Breeding of Gluconobacter oxydans with high PQQ-dependent D-sorbitol dehydrogenase for improvement of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production

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.