Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from structurally unrelated single carbon sources by newly isolated Pseudomonas sp. EL-2

Summary A Pseudomonas sp. EL-2 strain capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was isolated from activated sludge. For simulation of P(3HB-co-3HV) production in the cells, deficiency of nutrients such as NH₄ ⁺, SO₄ ^2- and Mg²⁺ was crucial and the maximum content of P(3HB-co-3HV) could reach 46% on NH₄ ⁺-deficient medium. This organism synthesized P(3HB-co-3HV) with 3HV monomer in the range from 1.9 to 49.3 mol% from unrelated single carbon sources such as glucose, fructose, propionate, or sorbitol. P(3HB-co-3HV)s containing a higher fraction of 3HV were produced by adding propionic acid to glucose medium.     

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20 g/L of glucose as a sole carbon source, P(3HB-co-2.3 mol% 3HV) was produced up to the polymer content of 61.7 wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5 mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.     

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose with elevated 3-hydroxyvalerate fraction via combined citramalate and threonine pathway in Escherichia coli

The biosynthesis of polyhydroxyalkanoate copolymers in Escherichia coli from unrelated carbon sources becomes attractive nowadays. We previously developed a poly(hydroxybutyrate-co-hydroxyvalerte) (PHBV) biosynthetic pathway from an unrelated carbon source via threonine metabolic route in E. coli (Chen et al., Appl Environ Microbiol 77:4886-4893, 2011). In our study, a citramalate pathway was introduced in recombinant E. coli by cloning a cimA gene from Leptospira interrogans. By blocking the pyruvate and the propionyl-CoA catabolism and replacing the β-ketothiolase gene, the PHBV with 11.5 mol% 3HV fraction was synthesized. Further, the combination of citramalate pathway with the threonine biosynthesis pathway improved the 3HV fraction in PHBV copolymer to 25.4 mol% in recombinant E. coli.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C₄ to C₆. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB-co-3HV).     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Engineering poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer composition in E. coli

A strain of Escherichia coli was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) of specified composition between 5% and 18% HV. A gene encoding propionyl-CoA synthetase (prpE from S. enterica) was placed under the control of the IPTG-inducible tac promoter (P(taclacUV5)) while the polyhydroxyalkanoate synthesis operon (phaBCA) from R. eutropha was expressed constitutively. A strain of E. coli harboring both plasmids was grown in defined medium and PHBV was produced with specified hydroxyvalerate (HV) molar content between 5% and 18%. The molecular weight of the copolymer was approximately 700,000 across various HV contents, and average polydispersity was approximately 1.3. The majority of the PHBV production occurred during the late exponential/stationary phase. The HV content of the copolymer generally peaked early in the incubation before falling to its final value. We found that the time profiles of PrpE activity, propionyl-CoA, and acetyl-CoA were well correlated to the HV content time profile. Despite an abundance of propionyl-CoA, incorporation of HV into the copolymer was inefficient. Therefore, both the PHA operon and conditions affecting the availability of propionyl-CoA must be chosen carefully to achieve the desired HV content. The ability to engineer copolymer composition control into an E. coli strain would be useful in cases where the feedstock composition is not adjustable.     

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from activated sludge

Poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) was produced using a co-culture of activated sludge. When butyric acid was used as sole carbon source, PHB was produced. When valeric acid was added to the medium, PHBV was produced. The 3-hydroxyvalerate (3HV) mole fraction in the PHBV reached a maximum of 54% when valeric acid was used as sole carbon source. When the 3HV units in the co-polymer increased from 0.0 to 54.0 mol%, the melting temperature ( T m ) decreased from 178 to 99°C. The composition, and hence the mechanical properties, of the co-polymer produced by activated sludge can be controlled by adjusting the medium composition.     

Production of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with varied composition using different nitrogen sources with Haloferax mediterranei

The extreme haloarchaea Haloferax mediterranei accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) without the need for specific precursors. In this study, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and, for the first time, nitrate. With excess nitrogen, ammonium and nitrate cultures generated 10.7 g/L biomass containing 4.6 wt% PHBV and 5.6 g/L biomass with 9.3 wt% PHBV, respectively. Copolymer composition varied with the nitrogen source used: PHBV from ammonium cultures had 16.9 mol% 3-hydroxyvalerate (HV), while PHBV from nitrate cultures contained 12.5 mol% HV. Nitrogen limitation was achieved with carbon-to-nitrogen (C/N) molar ratios of 25 or higher. Nitrogen limitation reduced biomass generation and polymer concentration, but polymer accumulation increased to 6.6 and 9.4% for ammonium and nitrate, respectively, with C/N 42. PHBV composition was also affected and cultures with lower C/N ratios produced richer HV polymers. Copolymer formation was not a uniform process: HV was only detected after a minimum accumulation of 0.45 g/L PHB and lasted for a maximum of 48 h. The understanding of copolymer synthesis and the influence of culture conditions such as the nitrogen source will help in designing novel strategies for the production of PHBV with more regular structure and material properties.

     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils

[This corrects the article on p. 3236 in vol. 59.].     

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.     

Viscoelastic relaxations and thermal properties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

3-Hydroxybutyrate-3-hydroxyvalerate (3HB-3HV) as well as 3-hydroxybutyrate-4-hydroxybutyrate (3HB-4HB) copolyesters have been investigated by differential scanning calorimetry, thermogravimetric analysis and dynamic mechanical spectroscopy, over a wide range of compositions (0-95 mol% 3HV; 0-82 mol% 4HB). Both series of isolated copolyesters are partially crystalline at all compositions. Quenched samples show a glass transition that decreases linearly with increasing co-monomer molar fraction, more markedly when the co-monomer is 4HB. Above Tg, all copolyesters, rich in 3HB units, show a cold crystallization phenomenon followed by melting, while at the other end crystallization on heating is observed only in 3HB-3HV copolymers. The viscoelastic spectrum, strongly affected by thermal history, shows two relaxation regions: the glass transition, whose location depends on copolymer type and composition, and a secondary dispersion region at low temperatures (-130/-80 degrees C). The latter results from a water-related relaxation analogous to that of P(3HB) and, in 3HB-4HB copolymers, from another overlapping absorption peak centered at -130 degrees C, attributed to local motion of the methylene groups in the linear 4HB units.     

Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate

Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, β-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, ¹H-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.