title: Reductive whole-cell biotransformation with Corynebacterium glutamicum: improvement of NADPH generation from glucose by a cyclized pentose phosphate pathway using pfkA and gapA deletion mutants.
abstract: In this study, the potential of Corynebacterium glutamicum for reductive whole-cell biotransformation is shown. The NADPH-dependent reduction of the prochiral methyl acetoacetate (MAA) to the chiral (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis (Lbadh) was used as model reaction and glucose served as substrate for the regeneration of NADPH. Since NADPH is mainly formed in the oxidative branch of the pentose phosphate pathway (PPP), C. glutamicum was engineered to redirect carbon flux towards the PPP. Mutants lacking the genes for 6-phosphofructokinase (pfkA) or glyceraldehyde 3-phosphate dehydrogenase (gapA) were constructed and analyzed with respect to growth, enzyme activities, and biotransformation performance. Both mutants showed strong growth defects in glucose minimal medium. For biotransformation of MAA to MHB using glucose as reductant, strains were transformed with an Lbadh expression plasmid. The wild type showed a specific MHB production rate of 3.1 mmol(MHB) h(-1) g                   (cdw)                             (-1)                  and a yield of 2.7 mol(MHB) mol                   (glucose)                             (-1)                 . The ∆pfkA mutant showed a similar MHB production rate, but reached a yield of 4.8 mol(MHB) mol                   (glucose)                             (-1)                 , approaching the maximal value of 6 mol(NADPH) mol                   (glucose)                             (-1)                  expected for a partially cyclized PPP. The specific biotransformation rate of the ΔgapA mutant was decreased by 62 % compared to the other strains, but the yield was increased to 7.9 mol(MHB) mol                   (glucose)                             (-1)                 , which to our knowledge is the highest one reported so far for this mode of NADPH regeneration. As one fourth of the glucose was converted to glycerol, the experimental yield was close to the theoretically maximal yield of 9 mol(NADPH) mol                   (glucose)                             (-1)                 .
authors.affiliation: Institut für Bio-und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
authors.forename: Solvej
authors.initials: S
authors.lastname: Siedler
authors.forename: Steffen N
authors.initials: SN
authors.lastname: Lindner
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D000090:Acetoacetates / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D052196:Levilactobacillus brevis / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D009249:NADP / Q000378:metabolism*; D010427:Pentose Phosphate Pathway / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C539306:3-hydroxybutyrate methyl ester; D000090:Acetoacetates; D006885:Hydroxybutyrates; D011994:Recombinant Proteins; D009249:NADP; C005217:methyl acetoacetate; D000426:Alcohol Dehydrogenase; D005947:Glucose