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 mmolMHB h−1 gcdw−1 and a yield of 2.7 molMHB molglucose−1. The ∆pfkA mutant showed a similar MHB production rate, but reached a yield of 4.8 molMHB molglucose−1, approaching the maximal value of 6 molNADPH molglucose−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 molMHB molglucose−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 molNADPH molglucose−1.
Highlights
Whole-cell biotransformation has become an important method in chemoenzymatic synthesis, e.g., for the production of amino acids and chiral alcohols (Ishige et al 2005)
Methyl acetoacetate (MAA), (R)-methyl 3-hydroxybutyrate (MHB), glucose, and extracellular metabolites were analyzed by HPLC as described previously (Siedler et al 2011)
In a C. glutamicum mutant lacking 6-phosphofructokinase, glucose catabolism is forced to proceed via the pentose phosphate pathway
Summary
Whole-cell biotransformation has become an important method in chemoenzymatic synthesis, e.g., for the production of amino acids and chiral alcohols (Ishige et al 2005). C. glutamicum was shown to be a suitable host for whole-cell biotransformation with resting cells for production of mannitol (Bäumchen and Bringer-Meyer 2007) and cyclohexanone derivatives (Doo et al 2009; Yun et al 2012) These reactions are often NAD(P)H dependent and cofactor recycling is crucial for profitable processes. Deletion of pfkA and pfkB encoding phosphofructokinase I and II, respectively, resulted in a partial cyclization of the PPP and a yield of 5.4 molMHB molglucose−1, which was near the theoretically maximal yield of 6 (Kruger and von Schaewen 2003). To determine whether this metabolic engineering strategy can be generalized, is e.g. transferable to C. glutamicum, was one major goal of this study. Oligonucleotides (listed in Table 2) were synthesized by Biolegio bv (Nijmegen, The Netherlands) and Eurofins MWG Operon (Germany)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
