Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation.
In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2'-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gram-negative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein. Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.
Fernández-Moreno
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MA
Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Canto Blanco, Madrid, Spain.
Vallín
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Malpartida
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D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D002965:Classification; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000235:genetics; D006867:Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D013309:Streptothricins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014158:Transcription, Genetic
D017077:Culture Media, Conditioned; D004269:DNA, Bacterial; D013309:Streptothricins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C044496:streptothricin acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C061369:macrolide 2'-kinase; D006867:Hydrolases