High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase

Abstract

2-Keto-L-gulonic acid (2-KLG) is the direct precursor for the production of L-ascorbic acid (L-Asc) on industrial scale. Currently, the production of L-Asc in the industry is a two-step fermentation process. Owing to many unstable factors in the fermentation process, the conversion rate of L-sorbose to 2-KLG has remained at about 90% for many years. In order to further improve the production efficiency of 2-KLG, a FAD-dependent sorbose dehydrogenase (SDH) has been obtained in our previous research. The SDH can directly convert L-sorbose to 2-KLG at a very high efficiency. However, the enzyme activity of the SDH is relatively low. In order to further improve the enzyme activity of the SDH, a high throughput screening platform the dehydrogenase is essential. By optimizing the promoter, host and sorbosone dehydrogenase (SNDH), knockout of the aldosterone reductases and PTS related genes, a reliable platform for high-throughput screening of more efficient FAD-dependent SDH has been established. By using the high-throughput screening platform, the titer of the 2-KLG has been improved by 14.1%. The method established here could be useful for further enhancing the FAD-dependent SDH, which is important to achieve the efficient one-strain-single-step fermentation production of 2-KLG.