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

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ID: 84169
2019
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Ranked #20 of 41 articles by views in Frontiers in bioengineering and biotechnology

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Abstract
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).
Reference Key
chen2019highthroughputfrontiers Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Chen, Yue;Liu, Li;Shan, Xiaoyu;Du, Guocheng;Zhou, Jingwen;Chen, Jian;
Journal Frontiers in bioengineering and biotechnology
Year 2019
DOI
10.3389/fbioe.2019.00385
URL
Keywords
high-throughput screening 2-keto-l-gulonic acid 2-ketoaldonate reductase gluconobacter oxydans l-sorbose fluorescence-activated cell sorting

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