Screening and Characterization of Novel Xylose Dehydrogenase for the Production of D-Xylonic Acid from D-Xylose in Escherichia coli

Authors: 
Valdehuesa, K. N. G., Myongji University
Ramos, K. R. M., Myongji University
Cabulong, R. B., Myongji University
Maza, P. A. M. A., Myongji University
Pangan, J. O., Myongji University
Bañares, A. B., Myongji University
Nisola, G. M., Myongji University
Lee, S. P., Myongji University
Lee, W. K., Myongji University
Chung, W. J., Myongji University

D-Xylonic acid has several industrial applications as chelator, as concrete dispersion agent, and as precursor for synthesis of polymers and polyalcohols. This compound is derived from D-xylose though enzymatic catalysis of xylose dehydrogenases (Xdh), which are found in a few Bacteria (Caulobacter and Gluconobacter) and Archaea (Haloarcula and Haloferax). The microbial production of D-xylonic acid has been well studied in different kinds of yeasts and bacteria. The commonly used Xdh is the xylB (or xdh), which was recruited from Caulobacter crescentus. In this study, novel xylose dehydrogenase enzymes were identified and characterized for their potential application in the production of D-xylonic acid or other valuable compounds. Candidate Xdh from other microorganisms mined from available databases for annotated ORFs with high amino acid sequence homology to C. crescentus Xdh. Three candidate genes were selected from Asticcacaulis, Brevundimonas, and Phenylobacterium genus. The Xdh ORFs were then codon optimized, cloned, and expressed in Escherichia coli. The purified Xdh were characterized for substrate specificity, co-factor preference, and enzyme kinetics. All three Xdhs showed good activity towards D-xylose oxidation. The best performing Xdh was then tested for efficient conversion of D-xylose to D-xylonic acid in metabolically engineered E. coli. This work was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (No. 2009-0093816), and Korea Research Fellowship Program through the NRF funded by the Ministry of Science, ICT and Future Planning (No. 2015H1D3A1062172).