Accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in Pseudomonas fluorescens BM07 utilizing saccharides and its inhibition by 2-bromooctanoic acid

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PRA) containing large amounts of 3-hyd roxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substr ates such as fructose, succinate, etc., was isolated from an activated slud ge in a municipal wastewater treatment plant. When it was grown on heptanoi c acid (C-7) to hexadecanoic acid (C-16) as the sole carbon source, the mon omer compositional characteristics of the synthesized PHA, were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C-18) led to no PRA accumulation , but instead free stearic acid was stored in the cell. The existence of th e linkage between fatty acid de novo synthesis and PRA synthesis was confir med by using inhibitors such as acrylic acid and two other compounds, 2-bro mooctanoic acid and 4-pentenoic acid, which are known to inhibit beta -oxid ation enzymes in animal cells. Acrylic acid completely inhibited PRA synthe sis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhib ition of PRA synthesis occurred in 70 mM fructose-grown cells in the presen ce of I to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid wer e found to much inhibit PRA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from I to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cel l growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA sy nthesis inhibition over a small range of concentrations below 100 muM, a fi nding indicative of a very specific inhibition, whereas 4-pentenoic acid sh owed a broad, featureless concentration dependence, suggesting a rather non specific inhibition. The apparent inhibition constant K-i (the concentratio n for 50% inhibition of PRA synthesis) for 2-bromooctanoic acid was determi ned to be 60 muM, assuming a single-site binding of the inhibitor at a spec ific inhibition site. Thus, it seems likely that a coenzyme A thioester der ivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest t hat 2-bromooctanoic acid can substitute for the far more expensive (2,000 t imes) and cell-growth-inhibiting PRA synthesis inhibitor, cerulenin.