title: D-mannitol production by resting state whole cell biotrans-formation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium.
abstract: An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.
authors.affiliation: Institute of Biotechnology 1, Research Centre Jülich GmbH, Jülich, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Bäumchen
authors.forename: Andreas H F J
authors.initials: AH
authors.lastname: Roth
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
authors.forename: Marco
authors.initials: M
authors.lastname: Malten
authors.forename: Martin
authors.initials: M
authors.lastname: Follmann
authors.forename: Hermann
authors.initials: H
authors.lastname: Sahm
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer-Meyer
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D004591:Electrophoresis, Polyacrylamide Gel; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D008353:Mannitol / Q000378:metabolism*; D008354:Mannitol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism
chemical_list: D009243:NAD; D005632:Fructose; D008353:Mannitol; D008354:Mannitol Dehydrogenases; D005560:Formate Dehydrogenases