulowski K, Fonstein L, Occi J L, Gewain K M, MacNeil D J, Hutchinson C R
School of Pharmacy, University of Wisconsin, Madison, WI 53706, USA.
Chem Biol. 2001 Jul;8(7):681-700. doi: 10.1016/s1074-5521(01)00043-6.
The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis.
A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose.
The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.
阿维菌素由阿维链霉菌产生，是一种强效驱虫剂，具有聚酮衍生的大环内酯骨架，与由两个α-连接的L-夹竹桃糖单元组成的二糖相连。八个相邻基因，avrBCDEFGHI（也称为aveBI - BVIII），位于阿维菌素生产基因簇内，先前已被定位到胸苷二磷酸 - 夹竹桃糖与阿维菌素苷元的生物合成和连接过程中。这个基因盒为研究2,6 - 二脱氧糖（即L - 夹竹桃糖）的生物合成以及通过组合生物合成改变阿维菌素的生物合成和结构提供了一种便捷的方法。
一个带有avrBCDEFGHI基因的单质粒的淡紫链霉菌菌株，其中avrBEDC和avrIHGF分别在actI和actIII启动子的控制下表达，能正确地将外源阿维菌素A1a苷元与相同的夹竹桃糖单元进行糖基化，生成阿维菌素A1a。这个最小基因集的修饰版本产生了新型的单糖和二糖阿维菌素。这些结果为L - 夹竹桃糖的生物合成提供了进一步的见解。
基于质粒的avr脱氧糖基因在异源系统中的表达重建与生物转化相结合，产生了关于2,6 - 脱氧糖生物合成机制的新信息。积累的双去甲基脱氧糖阿维菌素的结构表明，在夹竹桃糖途径中，C - 3位的立体化学最终由3 - O - 甲基转移酶决定，而不是由3 - 酮还原酶或可能的3,5 - 表异构酶决定。因此，AvrF蛋白是一种5 - 表异构酶，而不是3,5 - 表异构酶。从产生的新型阿维菌素的结构可以明显看出，AvrB（单）糖基转移酶能够容纳不同的脱氧糖中间体。
