Heterologous expression of membrane-bound alcohol dehydrogenase-encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives.

Autor: Habe H; Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8569, Japan. hiroshi.habe@aist.go.jp., Sato Y; Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8569, Japan., Tani H; Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8569, Japan., Matsutani M; NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo, 156-8502, Japan., Tanioka K; Joint Degree Program of Kasetsart University and Yamaguchi University, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan., Theeragool G; Joint Degree Program of Kasetsart University and Yamaguchi University, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan.; Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand., Matsushita K; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan., Yakushi T; Joint Degree Program of Kasetsart University and Yamaguchi University, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan. juji@yamaguchi-u.ac.jp.; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan. juji@yamaguchi-u.ac.jp.
Jazyk: angličtina
Zdroj: Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2021 Sep; Vol. 105 (18), pp. 6749-6758. Date of Electronic Publication: 2021 Aug 28.
DOI: 10.1007/s00253-021-11535-0
Abstrakt: 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.
(© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
Databáze: MEDLINE
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