Glycerol-3-phosphatase of Corynebacterium glutamicum.

Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg²âº or Mn²âº for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg⁻¹ with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and shown to significantly increase GPP activity.

The pstSCAB operon for phosphate uptake is regulated by the global regulator GlxR in Corynebacterium glutamicum.

The pstSCAB operon of Corynebacterium glutamicum, which encodes a high affinity transport system for uptake of the phosphorus source inorganic phosphate, is induced upon phosphate starvation involving activation by the two-component regulatory system PhoS-PhoR. Partial phosphate starvation induction of the pstSCAB operon in a ΔphoRS mutant indicated the involvement of (an) additional transcriptional regulator(s). Here, GlxR, a global cAMP-dependent transcriptional regulator, was shown to bind to the pstS promoter -133 bp to -117 bp upstream of the transcriptional start site as shown by gel shift and mutation experiments. Transcriptional fusion analysis revealed that GlxR activates the pstSCAB operon under phosphate limiting conditions in a carbon source dependent manner. Commensurate with these findings, overexpression of glxR was shown to stimulate growth under phosphate limiting conditions with glucose, but not with acetate, as carbon source. Thus, in C. glutamicum pstSCAB expression is regulated in response to the availability of phosphorus and carbon sources.