Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture

Abstract

<i>Streptomyces</i>, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of <i>Streptomyces coelicolor</i> A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of <i>S. coelicolor</i> due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from <i>S. coelicolor</i> was also heterologously cloned and expressed in <i>Escherichia coli</i> for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, k_cat/K_M, of 2.0 ± 0.1 mM^–1 s^–1. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, k_cat/K_M, of 1.9 ± 0.1 mM^–1 s^–1.