Functional characterization of the first two actinomycete 4-amino-4-deoxychorismate lyase genes

Abstract

In some antibiotic producers,p-aminobenzoic acid (PABA) or its immediate precursor, 4-amino-4-deoxychorismate (ADC), is involved in primary metabolism and antibiotic biosynthesis. InStreptomycessp. FR-008, a genepabC-1putatively encoding a fold-type IV pyridoxal 5′-phosphate (PLP)-dependent enzyme was found within the antibiotic FR-008/candicidin biosynthetic gene cluster, whose inactivation significantly reduced the productivity of antibiotic FR-008 to about 20 % of the wild-type level. Its specific role in PABA formation was further demonstrated by the successful complementation of anEscherichia coli pabCmutant. Moreover, a free-standing genepabC-2, probably encoding another fold-type IV PLP-dependent enzyme, was cloned from the same strain. Inactivation ofpabC-2reduced antibiotic FR-008 yield to about 57 % of the wild-type level in the mutant, and the complementation of theE. coli pabCmutant established its involvement in PABA biosynthesis. Furthermore, apabC-1/pabC-2double mutant only retained about 4 % of the wild-type antibiotic FR-008 productivity, clearly indicating thatpabC-2also contributed to biosynthesis of this antibiotic. Surprisingly, apparently retarded growth of the double mutant was observed on minimal medium, which suggested that bothpabC-1andpabC-2are involved in PABA biosynthesis for primary metabolism. Finally, both PabC-1 and PabC-2 were shown to be functional ADC lyases byin vitroenzymic lysis with the release of pyruvate.pabC-1andpabC-2appear to represent the first two functional ADC lyase genes identified in actinomycetes. The involvement of these two ADC lyase genes in both cell growth and antibiotic FR-008 biosynthesis sets an example for the interplay between primary and secondary metabolisms in bacteria.