2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is produced by a two-step fermentation route from D-sorbitol in industry. However, this route is a complicated mix-culture system which involves three bacteria. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. The one-step fermentation of 2-keto-L-gulonic acid (2-KLG) has been achieved in our previous study; 32.4 g/L of 2-KLG production was obtained by the one-step strain G. oxydans/pGUC-tufB-sdh-GGGGS-sndh after 168 h. In this study, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) were expressed in G. oxydans after the codon optimization. Furthermore, the trimeric protein CutA was used to improve the chemical structure stability of SDH and SNDH. The recombinant strain G. oxydans/pGUC-tufB-SH3-sdh-GGGGS-sndh-tufB-SH3lig-(GGGGS)2-cutA produced 40.3 g/L of 2-KLG after 168 h. In addition, the expression levels of the cofactor PQQ were enhanced to further improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 42.6 g/L. The efficient one-step production of 2-KLG was achieved, and the final one-step industrial-scale production of 2-KLG is drawing near.

中文翻译：

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基于蛋白质支架-三聚体 CutA 的氧化葡糖杆菌的有效优化，以提高用于直接生产 2-酮-L-古洛糖酸的酶的化学结构稳定性

2-Keto-L-古洛糖酸 (2-KLG) 是维生素 C 的直接前体，在工业上是通过两步发酵路线从 D-山梨糖醇生产的。然而，这条路线是一个复杂的混合培养系统，涉及三种细菌。因此，用一步法代替传统的两步发酵法在维生素 C 行业可能是革命性的。2-酮基-L-古洛糖酸（2-KLG）一步发酵在我们之前的研究中已经实现；168 小时后，一步菌株 G. oxydans/pGUC-tufB-sdh-GGGGS-sndh 获得 32.4 g/L 的 2-KLG 产量。在本研究中，L-山梨糖脱氢酶 (SDH) 和 L-山梨糖酮脱氢酶 (SNDH) 在密码子优化后在 G. oxydans 中表达。此外，三聚体蛋白 CutA 用于提高 SDH 和 SNDH 的化学结构稳定性。重组菌株 G. oxydans/pGUC-tufB-SH3-sdh-GGGGS-sndh-tufB-SH3lig-(GGGGS)2-cutA 在 168 小时后产生 40.3 g/L 的 2-KLG。此外，辅因子 PQQ 的表达水平得到增强，以进一步提高 2-KLG 的产量。通过 G. oxydans 的逐步代谢工程，最终的 2-KLG 产量提高到 42.6 g/L。实现了2-KLG的高效一步生产，2-KLG最终的一步工业化生产即将实现。