- Open Access
Poly(4-hydroxybutyrate) (P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth
© Le Meur et al.; licensee BioMed Central Ltd. 2013
- Received: 30 October 2013
- Accepted: 2 December 2013
- Published: 11 December 2013
Poly(4-hydroxybutyrate) (P4HB), belonging to the family of bacterial polyhydroxyalkanoates (PHAs), is a strong, flexible and absorbable material which has a large variety of medical applications like tissue engineering and drug delivery. For efficient production of P4HB recombinant Escherichia coli has been employed. It was previously found that the P4HB synthesis is co-related with the cell growth. In this study, we aimed to investigate the physiology of P4HB synthesis, and to reduce the total production cost by using cheap and widely available xylose as the growth substrate and sodium 4-hydroxybutyrate (Na-4HB) as the precursor for P4HB synthesis.
Six different E. coli strains which are able to utilize xylose as carbon source were compared for their ability to accumulate P4HB. E. coli JM109 was found to be the best strain regarding the specific growth rate and the P4HB content. The effect of growth conditions such as temperature and physiological stage of Na-4HB addition on P4HB synthesis was also studied in E. coli JM109 recombinant in batch culture. Under the tested conditions, a cellular P4HB content in the range of 58 to 70% (w w-1) and P4HB concentrations in the range of 2.76 to 4.33 g L-1 were obtained with a conversion yield (YP4HB/Na-4HB) of 92% w w-1 in single stage batch cultures. Interestingly, three phases were identified during P4HB production: the “growth phase”, in which the cells grew exponentially, the “accumulation phase”, in which the exponential cell growth stopped while P4HB was accumulated exponentially, and the “stagnation phase”, in which the P4HB accumulation stopped and the total biomass remained constant.
P4HB synthesis was found to be separated from the cell growth, i.e. P4HB synthesis mainly took place after the end of the exponential cell growth. High conversion rate and P4HB contents from xylose and precursor were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures with glucose.
- Bioprocess development
- Batch culture
Natural polyhydroxyalkanoates (PHAs) are synthesized by many microorganisms as carbon and energy storage compounds and deposited as granules in their cytoplasm. PHA accumulation appears when bacterial cells grow under conditions where nutrients other than carbon source, such as nitrogen or phosphorus, are limiting growth. Depending on the carbon substrate supplied, PHAs with different composition are produced. They are classified as short-chain, medium-chain and long-chain length PHAs according to the number of carbon atoms of the monomeric units . Over a hundred different carboxylic acid monomers were reported to be incorporated into PHAs, resulting in polymers with a wide range of material properties . These natural polymers have attracted particular attention due to their biodegradability and biocompatibility –. Among them, poly(4-hydroxybutyrate) (P4HB) is a highly interesting polymer for various biomedical applications .
P4HB biosynthesis has been studied for about 20 years and it was, and still is, the first and only PHA-based product approved by the FDA as an absorbable suture for clinical application. It is a strong, flexible thermoplastic material that can be processed easily to scaffolds, heart valves or cardiovascular tissue supports . The most remarkable property of P4HB is its very high elasticity and molecular weight, as both benchmark closely to ultra-high molecular weight polyethylene ; it can be stretched 10-times its original length before breaking . In addition, P4HB is biocompatible and extremely well tolerated in vivo because biological hydrolysis of P4HB yields 4HB, which is a common metabolite in the human body . When used in vivo, the degradation of P4HB implant takes place via surface erosion and does not lead to a burst release of acid, which is an immense advantage for medical applications . Thus, it is highly desired to obtain P4HB in large scale at a competitive cost. It was reported that up to 50% of the total cost of poly(3-hydroxybutyrate) (P3HB) arises from the carbon source . Therefore, to reduce the cost of the carbon source used for large scale P4HB production, agricultural derived feedstock such as processed hemicelluloses may be employed as a co-substrate to produce the bacterial biomass.
Annually, 60 billion tons of hemicelluloses are produced and remain mostly unused . Hemicellulose is the third most abundant polymer in nature and can be hydrolyzed into simple sugars by either chemical or enzymatic hydrolysis . The dominant building unit of hemicelluloses is xylose. In some plants, xylose polymer (xylan) comprises up to 40% of the total dry plant material. Xylose can be used as an industrially relevant carbon source for bacterial growth, for example, by Escherichia coli strains .
Up to now, several wild-type bacterial strains have been reported to be able to produce P(3HB-co-4HB) copolymer: Ralstonia eutropha, Alcaligenes latus, Comamonas acidovorans, Comamonas testosteroni and Hydrogenophaga pseudoflava[12, 13]. Saito and coworkers reported the production of P(3HB-co-4HB) copolymers by R. eutropha using different carbon sources with or without 4HB as precursor, however, only very low cellular polymer contents were obtained . It was also reported that a maximum of 21% w w-1 of P4HB can be achieved by C. acidovorans when using 4HB or 1,4-butanediol as precursor . Kim and colleagues performed fed-batch experiments with R. eutropha supplying in the first step fructose and in the second step only 4HB. They obtained a cell concentration of 33.6 g L-1 and a P(3HB-co-4HB) copolymer content of 41.7% w w-1 with 25 mol% 4HB . To produce P4HB homopolymers recombinant strains were mainly used.
It has been shown previously that microorganisms that do not produce PHA naturally are ideally suited for the manipulation of the levels of the PHA biosynthetic enzymes and, hence, allow to increase polymer productivity . Wild-type E. coli strains cannot synthesize any type of PHA, including P4HB. By introducing the P4HB synthesizing genes, recombinant E. coli strains are able to produce P4HB through the newly acquired biosynthetic pathway. It has been reported that the overexpression of PHA synthase (phaC) and β-ketothiolase (phaA) genes from R. eutropha allowed C. acidovarans to produce up to 51% w w-1 P4HB . By introducing phaC from R. eutropha and a 4-hydroxybutyric acid-coenzyme A transferase gene (orfZ) from Clostridium kluyveri, E. coli strain XL1-Blue was able to produce P4HB when 4HB was supplied as a precursor in the culture medium . A P4HB content of 58.5% w w-1 was achieved in 100 mL shake flasks, however, information on the biomass concentration was not mentioned . Recently, Zhou et al. reported that E. coli JM109 mutant carrying two plasmids reached about 1.9 g L-1 P4HB and 35% (w w-1) P4HB using LB medium containing glucose in a batch culture . There, LB rich medium was applied and two antibiotics were needed to keep the plasmids, which might be too expensive for large-scale production.
The importance of choosing a suitable E. coli host strain for recombinant culture cultivation was demonstrated by Luli and Strohl , who showed that specific growth rate, biomass yield, and acetate formation varied significantly among different strains tested. It has also been reported that among different E. coli strains E. coli JM109 was the only strain that allowed good production of poly(L-aspartyl-L-phenylalanine) . Up to now, little effort has been made to understand the physiology of P4HB synthesis in E. coli.
In this study, we compared P4HB production in different E. coli recombinants and identified the best E. coli strain regarding cell growth and P4HB accumulation. The effect of growth conditions in batch culture was studied for following parameters: temperature, the carbon source, and Na-4HB concentrations. Furthermore, the best physiological stage at which Na-4HB precursor should be added was investigated. P4HB productivity of 0.027 w w-1 h-1 with excellent conversion yield YP4HB/Na-4HB of 92% w w-1 was achieved.
Comparison of different E. coli recombinants for and influence of 4HB concentrations on P4HB production
Influence of Na-4HB concentrations on P4HB accumulation in E. coli JM109 (pKSSE5.3)
Na-4HB (g L-1)
%P4HB (w w-1)
1.77 ± 0.08
2 ± 0.1
0.32 ± 0.01
1.88 ± 0.02
21 ± 0.4
0.34 ± 0.02
1.94 ± 0.04
23 ± 0.7
0.33 ± 0.01
1.93 ± 0.06
21 ± 0.9
0.22 ± 0.01
Comparison of carbon sources for P4HB synthesis in JM109 (pKSSE5.3)
To produce P4HB under better controlled conditions, the selected JM109 (pKSSE5.3) was cultivated in a 1 L bioreactor using modified E2 minimal medium containing xylose and 4HB. For comparison, glucose and glycerol were used as growth substrates, respectively.
Comparison of carbon sources for growth and P4HB accumulation of E. coli JM109 (pKSSE5.3)
3.4 ± 1.4
3.9 ± 1.1
7.6 ± 0.4
CDW (g L-1)
2.16 ± 0.37
2.04 ± 0.60
3.80 ± 0.18
0.32 ± 0.09
0.38 ± 0.04
0.35 ± 0.01
P4HB content% (w w-1)
32 ± 3.7
19 ± 6.4
12 ± 3.6
P4HB concentration (g L-1)
0.65 ± 0.11
0.36 ± 0.05
0.41 ± 0.00
Influence of temperature on growth and P4HB accumulation
Impact of the precursor addition at different physiological growth stages on P4HB synthesis
The results obtained here demonstrate the following: 1) P4HB synthesis only started at the end of the exponential growth phase, regardless of the stage in which the precursor 4HB was added (i.e., either at the beginning or at the end of the exponential growth phase); 2) P4HB content and concentration increased exponentially once the P4HB synthesis was initiated; 3) The P4HB accumulation rate per cell dry weight was similar regardless when the precursor 4HB was added (i.e. at the beginning or the end of the exponential growth phase); 4) The increase of biomass after the exponential growth phase was mainly due to the P4HB accumulation; and 5) P4HB accumulation stopped due to either nutritional limitation and/or product(s) inhibition. To obtain more information, a more detailed analysis on substrate consumption and product formation was performed.
Batch culture for P4HB production
Despite the fact that bioprocesses for recombinant production of P3HB in E. coli have been studied extensively [22, 23], the biosynthesis of P4HB in E. coli has not been yet investigated in depth. Several reports have described the P4HB synthesis and accumulation in E. coli[16, 20, 24]. However, neither physiological and cultivation conditions, nor the external factors that may influence P4HB accumulation have been studied yet in detail. For this reason, we attempted to address two issues in this work. The first issue was whether or not P4HB can be produced from Na-4HB efficiently in combination with xylose as growth substrate. The second issue was to tackle how P4HB synthesis can be stimulated. Our results demonstrate that P4HB can be synthesized efficiently by combining xylose and 4HB and its production can be enhanced reproducibly by an unknown factor, either nutrient depletion or product inhibition.
To reach efficient P4HB production, cultures exhibiting high specific growth rate, high biomass concentration and high levels of P4HB content are desired. Since the metabolic status, including the concentrations of metabolites and the rate of metabolite formation may be different from one strain to another, it is very understandable that rates of P4HB synthesis and levels of P4HB accumulation will be different from one to another. Previously, it has been reported that P3HB production can differ dramatically by using different E. coli strains, e.g. the wild-type E. coli K12 synthesized 0.4 g L-1 P3HB, whereas XL1-Blue produced 7.2 g L-1 P3HB under the same conditions . In this study, we have chosen six E. coli strains originated from B strain (BL21(DE3)) and K12 strains including the wild type (W3110) and the K12 derivatives (DH5α, JM109, XL1-Blue, S17-1). JM109 seems to have the best physiological background for P4HB synthesis, whereas the worst performers were W3110 and BL21(DE3). The latter two strains grew fast, and used the carbon source mainly for biomass formation but produced little amount of P4HB (Figure 1).
Previously it has been reported that using E. coli XL1-Blue carrying pKSSE5.3, a P4HB concentration of about 4.0 g L-1 and P4HB content of 36% (w w-1) could be obtained by a fed-batch culture on M9 medium containing glucose and yeast extract and 18 g L-1 of 4HB . The conversion yield of the precursor 4HB to P4HB (g carbon : g carbon) was about 24%. Recently, Zhou et al. reported that E. coli JM109 mutant carry two plasmids reached about 1.9 g L-1 P4HB and 35% (w w-1) P4HB using LB rich medium containing glucose in a batch culture . Two antibiotics were needed to keep the plasmids and LB rich medium is costly. The authors also showed that in a fed-batch fermentation 7.5 g L-1 P4HB could be achieved by using LB medium containing a total of 90 g L-1 glucose after 52 hours . The conversion yield of the precursor glucose to P4HB (g carbon : g carbon) was about 10.5%. In the current study, we achieved 4.3 g L-1 P4HB and 67% (w w-1) P4HB in a batch culture using the described medium. The consumption of the precursor 4HB was almost complete with a conversion yield YP4HB/Na-4HB of 92% g g -1. Even though the cost of 4HB is higher than glucose, the price of 4HB can be significantly reduced by using gamma-butyrolactone as the precursor for chemical synthesis of 4HB (see Methods section). Hence, the process developed here is an efficient approach for P4HB production.
In earlier studies, addition of 4HB at the beginning of a cultivation was found to be the best for cell growth and P4HB production . Here, we observed no difference in cell growth and P4HB synthesis between adding 4HB at the beginning and at the end of the exponential growth phase (Figure 3). P4HB synthesis was initiated only at the end of exponential growth, even when 4HB was supplied right at the start. In contrast to P3HB accumulation in E. coli, where the polymer is synthesized during cell growth , P4HB production has been found to be distinctly separated from exponential cell growth in our experiments. The end of exponential growth caused by either product inhibition or nutrient limitation stimulated P4HB synthesis. It seems that the cell growth and P4HB production compete with each other for the same nutrients. As indicated from the results shown in Figure 1, both W3110 and BL21(DE3) strains grew fast and reached high final biomass but accumulated only a negligible amount of P4HB. Furthermore, when the conditions are favored for cell growth e.g. at 37°C, P4HB is disadvantaged (Figure 2). These results suggest that nutrients are directed mainly into the tricarboxylic acid (TCA) cycle for cell growth rather than into P4HB synthesizing pathway. We also did not observe the accumulation of acetic acid during the whole cultivation period. This seems to be due to the efficient utilization of excessive acetyl-CoA for the synthesis of P4HB, which would otherwise form acetic acid .
In this study, we compared for the first time different E. coli strains with respect to growth on xylose and P4HB accumulation. Different growth conditions were also investigated such as temperature and the cell physiological stages for P4HB synthesis. Unlike what has been reported previously, the P4HB synthesis was found to be separated from the cell growth, namely P4HB synthesis mainly takes place after the end of the exponential growth phase. Under the tested conditions, P4HB contents in the range of 58 to 70% (w w-1) and P4HB concentrations in the range of 2.8 to 4.3 g L-1 were obtained with a conversion yield YP4HB/Na4HB of 92% w w-1. These results were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures. However, to further improve the productivity of P4HB production process for practical industrial applications, high cell density cultures will need to be investigated and employed.
Bacterial strains and plasmids
E. coli strains and plasmid used in this study
F–, ø80dlacZ ΔM15, Δ(lacZYA-argF)U169, deo R, recA 1, endA 1, hsdR 17(rK–, mK+), gln V44, supE 44, λ-, thi-1, gyrA 96, relA 1, nup G
endA 1, glnV 44, thi-1, relA 1, gyrA 96, recA 1, mcrB+, Δ(lac-proAB), e14-, [F’ traD 36, proAB+, lacIq, lacZ ΔM15], hsdR 17(rK-mK+)
endA 1, gyrA 96(nalR), thi-1, recA 1, relA 1, lac, glnV 44, F’[ ::Tn10, proAB+, lacIq, Δ(lacZ)M15], hsdR 17(rK- mK+)
tmpR, spcR, strR, recA pro hsdR RP4-2-Tc::Mu-Km::Tn7
F, - λ - , rph-1, INV(rrnD, rrnE)
F - , ompT, gal, dcm, lon, hsdS B (r B - m B - ), λ(DE3), [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
phaC, orfZ, Ampr
Chemicals, media and cultivation conditions
All chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland).
Synthesis of Na-4HB
Gamma-butyrolactone + NaOH → Na-4HB.
E. coli strains were cultivated overnight in LB medium with 100 μg mL-1 ampicillin. This culture was used to inoculate the preculture containing modified E2 medium . Modified E2 medium contained the following constituents: NaNH4HPO4 · 4H2O 3.5 g L-1, KH2PO4 3.7 g L-1, K2HPO4 7.5 g L-1, dissolved in 1 L of water. One mL L-1 of 1 M MgSO4 · 7H2O was added to the medium. One mL L-1 of trace elements (TE) dissolved in 1 M HCl was also added. TE contained: FeSO4 · 7H2O 2.78 g L-1, CaCl2 · 2H2O 1.47 g L-1, MnCl2 · 4H2O 1.98 g L-1, CoCl2 · 6H2O 2.38 g L-1, CuCl2 · 2H2O 0.17 g L-1, ZnSO4 · 7H2O 0.29 g L-1. Xylose, glucose or glycerol (10 g L-1) was used as the sole carbon source.
Growth in shake flasks
Growth studies were performed in 1 L shake flasks containing 200 mL of modified E2 medium and 10 g L-1 of a carbon source. 1 g L-1 of NZ-amines and 100 μg mL-1 of ampicillin were added to the minimal medium. Na-4HB (1 to 6 g L-1 according to the experiment) was added as P4HB precursor as indicated in individual experiments.
Culture in 1 L bioreactors
Four 1 L reactor cultures were grown in parallel in Multifors benchtop bioreactors (Infors AG, Bottmingen, Switzerland). Temperature was controlled at 32°C with an external circulating water bath, and pH was maintained at 7.0 +/− 0.1 by automatic addition of 25% NaOH or 30% H3PO4. Dissolved oxygen tension was monitored continuously with an oxygen probe (Infors AG, Bottmingen, Switzerland) and kept always above 30% oxygen saturation. The agitation was set at 500 rpm. Each reactor was inoculated using a preculture prepared as described above in “Growth in shake flasks”. The initial OD600 value in bioreactors was between 0.10 and 0.30. The modified E2 medium was used to perform all the growth studies in 1 L reactors supplemented with 10 g L-1 of carbon source, 1 g L-1 of NZ-amines, 4 g L-1 of Na-4HB and 0.015 g L-1 of thiamine. Ampicillin was added to a final concentration of 100 μg mL-1 to maintain the pKSSE5.3 plasmid.
Growth of bacterial cells was followed by measuring optical density at 600 nm (OD600) using a UV spectrophotometer (Genesys 6, ThermoSpectronic, Switzerland).
Cell dry weight was determined either by using pre-weighed polycarbonate filters (pore size 0.2 μm, Whatman, Scheicher & Schuell, Dassel, Germany) or by pre-weighed 2 mL Eppendorf tubes. In the first method, an appropriate volume (0.5 to 5 mL) of culture was filtered in order to obtain a biomass dry weight of about 2 mg per filter. The filter was dried overnight at 100°C, cooled down to room temperature in a desiccator and then weighed. In the second method, 2 mL of culture broth was centrifuged at 12,000 g for 2 min in a 2 mL pre-weighed Eppendorf tube. The supernatant was discarded and the cell pellet was dried overnight at 100°C and cooled down to room temperature in a desiccator. The 2 mL Eppendorf tube was then weighed. For both methods, the weight difference was used to determine the dry biomass.
PHA content and composition were determined according to a method described previously . Methylene chloride containing benzoic acid (0.1 g L-1) was used as internal standard. Own lab purified P4HB was used for obtaining standard curves. Na2CO3 powder was added to dry the extracted chlorinated solvent phase. The samples were analyzed by gas chromatography (GC) (A200s, Trace GC 2000 series, Fisons Instruments, Rodano, Italy) equipped with a polar fused silica capillary column (Supelcowax-10: length 30 m; inside diameter 0.31 mm; film thickness 0.5 μm; Supelco, Buchs, Switzerland) . P4HB was depolymerized, esterified and methylated, leading to three different peaks in the GC chromatogram. These three peaks were also observed by Hein and coworkers when P4HB homopolymers were analyzed .
NH4+-nitrogen consumption was detected using an ammonium test kit following the manufacturer instruction (Merck KGaA, 64271 Darmstadt, Germany). The detection limit was 0.01 NH4+-nitrogen mg L-1. The method was linear up to 3.0 mg L-1, above which dilution with distilled water was needed. The results obtained are in mg L -1 of nitrogen.
Measurement of xylose, Na-4HB, acetate, pyruvate and lactate
Concentrations of xylose, Na-4HB and acetate in the culture medium were measured by HPLC/MS. Supernatant resulting from culture centrifugation at 12,000 g for 2 min was diluted to a concentration between 0.01 and 0.1 mM with distilled water, filtrated through a Titan HPLC filter (0.45 μm, Infochroma AG, Zug, Switzerland), and loaded on a reversed phase C18 column (Gemini C18 5 micron, 250 mm × 4.60 mm, Phenomenex, U.K.). A gradient of 100% of diluted formic acid (0.1 v% in water) to 100% of acetonitrile was applied as the mobile phase. The flow rate was 0.8 mL min-1 and the gradient was completed after 25 minutes. The peaks were detected by electrospray ionization (ESI) in negative mode . Standard curves for xylose, Na-4HB and acetate were recorded in the range of 0.01 to 1.00 g L-1, 0.01 g L-1 to 0.20 g L-1 and 0.01 to 1.00 g L-1, respectively.
Pyruvate and lactate in the culture supernatant were measured by ion chromatography (IC) (Metrosep A SUPP 5 250, 4 × 250 mm). A flow of 0.7 mL min-1 of eluent containing 1 mM NaHCO3 was used. Both acids were detected using a conductivity detector. A volume of 20 μL of sample diluted with water to a range of 50 to 250 ppm was injected and analyzed by IC system. Pure pyruvate and lactate were used to generate standard curves.
Calculation of conversion rate
Consumed Na-4HB was determined by the difference between the Na-4HB amount supplied at the beginning of a cultivation and Na-4HB content left over in the medium after the cultivation. The concentration of P4HB (g L-1) was determined from cell dry weight (CDW) in g L-1 and the cellular content of P4HB (w w-1) obtained at the end the cultivation. The conversion rate was calculated by dividing the mass of carbon in gram from P4HB with the mass of carbon in gram from Na-4HB (w w-1).
We thank Karl Kehl for IC measurements and Melisa Novelli for technical assistances. We thank Prof. Guoqiang Chen (Tsinghua University) for kindly providing the plasmid pKSSE5.3.
- Steinbüchel A, Valentin HE: Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett. 1995, 128: 219-228.View ArticleGoogle Scholar
- Gogolewski S, Jovanovic M, Perren SM, Dillon JG, Hughes MK: Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/PHV). J Biomed Mater Res. 1993, 27: 1135-1148. 10.1002/jbm.820270904.View ArticleGoogle Scholar
- Brandl H, Bachofen R, Mayer J, Wintermantel E: Degradation and applications of polyhydroxyalkanoates. Can J Microbiol. 1995, 41: 143-153. 10.1139/m95-181.View ArticleGoogle Scholar
- Rathbone S, Furrer P, Lubben J, Zinn M, Cartmell S: Biocompatibility of polyhydroxyalkanoate as a potential material for ligament and tendon scaffold material. J Biomed Mater Res. 2010, 93A: 1391-1403.View ArticleGoogle Scholar
- Martin DP, Williams SF: Medical applications of poly-4-hydroxybutyrate: a strong flexible absorbable biomaterial. Biochem Eng J. 2003, 16: 97-105. 10.1016/S1369-703X(03)00040-8.View ArticleGoogle Scholar
- Engelberg I, Kohn J: Physico-mechanical properties of degradable polymers used in medical applications: A comparative study. Biomaterials. 1991, 12: 292-304. 10.1016/0142-9612(91)90037-B.View ArticleGoogle Scholar
- Nelson T, Kaufman E, Kline J, Sokoloff L: The extraneural distribution of γ-hydroxybutyrate. J Neurochem. 1981, 37: 1345-1348. 10.1111/j.1471-4159.1981.tb04689.x.View ArticleGoogle Scholar
- Choi JI, Lee SY: Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng. 1997, 17: 335-342. 10.1007/s004490050394.View ArticleGoogle Scholar
- Xu F, Sun JX, Liu CF, Sun RC: Comparative study of alkali- and acidic organic solvent-soluble hemicellulosic polysaccharides from sugarcane bagasse. Carbohyd Res. 2006, 341: 253-261. 10.1016/j.carres.2005.10.019.View ArticleGoogle Scholar
- Sun Y, Cheng JY: Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol. 2002, 83: 1-11. 10.1016/S0960-8524(01)00212-7.View ArticleGoogle Scholar
- Song S, Park C: Utilization of D-ribose through D-xylose transporter. FEMS Microbiol Lett. 1998, 163 (2): 255-261.Google Scholar
- Kim JS, Lee BH, Kim BS: Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha. Biochem Eng J. 2005, 23 (2): 169-174. 10.1016/j.bej.2005.01.016.View ArticleGoogle Scholar
- Saito Y, Nakamura S, Hiramitsu M, Doi Y: Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Polym Int. 1996, 39 (3): 169-174. 10.1002/(SICI)1097-0126(199603)39:3<169::AID-PI453>3.0.CO;2-Z.View ArticleGoogle Scholar
- Choi JI, Lee SY: High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli. Appl Environ Microb. 1999, 65 (10): 4363-4368.Google Scholar
- Sudesh K, Fukui T, Taguchi K, Iwata T, Doi Y: Improved production of poly(4-hydroxybutyrate) by Comamonas acidovorans and its freeze-fracture morphology. Int J Biol Macromol. 1999, 25 (1–3): 79-85.View ArticleGoogle Scholar
- Hein S, Söhling B, Gottschalk G, Steinbüchel A: Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli. FEMS Microbiol Lett. 1997, 153: 411-418. 10.1016/S0378-1097(97)00282-6.View ArticleGoogle Scholar
- Zhou X-Y, Yuan X-X, Shi Z-Y, Meng D-C, Jiang W-J, Wu L-P, Chen J-C, Chen G-Q: Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli. Microb Cell Fact. 2012, 11 (1): 54-10.1186/1475-2859-11-54.View ArticleGoogle Scholar
- Luli GW, Strohl WR: Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microb. 1990, 56 (4): 1004-1011.Google Scholar
- Murata T, Horinouchi S, Beppu T: Production of poly(l-aspartyl-l-phenylalanine) in Escherichia coli. J Biotechnol. 1993, 28 (2–3): 301-312.View ArticleGoogle Scholar
- Song S, Hein S, Steinbüchel A: Production of poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant strains of Escherichia coli. Biotechnol Lett. 1999, 21 (3): 193-197. 10.1023/A:1005451810844.View ArticleGoogle Scholar
- Chang DE, Shin S, Rhee JS, Pan JG: Acetate metabolism in a pta mutant of Escherichia coli W3110: Importance of maintaining acetyl coenzyme a flux for growth and survival. J Bacteriol. 1999, 181 (21): 6656-6663.Google Scholar
- Wang FL, Lee SY: High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium. Biotechnol Bioeng. 1998, 58 (2–3): 325-328.View ArticleGoogle Scholar
- Lee SY, Chang HN: Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Can J Microbiol. 1995, 41 (13): 207-215. 10.1139/m95-189.View ArticleGoogle Scholar
- Zhang L, Shi Z-Y, Wu Q, Chen G-Q: Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms. Appl Microbiol Biotechnol. 2009, 84 (5): 909-916. 10.1007/s00253-009-2023-7.View ArticleGoogle Scholar
- Li ZJ, Cai L, Wu Q, Chen GQ: Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production. Appl Microbiol Biotechnol. 2009, 83 (5): 939-947. 10.1007/s00253-009-1943-6.View ArticleGoogle Scholar
- Riesenberg D: High cell-density cultivation of Escherichia coli. Curr Opin Biotech. 1991, 2 (3): 380-384. 10.1016/S0958-1669(05)80142-9.View ArticleGoogle Scholar
- Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Bio. 1983, 166 (4): 557-580. 10.1016/S0022-2836(83)80284-8.View ArticleGoogle Scholar
- YanischPerron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and Puc19 vectors. Gene. 1985, 33 (1): 103-119. 10.1016/0378-1119(85)90120-9.View ArticleGoogle Scholar
- Bullock W, Fernandez J, Short J: XL1-blue: high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques. 1987, 5: 376-378.Google Scholar
- Simon R, Priefer U, Puhler A: A broad host range mobilization system for invivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnol. 1983, 1 (9): 784-791. 10.1038/nbt1183-784.View ArticleGoogle Scholar
- Bachmann BJ: Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. Edited by: Neidhardt FC. 1996, Washington, DC: ASM Press, 2460-2488.Google Scholar
- Daegelen P, Studier FW, Lenski RE, Cure S, Kim JF: Tracing ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606 and BL21(DE3). J Mol Bio. 2009, 394 (4): 634-643. 10.1016/j.jmb.2009.09.022.View ArticleGoogle Scholar
- Marvel CS, Birkhimer ER: The preparation of the sodium salts of omega-hydroxybutyric,-valeric and-caproic acids. J Amer Chem Soc. 1929, 51 (1): 260-262. 10.1021/ja01376a032.View ArticleGoogle Scholar
- Ren Q, Grubelnik A, Hoerler M, Ruth K, Hartmann R, Felber H, Zinn M: Bacterial poly(hydroxyalkanoates) as a source of chiral hydroxyalkanoic acids. Biomacromolecules. 2005, 6 (4): 2290-2298. 10.1021/bm050187s.View ArticleGoogle Scholar
- Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B: Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microb. 1988, 54 (12): 2924-2932.Google Scholar
- le Meur S, Zinn M, Egli T, Thöny-Meyer L, Ren Q: Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440. BMC Biotechnol. 2012, 12 (1): 53-10.1186/1472-6750-12-53.View ArticleGoogle Scholar
- Braunegg G, Sonnleitner B, Lafferty R: A rapid gas chromatographic method for the determination of poly-3-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol. 1978, 6: 29-37. 10.1007/BF00500854.View ArticleGoogle Scholar
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