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.

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18373 Da) and 416 amino acids (45260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid-encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kil-kor system in autonomous replication of pMEA300.

(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica

Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-DL-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.