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| Hauptseite > Publikationsdatenbank > Identification of glyA (serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation with Corynebacterium glutamicum |
| Hauptseite > Publikationsdatenbank > Identification of glyA (serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation with Corynebacterium glutamicum |
| Journal Article | PreJuSER-24490 |
; ; ;
2002
Soc.
Washington, DC [u.a.]
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Please use a persistent id in citations: http://hdl.handle.net/2128/2410 doi:10.1128/AEM.68.7.3321-3327.2002
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 mumol 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.
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