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Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

2002; American Society for Microbiology; Volume: 68; Issue: 7 Linguagem: Inglês

10.1128/aem.68.7.3321-3327.2002

1098-5336

Petra Šimić, Juliane Willuhn, Hermann Sahm, Lothar Eggeling,

Amino Acid Enzymes and Metabolism

ABSTRACT l -Threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum . However, in the course of this process, some of the l -threonine is degraded to glycine. We detected an aldole cleavage activity of l -threonine in crude extracts with an activity of 2.2 nmol min −1 (mg of protein) −1 . In order to discover the molecular reason for this activity, we cloned glyA , encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA , SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with l -threonine as the substrate was 1.3 μmol min −1 (mg of protein) −1 , which was 4% of that with l -serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P tac , making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an l -threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while l -threonine was simultaneously increased by 49%. The intracellular availability of l -threonine to aldole cleavage was also reduced by overexpressing the l -threonine exporter thrE . In C. glutamicum DR-17, which overexpresses thrE , accumulation of 67 mM instead of 49 mM l -threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.