MUTANTS OF PSEUDOMONAS-PUTIDA AFFECTED IN POLY-3-HYDROXYALKANOATE SYNTHESIS

The generation and characterization of Pseudomonas putida KT2442 mutan ts affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. T he mutants from P. putida KT2442 carrying several copies of the PHA-po lymerase-encoding gene (phaC) were isolated via N-methyl-N'-nitro-N-ni trosoguanidine chemical mutagenesis and contained mutation(s) on genes that are involved in PHA accumulation other than the phaC genes. No P HA-free mutants were obtained, suggesting that there must be various r outes for the synthesis of PHA polymerase precursors. One of the isola ted mutants (GPp120) accumulated more PHA than the parental strain, an d there was virtually no down-regulation of PHA formation by growth in nonlimiting amounts of nitrogen, which normally block or reduce forma tion of PHA. Compared to the parental strain. GPp120 exhibited signifi cant changes in physiology and morphology when grown in minimal medium : the growth rate was reduced more than twofold and cells formed filam ents. The other four groups of isolated mutants, with P, putida strain s GPp121 to GPp124 as characteristic type strains, exhibited morpholog ical characteristics similar to those of the parental strain. However, they showed reduced PHA production compared to the parental PHA(+) st rain, and especially GPp121 and GPp122 showed PHA formation tightly co ntrolled by nutrient conditions. All of these mutants provide starting points for genetically dissecting the biosynthesis and regulation of PHA precursors.