High Yield Deregulated Plasmid DNA Production in Escherichia coli Characterized by Proteomics and Metabolic Flux Analysis

Plasmid DNA has long been used for transfection of eukaryotic cell lines for expression of heterologous protein. Whether at the lab scale (mg-g) for expression in mammalian- or insect- cell lines or at the industrial scale (g-kg) for therapeutic applications in human patients such as gene therapies or DNA vaccines, plasmid DNA has been established as an expression vector alongside viral agents. Plasmids continue to be widespread in basic biology research, and numerous clinical trials involve use of a plasmid either as the primary agent tested for efficacy or as an adjuvant to boost medicinal effect. It follows that there is a demand for frugal methods that achieve high yield plasmid DNA.;High yields of plasmid are currently obtained via bacterial fermentation with plasmid-containing cells. For biological research laboratories, these procedures typically are selected for the attributes of a relatively simple protocol, high titer, and microorganism non-pathogenicity. These qualities are also beneficial for investigators of human health and disease, but their additional concern for high purity product constitutes a significant barrier in downstream processing. To address this array of concerns, the strategy employed for this work was to increase plasmid production on a per-cell basis using Escherichia coli..;Outside of fermentation conditions, previous efforts toward higher plasmid yields have focused on identifying what enzymatic processes aid in the microorganism's resource allocation for the goal of increasing plasmid. By altering certain enzyme levels in order to route metabolic trafficking toward DNA's nucleotide or pentose precursors, metabolic engineers have found success in increasing plasmid yield and/or recovering growth rate while maintaining plasmid levels. This thesis approaches the problem from another angle --- deregulating the plasmid by loosening replication controls is shown to increase plasmid replication rates. Because no heterologous protein expression results from the plasmid used, increased plasmid production is possible without detrimental effects to specific growth rates. Investigation via proteomics and flux balance analysis of both the host strain and the plasmid-transformed construct illuminates the effects of the plasmid on cell protein expression and metabolism, providing insights for possible future metabolic engineering.;For this work we used a high copy number antibiotic-free system developed for therapeutic products such as DNA vaccines. Higher yields were obtained by altering the plasmid's replication control mechanism, aka "deregulating the plasmid." Briefly, pUC plasmid replication involves two pieces of antisense RNA whose genes are located in the ori region. Named RNA-I and RNA-II, these two molecules determine plasmid copy number. RNA-II is necessary as a primer for plasmid replication, and antisense RNA-I interacts with RNA-II preventing replication initiation. Specific point changes known as inc mutations significantly increased plasmid levels by four- to six-fold. For growth on minimal media, only a slight reduction in growth rate was observed.;During exponential growth on M9 minimal media, plasmid copy numbers of approximately 7,000 were achieved, and during the early-stationary phase the value increased to approximately 15,000. Separate experiments involving a temperature shift from 37 °C during exponential growth to 42 °C during late-log phase indicated plasmid yields increased from ∼2% of cellular biomass to ∼5%. This compares to the parent plasmid's yields of ∼0.5% of cellular biomass during exponential growth and ∼2% during early stationary phase after a temperature shift. These yields vastly outperform current laboratory techniques for which a typical plasmid copy number might be 300, and they are competitive with highly augmented fermentations that achieve plasmid levels of about 5% cell mass.;Quantitative proteomics showed plasmid production is associated with increased levels of Krebs cycle enzymes along with decreased levels of some glycolytic enzymes, though isozymes of these glycolytic enzymes often were not highlighted as down-regulated. Also, elevated levels of Ribonuclease E were observed in the high copy number construct. Ribonuclease E is known to be a central element of the cell's RNA degradosome, and specifically cleaves the control molecule RNA-I to render it ineffective.;These results were combined with modeling techniques for E. coli metabolism to calculate the feasible flux space available. By producing models for both plasmid-free cells as well as the mutation-based high copy number construct, it was shown that although a range of fluxes remain possible, certain features suggest coping strategies the cells may have used to maintain growth rate despite the burden of plasmid synthesis. By employing the proteomics obtained by experiment, solutions were generated that correspond to high maintenance energy as the cellular objective for plasmid-containing cells. Lastly, initial modeling work was performed for 13-C tracer studies. While experimental studies were not performed, mathematical possibilities for a separate plasmid-production line were established. Pre-analysis of the system used allows for the problem to be redefined from calculating the fluxes obtained from an experiment to distinguishing between only a few linearly independent extreme point fluxes that represent alternate (independent) metabolic strategies given experimentally available parameters such as glucose uptake or acetate production. This suggests future work could be performed by adaptation of the method to plasmid-producing systems.