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Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: application to production of toxic proteins.

ABSTRACT: In previous work transduction of Escherichia coli with phage ? particles carrying packaged plasmids was shown to provide complete off-to-on expression of plasmid-borne genes (Cronan, J.E., 2003. J. Bacteriol. 185, 6522-6529). The plasmids used contained the phage ?cos site (and hence are cosmids) and were very efficiently packaged into ? phage particles in vivo upon induction of ? lysogens having an inactivated cos site. However, a shortcoming was that the stocks contained a variable fraction of infectious ? phage which arose by recombinational repair of the inactive cos site. I report that the construction of E. coli strains that eliminate the background of infectious phage and show that the system can be utilized to express proteins by the phage T7 RNA polymerase/pET vector system.

SUBMITTER: Cronan JE

PROVIDER: S-EPMC3540202 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: application to production of toxic proteins.

Cronan John E JE

Plasmid 20120925 1

In previous work transduction of Escherichia coli with phage λ particles carrying packaged plasmids was shown to provide complete off-to-on expression of plasmid-borne genes (Cronan, J.E., 2003. J. Bacteriol. 185, 6522-6529). The plasmids used contained the phage λcos site (and hence are cosmids) and were very efficiently packaged into λ phage particles in vivo upon induction of λ lysogens having an inactivated cos site. However, a shortcoming was that the stocks contained a variable fraction of ...[more]

PMID: 23022297

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Project description:BackgroundPlasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.ResultsSuch a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date.ConclusionThese results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.

Project description:BackgroundMulti-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.ResultsThe engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-D-glucose biosynthesis genes under PT7 promoter led to the production of ~?439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose.ConclusionA synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.

Dataset Information

Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: application to production of toxic proteins.

Publications

Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: application to production of toxic proteins.

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