Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

A quantitatively repeatable protocol was developed for poly(3-hydroxybutyrate) (PHB) production by Escherichia coli XL1-Blue (pSYL107). Two constant-glucose fed-batch fermentations of duration 25 h were carried out in a 5-L bioreactor, with the measured oxygen volumetric mass-transfer coefficient (k(L)a) held constant at 1.1 min(-1). All major consumption and production rates were quantified. The intracellular concentration profiles of acetyl-CoA (300 to 600 mug.g RCM-1) and 3-hydroxy-butyryl-CoA (20 to 40 mug.g RCM-1) were measured, which is the first time this has been performed for E. coli during PHB production. The kinetics of PHB production were examined and likely ranges were established for polyhydroxyalkanoate (PHA) enzyme activity and the concentration of pathway metabolites. These measured and estimated values are quite similar to the available literature estimates for the native PHB producer Ralstonia eutropha. Metabolic control analysis performed on the PHB metabolic pathway showed that the PHB flux was highly sensitive to acetyl-CoA/CoA ratio (response coefficient 0.8), total acetyl-CoA + CoA concentration (response coefficient 0.7), and pH (response coefficient -1.25). It was less sensitive (response coefficient 0.25) to NADPH/NADP ratio. NADP(H) concentration (NADPH + NADP) had a negligible effect. No single enzyme had a dominant flux control coefficient under the experimental conditions examined (0.6, 0.25, and 0.15 for 3-ketoacyl-CoA reductase, PHA synthase, and 3-ketothiolase, respectively). In conjunction with metabolic flux analysis, kinetic analysis was used to provide a metabolic explanation for the observed fermentation profile. In particular, the rapid onset of PHB production was shown to be caused by oxygen limitation, which initiated a cascade of secondary metabolic events, including cessation of TCA cycle flux and an increase in acetyl-CoA/CoA ratio. (C) 2001 John Wiley & Sons. Inc. Biotechnol Bioeng 74: 70-80, 2001.

Recovery of poly-3-hydroxybutyrate from recombinant Escherichia coli by homogenization and centrifugation

Poly-3-hydroxybutyrate from recombinant E. coli was recovered using homogenization and continuous centrifugation with a purity of 94%. Final protein and DNA concentrations were 1.0% w/w and 1.9% w/w, respectively, when a hypochlorite treatment was employed prior to centrifugation. High fractional cell debris removal (94%) was achieved with two centrifugation steps.

Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli

Recombinant Escherichia coli strains harboring the genes from Alcaligenes eutrophus for polyhydroxyalkanoate biosynthesis were constructed and compared for their ability to synthesize poly(3-hydroxybutyrate) in a defined medium with whey as the sole carbon source. The highest PHB concentration and PHB content obtained were 5.2 g/L and 81% of dry cell weight, respectively.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-based scaffolds for tissue engineering

Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.

Inverse Metabolic Engineering of Synechocystis PCC 6803 for Improved Growth Rate and Poly-3-hydroxybutyrate Production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) with a broad range of 3HV content at high yields by Burkholderia sacchari IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y-3HV/prop) increasing from 1.10 to 1.34 g g(-1). Copolymer productivity of 1 g l(-1) h(-1) was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y-3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).

Biosynthesis and characterization of biodegradable Poly(3-hydroxybutyrate) from renewable sources

Poly(3-hydroxybutyrate) was produced in fed-batch cultures of Ralstonia eutropha DSM 428 and Alcaligenes latus ATCC 29712 on a mineral medium with different carbon sources such as sucrose, sodium lactate, lactic acid, soybean oil and fatty acid. The bacteria converted the different carbon sources supplied into P3HB. The best results were obtained when lactate or soybean oil were supplied as the sole carbon source. The range of number average molar mass (Mn) for the polymers, analyzed by Gel Permeation Chromatography was 1.65 to 0.79 x 10(5) g mol(-1). FTIR spectroscopy revealed a characteristic absorbance associated with polyester structures. The crystallinity degree, determinate from X-ray diffractograms, was about 69% in all synthesized polymers. The thermal properties associated to semicrystalline polymers indicated a glass transition at 0.1 degrees C and a melting point at about 175 degrees C and enthalpy of 63-89 J g(-1). The (1)H-NMR and (13)C-NMR spectra of the polymers were in agreement with the calculated chemical shifts associated with P3HB structures.

Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3-hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate in Burkholderia sacchari

The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus propionate. The efficiency of the mutants in converting propionate to 3HV units (Y(3HV/prp)) increased from 0.09 g.g(-1) to 0.81-0.96 g.g(-1), indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y(3HV/prp) (1.35 g.g(-1)). When increasing concentrations of propionate were supplied, decreasing values of Y(3HV/prp) were observed. The results obtained corroborate the hypothesis of the presence of other propionate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 g.L(-1) or higher. The efficiency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC.

Optical VOCs detection using poly(3-alkylthiophenes) with different side-chain lengths

The use of conjugated polymers in the gas and volatile organic compounds (VOCs) detections represents an advance in the development of the electronic noses. Polythiophenes show good thermal and environmental stability, are easily synthesized and they have been studied as gas and VOCs sensors using different principles or transduction techniques. Among these techniques, optical sensing has been attracted attention, mainly due to its versatility. However, conjugated polymer-based optical sensors are still less studied. This paper describes the use of two poly(3-alkylthiophenes) for VOCs optical detection. The sensing measurements were carried out using visible spectroscopy. Both polymers showed good sensitivity to the VOCs, showing fast and reversible responses with some hysteresis, and were unable to detect hydroxylated samples. Furthermore, it was demonstrated that the thickness of polymer films influences the intensity of the optical response. Although there is similarity in the superficial composition of the polymers films, demonstrated by their surface energies, they showed significant differences in their optical properties upon exposure to the VOCs. (c) 2009 Elsevier B.V. All rights reserved.

FT-Raman investigation of biodegradable polymers: Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This work presents a FT-Raman study (lambda(0) = 1064 nm) of naturally occurring polyester poly[(R)-3-hydroxybutyrate] (PHB) and its copolymer poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) with 5,8 and 12 mol % of HV (hydroxyvalerate). The FT-Raman spectra of films indicate that full width at half height of the band centered at 1725 cm(-1) and relative intensity of bands at 1443 and 1458 cm(-1) can be use to estimate the crystalline degree in film samples. The similarity between Raman spectra of molten PHB and PHBV and theirs CDCl(3) solutions suggested that molten polymers present similar conformation than polymers in solution. Raman data of these samples showed that bands at 1220, 1402, 1725, 2998 and 3009 cm(-1) are due to crystalline helical structure and the bands at 1453, 1740, 2881, 2938 and 2990 cm(-1) are originated from disordered domains. It is shown that composition of PHBV samples can be estimated by analyzing the ratio of the intensity of the bands at 2938 cm(-1) (nu C-H) and 1740 cm(-1) (nu C=O) in the spectra of solutions and of bands at 1354 (wCH(2)) and 1740 cm(-1) (nu C=O) in spectra of molten polymers. (C) 2010 Elsevier B.V. All rights reserved.

Blends of poly(3-hydroxybutyrate) and poly(p-dioxanone): miscibility, thermal stability and biocompatibility

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Electrical characterization of poly(amide-imide) for application in organic field effect devices

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bacterial cellulose/poly(3-hydroxybutyrate) composite membranes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Thermal characterization of the interaction of poly(3-hydroxybutyrate) with maleic anhydride

Poly(3-hydroxybutyrate), PHB, has been structurally modified with maleic anhydride, MA, in the presence of triethylamine, TEA. Glass transition, melting, and crystallization temperature, obtained from DSC curves, and thermal degradation temperatures obtained from TG ones, were employed to evaluate the influence of the MA proportion on the modification in the PHB chain. According to the results, most of chain modification reactions are the 80/20 and 90/10 proportions. Observations suggest that most chain modification reactions occur when the ratio of PHB/MA is 80/20 or 90/10. This suggests that modifications of PHB in the presence of MA involve main chain scission.

Kinetic model of poly(3-hydroxybutyrate) thermal degradation from experimental non-isothermal data

The non-isothermal data given by TG curves for poly(3-hydroxybutyrate) (PHB) were studied in order to obtain a consistent kinetic model that better represents the PHB thermal decomposition. Thus, data obtained from the dynamic TG curves were suitably managed in order to obtain the Arrhenius kinetic parameter E according to the isoconversional F-W-O method. Once the E parameters is found, a suitable logA and kinetic model (f(alpha)) could be calculated. Hence, the kinetic triplet (E +/- SD, logA +/- SD and f(alpha)) obtained for the thermal decomposition of PHB under non-isothermal conditions was E=152 +/- 4 kJ mol(-1), logA=14.1 +/- 0.2 s(-1) for the kinetic model, and the autocatalytic model function was: f(alpha)=alpha(m)(1-alpha)(n)=alpha(0.42)(1-alpha)(0.56).