Identification and characterization of a novel (−)-vibo-quercitol 1-dehydrogenase from Burkholderia terrae suitable for production of (−)-vibo-quercitol from 2-deoxy-scyllo-inosose

• Published in: Applied microbiology and biotechnology 2017-10, Vol.101 (20), p.7545-7555
• Main Authors: Itoh, Nobuya, Kurokawa, Junji, Toda, Hiroshi, Konishi, Kazunobu
• Format: Article
• Language: English
• Subjects: Amino acids; Arthrobacter; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Burkholderia; Burkholderiaceae - enzymology; Burkholderiaceae - genetics; Carbon sources; Cell culture; Chemical synthesis; Cloning, Molecular; Coenzymes - metabolism; Culture Media - chemistry; Dehydrogenase; Dehydrogenases; E coli; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Gene Expression Regulation, Bacterial - drug effects; Genomes; Homology; Honeydew; Inositol; Inositol - analogs & derivatives; Inositol - metabolism; Life Sciences; Microbial Genetics and Genomics; Microbiology; NAD; NAD - metabolism; Oak; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Pharmaceuticals; Polymerase Chain Reaction; Primers; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Stereoisomers; Substrate Specificity; Substrates
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-017-8483-2

(−)- vibo -Quercitol is a deoxyinositol (1 l -1,2,4/3,5-cyclohexanepentol) that naturally occurs in oak species, honeydew honey, wines aged in oak barrels, and Gymnema sylvestre and is a potential intermediate for pharmaceuticals. We found that (−)- vibo -quercitol is stereoselectively synthesized from 2-deoxy- scyllo -inosose by the reductive reaction of a novel (−)- vibo -quercitol 1-dehydrogenase found in the proteomes of Burkholderia , Pseudomonas , and Arthrobacter . Among them, Burkholderia terrae sp. AKC-020 (40-1) produced a (−)- vibo -quercitol 1-dehydrogenase appropriate for synthesizing (−)- vibo -quercitol with a high diastereomeric excess. The enzyme was strongly induced in Bu. terrae cells when quercitol or 2-deoxy- scyllo -inosose was used as carbon source in the culture medium. The enzyme is NAD(H)-dependent and shows highly specific activity for (−)- vibo -quercitol and myo -inositol among the substrates tested. The enzyme gene ( qudh ) was obtained by PCR using degenerate primers based on the N -terminal and internal amino acid sequences of the purified enzyme, followed by thermal asymmetric interlaced PCR. The qudh gene showed homology with inositol 2-dehydrogenase (sharing 49.5% amino acid identity with IdhA from Sinorhizobium meliloti 1021). We successfully produced several recombinant (−)- vibo -quercitol 1-dehydrogenases and related enzymes identified by genome database mining using an Escherichia coli expression system. This revealed that scyllo -inositol dehydrogenase (IolX) in Bacillus subtilis can catalyze the reduction of 2-deoxy- scyllo -inosose to yield scyllo -quercitol, a stereoisomer of (−)- vibo -quercitol. Thus, we successfully identified two enzymes to produce both stereoisomers of deoxyinositols that are rare in nature.