Zhang Keya, Nelson Kathryn M, Bhuripanyo Karan, Grimes Kimberly D, Zhao Bo, Aldrich Courtney C, Yin Jun
Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
Chem Biol. 2013 Jan 24;20(1):92-101. doi: 10.1016/j.chembiol.2012.10.020.
The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains.
非核糖体肽合成酶（NRPSs）的腺苷化（A）结构域激活芳酸或氨基酸，使其通过NRPS装配线进行转移，用于许多具有重要药用价值的天然产物的生物合成。为了扩大NRPSs的底物库，我们开发了一种基于酵母细胞表面展示的方法来改造A结构域的底物特异性。我们获得了DhbE的A结构域突变体，它们对非天然底物3-羟基苯甲酸和2-氨基苯甲酸的k(cat)/K(m)分别增加了11倍和6倍，而对天然底物2,3-二羟基苯甲酸的k(cat)/K(m)值相应降低了3倍和33倍，导致底物特异性发生了高达200倍的显著转变。我们的研究表明，酵母展示可作为一种高通量筛选平台，用于重新编程A结构域的“非核糖体密码”。
