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

Shanghai Jiao Tong University

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. In Streptomyces sp. FR-008, a gene pabC-1 putatively encoding fold-type IV of 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 ca. 20% of the wild-type level. Its specific role for PABA formation was further demonstrated by the successful complementation of an E. coli pabC mutant. Moreover, a free-standing gene pabC-2, probably encoding another fold-type IV of PLP-dependent enzyme, was cloned from the same strain. Inactivation of pabC-2 rendered antibiotic FR-008 yield to about 57% of the wild-type level in the mutant, and the complementation of the E. coli pabC mutant established its involvement in PABA biosynthesis. Furthermore, a pabC-1/pabC-2 double mutant only kept about 4% of the wild-type productivity, clearly indicating that pabC-2 also contributed to antibiotic FR-008 biosynthesis. Surprisingly, apparent retarded growth of the double mutant was observed on minimal medium, which suggested that both pabC-1 and pabC-2 were involved in PABA biosynthesis for primary metabolism. Finally, both PabC-1 and PabC-2 were shown to be functional ADC lyases through in vitro enzymatic lysis with the release of pyruvate. To date, pabC-1 and pabC-2 represent the first two functional ADC lyase genes indentified 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.

¹ E-mail: bailq{at}sjtu.edu.cn