Kwon Seok Joon, Lee Moo-Yeal, Ku Bosung, Sherman David H, Dordick Jonathan S
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
ACS Chem Biol. 2007 Jun 15;2(6):419-25. doi: 10.1021/cb700033s. Epub 2007 May 25.
The generation of biological diversity by engineering the biosynthetic gene assembly of metabolic pathway enzymes has led to a wide range of "unnatural" variants of natural products. However, current biosynthetic techniques do not allow the rapid manipulation of pathway components and are often fundamentally limited by the compatibility of new pathways, their gene expression, and the resulting biosynthetic products and pathway intermediates with cell growth and function. To overcome these limitations, we have developed an entirely in vitro approach to synthesize analogues of natural products in high throughput. Using several type III polyketide synthases (PKS) together with oxidative post-PKS tailoring enzymes, we performed 192 individual and multienzymatic reactions on a single glass microarray. Subsequent array-based screening with a human tyrosine kinase led to the identification of three compounds that acted as modest inhibitors in the low-micromolar range. This approach, therefore, enables the rapid construction of analogues of natural products as potential pharmaceutical lead compounds.
通过工程改造代谢途径酶的生物合成基因组装来产生生物多样性，已导致天然产物出现了广泛的“非天然”变体。然而，当前的生物合成技术不允许对途径组件进行快速操作，并且常常从根本上受到新途径的兼容性、其基因表达以及所得生物合成产物和途径中间体与细胞生长和功能的限制。为了克服这些限制，我们开发了一种完全体外的方法，以高通量方式合成天然产物类似物。我们将几种III型聚酮合酶（PKS）与PKS后氧化修饰酶一起使用，在单个玻璃微阵列上进行了192个单独的和多酶反应。随后用人类酪氨酸激酶进行基于阵列的筛选，鉴定出了三种在低微摩尔范围内起适度抑制作用的化合物。因此，这种方法能够快速构建作为潜在药物先导化合物的天然产物类似物。
