Abstract
2-Deoxy-d-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERAK12 (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards d-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-d-ribose yield from 0.41 mol/mol d-glyceraldehyde to 0.81 mol/mol d-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-d-robose (DR), with a yield of 0.71 mol/mol d-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-d-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-d-altrose from d-erythrose, as well as 2-deoxy-l-xylose and 2-deoxy-l-ribose from l-glyceraldehyde.
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Acknowledgments
This work was supported by the National High Technology Research and Development Program of China (No. 2012AA021403) and Science and Technology Projects of Tianjin (No. 13ZCZDSY05600).
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Jitao Li and Jiangang Yang contributed equally to this work.
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Li, J., Yang, J., Men, Y. et al. Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-d-ribose 5-phosphate aldolase. Appl Microbiol Biotechnol 99, 7963–7972 (2015). https://doi.org/10.1007/s00253-015-6740-9
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DOI: https://doi.org/10.1007/s00253-015-6740-9