No abstract

A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd--complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (M, 354848 was identified as the gene for Asd by comparing i t s sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the 5. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the 5. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in 5. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.

A high-performance liquid chromatographic method for the analysis of armentomycin (L-2-amino-4,4-dichlorobutanoic acid) in culture broths was developed and calibrated using DL-2-amino-4,4-dichlorobutanoic acid synthesized in five steps from methyl acrylate, carbon tetrachloride, and sodium azide. Armentomycin was produced optimally (5–10 mM during 8 days' fermentation) by Streptomyces armentosus in media containing starch, lysine, chloride ions, and a low concentration of phosphate. Production was markedly affected by carbon and nitrogen sources: of those tested, starch supported both the highest biomass yield and the highest armentomycin titre; lysine was the best source of nitrogen for growth and for achieving the maximal armentomycin titre. Increasing the concentration of lysine from 40 to 120 mM nitrogen increased armentomycin production, and gave no evidence of nitrogen catabolite repression. Growth and armentomycin biosynthesis increased as the initial concentration of phosphate in media was increased up to 3 mM; further additions of phosphate tended to increase biomass and decrease the armentomycin titre, but the responses were marginal. Chloride ions were absolutely required for armentomycin synthesis, and at their optimal concentration, more than 80% of the chloride ions in the medium were incorporated into the antibiotic. Excess chloride in cultures reduced armentomycin biosynthesis. Neither bromide nor iodide was incorporated; bromide interfered with the biosynthesis of armentomycin, but iodide had no effect. Analysis of fermentation parameters showed that armentomycin biosynthesis paralleled growth and suggested that secondary metabolism was derepressed by a nitrogen-related limitation of the growth rate.Key words: 2-amino-4,4-dichlorobutanoic acid, antibiotic, armentomycin, halide ions, Streptomyces armentosus, secondary metabolism.

Although over 1,500 halogenated natural products have been identified in animals, plants, and microorganisms,1'^only two biological routes for their formation are known: the direct methylation of halide ions,3) and haloperoxidase-catalyzed halogenation.2?3) The biosynthesis of armentomycin (L-2-amino-4,4-dichlorobutanoic acid, 6), a nonprotein amino acid with antibiotic properties40 is of particular interest because no mechanism is known for the chloroperoxidase-catalyzed insertion of chlorine substituents remote from other functional groups.2) Wepresent here the initial results from our investigation of armentomycin biosynthesis in Streptomycesarmentosusvar. armentosus. A starch, L-lysine and KC1 medium (100ml/500-ml Erlenmeyer flask) was inoculated with a 48-hour seed culture of S. armentosus var. armentosus UC2862and incubated at 27°C with rotary shaking (220rpm).5) Research Council of Canada (NSERC), and by an exchange fellowship (to J.-Y. H.) from the World University Services of Canada. Weare indebted to the Atlantic Region Magnetic Resonance Centre for providing NMRspectra.