Research Article: Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16

Date Published: July 13, 2011

Publisher: Springer

Author(s): Chlud Kaddor, Alexander Steinbüchel.


The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed.

Partial Text

Biodiesel (fatty acid methyl esters) is currently beside ethanol the major renewable energy source for substitution of petroleum. During production of biodiesel glycerol occurs as a main by-product (about 10%, w/w), thus saturating the glycerol market. Due to the huge surplus of glycerol that lowers its value, it is important to enlarge the field of its application e.g. as substrate for microbial growth and production of biodegradable polymers which in turn reduces the high production costs of polyhydroxyalkanoates (PHA) in industrial fermentation processes.

When comparing group A with group B, it is noticeable that the deletion of ptsM, ptsH, or ptsI exerted a significant change of the PHB synthesis phenotype. Besides the Tn5-induced mutants, the remaining mutants of group B harbored in addition the deletion of either ptsM, ptsH, ptsI or all three genes. Another mutant, H16 ΔptsHI behaved similar like mutant H16 ΔptsMHI (data not shown). The impact of the putative ptsMHI operon was observed during growth in presence of both, the PTS carbohydrate N-acetylglucosamine and the non-PTS carbon sources sodium gluconate, fructose, and glycerol. Particularly, during growth on glycerol in comparison to growth on the previously analyzed carbon sources (Kaddor and Steinbüchel 2011), mutants defective in the putative ptsMHI operon accumulated less PHB than the wild type. Pries et al. (1991) made similar observations with Tn5-induced ptsH/ptsI mutants exhibiting a PHB-leaky phenotype with a lower PHB content of the cells when grown on gluconate. However, a faster mobilization of PHB after exhausting the extracellular carbon source, as it occurred in presence of gluconate, was not noticed when cultivated in media containing glycerol. Despite the still unknown functions of ptsH and ptsI, an exclusively regulatory role in PHB and carbon metabolism was already proposed (Pries et al. 1991; Kaddor and Steinbüchel 2011).

The authors declare that they have no competing interests.