ANALYSIS OF GENES INVOLVED IN BIOSYNTHESIS OF CORONAFACIC ACID, THE POLYKETIDE COMPONENT OF THE PHYTOTOXIN CORONATINE

Coronafacic acid (CFA) is the polyketide component of coronatine (COR) , a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. The genes involved in CFA biosynthesis: are encoded by a sin gle transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb regi on located at the 3' end of the CFA biosynthetic gene cluster. Three o pen reading frames were identified and designated cfa8, cfa9, and tnp1 : the predicted translation products Of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. Th e translational products of cfa8 and cfa9 were overproduced in Escheri chia coli BL21; however, tnp1 was not translated in these experiments, Mutagenesis and complementation analysis indicated that cfa8 is requi red for the production of CFA and COR. Analysis of a cfa9 mutant indic ated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tn p1, how-ever, had no obvious function in CFA or COR biosynthesis. A ge netic strategy was used to produce CFA in a P. syringae strain which l acks the COR gene cluster; this approach will be useful in future stud ies designed to investigate biosynthetic products of the CFA gene flus ter.