Facile synthesis of bioactive Allitol from D-psicose by coexpression of ribitol dehydrogenase and formate dehydrogenase in Escherichia Coli

Coexpression of formate dehydrogenase (FDH) and ribitol dehydrogenase (RDH) in Escherichia coli was used for the synthesis of Allitol from D-psicose. FDH was coexpressed with RDH for continuous NADH regeneration. The results revealed that the optimum conditions for allitol production occurred at pH 7.0 and 30 °C. Allitol reached the maximum yield of 19.2 mg at 2.0% substrate concentration after 48 hours. Using D-psicose as a substrate, allitol was successfully produced using an engineered E. coli coexpressed with RDH and FDH.

Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

ABSTRACTRibitol dehydrogenase fromZymomonas mobilis(ZmRDH) catalyzes the conversion of ribitol tod-ribulose and concomitantly reduces NAD(P)+to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+(S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, whereKm,NADis theKmfor NAD+andKm,NADPis theKmfor NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.