DNA vaccines are becoming increasingly important as a way to confer immunity. Ultimately, DNA vaccines should be safer and more effective than traditional vaccines, however, currently, there are limitations in our ability to produce the necessary DNA. To meet the demand for DNA vaccines and other DNA products, it would be beneficial to increase the productivity of production processes. While reducing cost, faster processing would also enable DNA vaccine producers to more quickly respond to pathogen mutations and disease outbreaks. The proposed work addresses this problem. The main idea is when host and plasmid DNA are concurrently engineered, DNA production increases dramatically, while the host grows quickly on simple and inexpensive medium.
The investigators will use a combination of metabolic engineering, proteomics, NMR, bioprocessing, and mathematical modeling to achieve their goals. The work will extend metabolic engineering practice from the current foci of small molecules and proteins to DNA products, which is important in advancing the field of metabolic engineering. From a more fundamental standpoint, the integration of plasmid DNA synthesis with host cell metabolism, which is not now well understood, will be improved. The effect of combining mutations in the control of plasmid replication is also not yet well explored. The work will reveal the extent to which the effects are synergistic, which would be of interest to structural molecular biologists. The impact of redox cofactor balancing will also be elucidated, which is of current interest to a number of ongoing metabolic engineering efforts. A successful outcome will accelerate the production of therapeutic DNA products and facilitate clinical trials. This collaborative project would provide broad education and training for the graduate and undergraduate students (including minority students) involved in metabolic engineering, computation, NMR, proteomics, and the bioprocessing of DNA products.
Our main goal was to considerably increase the yield of plasmid DNA produced from the bacterium E. coli. The impact foreseen was in two areas: (i) increasing vaccine production and (ii) enabling transfection research. The second impact area pertains to a widely distributed method used in basic life science research. From a basic standpoint, we sought to determine just how much extra DNA synthesis E. coli would contend with. Also, we were interested in determining if and how the cell would adapt to deregulated plasmid synthesis. The work consisted of using molecular biology methods to insert what are known as the inc mutations into an already high copy number plasmid. These mutations relieve some of the negative constraints on plasmid replication thereby achieving what we refer to as deregulated plasmid synthesis. Modern proteomics and other methods were then used to characterize and understand the outcome(s) in terms of how what proteins within the cell were adjusted either upwards or downwards to allow the cells to produce plasmid DNA in a deregulated context. The results were better than we initially anticipated. We expected to initially find yield gains, but also some tradeoffs in that more time would be required to produce plasmid. Instead, we found very large enhancements in plasmid product yield and titer while a nil effect on the cell's growth rate occurred when hosting deregulated plasmids. An energy audit and modeling indicates that the low impact on growth rate can be traced to the plasmid's design. The plasmid does not encode the synthesis of an antibiotic-degrading protein. Rather, another selection mechanism is used that does not involve energy- and material-intensive protein synthesis. A delineation of the specifics we found are below: 1. The inc mutations were successfully introduced and verified by DNA sequencing. The plasmid copy number (PCN) attained ranges from 7,000 – 15,000 as determined by qPCR. Significant increases in plasmid yield result whether cells are cultured in minimal medium (glucose plus salts) or nutrient broth (contains amino acids and other constituents). The impact on growth rate was found to be minimal for cells cultured in minimal medium supplemented with glucose as an energy source. An 30% reduction was been found to occur for cells cultured in nutrient broth without glucose as a supplemental energy source, which is not a major change compared to order of magnitude-level changes in yield. Overall, when one uses a standard pUC plasmid as a bench mark, the gains we find in plasmid yield are 10- to 70-fold where the actual value depends on the cultivation context. 2. The proteomics showed upregulated and downregulated clusters of proteins arise in cells that are transformed with deregulated plasmids. Productive cells adapted by increasing the proteins related to ribosome assembly (protein synthesis machinery) and boosting energy production to presumably support the increased turnover of RNA associated with harboring and producing the plasmid product. 3. A small-scale fed-batch process was developed that utilizes enzymatic hydrolysis of the selection agent, sucrose. The use of this method triples the plasmid titer at the lab scale. It also brings industrial level yield capability in terms of titer to those at the small scale and on a lab bench. 4. We found that the fidelity of plasmid replication was not compromised. This means that the DNA sequence was found to be free from errors despite the high replication rate. 5. We found that primarily supercoiled topoisomers arise and not multimers. This is useful because the US FDA prefers the supercoiled form of plasmid DNA when use as a therapeutic product is sought. 6. No evidence of chromosomal integration was found. This means that the system is stable. 7. Proteomics (#2) plus modeling suggests that cells transformed with deregulated plasmids increase TCA cycles activity to contend with more energy use associated with RNA turnover. 8. We developed some explanations of why sometimes different flux distributions are found for some knockouts relevant to improving plasmid production. In terms of other outputs, this work led thus far to 4 peer-reviewed research articles of which 3 are either accepted or in press. The work contributed to composing 3 book chapters. An engineering chemical engineering student wrote a thesis based on this work and 2 post docs in life science gained additional training through his participation. Four undergraduates also gained research experience while working on this project.
