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

Abstract

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.

FIG. 1

Nuclease activity in nine P. putida nuclease integrants. P. putida KT2442 and derivatives with an integrated nuclease gene were grown in E₂–10 mM octanoate. Growth medium (top) and chloroform-permeabilized cell fractions representing the periplasm (bottom) were incubated with 4 μg of P. putida KT2442 genomic DNA at 37°C for 1 h. Lanes: 1, MBX926; 2, MBX925; 3, MBX924; 4, MBX923; 5, MBX922; 6, MBX921; 7, MBX920; 8, MBX919; 9, MBX918; 10, P. putida KT2442. chr. DNA, chromosomal DNA.

FIG. 2

Analysis of nuclease expression by P. putida KT2442 and P. putida MBX924 grown in a 20-liter fed-batch fermentation. Wild-type (wt) and nuclease-integrated (nuc) Pseudomonas strains were grown as described in the text, and at cell densities of 12 and 35 g/liter, samples were analyzed for nuclease activity (A) and protein (B) in the extracellular growth medium (EC) and periplasm (P). The arrows indicate chromosomal DNA (A) and the putative nuclease (B). Lane 1 contains molecular mass markers (either HindIII-digested λ DNA [A] or a combination of glutamic dehydrogenase [56 kDa], maltose binding protein [43 kDa], triosephosphate isomerase [27 kDa], and trypsin inhibitor [20 kDa] [B]).

FIG. 3

Nuclease activity in wild-type and transgenic PHA producers with a chromosomally integrated nuclease gene. Chromosomal (chr.) DNA was treated with periplasmic fractions of E. coli MBX245 (lane 1), E. coli MBX988 (::nuc-kan) grown on R medium (lane 2) or Luria-Bertani medium (lane 3), R. eutropha MBX917 (::nuc-kan) (lane 4), R. eutropha NCIMB40124HD (lane 5), Pseudomonas sp. strain MBX985 (::nuc-kan) (lane 6), Pseudomonas sp. strain MBX978 (lane 7), P. putida MBX924 (::nuc-kan) (lane 8), or P. putida KT2442 (lane 9).

FIG. 4

Viscosities of cell lysates. Nuclease-expressing and wild-type Pseudomonas sp. strain MBX978 were grown in fed-batch mode to a density of approximately 60 g/liter. Cell suspensions were subsequently homogenized with or without the addition of Benzonase. The viscosity of the lysate was determined as a function of the operating pressure of the homogenizer. Closed circles, Pseudomonas sp. strain MBX978 without added Benzonase; open circles, Pseudomonas sp. strain MBX978 with added Benzonase; closed squares, nuclease integrant Pseudomonas sp. strain MBX985 without added Benzonase.