Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol
Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol
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DOI：
10.1016/j.ymben.2014.04.003
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发表时间：
2014-07-01
影响因子：
8.4
通讯作者：
Chen, Jian
中科院分区：
文献类型：
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作者：
Gao, Lili;Hu, Yudong;Chen, Jian
2-酮基-L-古洛糖酸（2-KLG）是维生素C的直接前体，目前通过两步发酵路线从D-山梨糖醇生产。然而，这一途径涉及三种细菌，使得混合培养系统复杂且多余。因此，用一步法代替传统的两步发酵法可能是维生素C工业的革命性进展。本研究将来自Ketogulonicigenium vulgare WSH-001的5种L-山梨糖醇脱氢酶（SDH）和2种L-山梨糖酮脱氢酶（SNDH）的不同组合导入用于D-山梨糖醇转化为L-山梨糖的工业菌株氧化葡糖醛酸葡糖醇菌WSH-003中。最佳组合产生4.9g/L的2-KLG。此外，还利用10种不同的接头肽在G.氧化丹。重组菌G. oxydans/pGUC-k 0203-GS-k 0095）在168小时后产生32.4g/L的2-KLG。此外，吡咯喹啉奎宁（PQQ），这些酶的辅因子的生物合成得到加强，以提高2-KLG生产。利用G.氧化物，最终的2-KLG产量提高到39.2 g/L，这是8.0倍高于使用独立表达的酶获得的。这些结果使我们更接近最终的一步工业规模生产维生素C。(C)2014年国际代谢工程学会。爱思唯尔公司出版All rights reserved.
2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of D-sorbitol to L-sorbose. The optimum combination produced 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4 g/L of 2-KLG after 168 h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2 g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.