THE PHENOTYPE ENHANCEMENT METHOD IDENTIFIES THE XCP OUTER-MEMBRANE SECRETION MACHINERY FROM PSEUDOMONAS-ALCALIGENES AS A BOTTLENECK FOR LIPASE PRODUCTION

Pseudomonas alcaligenes M-1 has been selected from an intensive screen ing for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to al low high level secretion from Pseudomonas alcaligenes, via the introdu ction of multi-copy plasmids. In order to improve the lipase yield fur ther, the phenotype enhancement method has been developed. This idea c omprises the reintroduction of a cosmid library with random chromosoma l fragments in a P. alcaligenes strain with already high lipase produc tivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion gen es. These rep-genes are clustered in two divergently transcribed opero ns similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two rep gene clusters, two reading frames of unknown function-OrfV and OrfX-are present. For Orf X no equivalent can be found in the known protein data bases. On the o ther hand, OrfV shows homology to the regulatory proteins MalT and Aco K. Some evidence is provided that suggests that OrfV acts as a regulat or of the rep operons. A model is proposed for the regulation of the s ecretion system from P. alcaligenes. (C) 1998 Elsevier Science B.V. Al l rights reserved.