Huang Rui, Chen Hui, Upp David M, Lewis Jared C, Job Zhang Yi-Heng P
Biological Systems Engineering Department, Virginia Tech, Blacksburg, Virginia 24061, United States.
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.
ACS Catal. 2019 Dec 6;9(12):11709-11719. doi: 10.1021/acscatal.9b03840. Epub 2019 Nov 7.
Engineering flavin-free NAD(P)-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNADs) is of importance for eliminating the need for costly NAD(P) in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency ( / ) and a specific activity of 17.7 U/mg on NMN, which is comparable to the wild-type enzyme on its natural coenzyme, NADP. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNADs similar to the levels of natural enzyme/coenzyme pairs.
工程改造无黄素的NAD(P)依赖性脱氢酶以还原仿生烟酰胺类似物（mNADs）对于消除辅酶再生系统中对昂贵的NAD(P)的需求至关重要。当前基于氧化还原染料的用于工程改造脱氢酶mNAD特异性的筛选方法经常受到内源性NAD(P)和细胞内还原化合物的背景信号的阻碍，使得基于低mNAD的活性检测成为定向进化的限制因素。在此，我们开发了一种高通量筛选方法，即NAD(P)消除固相测定法（NESPA），它能够以最小的背景信号可靠地鉴定脱氢酶的mNAD活性突变体。该方法包括：（1）对菌落进行热裂解以通透细胞膜；（2）将菌落转移到滤纸上；（3）洗涤以去除内源性NAD(P)和还原化合物；（4）用于mNADH依赖性显色的酶联测定；（5）对菌落进行数字成像以鉴定mNAD活性突变体。该方法用于提高6-磷酸葡萄糖酸脱氢酶对烟酰胺单核苷酸（NMN）的活性。经过六轮定向进化获得的最佳突变体在催化效率（/）上提高了50倍，对NMN的比活性为17.7 U/mg，这与野生型酶对其天然辅酶NADP的活性相当。然后，将工程改造的脱氢酶用于构建NMNH再生系统以驱动烯还原酶催化。相对于使用野生型脱氢酶进行NADPH再生，使用工程改造系统观察到了相当水平的周转频率和产物产量。NESPA提供了基于mNAD活性的简单而准确的读数，并能进行高通量筛选（大约每轮数万），从而为具有与天然酶/辅酶对水平相似的对mNADs的特定活性的脱氢酶的进化开辟了一条途径。
