title: Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.
abstract: The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: S
authors.initials: S
authors.lastname: Guillouet
authors.forename: A A
authors.initials: AA
authors.lastname: Rodal
authors.forename: G
authors.initials: G
authors.lastname: An
authors.forename: P A
authors.initials: PA
authors.lastname: Lessard
authors.forename: A J
authors.initials: AJ
authors.lastname: Sinskey
mesh_terms: D018533:Biomass; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D007532:Isoleucine / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013913:Threonine Dehydratase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D007532:Isoleucine; D013913:Threonine Dehydratase