Abstract
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 strainG. oxydans/pGUC-tufB-sdh-GGGGS-sndhafter 168 h. In this study, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) were expressed inG. oxydansafter the codon optimization. Furthermore, the trimeric protein CutA was used to improve the chemical structure stability of SDH and SNDH. The recombinant strainG. oxydans/pGUC-tufB-SH3-sdh-GGGGS-sndh-tufB-SH3lig-(GGGGS)2-cutAproduced 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 ofG. 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.
Highlights
Vitamin C (L-ascorbic acid, L-AA) is widely used in pharmaceutical, food, beverages, cosmetics, and feed industries [1]. e most successful route for industrial production of vitamin C is the classical two-step fermentation process. e fermentation process contains one-step conversion from D-sorbitol to L-sorbose by Gluconobacter oxydans, followed by another step of converting L-sorbose to 2-keto-gulonic acid (2-KLG), the precursor of vitamin C, by a mixed culture system of Ketogulonicigenium vulgare and Bacillus megaterium
With the stepwise metabolic engineering of G. oxydans, the final 2-Keto-L-gulonic acid (2-KLG) production was improved to 42.6 g/L, which was 5.7% higher than that by pGUC-tufB-SH3-sdh-GGGGSsndh-tufB-SH3lig-(GGGGS)2-cutA
Five sorbose dehydrogenase (SDH) (KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, and KVU_0203) and two sorbosone dehydrogenase (SNDH) (KVU_0095 and KVU_pmdB_0115) in K. vulgare WSH-001 were identified [17], which were introduced into G. oxydans WSH-003 in different combinations to construct the one-step strain
Summary
Vitamin C (L-ascorbic acid, L-AA) is widely used in pharmaceutical, food, beverages, cosmetics, and feed industries [1]. e most successful route for industrial production of vitamin C is the classical two-step fermentation process. e fermentation process contains one-step conversion from D-sorbitol to L-sorbose by Gluconobacter oxydans, followed by another step of converting L-sorbose to 2-keto-gulonic acid (2-KLG), the precursor of vitamin C, by a mixed culture system of Ketogulonicigenium vulgare and Bacillus megaterium. E most successful route for industrial production of vitamin C is the classical two-step fermentation process. E fermentation process contains one-step conversion from D-sorbitol to L-sorbose by Gluconobacter oxydans, followed by another step of converting L-sorbose to 2-keto-gulonic acid (2-KLG), the precursor of vitamin C, by a mixed culture system of Ketogulonicigenium vulgare and Bacillus megaterium. K. vulgare is difficult to culture alone and it possesses a rather low production capacity of 2-KLG. Researchers have attempted to improve fermentation processes in many ways; tremendous advances have been achieved in microbial production of vitamin C [2,3,4,5]. The mix-culture system makes the process optimization difficult. In consideration of problems presented above in the two-step fermentation process, using D-glucose or Journal of Chemistry
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