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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.
Polyhydroxyalkanoates (PHAs) are
biodegradable and biocompatible polyesters that accumulate as
intracellular inclusion bodies in a variety of bacteria (24,
27). Because these polymers are produced from renewable resources
such as fatty acids and sugars, they provide a new resource for plastic
materials and small molecules derived from their monomers
(45). The properties of these polyesters range from stiff
and brittle materials, such as poly(3-hydroxybutyrate) (PHB), to
elastomers, such as poly(3-hydroxyoctanoate). The monomeric composition
of a PHA depends on the bacterial strain, the culture conditions, and
the carbon source used for growth, but generally, bacteria synthesizing
PHAs can be subdivided into two groups. One group, including
Ralstonia eutropha, produces short-side-chain PHAs with
C3 to C5 monomers, while the second group,
including Pseudomonas putida, synthesizes medium-side-chain PHAs with C6 to C16 monomers (27).
PHAs can be recovered and purified from biomass by a number of
different techniques. One technique involves extraction of the polymer
from lyophilized cells with organic solvents (5). Most other
techniques involve mechanical or chemical cell disruption followed by
chemical or enzymatic treatment. During such processes, the PHA
granules are released from the cells and are subsequently purified by
repeated centrifugation and/or filtration steps. Cell disruption,
however, also results in liberation of chromosomal DNA, which causes a
rapid increase in the viscosity of the cell lysate, thereby impeding
subsequent filtration and centrifugation (26, 39, 41). Since
the efficiencies of filtration and centrifugation are inversely
proportional to the viscosity and a high viscosity directly affects
pumping, mixing, and heat transfer, quick removal of the chromosomal
DNA is critical (3). Previously described methods for DNA
degradation in PHA processes included the use of hypochlorite
(4), heat treatment (8, 9), or enzyme cocktails
(18). Even though these three methods may seem applicable in
small-scale fermentation systems, they have some drawbacks for the
envisioned 100 million-lb production scale for PHAs. Besides the
limitations at the larger scale, these procedures have additional disadvantages, since hypochlorite causes limited hydrolysis of the PHA
while heat treatment and the use of enzyme cocktails are costly.
To provide a commercially attractive solution to the viscosity problem,
we integrated the staphylococcal nuclease gene into the chromosomes of
different PHA producers. Staphylococcal nuclease has been shown to
hydrolyze DNA and RNA to fragments of less than 100 nucleotides
(2, 6). The described recombinant strains were stable in
high-cell-density fermentations and during recovery of PHA granules.
The viscosity of the lysates from such strains was reduced compared to
the viscosity of a lysate from the wild-type strain.
Bacterial strains and growth media.
The strains used in this
study are listed in Table 1.
Escherichia coli and P. putida strains were
routinely grown in Luria-Bertani medium or R medium (34).
R. eutropha was grown in either Trypticase soy broth (Becton
Dickinson, Cockeysville, Md.) or PCT medium (31)
supplemented with 1% glucose. Media were supplemented with chloramphenicol (32 µg/ml), nalidixic acid (30 µg/ml), or kanamycin (50 µg/ml) as required. Benzonase was obtained from American
International Chemical (Natick, Mass.).
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
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
Primers and DNA amplification. The nuc gene, encoding the nuclease from Staphylococcus aureus, was obtained by PCR, using plasmid pNuc1 (25) as a template. Reactions mixtures (50 µl) contained 10 pmol each of primers nucA (5' - T TC TC TAGAAT TCAGGAGGT T T T TATGGC TATCAGTAATGT T TCG) and nucB (5'-GCCGGTACCTTATTGACCTGAATCAGCGTTG) and the template in PCRmix (Gibco BRL, Gaithersburg, Md.), and reactions were performed in a thermocycler (Ericomp, San Diego, Calif.), using a program comprising 30 cycles of incubation at 95°C (30 s), at 55°C (45 s), and at 72°C (45 s). PCR products were gel purified and cloned into the pCR2.1 cloning vector (Invitrogen, Carlsbad, Calif.). The insert of the resulting plasmid, pCR2.1-nuc, was confirmed by DNA sequencing to be identical to the reported sequence of the nuc gene from S. aureus (37) (GenBank accession no. J01785).
Plasmid construction. pCR2.1-nuc was digested with EcoRI and Acc65I according to the manufacturer's (New England Biolabs, Beverly, Mass.) recommendations, and the nuc gene fragment was purified and cloned into the corresponding sites of pUC18Not (16). A promoterless, blunt-ended kanamycin gene from Tn903 (obtained by PCR from pBGS18, using primers linkK1 [5'-TGCATGCGATATCAATTGTCCAGCCAGAAAGTGAGG] and linkK2 [5'-ATTTATTCAACAAAGCCGCC]), was inserted into the SmaI site to generate pMNX-nuc-kan. The NotI fragment containing the promoterless nuc-kan operon was then cloned into the NotI sites of the integration vector pUTkan (16), a process which deleted the original kanamycin resistance marker, generating pMUX-nuc-kan.
Transposon mutagenesis and selection of integrants.
Plasmid
pMUX-nuc-kan was transformed into E. coli S17-1 
pir and
then conjugated into PHA-producing strains such as P. putida, Pseudomonas sp. strain MBX978, and R. eutropha as described elsewhere (16). P. putida and Pseudomonas sp. strain MBX978 integrants were selected on plates of minimal E2 medium
(22) containing 10 mM octanoate as the carbon source and
kanamycin as the selective agent. Integrants were replica plated onto
DNase agar plates (Difco Laboratories, Detroit, Mich.) supplemented
with kanamycin, and clones that expressed nuclease were identified by
the presence of zones of clearing around the colonies (37).
For R. eutropha, integrants were selected on PCT medium
supplemented with 1% glucose, nalidixic acid, and kanamycin.
Integrants were subsequently identified by replica plating onto DNase
agar plates supplemented with 10 g of Trypticase soy broth per
liter and kanamycin. Integrants of E. coli MBX245 were
selected on minimal E2 plates supplemented with 10 mM
octanoate, 0.5% corn steep liquor, kanamycin, and nalidixic acid.
After replica plating of resulting colonies onto DNase agar plates and
subsequent incubation, colonies exhibiting zones of clearing were selected.
DNA sequencing.
Transposon insertion sites were determined
from genomic fragments that contain the nuc-kan operon and
adjacent chromosomal DNA. Chromosomal DNA was digested to completion
with EcoRI and ligated into the corresponding site of pUC19
(36). After transformation of the ligation mixture into
E. coli DH5
, kanamycin-resistant mutants were selected
and the insertion site was determined, using the oligonucleotide
kan-up3 (5'-CGCACTTGTGTATAAGAGTC) as a primer. This primer
allows the determination of the nucleotide sequence directly downstream
of the insertion locus. Automated DNA sequencing was performed at
Boston University Medical Center (Boston, Mass.).
Detection of nuclease activity. Nuclease expression was routinely examined by observing the appearance of zones of clearing around colonies grown on DNase agar plates (25). For convenient estimation of nuclease activity, agarose gel electrophoresis with high-molecular-weight DNA was used as follows. PHA-producing strains were grown in their corresponding minimal media, and at various times were removed 500-µl samples, to each of which was added 100 µl of chloroform to release periplasmic nuclease. After centrifugation, 16 µl of the aqueous supernatant was mixed with 4 µg of P. putida KT2442 genomic DNA, and the mixture was incubated at 37°C for 1 h after CaCl2 was added to 1 mM (6). DNA was subsequently separated by agarose gel electrophoresis (36), and nuclease activity was assessed by determining the decrease in the molecular weight of the genomic DNA.
PHA analysis. PHA-containing cells (5 to 20 mg) were subjected to hydrolysis in dichloroethane-propanol-HCl (5:4:1) for 2 h at 100°C (35). Resulting propyl esters of hydroxyalkanoic acids were analyzed by gas chromatography as described previously (22).
Viscosity assay. Pseudomonas sp. strains MBX978 and MBX985 were grown in 20-liter computer-controlled fermentors (Applicon, Schiedam, The Netherlands) on R medium with a dissolved oxygen-controlled (DO-stat) octanoate feed (a detailed report on the fermentation procedures will be reported elsewhere). At the end of the fermentation, cultures were supplemented with 1 mM CaCl2 and lysed by passage through a microfluidizer (model M110EH; Microfluidics International Corp., Newton, Mass.) operating at pressures ranging from 8,000 to 20,000 lb/in2. The homogenized cultures were incubated at room temperature for 1 h, and the viscosities of the lysates were determined at room temperature with an LVF viscometer (Brookfield, Stoughton, Mass.). In control experiments, a commercial preparation of nuclease from Serratia marcescens (Benzonase; American International Chemical Inc.) was added.
SDS-PAGE. Cell extracts, obtained by sonicating cells from 50-ml cultures and resuspending them in 2 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM 2-mercaptoethanol, 5% glycerol), were subjected to sodium dodecyl sulfate (SDS)-12.5% polyacrylamide electrophoresis (PAGE) (Bio-Rad, Richmond, Calif.) as described elsewhere (36).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nuclease integrants of P. putida KT2442. P. putida KT2442 strains that express staphylococcal nuclease were constructed by random integration of a nuc-kan cassette. Of 12,000 kanamycin-resistant integrants, 1,500 colonies were replica plated onto DNase agar plates to screen for the clones with the highest levels of nuclease expression. The presence of nuclease activity in both the extracellular medium and the periplasm of nine different isolates was subsequently determined (Fig. 1). Nuclease is primarily secreted into the periplasm, suggesting that the P. putida protein secretion apparatus recognizes the signal peptide of staphylococcal nuclease. Because the nuc gene in the transposon is not preceded by a promoter element, its expression is driven from promoter sequences located on the chromosome. This is expected to result in various levels of nuclease for different nuclease integrants, as was demonstrated by the low level of activity in MBX920 (lane 7), the high level of activity in MBX924 (lane 3), and intermediate levels of activity for the other strains. For P. putida MBX924, nuclease activity was detected in both the growth medium and the periplasm. The chromosomal DNA fragment containing the nuc-kan operon from P. putida MBX924 was cloned as a 4-kb EcoRI fragment into pUC19. The nucleotide sequence of the DNA downstream of the nuc-kan operon showed that the insertion was in a gene encoding a homolog of the 23S rRNA from Pseudomonas aeruginosa (96.4% identity over 362 nucleotides), suggesting that the corresponding promoter for this rRNA operon directs the transcription of nuc in MBX924. Unfortunately, a definite assignment of the nuc insertion site to this rRNA locus cannot be made because the complete sequence of the corresponding P. putida gene is unknown at this time.
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Construction of nuclease integrants of other PHA producers. By the use of similar methods, nuc was integrated into the chromosome of Pseudomonas sp. strain MBX978. For Pseudomonas sp. strain MBX978, 50 mutants were selected on DNase agar plates, of which 9 were grown in minimal E2-octanoate medium and tested for relative nuclease activity levels by agarose gel electrophoresis. All nine clones secreted nuclease into the periplasm (results not shown).
Nuclease integrants for R. eutropha NCIMB40124HD and E. coli MBX245 were subsequently generated. R. eutropha, an exceptional PHB producer, was the strain of choice for commercial PHB production by ICI, Ltd. (5). E. coli generally does not synthesize PHAs but is regarded as a suitable host for improved recombinant PHA production processes (27). For R. eutropha, 10 random colonies were grown and tested for nuclease activity; only one (MBX917) exhibited nuclease activity. For E. coli, 75 colonies were screened; 4 mutants exhibited nuclease activity (data not shown). The transgenic nuclease-producing strains derived from both E. coli and R. eutropha secreted nuclease into the periplasm but not into the growth medium. Figure 3 shows a summary of the nuclease activities for the different transgenic nuclease-secreting species and their corresponding parent strains. These data indicate that transgenic nuclease expression can be achieved by different PHA-producing strains and that it is a generally applicable procedure to prevent processing problems related to viscosity caused by chromosomal DNA.
|
Evaluation of nuclease integrants of Pseudomonas sp.
strain MBX978 for PHA production.
In order to successfully replace
wild-type strains as PHA producers, the integrated strains should
demonstrate the same stability and PHA productivity as the wild-type
strain. All integrants derived from Pseudomonas sp. strain
MBX978 were tested for PHA content, growth rate, and relative nuclease
activity in E2-10 mM octanoate medium before being
analyzed in a large-scale fermentor for PHA production and processing.
Table 2 lists the characteristic growth rates and PHA contents of these strains. Most of these nuclease integrants exhibited the same growth rate as the parental strain. Except for Pseudomonas sp. strain MBX984, the PHA contents
and the compositions of the accumulated PHAs were similar. However, similar to what was observed for the nuclease integrants derived from
P. putida KT2442 (Fig. 1), the nuclease levels in these
strains differed. Because Pseudomonas sp. strain MBX985
exhibits a relatively high level of nuclease activity and growth
characteristics similar to those of the wild-type strain, it was
further evaluated for its ability to improve the PHA extraction process
in a 20-liter fermentation-downstream processing protocol.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The nuclease gene from S. aureus was integrated into the genomes of several well-known PHA producers. Most pseudomonads from rRNA homology group I produce PHAs composed of medium-side-chain 3-hydroxy fatty acids when grown on fatty acids or on carbohydrates (15, 19, 20, 40). R. eutropha is a well-known producer of PHB and related short-side-chain PHAs (5); E. coli is a bacterium that is considered to be a potential recombinant PHA producer, and both short- and medium-side-chain PHAs are produced by this organism (23, 33, 42, 43). Here the effect of nuclease expression on ease of downstream processing was demonstrated for a Pseudomonas sp. strain MBX978 derivative, and nuclease-expressing strains were also derived from P. putida KT2442, R. eutropha NCIMB40124HD, and E. coli MBX245.
The production cost of a fermentation product depends to a large extent on the combined costs of fermentation and downstream processing (8). While molecular genetics has frequently been applied to improve the productivity (10, 11, 29) and fitness (17, 44) of a microorganism, its application to downstream processing is rather uncommon. In the aqueous processing of PHA-containing cells, it is necessary to lyse the cells, and this process is accompanied by a dramatic increase in sample viscosity, due to chromosomal DNA, that reduces the efficiency of the subsequent centrifugation, filtration, and washing steps (3). The PHA-producing strains that are described here express an endogenous nuclease whose use leads to significant cost savings. Such strains are useful in processes besides the production of PHAs, since many industrial fermentation processes deal with similar viscosity problems. The production of high-value pharmaceutical proteins frequently involves isolation of inclusion bodies, which should be essentially free of nucleic acids to allow efficient purification and formulation (14). Furthermore, we can expect a dramatic increase in the use of recombinant organisms for the production of chemicals, proteins, and polymers, and it is desirable to prevent proliferation of heterologous genes by inclusion of a procedure for DNA degradation.
The improvement of PHA-accumulating microorganisms for PHA production by integration of a nuclease-encoding gene is potentially of great economic value. Although future PHA production is envisioned to be a major agricultural process (30, 32, 45), the use of fermentation systems for the production of PHA latex as well as specialty PHAs will continue. Such specialty PHAs may contain monomers that are strictly derived from the feedstock and contain, for instance, aromatic, halogenated, or unsaturated functionalities (1, 12, 13, 21, 38), while the PHA latex can be used for making PHA films with potential applications for paper and food coating (28). The developments described here are therefore expected to contribute to the efficiency of future PHA production facilities.
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ACKNOWLEDGMENTS |
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We thank Anthony Sinskey for plasmid pNuc1 and David Martin and Lara Madison for critical reading of the manuscript.
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FOOTNOTES |
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* 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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Materials and Methods
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Discussion
References
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Applied and Environmental Microbiology, April 1999, p. 1524-1529, Vol. 65, No. 4
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