Imperial College Centre for Synthetic Biology, Imperial College London, London, SW7 2AZ, UK.
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Nat Commun. 2021 Nov 25;12(1):6859. doi: 10.1038/s41467-021-27266-9.
The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.
工业应用的非自然需求通常需要新的或改进的酶。酶的结构和性质很难预测或从头设计。相反，模仿进化的半理性方法需要对亲本酶进行多样化，然后对分离的变体进行评估。将所需酶特性与细胞生长相耦合的人工选择压力可以克服这一关键瓶颈，但通常范围较窄。在这里，我们展示了使用普遍存在的辅因子烟酰胺腺嘌呤二核苷酸（NAD）或烟酰胺腺嘌呤二核苷酸磷酸（NADP）的各种酶可以替代有缺陷的 NAD 再生，这代表了一种非常广泛适用的人工选择。大肠杆菌中用于厌氧 NAD 再生的基因失活会导致条件生长缺陷。只有当提供其底物时，通过代谢网络仅与 NAD 或 NADP 连接的外源酶才能拯救细胞。利用这一原理，从数百万个变体的文库中，在具有典型有限信息以指导设计的单轮实验中，分离出具有所需选择性修饰的醇脱氢酶、亚胺还原酶和硝基还原酶变体，以及高性能异丙醇代谢途径。
