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Applied and Environmental Microbiology, April 1999, p. 1524-1529, Vol. 65, No. 4
Departments of Molecular
Biology,1
Fermentation,2 and Downstream
Processing,3 Metabolix Inc., Cambridge,
Massachusetts 02142
Received 12 November 1998/Accepted 25 January 1999
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.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Reduction of Cell Lysate Viscosity during
Processing of Poly(3-Hydroxyalkanoates) by Chromosomal Integration of
the Staphylococcal Nuclease Gene in Pseudomonas
putida
*
Corresponding author. Present address: Maxygen, 3410 Central Expressway, Santa Clara, CA 95051. Phone: (408) 522-6001. Fax: (408) 481-0385. E-mail: gjalt_huisman{at}maxygen.com.
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