Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose

The miglitol intermediate, 6-(N -hydroxyethyl)-amino-6-deoxy-α- l -sorbofuranose (6NSL), is catalyzed from N -2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans /pBBR1 -sldAB-pqqABCDE-tldD (G. oxydans /AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans /AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans /AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans /AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans /AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%. • Vitreoscilla hemoglobin (VHb) was engineered to enhance oxygen transfer efficiency. • The transcription levels of respiratory chain-related enzyme genes were up-regulated. • The DO level of 20% was the optimal condition for cell proliferation. • The biotransformation of NHEG to 6NSL by resting cells was performed at 30% DO. [ABSTRACT FROM AUTHOR]