1,721,029 research outputs found
Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli
No full textRecombinant 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.We thank Mary Berlyn for kindly providing the GCSC
strains. This work was supported by KAIST and
KOSEF. Anton Middelberg thanks KOSEF and the
Australian Academy of Science for support under the
joint exchange program
Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli
No full textA 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
Control of fed-batch fermentations
No full textFed-batch fermentation is used to prevent or reduce substrate-associated growth inhibition by controlling nutrient supply. Here we review the advances in control of fed-batch fermentations. Simple exponential feeding and inferential methods are examined, as are newer methods based on fuzzy control and neural networks. Considerable interest has developed in these more advanced methods that hold promise for optimizing fed-batch techniques for complex fermentation systems. (C) 1999 Elsevier Science Inc. All rights reserved.This work was supported by the Korea-Australia joint research project (MOST) and partially
by the BPERC and Automation Research Center (POSTECH)
Metabolic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli
No full textPoly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYL107 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78% of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95%) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs
Size analysis of poly(3-hydroxybutyric acid) granules produced in Escherichia coli
No full textSize distributions of PHB granules synthesized in recombinant Escherichia coli are determined by photosedimentation. Mean granule Stokes diameters are in the range 1.13-1.25 mu m, which is larger than reported values for wild type microorganisms. Treatment with 1.5% hypochlorite and mild centrifugation did not affect granule size distribution. Treatment with 10% hypochlorite led to a significant reduction in mean diameter and total PHB.This work was partially supported by KOSEF and the ARC Small Grants Scheme
The influence of protein refolding strategy on cost for competing reactions
No full textProtein refolding is a key operation in many bioprocesses utilizing Escherichia coli. Most laboratory studies into refolding are conducted in batch systems at "infinite" dilution to avoid the formation of aggregates. This policy will be grossly suboptimal if adopted for scale-up. The impact of refolding policy on dimensionless annual operating cost for a simplified kinetic scheme is therefore investigated in this work. For competing refolding and aggregation in a continuous stirred-tank reactor, high conversion (X> 99.9%) and high dimensionless concentration minimize the annual cost. However, this policy results in a significant cost penalty when the native protein is in equilibrium with an aggregating intermediate. In such cases, a distinct optimal conversion and concentration exist. The precise location of the optimum depends on the extent of the back reaction, with a trend to lower conversions and concentrations as the equilibrium distribution shifts towards the intermediate. This work highlights the need to establish critical design parameters at an early stage in the scale-up of bioprocesses involving refolding. It also highlights the need to optimize the process in a global rather than unit-wise fashion
Large-scale recovery of recombinant protein inclusion bodies expressed in Escherichia coli
No full textThe production of recombinant proteins in Escherichia coli often leads to the formation of an intracellular inclusion body. Key process steps that can determine the economics of large-scale protein production from inclusion bodies are fermentation, inclusion body recovery, and protein refolding. Compared with protein refolding and fermentation, inclusion body recovery has received scant research attention. Nevertheless, it can control the final product yield and hence process cost for some products. Optimal separation of inclusion bodies and cell debris can also aid subsequent operations by removing contaminant particulates that foul chromatographic resins and contain antigenic pyrogens. In this review, the properties of inclusion bodies and cellular debris are therefore examined. Attempts to optimise the centrifugal separation of inclusion bodies and debris are also discussed
Numerical and experimental study of a homogenizer impinging jet
No full textHigh-pressure homogenization is a key unit operation used to disrupt cells containing intracellular bioproducts. Modeling and optimization of this unit are restrained by a lack of information on the flow conditions within a homogenizer value. A numerical investigation of the impinging radial jet within a homogenizer value is presented. Results for a laminar and turbulent (k-epsilon turbulent model) jet are obtained using the PHOENICS finite-volume code. Experimental measurement of the stagnation region width and correlation of the cell disruption efficiency with jet stagnation pressure both indicate that the impinging jet in the homogenizer system examined is likely to be laminar under normal operating conditions. Correlation of disruption data with laminar stagnation pressure provides a better description of experimental variability than existing correlations using total pressure drop or the grouping 1/Y(2)h(2)
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