Acetyl-CoA Production and Utilization during Growth of the Facultative Methylotroph Pseudomonas AM1 on Ethanol, Malonate and 3-Hydroxybutyrate

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Abstract
In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase [EC 1.1.1.30], an acetoacetate:succinate CoA transferase [EC 2.8.3.5] (specific for succinyl-CoA) and an inducible .beta.-ketothiolase [EC 2.3.1.9]. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase [EC 6.2.1.1] was partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51) lacking malate synthase activity was isolated, and its characteristics indicate that this activity is normally essential for growth of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by acetyl-CoA.