Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus
Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus
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DOI：
10.1016/j.synbio.2019.07.001
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发表时间：
2019-07
影响因子：
4.8
通讯作者：
Weizhu Zeng;W. Cai;Li Liu;G. Du;Jian Chen;Jingwen Zhou
中科院分区：
文献类型：
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作者：
Weizhu Zeng;W. Cai;Li Liu;G. Du;Jian Chen;Jingwen Zhou
2-酮基-d-葡萄糖酸（2-KGA）是合成维生素C和异维生素C的关键前体。然而，在利用荧光假单胞菌生产2-KGA的工业化生产中，噬菌体污染一直是一个问题。本研究通过调控2-KGA合成途径，提高2-KGA的产量，减少副产物5-酮基-d-葡萄糖酸（5-KGA）和d-葡萄糖酸（D-GA）的积累。敲除竞争途径中的ga 5dh-1基因，通过同源重组过量表达2-KGA合成途径中的ga 2dh-A基因，摇瓶效价提高63.81%。此外，5-KGA的积累减少了63.52%。japonicas-Δga5dh-1-ga2dh-Astrain.在3 L发酵罐中使用间歇补料分批模式，2-KGA达到235.3 g L− 1，葡萄糖转化率为91.1%。在15 L发酵罐中放大导致稳定的2-KGA滴度，生产率为2.99 g L−1h−1，比3 L发酵罐高11.99%，并且D-GA和5-KGA副产物完全转化为2-KGA。
2-keto-d-gluconic acid (2-KGA) is a key precursor for synthesising vitamin C and isovitamin C. However, phage contamination is as constant problem in industrial production of 2-KGA usingPseudomonas fluorescens.Gluconobacterholds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids, and high utilisation of glucose. In this study, the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-d-gluconic acid (5-KGA) andd-gluconic acid (D-GA) in the 2-KGA producerGluconobacter japonicusCGMCC 1.49. Knocking out thega5dh-1gene from a competitive pathway and overexpressing thega2dh-Agene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81% in shake flasks. Additionally, accumulation of 5-KGA was decreased by 63.52% with the resultingG. japonicas-Δga5dh-1-ga2dh-Astrain. Using an intermittent fed-batch mode in a 3 L fermenter, 2-KGA reached 235.3 g L−1with a 91.1% glucose conversion rate. Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L−1h−1, 11.99% higher than in the 3 L fermenter, and D-GA and 5-KGA by-products were completely converted to 2-KGA.