Research Article
20 April 2011

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

ABSTRACT

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

INTRODUCTION

Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria as carbon and energy storage compounds (2). The first PHA discovered was the homopolymer poly(3-hydroxybutyrate) (PHB) (22). It was established that other types of PHAs also exist in nature when Wallen and Rohwedder extracted PHA copolymers from sewer sludge (39) and when de Smet et al. observed that Pseudomonas oleovorans can synthesize poly(3-hydroxyoctanoate) (10). Today, PHAs are characterized as containing short-chain-length (SCL; C3 to C5) and/or medium-chain-length (MCL; C6 and longer) monomers (30).
There has long been interest in using PHAs as biodegradable bioplastics that could serve as alternatives to petrochemical plastics. The commercial potential of PHB was first investigated by W. R. Grace and Company (3), who determined that this polymer has several issues that limit its value. PHB is a highly crystalline polymer that lacks toughness and begins to decompose near its melting temperature, making it difficult to process (21). Many studies were later conducted with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)], but it was found that introduction of HV units into the polymer had limited impact on the material properties (17). This is due to the fact that HB and HV units are able to cocrystallize (4). Copolymerization of MCL monomers with HB leads to more dramatic changes to the properties of the plastic (27). The best-studied member of this class of PHA is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)]. P(HB-co-HHx) has a lower melting temperature, lower Young's modulus, and longer elongation to break than PHB (11, 27). This means that P(HB-co-HHx) is a tougher, more flexible plastic than PHB.
The model organism for studying PHA synthesis and accumulation is Ralstonia eutropha H16, because it accumulates large quantities of polymer when grown in nutrient-limited conditions (29). R. eutropha has also been shown to grow efficiently with plant oil as the sole carbon source (6). The wild type produces only SCL PHA, however, and thus is limited as an industrial PHA production organism. One of the first organisms identified that synthesizes P(HB-co-HHx) was Aeromonas caviae (33). This bacterium and related species store P(HB-co-HHx) when grown on plant oils and fatty acids but exhibit a low level of PHA accumulation (11). Investigations of A. caviae have revealed how this organism is able to synthesize P(HB-co-HHx). The PHA synthase from A. caviae (PhaCAc) efficiently polymerizes both HB-coenzyme A (HB-CoA) and HHx-CoA (12). A. caviae also has a gene encoding an (R)-specific enoyl-CoA hydratase (phaJAc), which allows for conversion of fatty acid β-oxidation intermediates to PHA precursors (14, 16).
Plant oils and fatty acids are appealing feedstocks for industrial PHA production because of their high carbon contents and because metabolism of these compounds can influence the monomer composition of the resulting PHA (1). Several groups have produced P(HB-co-HHx) by using Aeromonas strains or recombinant R. eutropha expressing phaCAc (9, 18, 23, 26). In these cases, however, production of PHA with high HHx content (>5 mol%) required feeding the cells short-chain-length fatty acids (≤12 carbons), which is undesirable because these compounds are more costly than raw plant oil. Mifune and coworkers recently reported engineered R. eutropha strains that expressed evolved phaCAc and phaJAc (25). Strains from this study were able to accumulate high levels of PHA (>75% of the cell dry weight [CDW; i.e., wt%]) with up to 9.9 mol% HHx content when grown on soybean oil.
Our group hypothesized that the high levels of HB-CoA produced by R. eutropha when it is grown on plant oil could limit incorporation of other monomers into the PHA, even if the strain expressed a PHA synthase that could polymerize both HB-CoA and HHx-CoA. Recently, we reported an R. eutropha strain in which genes encoding acetoacetyl-CoA reductases were deleted, and this strain makes significantly less PHB than the wild type (8). We planned to engineer this strain to produce P(HB-co-HHx). A series of strains was constructed in which PHA synthase genes from A. caviae and Rhodococcus aetherivorans I24 (7) were integrated into the genomes of R. eutropha strains with different levels of acetoacetyl-CoA reductase activity. Rhodococcus species have been shown to synthesize PHA copolymers (41), and analysis of a draft genome of R. aetherivorans revealed two putative PHA synthase genes that had not been described in the literature. The strains were further improved by incorporating phaJ genes and by increasing gene expression with a stable plasmid expression system. Our work resulted in the construction of two stable R. eutropha strains that accumulate high levels of P(HB-co-HHx) when grown on plant oil, in which the HHx contents of the PHAs from both strains are >12 mol%.

MATERIALS AND METHODS

Bacterial strains and cultivation conditions.

All PHA production experiments in this study were conducted with Ralstonia eutropha H16 and mutants derived from this strain (Table 1). Strain genotypes are also illustrated in Fig. S1 in the supplemental material. The rich medium used for growth of R. eutropha was dextrose-free tryptic soy broth (TSB) medium (Becton Dickinson, Sparks, MD). The concentrations of salts in the R. eutropha minimal medium have been reported previously (8). Carbon and nitrogen sources were added to the minimal medium as described in the text. The carbon sources used in this study were fructose and palm oil (Wilderness Family Naturals, Silver Bay, MN). All media contained 10 μg/ml gentamicin sulfate. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. R. eutropha strains were always grown aerobically at 30°C. In shake flask experiments, 50 ml medium was used in 250-ml flasks. The shaker was set to 200 rpm.
Table 1.
Table 1. Strains and plasmids used in this study
Strain or plasmidDescriptionaReference or source
R. eutropha strains  
    H16Wild-type strain, Gm resistantATCC 17699
    Re1034H16 ΔphaC142
    Re2000Re1034::phaC1Ra, made with pINY3This study
    Re2001Re1034::phaC2Ra, made with pINY4This study
    Re2058Re1034 ΔproC, made with pCB110This study
    Re2115H16 ΔphaB1 ΔphaB2 ΔphaB38
    Re2133Re2115 ΔphaC1, made with pGY46This study
    Re2135Re2133::phaC2Ra, made with pINY4This study
    Re2136Re2133::phaCAc, made with pLW484This study
    Re2151Re2135::phaB2, made with pCB66This study
    Re2152Re2135::phaJ1Pa, made with pCB69This study
    Re2153Re2135::phaJAc, made with pCB72This study
    Re2154Re2136::phaB2, made with pCB66This study
    Re2155Re2136::phaJ1Pa, made with pCB69This study
    Re2156Re2136::phaJAc, made with pCB72This study
    Re2160Re2133 ΔproC, made with pCB110This study
E. coli strains  
    DH5αGeneral cloning strainInvitrogen
    S17-1Strain for conjugative transfer of plasmids to R. eutropha34
Other strains  
    R. aetherivorans I24Source of phaC1Ra and phaC2Ra7
    P. aeruginosa PAO1Source of phaJ1Pa36
Plasmids  
    pGY46Plasmid for deletion of phaC1 from R. eutropha genome; the backbone was used to make other plasmids for gene deletion/insertion in R. eutropha genome; confers Km resistance42
    pJV7pGY46 with SwaI site between regions of DNA upstream and downstream of phaC1, used to insert new genes at the phaC1 locusThis study
    pINY3pJV7 with phaC1Ra cloned into SwaI siteThis study
    pINY4pJV7 with phaC2Ra cloned into SwaI siteThis study
    pLW484pJV7 with phaCAc cloned into SwaI siteThis study
    pCB42Plasmid for insertion of genes at the phaB1 locus in the R. eutropha genome, confers Km resistance8
    pCB66pCB42 with R. eutropha phaB2 cloned into SwaI site8
    pCB69pCB42 with phaJ1Pa cloned into SwaI siteThis study
    pCB72pCB42 with phaJAc cloned into SwaI siteThis study
    pCB110Plasmid for deletion of R. eutropha proCThis study
    pBBR1MCS-2Vector for plasmid-based gene expression in R. eutropha, confers Km resistance19
    pCB81pBBR1MCS-2 with the PHA operon from Re2152 cloned between KpnI and HindIII sitesThis study
    pCB113pCB81 with R. eutropha proC region cloned into AgeI siteThis study
a
Abbreviations: Gm, gentamicin; Km, kanamycin.

Plasmid and strain construction.

In this study, DNA was routinely amplified by using high-fidelity DNA polymerase (Qiagen, Valencia, CA) and digested using restriction enzymes from New England BioLabs (Ipswich, MA). Plasmids were transformed into R. eutropha via transconjugation with Escherichia coli S17-1. Markerless gene deletions and insertions in the R. eutropha genome were achieved following the protocol described in reference 8, which is based on the work of York et al. (42). The strains and plasmids used in this study are described in Table 1. The sequences of all oligonucleotide primers used in this study are provided in Table S1 in the supplemental material.
An R. eutropha strain with the phaC1 gene deleted (Re1034) was previously constructed in our lab (42). This strain is unable to synthesize PHA. The plasmid used to make the phaC1 deletion (pGY46) contained a section of DNA in which the region of the genome immediately upstream of phaC1 was connected to the region of the genome downstream of phaC1. In order to insert new synthase genes at the phaC1 locus, pGY46 was altered via site-directed mutagenesis using the Invitrogen GeneTailor kit (Carlsbad, CA). An SwaI site was inserted between the upstream and downstream sequences, allowing synthase genes to be cloned into this site in the mutated plasmid (pJV7). We investigated in this study two novel PHA synthase genes from R. aetherivorans I24, which were named phaC1Ra and phaC2Ra. These genes were identified by analyzing a draft copy of the R. aetherivorans I24 genome, provided by John Archer (University of Cambridge, United Kingdom). When amplifying phaC1Ra from the R. aetherivorans I24 genome, a primer was used such that the start codon in the cloned gene was ATG, rather than the TTG found in the genome. A version of the phaC gene from A. caviae in which the DNA sequence was codon optimized for expression in R. eutropha was purchased from Codon Devices (Cambridge, MA). The optimized phaCAc was designed with SwaI sites on both ends of the gene so that it could also be cloned into pJV7.
Many strains were constructed based on Re2115, an R. eutropha strain in which the three phaB genes in the R. eutropha genome had been deleted (8). New genes were inserted into the phaB1 locus to alter production of PHA monomers by using plasmids based on pCB42. The gene inserted into this locus was phaB2 from R. eutropha (8), phaJ1 from Pseudomonas aeruginosa (phaJ1Pa) (38), or phaJ from A. caviae (14). The phaJ1Pa gene was cloned via colony PCR from P. aeruginosa PAO1. The phaJAc gene was synthesized by Integrated DNA Technologies (Coralville, IA) and had EcoRV sites located at both ends of the gene, allowing it to be cloned into pCB42.
In order to increase the gene copy number, and thus gene expression, the engineered PHA biosynthesis operon from strain Re2152 (Table 1) was amplified via PCR and cloned into plasmid pBBR1MCS-2, creating pCB81. This plasmid was maintained in R. eutropha by adding 200 μg/ml kanamycin to the growth media. In order to improve plasmid stability in the absence of kanamycin, R. eutropha strains in which the proC gene was deleted from their genomes were constructed. The gene proC (locus tag h16_A3106; GeneID number 4250351) encodes pyrroline-5-carboxylate reductase, which is part of the proline biosynthesis pathway. When constructing the ΔproC strains, 0.2% proline was added to all selection plates. The region of the R. eutropha genome containing proC and h16_A3105 was amplified via colony PCR and cloned into pCB81, creating plasmid pCB113. This plasmid was transformed into ΔproC R. eutropha strains.

Fermentation conditions.

Strains Re2058/pCB113 and Re2160/pCB113 were grown to higher densities than is possible in shake flasks by using an Infors Sixfors multiple fermentor system (Bottmingen, Switzerland). Cultures were prepared by first growing the strains overnight in TSB containing 200 μg/ml kanamycin. These cultures were used to inoculate 50 ml minimal medium flask precultures containing 2% fructose and 0.1% NH4Cl. The minimal medium precultures were used to inoculate the fermentors so that the initial optical density at 600 nm (OD600) of each 400 ml culture was 0.1. Each fermentor contained either 4% (Re2160/pCB113) or 4.5% (Re2058/pCB113) palm oil and 0.4% NH4Cl. Neither the fructose nor palm oil minimal medium cultures contained kanamycin.
The temperature of each fermentor was kept constant at 30°C. The pH of each culture was maintained at 6.8 ± 0.1 through controlled addition of 2 M sodium hydroxide. Stirring was provided by two six-blade Rushton impellers at speeds of 500 to 1,000 rpm. Air was supplied at 0.5 to 1 vol/vol/min, and the dissolved oxygen concentration was maintained above 40% through controlled addition of pure oxygen. Sterile silicone oil AR200 was used as an antifoam in these experiments and was added to cultures by hand as necessary.

Analytical methods.

The cell dry weights (CDWs) of cultures were measured by taking 8- to 14-ml samples in preweighed plastic test tubes. The samples were centrifuged, and the pellets were washed with 5 ml cold water. For experiments using palm oil as the carbon source, 2 ml cold hexane was also included during the wash step to remove unused oil from the samples. Samples were then centrifuged again, resuspended in 1 ml cold water, frozen at −80°C, and lyophilized. The dried samples were weighed, and CDWs were determined. Residual cell dry weight (RCDW) values were calculated for each sample, and these were defined as the total CDW minus the mass of PHA. Ammonium concentrations in clarified culture supernatants were measured with an ammonium assay kit (catalog no. AA0100; Sigma-Aldrich) following the manufacturer's instructions.
The PHA contents and compositions from dried samples were determined using a methanolysis protocol adapted from reference 5. Dried cells were weighed in screw-top glass test tubes and reacted with methanol and sulfuric acid in the presence of chloroform for 2.5 h at 100°C. This reaction converts PHA monomers into their related methyl esters. The concentrations of methyl esters were determined via gas chromatography with an Agilent 6850 gas chromatograph (GC) (Santa Clara, CA) equipped with a DB-Wax column (30 m by 0.32 mm by 0.5 μm; Agilent) and a flame ionization detector. Two microliters of each sample was injected into the GC with a split ratio of 30:1 (vent:column). Hydrogen was used as the carrier gas at a flow rate of 3 ml/min. The oven was held at 80°C for 5 min, heated to 220°C at 20°C/min, and held at 220°C for 5 min. Pure standards of methyl 3-hydroxybutyrate and methyl 3-hydroxyhexanoate were used to generate calibration curves for the methanolysis assay.
PHA was extracted from dried cells by using chloroform for measurement of polymer molecular weights. Molecular weight measurements were made via gel permeation chromatography (GPC) relative to polystyrene standards as described previously (8). The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) were measured for each sample.
Additional polymer characterization was performed with purified PHA recovered from samples of Re2058/pCB113 and Re2160/pCB113 taken at the end of palm oil fermentations (120 h of growth). PHA was isolated from lyophilized cells by extracting the polymer with methyl isobutyl ketone (MIBK). For each extraction, 100 ml of MIBK was added to 1.5 g of dried cells and stirred at 100°C for 4 h under reflux conditions. Cell debris was removed by centrifugation, and PHA was precipitated from solution by the addition of 3 volumes of hexane. The resulting precipitate was collected by centrifugation, washed with additional hexane, and dried. The monomer composition of the purified PHA was determined by proton nuclear magnetic resonance (NMR) spectroscopy. Polymer was dissolved in deuterated chloroform, and 1H NMR spectra were collected with a Varian Mercury 300 MHz spectrometer.

Nucleotide sequence accession numbers.

The sequences of phaC1Ra and phaC2Ra have been deposited in GenBank under accession numbers HQ130734 and HQ130735. The sequence of the optimized phaCAc was deposited in GenBank under accession number HQ864571.

RESULTS

Characterization of R. aetherivorans I24 synthases.

It was confirmed that the putative synthase genes from R. aetherivorans I24 encoded active enzymes by inserting the genes into the Re1034 genome at the phaC1 locus. Insertion of either gene restored production of PHB from fructose (Table 2). Re2000 accumulated approximately the same amount of PHB as H16, while Re2001 made significantly less. It has been demonstrated that if a synthase capable of polymerizing MCL monomers is expressed in recombinant R. eutropha, the strain will accumulate MCL PHA when grown on fatty acids (24). The pathway through which MCL hydroxyacyl-CoA molecules are synthesized in wild-type R. eutropha has not yet been identified. Re2000 and Re2001 were therefore grown on a series of fatty acids in order to characterize the substrate specificities of the R. aetherivorans PHA synthases (Table 2). The cultures contained 0.05% NH4Cl and an initial fatty acid concentration of 0.2%. An additional 0.2% fatty acid was added to the cultures after 24 h of growth. It was found that both recombinant strains were able to incorporate more HHx into PHA than H16 and that PHA from the strain harboring phaC2Ra also included 3-hydroxyheptanoate (HHp) when heptanoate was used as the carbon source. No PHA monomers longer than HHp were detected in any of the samples.
Table 2.
Table 2. Compositions of PHAs from strains grown on different carbon sourcesb
Carbon sourceStrainPHA (% of CDW)PHA composition (mol%)a
HBHVHHxHHp
FructoseH1675 ± 3100   
 Re200079 ± 2100   
 Re200139 ± 1100   
HexanoateH1649 ± 299.61 ± 0.01 0.39 ± 0.01 
 Re200051 ± 188.5 ± 0.2 11.5 ± 0.2 
 Re200148 ± 281.1 ± 0.4 18.9 ± 0.4 
HeptanoateH1652 ± 362.6 ± 0.537.4 ± 0.5 0
 Re200062 ± 140.4 ± 0.359.6 ± 0.3 tr
 Re200148 ± 625.2 ± 1.172.9 ± 1.6 1.9 ± 0.5
OctanoateH1666 ± 3100 tr 
 Re200066 ± 293.44 ± 0.08 6.56 ± 0.08 
 Re200142 ± 489.6 ± 0.3 10.4 ± 0.3 
a
Abbreviations: HB, 3-hydroxybutyrate; HV, 3-hydroxyvalerate; HHx, 3-hydroxyhexanoate; HHp, 3-hydroxyheptanoate; tr, trace amounts.
b
PHA produced by H16 and recombinant R. eutropha strains expressing phaC1Ra and phaC2Ra was analyzed after the strains were grown for 60 h on 2% fructose or 0.4% fatty acids. All media contained 0.05% NH4Cl. The values reported are averages from triplicate cultures ± SDs.

Analysis of R. eutropha strains with engineered genomes.

The goal of this study was to produce SCL/MCL PHA copolymers by using palm oil as the carbon source. Palm oil is an important agricultural product in Southeast Asia, with a high yield of oil per acre of land (40). Re2000 and Re2001 were therefore grown in minimal medium with palm oil as the sole carbon source. While these strains accumulated P(HB-co-HHx) with significant HHx content when grown on hexanoate and octanoate, the PHA made from palm oil consisted of <2 mol% HHx (Table 3). We hypothesized that high intracellular concentrations of HB-CoA may limit HHx incorporation into the PHA made by the recombinant strains. Our group previously constructed a strain with low acetoacetyl-CoA reductase activity that accumulates significantly less PHB than H16 (Re2115). The phaC1 gene from the genome of Re2115 was deleted, and phaC2Ra (Re2135) or phaCAc (Re2136) was inserted in its place. The synthase gene phaC2Ra was chosen because the primary focus was to produce PHA with high HHx content, and the strain containing phaC2Ra synthesized PHA with the highest levels of HHx during fatty acid growth (Table 2, compare Re2001 to Re2000 and H16). The gene phaCAc was also investigated because it has been used in most of the P(HB-co-HHx) production studies in the literature. Both Re2135 and Re2136 made PHA with high HHx content from palm oil (Table 3), but these strains did not accumulate significant polymer (∼25% of the CDW after 72 h).
Table 3.
Table 3. Cell dry weights and levels of PHA and HHxa
Strain48 h72 h
CDW (g/liter)PHA (% of CDW)HHx (mol%)CDW (g/liter)PHA (% of CDW)HHx (mol%)
H165.3 ± 0.471 ± 106.0 ± 0.279.2 ± 0.90
Re20006.1 ± 0.175.3 ± 0.31.5 ± 0.17.3 ± 0.182 ± 41.1 ± 0.3
Re20011.89 ± 0.0449 ± 21.6 ± 0.22.19 ± 0.0950 ± 31.5 ± 0.2
Re21150.78 ± 0.0416.9 ± 0.21.68 ± 0.011.13 ± 0.0622 ± 31.7 ± 0.3
Re21351.0 ± 0.122.3 ± 0.231.4 ± 0.21.22 ± 0.0826 ± 231.4 ± 0.8
Re21360.72 ± 0.0421.3 ± 0.215.01 ± 0.021.05 ± 0.0125.5 ± 0.713.9 ± 0.5
Re21510.83 ± 0.0128.63 ± 0.0115.04 ± 0.011.01 ± 0.0733 ± 312 ± 1
Re21521.15 ± 0.0735.27 ± 0.0723.29 ± 0.021.40 ± 0.0240.4 ± 0.422.44 ± 0.08
Re21531.1 ± 0.131.5 ± 0.822.29 ± 0.011.32 ± 0.0937 ± 222.29 ± 0.07
Re21541.26 ± 0.0845.8 ± 0.85.8 ± 0.21.92 ± 0.0453 ± 34.83 ± 0.01
Re21551.87 ± 0.0154.9 ± 0.53.85 ± 0.072.55 ± 0.0663 ± 34.00 ± 0.04
Re21562.2 ± 0.253 ± 33.8 ± 0.32.45 ± 0.0957 ± 22.8 ± 0.4
Re1034/pCB813.3 ± 0.268.8 ± 0.813.6 ± 0.24.0 ± 0.273.0 ± 0.911.6 ± 0.2
Re2058/pCB1133.24 ± 0.0368 ± 215.3 ± 0.43.6 ± 0.373.1 ± 0.212.7 ± 0.3
Re2133/pCB812.3 ± 0.160 ± 424.3 ± 0.82.9 ± 0.167.0 ± 0.323.3 ± 0.2
Re2160/pCB1132.00 ± 0.0156.0 ± 0.525.32 ± 0.092.74 ± 0.0663.99 ± 0.0324.13 ± 0.02
a
R. eutropha strains were grown in minimal medium with 1% palm oil and 0.05% NH4Cl. Samples were harvested after 48 and 72 h of growth to analyze CDW and P(HB-co-HHx) content. Re1034/pCB81 and Re2133/pCB81 cultures contained kanamycin. All values represent means from duplicate or triplicate cultures, with the uncertainties indicating the range of observed values.
Additional genes were therefore inserted into the genomes of these strains at the phaB1 locus, with the goal of increasing total polymer accumulation. One of these genes was phaB2, which encodes a low-activity acetoacetyl-CoA reductase (8). It was hypothesized that expression of this gene would increase HB-CoA production but not to the level of H16. We also inserted the phaJ genes from P. aeruginosa and A. caviae, which would allow the strains to convert intermediates of fatty acid β-oxidation into 3-hydroxyacyl-CoA molecules. All of these strains exhibited greater PHA production than Re2135 and Re2136 when grown on palm oil (Table 3). The strains containing phaCAc (Re2154, Re2155, and Re2156) made the most polymer, but the HHx content of the PHA was reduced to 4 to 5 mol% at 72 h. The strains containing phaC2Ra (Re2151, Re2152, and Re2153) made more PHA than Re2135, and the polymer still contained significant HHx. Of these strains, Re2152 was the most promising, as it accumulated 40 wt% P(HB-co-HHx) with 22 mol% HHx.

Analysis of engineered R. eutropha strains harboring plasmids.

It was hypothesized that polymer accumulation could be increased in the engineered R. eutropha strains by increasing expression of the PHA biosynthesis genes. To accomplish this, the engineered PHA operon from Re2152 (phaC2Ra-phaA-phaJ1Pa) was amplified and cloned into pBBR1MCS-2. The cloned region included 460 bp from the genome upstream of the start codon of phaC2Ra so that the operon in the plasmid would be expressed from the native R. eutropha promoter. The resulting plasmid (pCB81) was transformed into Re1034 and Re2133 to determine how the different acetoacetyl-CoA reductase activity levels of the two host strains would influence PHA synthesis. When these strains were grown in palm oil minimal medium containing kanamycin, both accumulated >65 wt% P(HB-co-HHx) (Table 3) at 72 h. At this time point, the PHA from Re1034/pCB81 contained 12 mol% HHx, while the PHA from Re2133/pCB81 contained 23 mol% HHx.
While both strains harboring pCB81 accumulated significant P(HB-co-HHx) with high HHx content, these strains are not suitable for industrial PHA production from palm oil. The use of plasmid pCB81 would require the addition of expensive antibiotics to fermentations, which would add excessive cost at the industrial scale. A common strategy for maintaining plasmid stability without the use of antibiotics is to create an auxotrophic mutant through a genome mutation and then to complement the mutation with a plasmid containing the deleted gene (20). We deleted the proC gene from Re1034 and Re2133 to create Re2058 and Re2160, respectively. These strains were unable to grow in minimal medium that did not contain proline (data not shown). Plasmid pCB113 was created by cloning the proC region of the R. eutropha genome into pCB81. When pCB113 was transformed into Re2058 and Re2160, the ability of these strains to grow in minimal medium without proline was restored. PHA production from palm oil in kanamycin-free medium by Re2058/pCB113 and Re2160/pCB113 closely matched the results observed for Re1034/pCB81 and Re2133/pCB81 (Table 3). It was also found that these strains made the desired PHA copolymers only when oil or fatty acids were provided as carbon sources. When Re2058/pCB113 and Re2160/pCB113 were grown in fructose minimal medium, the strains accumulated only 40 wt% and 17 wt% PHA, respectively, and no HHx was detectable in the polymer.
The performances of Re2058/pCB113 and Re2160/pCB113 in higher-density palm oil cultures were evaluated by growing these strains in fermentors, using medium with an NH4Cl concentration eight times that of the medium in the flask cultures (Fig. 1). No kanamycin was added to the fermentation medium or the minimal medium precultures in these experiments. Both strains grew in the high-nitrogen medium, although Re2160/pCB113 exhibited a lag phase of 24 h. By the end of the fermentations, Re2058/pCB113 accumulated 71 wt% PHA with 17 mol% HHx, while Re2160/pCB113 accumulated 66 wt% PHA with 30 mol% HHx. The PHA contents of the cells in both fermentations closely matched the values measured in the low-density flask cultures, suggesting that plasmid loss did not occur at the higher cell densities. When samples taken from similar fermentations were diluted and plated onto solid TSB with and without kanamycin, equal numbers of colonies were observed (data not shown), further indicating that plasmid loss does not occur with these strains.
Fig. 1.
Fig. 1. Re2058/pCB113 (A) and Re2160/pCB113 (B) fermentations were carried out using palm oil as the sole carbon source. Plasmid pCB113 was retained by the cells without the use of kanamycin. Data points are means from triplicate fermentations, and error bars indicate standard deviations (SDs). Note that different scales are used for the y axes in panels A and B.
Several interesting observations were made when analyzing the PHA made in these experiments. In both fermentations, the HHx content of the polymer was extremely high (>40 mol%) early in the cultures. Over time, the HHx contents decreased and then remained stable over the final 48 h of each experiment. The final HHx content in the PHA in the fermentor cultures was higher than that in the low-density flask cultures (Table 3). When analyzing the gas chromatograms of the methanolysis samples from both fermentations, small peaks with the same retention time as methyl 3-hydroxyoctanoate were observed (data not shown). These peaks were also present when polymer purified from dried cells of both strains was subjected to the methanolysis assay. This suggests that the PHA produced in these fermentations contained trace amounts of 3-hydroxyoctanoate in addition to HB and HHx.
In order to confirm the HHx content of the PHA produced by these strains, polymer was extracted from dried cells harvested at the end of the fermentations. PHA was dissolved using MIBK and precipitated by the addition of hexane. Proton NMR spectra were taken for PHA from each strain (see Fig. S2 in the supplemental material). The NMR spectroscopy data indicated that PHA from Re2058/pCB113 contained 21 mol% HHx, while the PHA from Re2160/pCB113 contained 28 mol% HHx. These values agree well with the methanolysis results.
PHA was extracted from lyophilized fermentation samples taken after 48 h and 96 h of growth, and the number-average (Mn) and weight-average (Mw) molecular weights were measured relative to polystyrene standards (Table 4). PHAs from both Re2058/pCB113 and Re2160/pCB113 had similar molecular weights at each time point. In both cases, the polymers had significantly shorter chain lengths than PHB made by wild-type R. eutropha H16, which has an Mw of ∼3 × 106 (8). For both Re2058/pCB113 and Re2160/pCB113, the average PHA molecular weight decreased and the polydispersity increased from 48 to 96 h. This agrees with previous work that showed that PHA is continuously turned over by R. eutropha, even under PHA storage conditions, and that this turnover is accompanied by a decrease in average polymer molecular weight (37). This means that it is important to harvest the biomass from R. eutropha fermentations as soon as maximum PHA accumulation has been reached, as additional time will lead to a decrease in average polymer chain length.
Table 4.
Table 4. Molecular weights and polydispersitiesa
StrainTime point (h)Mn (103)Mw (103)PDI
Re2058/pCB11348191 ± 27362 ± 381.9
 96105 ± 40260 ± 522.5
Re2160/pCB11348192 ± 15350 ± 171.8
 96108 ± 12276 ± 192.6
a
PHA was extracted from Re2058/pCB113 and Re2160/pCB113 samples with chloroform, and the molecular weights were measured by GPC relative to polystyrene standards. Values reported represent means from three independent samples ± SDs.

DISCUSSION

Two novel PHA synthases from the bacterium R. aetherivorans I24 were identified and characterized. When these PHA synthase genes were integrated into the Re1034 genome, the recombinant strains accumulated P(HB-co-HHx) when grown on even-chain-length fatty acids, with the strain containing phaC2Ra synthesizing polymer with the highest HHx content (Table 2). These strains also accumulated P(HB-co-HHx) when grown on palm oil, but the HHx content of the PHA was significantly lower. For example, the PHA from Re2001 contained 10 mol% HHx when the strain was grown on octanoate but only 1.5 mol% HHx when the strain was grown on palm oil. It has previously been demonstrated that HHx content in PHA decreases as the lengths of the fatty acids fed to recombinant R. eutropha increase (26). As the most abundant fatty acids in palm oil are palmitic acid (C16:0) and oleic acid (C18:1) (31), our results agree with this observation.
In order to increase the HHx content of the PHA, R. eutropha strains that expressed recombinant PHA synthases and had low acetoacetyl-CoA reductase activity were constructed. It was previously discovered that R. eutropha strains in which the acetoacetyl-CoA reductase genes (phaB) had been deleted made significantly less PHB than the wild type, presumably because the HB-CoA synthesis pathway had been disrupted (8). The PHA made by the phaB deletion strains with recombinant synthases had high HHx contents, but the strains stored little polymer (Table 3). Notably, the strain containing phaC2Ra (Re2135) made PHA with an HHx content much higher than that of the analogous strain containing phaCAc (Re2136). The PhaCAc synthase has been the most widely studied enzyme for synthesis of P(HB-co-HHx) (12, 13, 23, 25, 26).
In order to increase synthesis of HB-CoA and HHx-CoA from fatty acid β-oxidation intermediates, phaJ genes were inserted into the genomes of the recombinant strains. PhaJ enzymes from A. caviae and P. aeruginosa have been shown to hydrate crotonyl-CoA and 2-hexenoyl-CoA at similar rates, leading to synthesis of both HB and HHx monomers (14, 38). It was found that insertion of either phaJAc or phaJ1Pa into our recombinant strains led to increased PHA accumulation, with the strains expressing phaJ1Pa generating polymer with slightly higher HHx contents (Table 3).
Expression of the PHA biosynthesis genes was increased using a plasmid-based expression system. Plasmid stability issues have been reported in high-density R. eutropha cultures, even in the presence of antibiotics (35). In order to ensure that our strains would produce PHA in high-density cultures without the need for kanamycin, we adapted a plasmid stability system that has been used successfully with other species of bacteria (32). The proC gene was deleted from the genomes of R. eutropha strains and expressed from plasmid pCB113. One scenario that could potentially lead to plasmid loss with this system is if some cells produced excess proline and excreted it into the medium, which would allow other cells to grow and replicate without pCB113. Plasmid loss was not observed in Re2058/pCB113 or Re2160/pCB113 cultures, suggesting that proline excretion does not occur under the conditions tested.
The data presented in Fig. 1 show that the polymers produced by Re2058/pCB113 and Re2160/pCB113 varied over time in the fermentations. The HHx content in the PHA is very high early in the cultures, then it decreases, and eventually it stabilizes. This means that late in the cultures, newly synthesized polymer has an HHx content lower than the overall average. For example, Re2058/pCB113 produced 10.1 g/liter PHA with 22.0 mol% HHx by the 48-h time point. By the 96-h time point, this strain had produced 17.5 g/liter PHA with 17.3 mol% HHx. Therefore, from 48 to 96 h, 7.4 g/liter PHA accumulated with an average HHx content of 10.9 mol%. The reason for higher HHx contents in the PHA early in cultures is not completely understood. Some of the HB-CoA made by the strains is produced from acetyl-CoA through the actions of a β-ketothiolase (PhaA) and an acetoacetyl-CoA reductase (PhaB1 in Re2058/pCB113 and unknown reductases in Re2160/pCB113). It has been shown that during the R. eutropha growth phase, the high intracellular concentration of free CoA inhibits PhaA, decreasing the rate of HB-CoA synthesis (28). This suggests that early in the cultures, the ratio of HHx-CoA to HB-CoA is high, causing more HHx to be incorporated into the PHA. This could also explain the HHx content observed in the fermentor cultures being higher than that in the flask cultures. The fermentation medium contained more NH4Cl than the flask medium, leading to a longer growth phase in which more HHx was included in the PHA.
Interesting questions concerning MCL PHA formation in R. eutropha remain. Strains Re2000 and Re2001 expressing R. aetherivorans PHA synthases produced P(HB-co-HHx). As PhaB1 can reduce 3-ketohexanoyl-CoA in addition to acetoacetyl-CoA (15), this enzyme likely contributed to some of the HHx-CoA formation in these strains. It is unclear, however, how HHx-CoA is synthesized in Re2135 and Re2136, as these two strains lack both phaB and phaJ genes.
Moving forward, our group will scale up the size and density of Re2058/pCB113 and Re2160/pCB113 palm oil cultures in order to characterize the PHA made under these conditions. High-density fermentations will likely require an oil feeding strategy to prevent excess substrate in the bioreactors. We are also exploring routes to further increase the amount of bioplastic accumulated by our engineered strains and the average molecular weights of the polymers.

ACKNOWLEDGMENTS

We thank Phil Lessard for analysis of the R. aetherivorans I24 genome and identification of PHA synthase genes. Jessica Vanessendelft and Iny Jhun assisted with plasmid and strain construction. Mimi Cho assisted with NMR measurements. We thank our Malaysia MIT Biotechnology Partnership Programme (MMBPP) collaborators for their helpful suggestions during this study.
Funding for this research was provided by the MMBPP.

Supplemental Material

File (supplemental_figure_s1___summary_of_engineered_strains.pdf)
File (supplemental_figure_s2___pha_nmr_spectra.pdf)
File (supplemental_table_s1___primer_sequences.pdf)
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

REFERENCES

1.
Akiyama M., Tsuge T., and Doi Y. 2003. Environmental life cycle comparison of polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation. Polym. Degrad. Stab. 80:183–194.
2.
Anderson A. J. and Dawes E. A. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Mol. Biol. Rev. 54:450–472.
3.
Baptist J. May 1962. Process for preparing poly-β-hydroxy-butyric acid. U.S. patent 3,036,959.
4.
Bluhm T. L., Hamer G. K., Marchessault R. H., Fyfe C. A., and Veregin R. P. 1986. Isodimorphism in bacterial poly(β-hydroxybutyrate-co-β-hydroxyvalerate). Macromolecules 19:2871–2876.
5.
Brandl H., Gross R. A., Lenz R. W., and Fuller R. C. 1988. Pseudomonas oleovorans as a source of poly(β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl. Environ. Microbiol. 54:1977–1982.
6.
Brigham C. J. et al. 2010. Elucidation of β-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression. J. Bacteriol. 192:5454–5464.
7.
Buckland B. C. et al. 1999. Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN. Metab. Eng. 1:63–74.
8.
Budde C. F., Mahan A. E., Lu J., Rha C., and Sinskey A. J. 2010. Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16. J. Bacteriol. 192:5319–5328.
9.
Chen G. Q., Zhang G., Park S. J., and Lee S. Y. 2001. Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Appl. Microbiol. Biotechnol. 57:50–55.
10.
de Smet M. J., Eggink G., Witholt B., Kingma J., and Wynberg H. 1983. Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane. J. Bacteriol. 154:870–878.
11.
Doi Y., Kitamura S., and Abe H. 1995. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28:4822–4828.
12.
Fukui T. and Doi Y. 1997. Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae. J. Bacteriol. 179:4821–4830.
13.
Fukui T. and Doi Y. 1998. Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Appl. Microbiol. Biotechnol. 49:333–336.
14.
Fukui T., Shiomi N., and Doi Y. 1998. Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J. Bacteriol. 180:667–673.
15.
Haywood G. W., Anderson A. J., Chu L., and Dawes E. A. 1988. The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus. FEMS Microbiol. Lett. 52:259–264.
16.
Hisano T. et al. 2003. Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis. J. Biol. Chem. 278:617–624.
17.
Holmes P. A. 1985. Applications of PHB—a microbially produced biodegradable thermoplastic. Phys. Technol. 16:32.
18.
Kahar P., Tsuge T., Taguchi K., and Doi Y. 2004. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym. Degrad. Stab. 83:79–86.
19.
Kovach M. E. et al. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176.
20.
Kroll J., Klinter S., Schneider C., Voß I., and Steinbüchel A. 2010. Plasmid addiction systems: perspectives and applications in biotechnology. Microb. Biotechnol. 3:634–657.
21.
Lehrle R. S. and Williams R. J. 1994. Thermal degradation of bacterial poly(hydroxybutyric acid): mechanisms from the dependence of pyrolysis yields on sample thickness. Macromolecules 27:3782–3789.
22.
Lemoigne M. 1927. Études sur l'autolyse microbienne origine de l'acide β-oxybutyrique formé par autolyse. Ann. Institut Pasteur 41:148–165.
23.
Loo C.-Y., Lee W.-H., Tsuge T., Doi Y., and Sudesh K. 2005. Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnol. Lett. 27:1405–1410.
24.
Matsusaki H., Abe H., Taguchi K., Fukui T., and Doi Y. 2000. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3. Appl. Microbiol. Biotechnol. 53:401–409.
25.
Mifune J., Nakamura S., and Fukui T. 2010. Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polym. Degrad. Stab. 95:1305–1312.
26.
Mifune J., Nakamura S., and Fukui T. 2008. Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can. J. Chem. 86:621–627.
27.
Noda I., Green P. R., Satkowski M. M., and Schechtman L. A. 2005. Preparation and properties of a novel class of polyhydroxyalkanoate copolymers. Biomacromolecules 6:580–586.
28.
Oeding V. and Schlegel H. G. 1973. β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism. Biochem. J. 134:239–248.
29.
Pohlmann A. et al. 2006. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat. Biotechnol. 24:1257–1262.
30.
Rehm B. H. A. 2003. Polyester synthases: natural catalysts for plastics. Biochem. J. 376:15–33.
31.
Sambanthamurthi R., Sundram K., and Tan Y.-A. 2000. Chemistry and biochemistry of palm oil. Prog. Lipid Res. 39:507–558.
32.
Schneider J. C., Jenings A. F., Mun D. M., McGovern P. M., and Chew L. C. 2005. Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation. Biotechnol. Prog. 21:343–348.
33.
Shimamura E. et al. 1994. Physical properties and biodegradability of microbial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 27:878–880.
34.
Simon R., Priefer U., and Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology (NY) 1:784–791.
35.
Srinivasan S., Barnard G. C., and Gerngross T. U. 2003. Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha. Biotechnol. Bioeng. 84:114–120.
36.
Stover C. K. et al. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964.
37.
Taidi B., Mansfield D. A., and Anderson A. J. 1995. Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture. FEMS Microbiol. Lett. 129:201–205.
38.
Tsuge T. et al. 2000. Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiol. Lett. 184:193–198.
39.
Wallen L. L. and Rohwedder W. K. 1974. Poly-β-hydroxyalkanoate from activated sludge. Environ. Sci. Technol. 8:576–579.
40.
Waltz E. 2009. Biotech's green gold? Nat. Biotechnol. 27:15–18.
41.
Williams D. R., Anderson A. J., Dawes E. A., and Ewing D. F. 1994. Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations. Appl. Microbiol. Biotechnol. 40:717–723.
42.
York G. M., Junker B. H., Stubbe J., and Sinskey A. J. 2001. Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J. Bacteriol. 183:4217–4226.

Information & Contributors

Information

Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 77Number 91 May 2011
Pages: 2847 - 2854
PubMed: 21398488

History

Received: 13 October 2010
Accepted: 28 February 2011
Published online: 20 April 2011

Permissions

Request permissions for this article.

Contributors

Authors

Charles F. Budde
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Sebastian L. Riedel
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Laura B. Willis
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
ChoKyun Rha
Biomaterials Science & Engineering Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Anthony J. Sinskey [email protected]
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy