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Harris DM van der Krogt ZA van Gulik WM van Dijken JP Pronk JT 《Applied and environmental microbiology》2007,73(15):5020-5025
Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams. 相似文献
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Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation 总被引:3,自引:0,他引:3
Kuyper M Hartog MM Toirkens MJ Almering MJ Winkler AA van Dijken JP Pronk JT 《FEMS yeast research》2005,5(4-5):399-409
After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate. 相似文献
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Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast 总被引:3,自引:0,他引:3
Ifoeng Chin-Joe Adrie J. J. Straathof Jack T. Pronk Jaap A. Jongejan Joseph J. Heijnen 《Biocatalysis and Biotransformation》2002,20(5):337-345
Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l -1 , a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l -1 h -1 , using 2.1 kg pressed yeast kg product -1 and 0.52 kg glucose kg product -1 . During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions. 相似文献
79.
F C Van der Helm H E Veeger G M Pronk L H Van der Woude R H Rozendal 《Journal of biomechanics》1992,25(2):129-144
A dynamical finite-element model of the shoulder mechanism consisting of thorax, clavicula, scapula and humerus is outlined. The parameters needed for the model are obtained in a cadaver experiment consisting of both shoulders of seven cadavers. In this paper, in particular, the derivation of geometry parameters from the measurement data is described. The results for one cadaver are presented as a typical example. Morphological structures are modelled as geometrical forms. Parameters describing this form are estimated from 3-D position coordinates of a large number of datapoints on the morphological structure, using a least-squares criterion. Muscle and ligament attachments are represented as a plane or as a (curved) line. Muscle paths are determined by a geometrical form of the bony contour around which the muscle is wrapped. Muscle architecture is determined by the distribution of muscle bundles over the attachment area, mapping the distribution of the origin to the insertion. Joint rotation centers are derived from articular surfaces. Hence, muscle moment arms can be calculated. The result of this study is a set of parameters for each cadaver, describing very precisely the geometry of the shoulder mechanism. This set allows positioning of muscle force vectors a posteriori, and recalculation of position coordinates and moment arms for any position of the shoulder. 相似文献
80.
M. C. M. Hensing K. A. Bangma L. M. Raamsdonk E. de Hulster J. P. van Dijken J. T. Pronk 《Applied microbiology and biotechnology》1995,43(1):58-64
Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale. 相似文献