Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-d-ribose 5-phosphate aldolase
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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.
Keywords2-Deoxy-d-ribose 5-phosphate aldolase (DERA) Deoxysugars Whole-cell transformation Directed mutagenesis
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).
Conflict of interest
The authors declare that they have no competing interests.
- Ben Sahra I, Laurent K, Giuliano S, Larbret F, Ponzio G, Gounon P, Le Marchand-Brustel Y, Giorgetti-Peraldi S, Cormont M, Bertolotto C, Deckert M, Auberger P, Tanti JF, Bost F (2010) Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells. Cancer Res 70:2465–2475CrossRefPubMedGoogle Scholar
- Fessner WD, Sinerius G, Schneider A, Dreyer M, Schulz GE, Badia J, Aguilar J (1991) Diastereoselective enzymatic aldol addition: L-rhamnulose and L-fuculose-1-phosphate aldolases from E. coli. Angew Chem Int Ed 30:555–558Google Scholar
- Horinouchi N, Ogawa J, Sakai T, Kawano T, Matsumoto S, Sasaki M, Mikami Y, Shimizu S (2003) Construction of deoxyriboaldolase expressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2’-deoxyribonucleoside production. Appl Environ Microbiol 69:3791–3797PubMedCentralCrossRefPubMedGoogle Scholar