6-(N-hydroxyethyl)-amino-6-deoxy-l-sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ± 3.0 g/L of 6NSL production with 94.5 ± 3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess.

中文翻译：

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D-山梨糖醇脱氢酶和吡咯喹啉醌的组合表达通过辅因子操作增加氧化葡糖杆菌产生的 6-(N-羟乙基)-amino-6-deoxy-α-L-sorbofuranose

6-(N-羟乙基)-氨基-6-脱氧-1-山梨呋喃糖 (6NSL) 是米格列醇合成中的关键前体，它是通过氧化葡糖杆菌的区域选择性氧化从 N-2-羟乙基-葡糖胺 (NHEG) 产生的. PQQ 生物合成的局限性成为提高 PQQ 依赖性 D-山梨糖醇脱氢酶 (mSLDH) 活性的瓶颈。以pBBR1-gHp0169-sldAB为基础，构建了5个表达质粒，共表达pqqABCDE基因簇和tldD基因，以增加PQQ的生物合成。G. oxydans/pGA004，其中 pqqABCDE 和 tldD 在 gHp0169 启动子的控制下表达为簇，表现出最佳性能。与 G. oxydans/pBBR-sldAB 相比，细胞内 mSLDH 的细胞内 PQQ 浓度和比活性分别增加了 79.3% 和 53.7%。然后，进行了通过 G. oxydans/pGA004 将 NHEG 重复分批生物转化为 6NSL。运行四个循环后，6NSL 的产量高达 75.0 ± 3.0 g/L，NHEG 到 6NSL 的平均转化率为 94.5 ± 3.6 %。这些结果表明，具有高生产力的 G. oxydans/pGA004 在工业生物过程中具有巨大的 6NSL 生产潜力。