THE USE OF A HYBRID GENETIC SYSTEM TO STUDY THE FUNCTIONAL-RELATIONSHIP BETWEEN PROKARYOTIC AND PLANT MULTIENZYME FATTY-ACID SYNTHETASE COMPLEXES

Fatty acid synthesis in bacteria and plants is catalysed by a multi-en zyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS component s of plant and bacterial origin. The putative bacterial enoyl-ACP redu ctase gene (envM) was identified on the basis of amino acid sequence s imilarities with the recently cloned plant enoyl-ACP reductase. Subseq uently, it was unambiguously demonstrated by overexpression studies th at the envM gene encodes the bacterial enoyl-ACP reductase. An anti-ba cterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was ins ensitive to this synthetic antibiotic. The close functional relationsh ip between the plant and bacterial enoyl-ACP reductases was inferred f rom genetic complementation of an envM mutant of Escherichia coli. Ult imately, envM gene-replacement studies, facilitated by the use of diaz aborine, demonstrated for the first time that a single component of th e plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinant E. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty;acids.