State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
J Agric Food Chem. 2015 Jun 10;63(22):5492-8. doi: 10.1021/acs.jafc.5b01652. Epub 2015 Jun 2.
2-Keto-D-gluconic acid (2KGA), a precursor of the important food antioxidant erythorbic acid, can be produced by Gluconobacter oxidans. To genetically engineer G. oxidans for improved 2KGA production, six new expression vectors with increased copy numbers based on pBBR1MCS-5 were constructed via rational mutagenesis. The utility of the mutant vectors was demonstrated by the increased ga2dh mRNA abundance, enzyme activity, and 2KGA production when the ga2dh gene was overexpressed using these vectors. Among the obtained constructs, G. oxidans/pBBR-3510-ga2dh displayed the highest oxidative activity toward gluconic acid (GA). In a batch biotransformation process, the G. oxidans/pBBR-3510-ga2dh strain exhibited 2KGA productivity (0.63 g/g CWW/h) higher than that obtained using strain G. oxidans/pBBR-ga2dh (0.40 g/g CWW/h). When sufficient oxygen was supplied during the biotransformation, up to 480 g/L GA was exhausted in 45 h by the G. oxidans/pBBR-3510-ga2dh strain and approximately 486 g/L 2KGA was produced, generating the productivity of 0.54 g/g CWW/h.
2-酮基-D-葡萄糖酸（2KGA）是重要食品抗氧化剂赤藻糖酸的前体，可以由氧化葡萄糖酸杆菌生产。为了通过遗传工程改造氧化葡萄糖酸杆菌以提高 2KGA 的产量，通过合理的诱变构建了六个基于 pBBR1MCS-5 的拷贝数增加的新表达载体。当使用这些载体过表达 ga2dh 基因时，ga2dh mRNA 丰度、酶活性和 2KGA 产量的增加证明了突变载体的实用性。在所获得的构建体中，氧化葡萄糖酸杆菌/pBBR-3510-ga2dh 对葡萄糖酸（GA）表现出最高的氧化活性。在分批生物转化过程中，氧化葡萄糖酸杆菌/pBBR-3510-ga2dh 菌株的 2KGA 生产力（0.63 g/g CWW/h）高于使用氧化葡萄糖酸杆菌/pBBR-ga2dh 菌株（0.40 g/g CWW/h）获得的生产力。当生物转化过程中提供足够的氧气时，氧化葡萄糖酸杆菌/pBBR-3510-ga2dh 菌株在 45 小时内耗尽了 480 g/L 的 GA，产生了约 486 g/L 的 2KGA，产生了 0.54 g/g CWW/h 的生产力。
