Abstract
In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L−1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L−1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L−1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly.
Key points
• Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp.
• D-GA production from glycerol was performed using recombinant Gluconobacter sp.
• Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.
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Data availability
Gluconobacter sp. (formerly Gluconobacter frateurii) CHM43 has been deposited in the National Institute of Technology and Evaluation (NITE) NBRC under accession number NBRC 101,659.
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Acknowledgements
The authors thank Shohei Kawamoto and Yukihiro Kaneko for their technical support.
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This work was supported in part by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) of the Japan Science and Technology Agency (JST).
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All authors contributed to the writing of the manuscript and have read and approved the final manuscript. HH, KM, and TY conceived and designed research. YS and HT performed GA production and RT-qPCR. MM contributed data analysis. KT and GT contributed mADH enzyme assay.
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Habe, H., Sato, Y., Tani, H. et al. Heterologous expression of membrane-bound alcohol dehydrogenase–encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives. Appl Microbiol Biotechnol 105, 6749–6758 (2021). https://doi.org/10.1007/s00253-021-11535-0
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DOI: https://doi.org/10.1007/s00253-021-11535-0