Inactivation of type I polyhydroxyalkanoate synthase allowed the finding of other possible type II synthases.

CATONE, M.V.; AYUB, N.D.; LÓPEZ, N.I.

Rosario, Santa Fe

Congreso; V Congreso Argentino de Microbiología General (SAMIGE).; 2008

Sociedad Argentina de Microbiología General

Pseudomonas sp. 14-3 produces high levels of polyhydroxybutyrate (PHB). The cluster of genes involved in PHB biosynthesis (phaRBAC) arelocated on a genomic island being the PHB synthase (PhaC type I) the key enzyme. The wild type strain produces only PHB, the most commonpolyhydroxyalkanoate (PHA), from octanoic acid, but not from glucose. Inactivation of the phaC gene showed the ability to produce mediumchain lengh PHA (PHAmcl) in this mutant, indicating the existence of another PHA synthase. Using PCR techniques, two putative PHA synthasegenes (phaCmcl) were obtained from the wild type genomic DNA. One of them presented strong homology to type II PHA synthase genes ofPseudomonas, and the second one showed no homology with any of the synthases characterized until now. To analyze the functionality of thesegenes we followed two different strategies. In first place, the phaC genes were cloned into plasmid pBBR1MCS2 to complement a PHA-negativemutant of Pseudomonas putida. The second strategy consisted in mutate the two candidate genes using crossover PCR deletion techniques andscreen for the lost of the ability to produce PHA. For the screening of acquisition or lost of the capability to synthesize PHA we used Nile red andNile blue staining. Complementation of Pseudomonas putida KT2440 with phaC type I gene of Pseudomonas sp. 14-3 lead to the production ofPHB instead of PHAmcl, showing a hierarchical control of type I synthase.