title: Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.
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 KDERA(K12) (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.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Jitao
authors.initials: J
authors.lastname: Li
authors.forename: Jiangang
authors.initials: J
authors.lastname: Yang
authors.forename: Yan
authors.initials: Y
authors.lastname: Men
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
authors.forename: Yueming
authors.initials: Y
authors.lastname: Zhu
authors.forename: Caixia
authors.initials: C
authors.lastname: Dong
authors.forename: Yuanxia
authors.initials: Y
authors.lastname: Sun
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D012267:Ribosemonophosphates / Q000096:biosynthesis*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012267:Ribosemonophosphates; C058245:2-deoxyribose 5-phosphate; D005634:Fructose-Bisphosphate Aldolase