Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

Abstract

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.

Marker-free plasmids for gene therapeutic applications—Lack of antibiotic resistance gene substantially improves the manufacturing process

“…This huge increase seems to be caused by one, more or a combination of all effects: (i) the decrease in metabolic load and consequently the increased availability of metabolic precursors, resulting from deletion of the constitutively expressed nptII gene, (ii) the decrease of plasmid size and (iii) the increase in pDNA replication rate. This increase in specific plasmid yield is in accordance with a recent publication based on stoichiometric modelling of E. coli (Cunningham et al, 2009a). Although some therapeutic plasmids might be larger due to introns or multiple genes, the effect of decreased size is expected to be the same, in fact might even be more pronounced due to increased availability of metabolic precursors available for plasmid replication.…”

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

“…Interestingly, previous studies have indicated that constitutive expression of antibiotic resistance markers negatively affects host cells and in fact marker accumulation up to levels as high as 20% of total cellular protein29 and even inclusion body formation have been reported23. This imposes a metabolic load on host cells and it has been shown that its elimination can improve process performance, namely for plasmid production processes23.…”

“…Furthermore, it is expected to impose a reduced metabolic burden on host cells due to the small sizes of the toxin (100 amino acids) and anti-toxin (90 amino acids) as well as the regulated expression of these (toxin production only in the absence of anti-toxin)252627. In contrast, the relatively large antibiotic-resistance proteins (β-lactamase: 263 amino acids + 23 amino acid signal sequence; aminoglycoside 3′-phosphotransferase: 271 amino acids) are constitutively expressed from the pET expression vectors and indeed have already been shown to have a deleterious effect on host cells242829.…”

Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

“…To test the inducible cell lysis system, we first introduced the genetic circuits L1, L2, L3, L12, L13, and L123 into strain Ec(Ri) on plasmids pSB1K3(FRTL1), pSB1K3(FRTL2), pSB1K3(FRTL3), pSB1K3(FRTL12), pSB1K3(FRTL13), and pSB1K3(FRTL123), respectively. Integration of genetic circuits into the chromosome is preferable to their maintenance on the plasmids [40, 41], therefore, in the next step we also integrated L1, L2, L3, L12, L13, and L123 into the chromosome of the strain Ec(Ri). The correct assemblies of lysis genes on plasmids and the chromosomally integrated genetic circuits were verified by diagnostic PCR with flanking primers (S1 Fig and S1 Table) and sequencing.…”