OVERPRODUCTION OF A FUNCTIONAL FATTY-ACID BIOSYNTHETIC ENZYME BLOCKS FATTY-ACID SYNTHESIS IN ESCHERICHIA-COLI

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of gr owing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS i sozymes, KAS I or KAS III. The immediate effect of KAS II overproducti on is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters, Although no signific ant changes were detected in the acyl-ACP pools, the CoA pools were dr amatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction fr om a steady-state level of around 0.5% in the absence of KAS II overpr oduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the con version of malonyl-CoA to malonyl ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-Co A:ACP transacylase, the enzyme catalyzing the conversion of malonyl-Co A to malonyl-ACP, partially relieved the toxicity of KAS II overproduc tion, consistent with a model in which high levels of KAS II blocks ac cess of the other KAS isozymes to malonyl-CoA:ACP transacylase.