From plasmid sequence to process design: A computational analysis of metabolism in the context of plasmid DNA manufacturing

Detailed understanding of plasmid-host physiological interactions and recombinant protein expression is crucial for the design and optimization of plasmid DNA (pDNA) manufacturing processes. Successfully transformed host cells carrying one or more copies of a particular plasmid exhibit modified metabolic behavior which may include reduced specific growth rate, increased metabolic resource cycling (ATP/ADP, NAD/NADH, NADP/NADPH) and, in some cases, even modified nutrient uptake and metabolite secretion rates. However, to the extent of our knowledge, there have been no attempts to map the impact of plasmid design directly to critical process parameters (CPPs) such as the specific growth rate and/or productivity. Herein we present, a comprehensive computational analysis based on carbon constrained Flux Balance Analysis (ccFBA) to precisely calculate the metabolic burden imposed by plasmid replication and recombinant protein expression. Explicit stoichiometric coefficients for nucleotides and amino acids were derived directly from the plasmids sequence and were used to formulate biosynthetic reactions tailored to the components of individual plasmids. Three tunable parameters were introduced to map the impact of promoter strength, copy number and choice of selection marker on the host cells metabolism. Literature derived experimental data from E. coli cultures producing three different plasmids were used to constrain the iJR904 Genome Scale Model (GeM). Our analysis revealed correlations between cellular growth rate, promoter strength and pDNA productivity with significant implications for the design of recombinant technology based manufacturing processes.