The Biosynthesis of Polymyxin B by Growing Cultures of Bacillus poZyrnyxa*

In the last two decades, numerous oligopeptides have been isolated from sporogenic bacteria and fungi. These peptides are almost invariably characterized by the presence of macrocyclic structures, linkages other than cr-peptide bonds, amino acids that do not occur in protein, n-amino acids, and nonamino acid moieties. The formation of some of these peptides by microorganisms has been studied in considerable detail. The biosynthesis of the actinomycins has been investigated in the laboratory of Katz (l-4), that of bacitrarin A by Bernlohr (5, 6) and by Snoke (7, 8), that of gramicidm S by Winnick (9, lo), that of valinomycin by MacDonald (ll), and that of penicillin by several groups (12 15). In all cases, the production of the peptide is related in a characterist,ic manner to the growth cycle of the microorganism, and it can proceed in the absence of protein synthesis. The origin of the n-amino acid moieties has in all instances remained ambiguous. The investigation described in this paper deals with the formation of polymyxin l3 by Bacillus polymyxa. The amino acid sequence of this branched cyclic decapeptide has been elucidated by Hausmann (16) and Biserte and Dautrevaux (17) and has been confirmed by the elegant synthetic work of Vogler et al. (18). The structure is shown in Fig. 1. The peptide contains 6 residues of a,y-diaminobutyric acid, 2 of threonine, and 1 each of leucine and phenylalanine. The amino-terminal diaminobutyric acid and the phenylalanine residue are of the D configuration; all other amino acids belong to the L series. The side cham is attached to a y-amino group of the cyclic hcptapeptide, and d-6-methyloctanoic acid is present in amide linkage with the a-amino group of the terminal diaminobutyric acid residue. In view of the intriguing chemical structures of these peptides, the enzymic mechanisms involved in their biosynthesis should be of considerable interest. However, difficulty has been encountered in the search for cell-free systems capable of carrying out the biosynthesis of peptide antibiotics. A study of the biosynthesis of polymysin B was undertaken because the formation of this peptide appeared to be more amenable to investigation than that of most other antibiotics. Polymyxin B is released into the growth medium and is not accompanied by a family of closely related peptides when it is produced by pure strains of Bacillus polymyxa; moreover, it is a highly basic substance that can be obtained in a pure state by relatively simple procedures. It has a known structure that has been