High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Strain Based on Related Dehydrogenases.

High-throughput screening is a powerful tool for discovering strains in the natural environment that may be suitable for target production. Herein, a novel enzyme-based high-throughput screening method was developed for rapid screening of strains overproducing 2-keto-L-gulonic acid (2-KLG). The screening method detects changes in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm using a microplate reader when 2-KLG is degraded by 2-KLG reductase. In this research, three different 2-KLG reductases were expressed, purified, and studied. The 2-KLG reductase from were selected as the best appropriate reductase to establishment the method for its high activity below pH 7. Using the established method, and coupled with fluorescence-activated cell sorting, we achieved a high 2-KLG-producing strain of WSH-004 from soil. When cultured with D-sorbitol as the substrate, the 2-KLG yield was 2.5 g/L from 50 g/L D-sorbitol without any side products. Compared with other reported screening methods, our enzyme-based method is more efficient and accurate for obtaining high-producing 2-KLG strains, and it is also convenient and cost-effective. The method is broadly applicable for screening keto acids and other products that can be oxidized via nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP).