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 granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings 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 generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.
Boynton
Z L
ZL
Departments of Molecular Biology, Metabolix Inc., Cambridge, Massachusetts 02142, USA.
Koon
J J
JJ
Brennan
E M
EM
Clouart
J D
JD
Horowitz
D M
DM
Gerngross
T U
TU
Huisman
G W
GW
D003470:Culture Media; D004251:DNA Transposable Elements; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011091:Polyesters / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D014783:Viscosity
D003470:Culture Media; D004251:DNA Transposable Elements; D011091:Polyesters; D008836:Micrococcal Nuclease