(449a) Redox Balance-Based Growth Selection As a Universal Tool for Enzyme Engineering

Over 15,000 NAD(P)H-dependent enzymes discovered in Nature represents a rich yet largely untapped resource for catalyst development, with many of these enzymes outperforming any man-made catalysts. To meet the needs of in vitrobiotransformation and in vivo metabolic engineering, these enzymes often need their substrate and cofactor specificity tuned, and their stability and turnover rate improved. However, there's currently no facile ways to accomplish these engineering goals. Traditional methods which typically rely on 96 well-based in vitro assays do not provide sufficient throughput to deeply search a large protein sequence space, and do not directly evaluate enzyme's functionality in vivo. Here, we report the development of multiple ultrahigh-throughput (~10⁹ candidates per round of selection), universal, in vivo selection platforms which use cell growth as an easy readout of NAD(P)H-dependent enzyme's activity. These selection platforms are designed based on the general principle of redox balance, where NAD(P)⁺ to NAD(P)H ratio in Escherichia coli cells is drastically perturbed so that cell survival requires a heterologous enzyme that can utilize the desired substrate with sufficiently high activity in vivo and concomitantly restore the cofactor redox balance. Using these selection platforms, we have achieved, through just a single round of selection, remodeling of 4-hydroxybenzoate hydroxylase (PobA) to accept a nonnative substrate for natural product biosynthesis, improving the coupling efficiency of cytochrome p450 BM3 for environmental pollutant degradation, and switching the cofactor specificity and enhancing the thermal stability of an industrially important enzyme cyclohexanone monooxygenase (CHMO). Our platforms complete the scope of redox balance-based in vivo selection by covering both NADH and NADPH cofactors, and both aerobic and anaerobic conditions. We envision their broad applications in accelerating enzyme engineering.