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

Xiaoyu Shan,Jingwen Zhou,Weizhu Zeng,Li Liu,Jian Chen

doi:10.3389/fbioe.2020.00194

Xiaoyu Shan, Jingwen Zhou + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2020.00194

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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.

Highlights

Vitamin C, known as L-ascorbic acid (L-Asc), is an essential nutrient for human beings and occupies the largest global market among vitamins
By expressing the sorbose dehydrogenase (SDH) with the optimum promoter PcspA in different E. coli strains, it was found that SDH could convert L-sorbose to 2-Keto-L-gulonic acid (2-KLG) in E. coli K-12 substr
The results showed that the optimum temperature for 2-KLG production was 37◦C, under which the 2-KLG titer could reached to 2.83 g/L

Summary

Introduction

Vitamin C, known as L-ascorbic acid (L-Asc), is an essential nutrient for human beings and occupies the largest global market among vitamins. It can affect cells by participating in the elimination of reactive oxygen species (Wenzel et al, 2004), promoting collagen production (May and Qu, 2005), and helping immune defense (Carr and Maggini, 2017). The process has two fermentation steps and requires two times of sterilization, resulting in high energy consumption and long production period (Liu et al, 2011a,b). To achieve the one-step-single-strain production of 2-KLG is a long pursued goal for the vitamin C industry

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Conclusion