Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application
© Wang et al; licensee BioMed Central Ltd. 2009
Received: 23 June 2009
Accepted: 31 August 2009
Published: 31 August 2009
Poly-β-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions. Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.
The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion. Starvation experiment demonstrated that the complete PHB mobilization system in E. coli served as an intracellular energy and carbon storage system, which increased the survival rate of the host when carbon resources were limited. Stress tolerance experiment indicated that E. coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.
This engineered E. coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.
A wide variety of microorganisms are able to accumulate polyhydroxyalkanoates (PHAs) as intracellular carbon/energy storage compounds or reducing power for coping with changing, often oligotrophic environments [1, 2]. Various PHAs, as well as the best-known poly 3-β-hydroxybutyrate (PHB), were found to be accumulated and degraded as required under environmental conditions by most natural PHAs producing bacteria . When the environment is sufficient with carbon source or the C/N ratio is quite high (>20), the PHAs accumulation is much faster than degradation [4, 5]. While facing different stresses, such as low nutrient availability and detrimental physical, chemical, or biological factors, these bacteria begin to mobilize PHAs to conquer those unfavorable environments. The biosynthesis and degradation of PHAs is a cyclic mechanism that has already been found in many bacteria, such as Ralstonia eutropha, Azotobacter beijerinckii and Hydrogenomonas eutropha [6–8]. The in vivo PHB biosynthesis pathway is conducted by the successive action of β-ketoacyl-CoA thiolase (phb A), acetoacetyl-CoA reductase (phb B) and PHB polymerase (phb C). However, PHB degradation, which has been investigated for years, was divided into intracellular mobilization and extracellular degradation. Intracellular PHB mobilization is initialized by the hydrolization action of intracellular PHB depolymerase [9, 10]. The depolymerized product, (R)-3-hydroxybutyric acid (3HB) , is then metabolized in vivo as carbon and energy source by cells or excreted into the environment. To metabolize (R)-3-hydroxybutyric acid, cells have to convert it to acetoacetate by (R)-3-hydroxybutyric acid dehydrogenase  or activate it to a CoA derivative by enzymes such as acyl-CoA synthetase or thioesterase [13–15]. Acetoacetate can be converted to two molecules acetyl-CoA under the function of β-ketothiolase by primarily activated to acetoacetyl-CoA , then acetyl-CoA is further metabolized via the tricarboxylic acid (TCA) cycle or the glyoxylate cycle; while (R)-3-hydroxybutyl-CoA can be immediately epimerized to the (S)-isomer in order to be catabolized by β-oxidation pathway for energy release.
Escerichia coli, which possesses neither PHB synthase nor depolymerase genes, was thought to be one of the best PHA production candidates. By metabolic engineering, recombinant E. coli was confirmed to accumulate PHB up to 90% of dry cell weight [6, 17, 18]. When co-expressed with phaZ 1 gene from R. eutropha, recombinant E. coli was able to depolymerize PHB and secrete 3HB into the medium [9, 19]. However, can E. coli realize the complete PHB mobilization in vivo? Can E. coli obtain any benefit on survival in stress conditions from the PHB mobilization as described for several wild type species e.g. Azospirillum brasilense and Sinorhizobium meliloti? To answer these questions, we constructed a stress induced PHB mobilization system in E. coli by mimicking the natural PHB producer. The stress-induced system was developed by introducing 5'-untranslated region of rpoS . This stress-induced region (SIR) fragment promotes the transcription of rpoS gene, which is induced under stationary phase or under stress conditions . The engineered E. coli was then investigated for its stress resistance capability.
The complete PHB mobilization in engineered E. coli
(R)-3-hydroxybutyric acid (3HB) secretion was detected in the culture supernatant of E. coli DH5α (pQWQ2/pSCP-CAB), indicating the realization of PHB mobilization in the engineered E. coli (Fig. 1). The 3HB appeared in the medium after 20 hours cultivation along with the PHB formation without additional induction; then the 3HB concentration increased to 0.72 g/L at 40 h. However, the 3HB secretion rate is lower than PHB accumulation rate since the intracellular PHB was accumulated. After that, the 3HB concentration decreased slowly to 0.45 g/L. The slow decrease of 3HB concentration in the medium implied the re-utilization of 3HB by the cells at certain conditions. In recombinant E. coli without a phaZ 1 gene, only little 3HB was detected (0.003 g/L) in culture medium.
In vitro 3-hydroxybutyral-CoA synthetase activity assay
Concentration of 3HB (mM) at:
E.coli enzyme extract, 3HB, CoA
3.99 ± 0.06
3.86 ± 0.10
3.86 ± 0.06
E.coli enzyme extract, 3HB, ATP
3.33 ± 0.02
3.41 ± 0.04
3.29 ± 0.05
E.coli enzyme extract, 3HB, ATP, CoA
4.39 ± 0.03
2.02 ± 0.02
0.15 ± 0.10
PHB mobilization enabled the host for long term starvation
Resistance of engineered E. coli to different environmental stress
Many studies showed that PHA biosynthesis would be promoted under nitrogen-deficient conditions in micro-organisms . Under nitrogen deficient conditions, the metabolism of TCA cycle will be repressed, resulting in an increase of acetyl-CoA. Thus, the surplus of acetyl-CoA would be redirected to the PHA biosynthesis. While facing different stresses, such as low nutrient availability, especially carbon source limitation, these bacteria begin to mobilize PHA to conquer those unfavorable environments. In this study, we constructed a PHB mobilization system in E. coli using the stress induced promoter. The rpoS promoter in E. coli was confirmed previously to be induced under various stress conditions, such as cold shock, pH stress as well as cell density . Some reports also pointed out that in E. coli the levels of sigma factor, RpoS, increase in response to starvation for carbon, nitrogen, or phosphate sources as well as for amino acids . Here we confirmed that this stress induced promoter of rpoS can be induced under nitrogen limitation, which is a PHA accumulation condition in PHA producing micro-organisms. Thus, the PHB accumulation process in engineered E. coli under nitrogen limitation is similar as that in natural PHB producer.
In micro-organisms, PHA formation and mobilization is an important process for stress survival [24–27]. PHA formation provided the host with carbon and energy storage [2, 28], while PHB mobilization is also of great importance. Previous studies indicated that incomplete PHB mobilization system, like a phaZ mutant (lacking PHB depolymerase) of Azospirillum brasilense, showed low stress endurance in various challenges [29, 30]. In this study, we constructed a PHB mobilization system in engineered E. coli. We found that a stress induced PHB mobilization remarkably improved the carbon starvation tolerance of the E. coli host cell. Meanwhile, the PHB mobilization in engineered E. coli also provided the host with some other stress resistance. The mechanisms by which the PHA cycle favors stress alleviation are not yet fully understood [26, 29]. We supposed that at least two reasons are responsible for the improved stress resistance in engineered E. coli strains. First, PHA mobilization may work at the small-molecule levels. It was found that a rise in ATP and guanosine tetraphosphate (ppGpp) levels was concomitant with PHA degradation in Pseudomonas oleovorans . While the ppGpp was found to increase mRNA translation of the central stationary phase regulator rpoS , which up-regulates resistance to environmental stress [32–34]. Recently, it was found that the enhanced cross-tolerance to different stress agents during PHA-depolymerization in P. oleovorans is related to an increase in the intracellular concentration of RpoS . Second, PHA mobilization may influence the chaperone protein levels. It was demonstrated that the large amount PHB accumulation in recombinant E. coli acted as a stress on the cells, which reduced the cells' ability to synthesize metabolic proteins and induced the expression of various protective proteins. Three heat shock proteins (GroEL, GroES, and DnaK) were significantly up-regulated in PHB-accumulating cells of E. coli as it was shown by proteome analysis . The heat shock protein HspA was reported to be synthesized and bound to the PHB granule surface, indicating that E. coli also synthesizes protective proteins to reduce stress by binding these proteins to recombinant expressed inclusion bodies . These protective proteins are helpful to the overall stress resistance of the host.
The PHB mobilization in E. coli that served as an intracellular energy and carbon storage changed the stress resistance of the host, which can enhance survival of E. coli when these sources are limited. Accumulation of PHB in succinate producing E. coli was confirmed to increase host resistance and showed a beneficial effect on succinate production (data submitted). Recombinant E. coli with PHB mobilization can also serve as immobilized cell factories for non-substrate/energy related biocatalysis and biotransformation since the cell number was not reduced even after long term incubation without carbon resource.
Methods and materials
Media and culture conditions
Escherichia coli strains were grown in Luria-Bertani medium (LB) at 37°C. Antibiotics were added to the corresponding cultures at a final concentration of 100 μg/mL ampicillin and 50 μg/mL spectinomycine for the maintenance of plasmids when necessary. For PHB production, E. coli pre-culture (0.5 mL) was inoculated into 50 mL LB or M9 medium supplemented with 2% (w/v) glucose as the sole carbon source in a 300-mL shake flask for 72 h. To analyze the PHB accumulation of recombinant E. coli under different C/N ratio (nitrogen limitation condition), various amount of nitrogen (0.2 g/L NH4Cl, 1 g/L NH4Cl, 2.5 g/L NH4Cl and 5 g/L NH4Cl) were used. The basal medium was 15.138 g/L Na2HPO4·H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 mg/L Thiamine VB1, 1 mM MgSO4 and 0.1 mM CaCl2 supplemented with 16 g/L glucose as carbon source.
Construction of recombinant E. coli strains
DNA manipulation was performed by standard procedures . Chromosomal DNA of R. eutropha was prepared by genomic DNA purification kit (Fermentas Inc.). PCR was carried out with an automatic thermal cycler (Takara Shuzo Co., Kyoto, Japan). Primer pair 5'- AAAGGATCCGCCTGCACAAAATTCCACCGTTGCTG and 5'-TTTGGGCCCCCCCTCGAGGTCGACGGTAT were used to amplify the stress-induced region (SIR) fragment from pQKZ100 . The SIR fragment included rpoS promoter and the 5'-untranslated regulation region of rpoS gene. Primer pair 5'-TTTGGATCCCGACAGTAAGACGGGTAAGCCTGTTGATGAT and 5'-ACTGGGCCCGAGCTCCTTGAACGAATTGTTAGACATTATTTG were used to amplify the stable low copy number plasmid vector pCL1920 . The two PCR products were digested with Bam HI and Apa I, and ligated to form plasmid pSCP. Fragment of 5.4 kb Sma I - Eco RI phbCAB operon from pBHR68  was subcloned into pSCP, which resulted in plasmids pSCP-CAB. To construct the phaZ 1 expression plasmid, the intact phaZ 1 gene was amplified by PCR using the genomic DNA from R. eutropha as template with a pair of primers (5'-ATAAGCTTAAGGAGAATGCTCTACCAATTGCATGAGTT and 5'-ATCTCGAGTTACCTGGTGGCCGAGGCCT). The PCR product was digested with Hin dIII and Xho I and inserted into corresponding sites of pQKZ100, resulting plasmid pQWQ2.
After cultivation in M9 medium supplemented with 2% (w/v) glucose, cells were collected and washed twice by centrifugation at 4,000 g for 10 min and re-suspended in M9 medium without carbon source. The number of viable cells (CFU/mL) was determined by dilution plating prior to and at the end of each experiment (three replicates). For each experiment, the same initial number of cells was used (between 1 × 107 and 1 × 108 cells/mL). In heat resistance experiment, cells were incubated in a water-bath at 65°C for a total of 60 min. Samples (10 μL) were taken every 20 min to test the survival rate. The resistance test of UV irradiation was performed by placing 10 mL of cells in 90 mm Petri dishes and by exposing to short-wave UV irradiation (254 nm) from a Lourmat VL-6 LC UV lamp for 60 s. The UV treated cells were collected and re-suspended in potassium phosphate buffer (0.06 M, pH 6.8) for checking the surviving rate. In the acid resistance experiment, cells were maintained in 0.05 M potassium phosphate buffer (adjusted pH value to 3.0). The sensitivity of cells to osmotic pressure was estimated by adding 25 mL 50% glucose (w/v) solution to 25 mL cell suspension to give a final glucose concentration of 25% (w/v).
After cultivation in M9 medium supplemented with 2% (w/v) glucose, cells were collected and washed twice by centrifugation at 4,000 g for 10 min and re-suspended in M9 medium without carbon source (pH 6.8). Cells were then incubated on a shaker at 200 r.p.m for 30 days under starvation conditions . Bacterial density (CFU/mL) was determined by dilution plating (three replicates). Cells were diluted to about 1 × 107 cells/mL.
Determination of PHB and 3HB
Samples (5 mL) were taken out from culture medium every 2 or 4 hours (before 72 h) or 4 days (after 72 h), and were lyophilized overnight. The PHB content was determined by gas chromatography (GC) after methanolysis of the lyophilized cells in the presence of 15% sulfuric acid. Purified PHB polymer was obtained by chloroform extraction for 72 h and ethanol precipitation at room temperature . Culture supernatants were used to detect the 3HB monomer by high-pressure liquid chromatography (HPLC) on a reversed-phase column (Waters C18; 5 mm, 4.6 mm by 15 cm). 0.02 M NaH2PO4 (pH 2~3) solution was used as the mobile phase at the flow rate of 0.8 mL/min.
3-HA-CoA synthase activity
The acyl-CoA synthetase activity was measured in 100 mM potassium phosphate buffer (pH 7.5) containing 5 mM ATP, 10 mM organic acid, 1.25 mM CoASH, 5 mM dithiothreitol (DTT), 5 mM magnesium chloride. To start the reaction, enzyme sample (20 μL) was added to the assay mixture to form a final volume of 200 μL. After 15 min of incubation at 30°C, the reaction was quenched by adding 20 μL of 10% (v/v) formic acid. 3HB consumption was detected by HPLC as described above.
This work was financially supported by research grants from the National High-Tech Research and Development Plan of China (2006AA02Z218), the National Basic Research Program of China (2007CB707803) and a grant from the National Natural Science Foundation of China (30870022).
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