The serine hydroxymethyltransferase gene glyA in Corynebacterium glutamicum is controlled by GlyR

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon metabolism, and we here study its regulation in Corynebacterium glutamicum. Enzyme quantifications revealed an about 3-fold increase of SHMT activity during exponential growth with a further increase at the onset of the stationary phase. The SHMT encoding glyA gene was shown to be transcribed as a monocistronic mRNA, and its transcriptional start site was determined. Using DNA affinity chromatography the regulator GlyR (Cg0527) was identified and its chromosomal deletion shown to abolish the increase in SHMT activity in the stationary phase. The involvement of GlyR in glyA control was further confirmed by a transcriptional fusion of the glyA promoter with cat and chloramphenicol acetyltransferase quantifications. GlyR was isolated and mutational studies together with electrophoretic mobility shift assays showed that it binds to the imperfect palindromic motif CACT-N(2)-AATG in the -119 to -96 upstream region of the glyA promoter. These and further data illustrate that the essential SHMT has highest activity in the stationary phase and that GlyR acts as an activator of glyA transcription in this growth phase

Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production

L-Threonine is an important biotechnological product and Corynebacterium glutamicum is able to synthesize and accumulate this amino acid to high intracellular levels. We here use four exporters of Escherichia coli and show that three of them operate in C. glutamicum, with RhtA and RhtC being the most effective. Whereas RhtA was unspecific, resulting in L-homoserine together with L-threonine excretion, this was not the case with RhtC. Expression of rhtC reduced the intracellular L-threonine concentration from 140 to 11 mM and resulted in maximal excretion rates of 11.2 nmol min(-1) mg(-1) as compared to 2.3 nmol min(-1) mg(-1) obtained without rhtC expression. In combination with an ilvA mutation generated and introduced into the chromosome, an accumulation of up to 54 mM L-threonine was achieved as compared to 21 mM obtained with the ancestor strain. This shows that expression of rhtC is the pivotal point for industrial relevant L-threonine production with C. glutamicum, and might encourage in general the use of heterologous exporters in the field of white biotechnology to make full use of biosynthesis pathways