RESUMEN
Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (ß-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7.