Abstract 6-(N-hydroxyethyl)-amino-6-deoxy-L-sorbofuranose (6NSL), a key intermediate in the synthesis of miglitol, was produced from N-2-hydroxyethyl glucamine (NHEG) by biotransformation with whole cells of Gluconobacter oxydans. The main troubles in 6NSL production were the low activity of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) in G. oxydans and the high cost of cell preparation. To solve these problems, a combined mutagenesis of 60Co-γ irradiation and microwave treatment with a high-throughput screening method by cultivation in a 96-well microtiter plate was conducted. After several cycles of mutagenesis, a stable mutant H-8 with high mSLDH activity was obtained, and the cell biomass increased by 11.6% when cultivated in a 5 L bioreactor. The transcription levels of the mSLDH subunit sldA and sldB in G. oxydans H-8 increased by 1.4- and 2.0-fold, respectively. Meanwhile, the intracellular PQQ concentration in G. oxydans H-8 was 16.1% higher than that of the parent strain, and qRT-PCR analysis showed that the genes pqqB and pqqC played an important role in PQQ biosynthesis (transcription levels increased by 2.4- and 1.8-fold, respectively). Relying on the advantages of the above, G. oxydans H-8 could produce 64.3 ± 2.2 g/L 6NSL after 36 h of bioconversion with resting cells, showing a 33.7% increase in the product yield.

中文翻译：

---

用高 PQQ 依赖性 D-山梨糖醇脱氢酶培育氧化葡糖杆菌以提高 6-(N-羟乙基)-氨基-6-脱氧-α-L-山梨呋喃糖产量

摘要 6-(N-羟乙基)-氨基-6-脱氧-L-山梨呋喃糖(6NSL)是米格列醇合成的关键中间体，是由N-2-羟乙基葡糖胺(NHEG)通过葡糖杆菌全细胞生物转化制备的。氧化酶。6NSL 生产的主要问题是 G. oxydans 中 PQQ 依赖性 D-山梨糖醇脱氢酶 (mSLDH) 的低活性和细胞制备的高成本。为了解决这些问题，通过在 96 孔微量滴定板中培养，进行了 60Co-γ 辐射和微波处理的联合诱变以及高通量筛选方法。经过数轮诱变，获得了具有高mSLDH活性的稳定突变体H-8，在5L生物反应器中培养时细胞生物量增加了11.6%。G. oxydans H-8 中 mSLDH 亚基 sldA 和 sldB 的转录水平增加了 1。分别为 4 倍和 2.0 倍。同时，G. oxydans H-8 胞内 PQQ 浓度比亲本高 16.1%，qRT-PCR 分析表明 pqqB 和 pqqC 基因在 PQQ 生物合成中起重要作用（转录水平提高了 2.4-和 1.8 倍）。凭借上述优势，G. oxydans H-8 与静息细胞生物转化 36 h 后可产生 64.3 ± 2.2 g/L 6NSL，产品收率提高 33.7%。