Recently, Pseudomonas putida revealed excellent properties as production host for the expression of a natural product gene cluster from myxobacteria, encoding the myxochromide biosynthetic pathway. Here we present a physiological study of the mutant P putida::CMch37a for the production of this compound. Feeding of alanine, leucine, glutamine, and threonine as building blocks of the cyclic peptide of myxochromides S led to 5-fold increase of production. This improvement was due to increased intracellular levels of these amino acids, as response to the feeding, thus optimizing their availability for the non-ribosomal peptide synthetases from the myxochromide assembly line. As shown by C-13 labelling studies, amino acids incorporated into myxochromides S originated predominantly from the externally added pools, whereas de novo synthesis from glucose was very low. Alanine, leucine and glutamine were, however, catabolized to a large extent, which reduces the efficiency of their feeding. Acetate and malonate, as building blocks of the polyketide chain of myxochromide, however, inhibited growth of P. putida::CN1637a and partly reduced myxochromide production. The effective co-utilization of acetate in a mixture with glucose indicated the presence of a highly active acetyl-CoA synthase. The fact that this was not reflected by an increased myxochromide production speaks against a limitation of production, caused by this enzyme. Malonate was not co-consumed with glucose so that malonyl-CoA synthase remains as a potential bottleneck for myxochromide production in P. putida. (c) 2006 Elsevier Ltd. All rights reserved.