Plasmid addiction systems: perspectives and applications in biotechnology

Krøll, Jens; Klinter, Stefan; Schneider, Cornelia; Voß, Isabella; Steinbüchel, Alexander

doi:10.1111/j.1751-7915.2010.00170.x

Cited by 107 publications

(77 citation statements)

References 268 publications

(277 reference statements)

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“…The use of plasmid pCB81 would require the addition of expensive antibiotics to fermentations, which would add excessive cost at the industrial scale. A common strategy for maintaining plasmid stability without the use of antibiotics is to create an auxotrophic mutant through a genome mutation and then to complement the mutation with a plasmid containing the deleted gene (20). We deleted the proC gene from Re1034 and Re2133 to create Re2058 and Re2160, respectively.…”

Section: Resultsmentioning

confidence: 99%

Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

Budde

Riedel

Willis

et al. 2011

Appl Environ Microbiol

197

145

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The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-coHHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chainlength fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

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Section: Resultsmentioning

confidence: 99%

Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

Budde

Riedel

Willis

et al. 2011

Appl Environ Microbiol

197

145

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“…Implementation of plasmid addiction. The use of antibiotics to reduce contamination and select for bacteria with plasmids is not economically feasible in a commercial-scale process (20). To address this issue, we implemented a plasmid addiction system based on glycerol addiction.…”

Section: Resultsmentioning

confidence: 99%

“…Chromosomal integration of foreign genes is applicable in some cases, but important factors such as polar effects and limited gene copy number are disadvantages (20). Kroll et al described, as a possible solution to these problems, several plasmid addiction systems (20). We chose to implement a catabolism-based addiction system.…”

Section: Discussionmentioning

confidence: 99%

Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

Wong¹,

Li²,

Black³

et al. 2014

Appl Environ Microbiol

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Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.

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“…Plasmid loss has been recognized as a limiting factor for large-scale recombinant gene expression and for bioremediation because the use of antibiotics to ensure plasmid retention is not practical in these settings (13, 14). Available approaches for increasing plasmid stability depend on a particular strain or culture condition (plasmid addiction systems) (93, 94), or on inducing growth arrest through massive metabolic burden (95, 96), or through induction of a quiescent state (97). Approaches aimed at increasing plasmid stability based on modifying the plasmid origin of replication are particularly appealing because they would be largely autonomous, i.e.…”

Section: Alternative Hypothesismentioning

confidence: 99%

Mechanisms of plasmid segregation: Have multicopy plasmids been overlooked?

Million-Weaver

Camps

2014

Plasmid

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Plasmids are self-replicating pieces of DNA that can help dissemination of non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

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Plasmid addiction systems: perspectives and applications in biotechnology

Cited by 107 publications

References 268 publications

Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

Mechanisms of plasmid segregation: Have multicopy plasmids been overlooked?

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