A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacylACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida AfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the AfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida n-oxidation pathway gave increased accumulation. The AfabB Delta fabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the AfabF2 APput_2425 double mutant strain still accumulated suppressors at low UFA concentrations.

Automated summary

The genome of Pseudomonas putida F1 contains multiple genes encoding 3-ketoacyl-acyl carrier protein synthases, some of which only show physiological significance under specific conditions. Experimental results with Escherichia coli demonstrate that certain genes in P. putida can substitute for functions in E. coli, particularly in relation to their roles in fatty acid synthesis pathways.