Cellular plasmid content and cloned‐gene expression: Some useful equations

Satyagal, Vasuki N.; Agrawal, Piyush

doi:10.1002/bit.260350105

Cited by 14 publications

(3 citation statements)

References 22 publications

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“…Biosynthetic Burden: a Unifying Hypothesis. The diversion of biosynthetic resources from native functions to the replication of plasmids and the expression of plasmid-borne genes typically results in a decrease in growth rate and vigor of the recombinant, that is, plasmid burden (19,21,28,32). In Z. mobilis, for example, overexpression of a single recombinant gene reduced the specific activities of chromosomally encoded glycolytic enzymes, resulting in slower glycolysis (31).…”

Section: Resultsmentioning

confidence: 99%

Biosynthetic Burden and Plasmid Burden Limit Expression of Chromosomally Integrated Heterologous Genes (pdc,adhB) in Escherichiacoli

Martı́nez

York

Yomano

et al. 1999

Biotechnology Progress

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Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma(28). The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation. From this work, we propose that the activation of biosynthetic genes in nutrient-poor media creates a biosynthetic burden that reduces the expression of chromosomal pdc and adhB by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.

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Section: Resultsmentioning

confidence: 99%

Biosynthetic Burden and Plasmid Burden Limit Expression of Chromosomally Integrated Heterologous Genes (pdc,adhB) in Escherichiacoli

Martı́nez

York

Yomano

et al. 1999

Biotechnology Progress

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“…There have been a number of studies of a "plasmid burden" effect on the metabolism of recombinant bacteria, viz., the effect of an extrachromosomal plasmid(s) in reducing the growth rate, cell yield, and cell viability of these recombinant strains (3,6,24). In a recent report, an average biomass yield on xylose of 0.034 g/g of xylose for recombinant Z. mobilis CP4(pZB5) was compared with yields on glucose for the recombinant and wild-type cultures, viz., 0.055 and 0.058 g/g of glucose, respectively (Lawford and Rousseau, Abstr.…”

Section: Figmentioning

confidence: 99%

Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5)

Kim¹,

Barrow

Rogers³

2000

Appl Environ Microbiol

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The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucosexylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by 13 Cnuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo 31 P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.Zymomonas mobilis has attracted widespread interest for fuel ethanol production because of its higher specific rates of sugar uptake and ethanol production, higher ethanol tolerance, and higher ethanol conversion efficiencies when compared to the traditionally used yeasts (10,14,21,22,26). However, wild-type strains of Z. mobilis can only utilize glucose, fructose, and sucrose, and they lack the pentose metabolism pathway necessary to ferment such sugars as xylose or arabinose. The cloning of enzymes for xylose assimilation and metabolism in Z. mobilis has now been reported (30), and a subsequent study has resulted in the successful integration of the requisite genes into the Zymomonas genome (M. Zhang, Y. C. Chou, X. K. Lai, S. Milstrey, N. Danielson, K. Evans, A. Mohagheghi, and M. Finkelstein, Abstr. 21st Symp. Biotechnol. Fuels Chem., abstr. 2-16, 1999). These genetically engineered strains can now convert xylose to ethanol by the combined use of the Entner-Doudoroff and pentose pathways facilitated by the cloned enzymes xylose isomerase and xylulokinase for xylose assimilation and by transketolase and transaldolase for pentose metabolism. In a further study, the cloning of three additional enzymes for arabinose utilization has been reported (4). However, when xylose is the sole carbon source, lower biomass yields and slower growth rates as well as lower ethanol yields for recombinant strains have been reported (9, 11, 12, 23; H. G. Lawford and J. D. Rousseau, Abstr. 21st Symp. Biotechnol. Fuels Chem., abstr. 2-24, 1999).In this study, the fermentation characteristics of the recombinant Z. mobilis ZM4(pZB5) on xylose or glucose alone, or on xylose-glucose mixtures, have been investigated to determine possib...

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“…Most lumped parameter models have been proposed in the past with the common feature that they are based on the “metabolic burden” approach (Bentley and Quiroga, 1993; Lee et al, 1985; Satyagal and Agrawal, 1990). A lot of datasets presented in the literature can be fitted to these models.…”

Section: Theoretical Aspectsmentioning

confidence: 99%

Plasmid DNA production for pharmaceutical use: Role of specific growth rate and impact on process design

Bohle

Ross

2011

Biotech & Bioengineering

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Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide sufficient material that is in accordance with quality of pharmaceutical grade it is mandatory to gain comprehensive process knowledge which is even requested by regulatory agencies. Regarding plasmid DNA production the specific growth rate has been identified as one of the key parameters. The reduction of specific growth rate results in an increase of plasmid DNA formation. However, quantitative explanations that allow for efficient process development and design are still missing. The presented study proposes a model that clearly demonstrates the relationship between specific growth rate and plasmid formation due to identification of the specific plasmid production rate as relevant key parameter. In addition the model is proved to serve as a useful tool in process development and design.

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Cellular plasmid content and cloned‐gene expression: Some useful equations

Cited by 14 publications

References 22 publications

Biosynthetic Burden and Plasmid Burden Limit Expression of Chromosomally Integrated Heterologous Genes (pdc,adhB) in Escherichiacoli

Biosynthetic Burden and Plasmid Burden Limit Expression of Chromosomally Integrated Heterologous Genes (pdc,adhB) in Escherichiacoli

Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5)

Plasmid DNA production for pharmaceutical use: Role of specific growth rate and impact on process design

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