Analysis of the Growth and Metabolites of a Pyruvate Dehydrogenase Complex- Deficient Klebsiella pneumoniae Mutant in a Glycerol-Based Medium.

To determine the role of pyruvate dehydrogenase complex (PDHC) in Klebsiella pneumoniae, the growth and metabolism of PDHC-deficient mutant in glycerol-based medium were analyzed and compared with those of other strains. Under aerobic conditions, the PDHC activity was fourfold higher than that of pyruvate formate lyase (PFL), and blocking of PDHC caused severe growth defect and pyruvate accumulation, indicating that the carbon flux through pyruvate to acetyl coenzyme A mainly depended on PDHC. Under anaerobic conditions, although the PDHC activity was only 50% of that of PFL, blocking of PDHC resulted in more growth defect than blocking of PFL. Subsequently, combined with the requirement of CO2 and intracellular redox status, it was presumed that the critical role of PDHC was to provide NADH for the anaerobic growth of K. pneumoniae. This presumption was confirmed in the PDHC-deficient mutant by further blocking one of the formate dehydrogenases, FdnGHI. Besides, based on our data, it can also be suggested that an improvement in the carbon flux in the PFL-deficient mutant could be an effective strategy to construct highyielding 1,3-propanediol-producing K. pneumoniae strain.

Deleting pck improves growth and suppresses by-product formation during 1,3-propanediol fermentation by Klebsiella pneumoniae.

AIMS: To investigate the role of phosphoenolpyruvate (PEP) carboxylation in cell metabolism in Klebsiella pneumoniae. METHODS AND RESULTS: The effects of deleting pck, which encodes PEP carboxykinase (PCK), and/or ppc, which encodes PEP carboxylase (PPC), on growth, enzyme activity and metabolite formation of Kl. pneumoniae were investigated. A self-regulatory mechanism of PEP carboxylation was found in ppc- or pck-deficient mutants, which resulted in almost no change in succinate formation. However, almost no growth was observed in a ppc- and pck-deficient mutant. Interestingly, only deleting pck affected the energy metabolism and promoted aerobic cell growth. Under micro-aerobic conditions, although there was only a small (8·1%) increase of 1,3-propanediol production by such pck-deficient mutant during a 2-l fed-batch process, the by-products 2,3-butanediol and acetate significantly decreased by 73·0% and 54·8%, respectively, compared with those in the parent strain. CONCLUSIONS: PEP carboxylation could be a critical anaplerotic reaction for converting C3 to C4 metabolites in the central metabolism of Kl. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to identify the critical role of PEP carboxylation, as well as those of PPC and PCK, which are responsible for this reaction, in Kl. pneumoniae. In addition, the pck-deficient mutant was proven to be a valuable 1,3-propanediol producer.

Deletion of poxB, pta, and ackA improves 1,3-propanediol production by Klebsiella pneumoniae.

To date, few studies have focused on reducing the toxic by-product acetate during 1,3-propanediol production by Klebsiella pneumoniae. In this study, the effects of deleting the poxB, pta, and ackA genes, which are involved in the two main acetate synthesis pathways, on cell growth and 1,3-propanediol production were investigated. Although acetate synthesis via pyruvate oxidase (PoxB, encoded by poxB) generally seems unnecessary and wasteful, PoxB was shown to play an important role in K. pneumoniae. Deletion of poxB severely inhibited cell growth, and the poxB mutant exhibited an anomalously high accumulation of acetate in aerobic cultures and failed to produce an endogenous supply of carbon dioxide (CO2) in anaerobic cultures. It is interesting that both the aerobic and anaerobic growth defects of the poxB mutant were corrected by further deleting pta and ackA, which blocked the other main acetate synthesis pathway. The poxB-pta-ackA mutant excreted less acetate and showed an excellent ability to produce 1,3-propandiol. The final 1,3-propanediol yield and concentration in a 2-L fed-batch fermentation reached 0.66 (mol/mol) and 76.8 g/L, respectively, which were 16 and 15 % greater, respectively, than those of the parent strain.