Improvement of the Stability of Recombinant Plasmids Carrying the Threonine Operon in an L-Threonine-hyperproducing Strain of Escherichia coli W

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An L-threonine-hyperproducing strain of E. coli W, KY8366, which constitutively expresses L-threonine-sensitive aspartokinase (AKI) and L-lysine-sensitive aspartokinase (AKIII) was constructed [T. Mizukami et al., Agric. Biol. Chem., 50, 0000 (1986)]. In order to further improve the L-threonine productivity of KY8366, we cloned the E. coli thr operon from an α-amino-β-hydroxyvaleric-acid resistant (AHV^r) mutant of E. coli K-12 and introduced various types of recombinant plasmids carrying the AHV^r-thr operon into KY8366. Most ol them were unstable in KY8366; the loss of the whole plasmid molecules from the host cells or the deletion of the thr operon from the plasmids was observed. However, pGH2B1, a recombinant plasmid which is composed of the whole thr operon, the kanamycin-resistance (Km^r) gene and the replication origin of p15A, was stably maintained in KY8366 in the presence of kanamycin. KY8366 harboring pGH2B1 expressed 4.5-fold higher AKI activity than parental strain KY8366, and its L-threonine productivity was elevated to 20% higher than that of KY8366.