Isolation and analysis of mutants of the methylotrophic actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis

Mutants of the actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis were isolated using brief ultrasonic treatments to obtain single cells. After UV irradiation, auxotrophic mutants were selected as pinpoint colonies on mineral agar with only 1 mg l-1 of amino acid supplements. Mutant characterization provided unambiguous evidence that L-tyrosine is synthesized via arogenate and that L-phenylalanine is synthesized via phenylpyruvate. The efficiency of chromosomal DNA marker exchange was highest in matings with mutant strains that lacked the previously characterized 13.3-kb integrative plasmid pMEA300