Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granule s and which are thought to serve as reserves of carbon and energy, Pseudomo nas putida accumulates a polyester, composed of medium-side-chain 3-hydroxy alkanoic acids, which has excellent film-forming properties, Industrial pro cessing of PHA involves purification of the PHA granules from high-cell-den sity cultures. After the fermentation process, cells are lysed by homogeniz ation and PHA granules are purified by chemical treatment and repeated wash ings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is genera lly achieved by the supplementation of commercially available nuclease prep arations or by heat treatment; however, both procedures add substantial cos ts to the process, As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA p roducers. Staphylococcal nuclease is readily expressed in PHA-producing Pse udomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. Durin g downstream processing, the viscosity of the lysate from a nuclease-integr ated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nucleas e gene was also functionally integrated into the chromosomes of other PHA p roducers, including Ralstonia eutropha.