REDUCTION OF CELL LYSATE VISCOSITY BY CLONING STAPHYLOCOCCUS AUREUS NUCLEASE GENE IN POLYHYDROXYALKANOATES PRODUCING BACTERIA

Downstream processing may significantly affect the price of polyhydroxyalkanoates (PHA); hence, the development of an economical and efficient recovery processes is needed. During PHA production, the high viscosity of the PHA-synthesizing bacterial cell lysate, which is due to the high content of nucleic acids, could represent a technological problem. In order to reduce viscosity during this stage, a nuclease enzyme could be used. The aim of this work was to introduce in highly efficient PHA producing selected bacteria ( Cupriavidus necator DSM 545 and Pseudomonas oleovorans DSM 1045) the nuclease gene (nuc), encoding for a staphylococcal extracellular thermo-stable nuclease (SNase), deriving from Staphylococcus aureus . Since plasmid pNuc, containing the nuc gene, is unable to replicate in E. coli , an amplified fragment of 700 bp was obtained and cloned in a broad host range plasmid. Once transferred into E. coli the nuclease activity was tested on chloroform-permeabilized cells incubated with -phage DNA, and found to be effi ciently expressed and therefore suitable for subsequent cloning purposes. After conjugation of the obtained plasmid into C. necator DSM 545 and P. oleovorans DSM 1045 nuclease activity and PHA production were analyzed. The results obtained indicated that nuc gene can proficiently be expressed in the two recipient strains without significant limitations of PHA production.