The two main contributions of this are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of bansal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a through literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support fluxes during growth and lysine overproduction branch point does not appear to limit lysine synthesis.

Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction.

The present invention describes an integrated computer-based graphical interface, methods and systems providing a shell environment for development and deployment for graphic information storage and retrieval, visual modeling and dynamic simulation of complex systems. In the current implementation the system comprises libraries of knowledge-based building-blocks that include sets of icons representing chemical processes, the pools of entities that participate in those processes, and the graphical description of those entities, encapsulating both information and mathematical models within the modular components, in the form of tables and in the form of component icons, and a plurality of methods are associated with each of the icons. The models are built by interconnecting each pool to one or several processes, and each process to one or several pools, resulting in complex networks of multidimensional pathways. A number of functions and graphical interfaces can be selected from the menus associated with each icon, to extract in various forms the information contained in the models build with those building blocks. Those functions include the creation of interactive networks of pathways, graphic selection of complex predefined queries based on the relative position of pools of entities in the pathways, the role that the pools play in the processes, and the structural components of the entities of those pools, and quantitative simulations. The system integrates inferential control with quantitative and semi-quantitative simulation methods, and provides a variety of alternatives to deal with complex dynamic systems and with incomplete and constantly evolving information and data.

Computer-based system, methods and graphical interface for information storage, modeling and stimulation of complex systems

Modelling of microbial kinetics

Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established. Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.

https://link.springer.com/content/pdf/10.1186%2F1475-2859-7-6.pdf

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria.

A model was formulated to examine specific experimental data of growth and heterologous product formation with recombinant Saccharomyces cerevisiae while incorporating available literature. The model simulated dry cell weight, glucose, ethanol, dissolved oxygen, human Epidermal Growth Factor (hEGF) production, fraction of recombinant cells, oxygen uptake rate, and carbon dioxide production rate for batch, fed batch, and hollow fiber bioreactor configurations. Nineteen differential equations, 24 analytical equations, and 48 parameters were required. Due to the lack of detailed studies needed for the ADH-II and the TCA enzyme pool, 8 of the 48 parameters were adjustable. Simulation results are presented for verification of the model which successfully described the observed phenomena for the fermentations of S. cerevisiae strain AB103. 1 pYalphaEF-25. Also presented is a statistical analysis of the model's fit and model parameter sensitivity.

A mathematical description of recombinant yeast.

Expression kinetics of the human Epidermal Growth Factor (hEGF) from the alpha-factor prepro region in a 2-mum based plasmid was studied in Saccharomyces cerevisiae. Production of hEGF was highly medium de pendent as a chemically defined, nonenriched media had a significantly lower yield than did enriched media. Also cells grown on yeast nitrogen base without amino acids with casamino acids degraded the hEGF after cell growth as opposed to a yeast extract, peptone, and dextrose (YEPD) medium, which elicited no measurable extracellular proteolysis of the hEGF. alpha-factor directed production kinetics of hEGF on the YEPD medium were growth associated, secretion limitations and extracellular degradation were negligible, and the hEGF was nearly 100% selectively secreted. With sufficient agitation, shake flask experiments were representative of aerated controlled batch fermentations. No effect of high cell density was observed on cell growth or hEGF production kinetics. The hollow fiber bioreactor had no direct effect on the substrate or protein yields of S. cerevisiae, however the low oxygen transfer capacity of the membrane was not sufficient to support respiration.

Steven J. Coppella

Papers

A mathematical description of recombinant yeast.

α‐Factor directed expression of the human epidermal growth factor in Saccharomyces cerevisiae

Low-cost computer-coupled fermentor off-gas analysis via quadrupole mass spectrometer.

Isolation of high-molecular-weight nucleic acids for copy number analysis using high-performance liquid chromatography.

A detailed analysis of Saccharomyces cerevisiae growth kinetics in batch, fed-batch, and hollow-fiber bioreactors