A Fermentation State Marker Rule Design Task in Metabolic Engineering.

There are several ways in which mathematical modeling is used in fermentation control, but mechanistic mathematical genome-scale models of metabolism within the cell have not been applied or implemented so far. As part of the metabolic engineering task setting, we propose that metabolite fluxes and/or biomass growth rate be used to search for a fermentation steady state marker rule. During fermentation, the bioreactor control system can automatically detect the desired steady state using a logical marker rule. The marker rule identification can be also integrated with the production growth coupling approach, as presented in this study. A design of strain with marker rule is demonstrated on genome scale metabolic model iML1515 of Escherichia coli MG1655 proposing two gene deletions enabling a measurable marker rule for succinate production using glucose as a substrate. The marker rule example at glucose consumption 10.0 is: IF (specific growth rate µ is above 0.060 h-1, AND CO2 production under 1.0, AND ethanol production above 5.5), THEN succinate production is within the range 8.2-10, where all metabolic fluxes units are mmol ∗ gDW-1 ∗ h-1. An objective function for application in metabolic engineering, including productivity features and rule detecting sensor set characterizing parameters, is proposed. Two-phase approach to implementing marker rules in the cultivation control system is presented to avoid the need for a modeler during production.

OptEnvelope: A target point guided method for growth-coupled production using knockouts.

Finding the best knockout strategy for coupling biomass growth and production of a target metabolite using a mathematic model of metabolism is a challenge in biotechnology. In this research, a three-step method named OptEnvelope is presented based on finding minimal set of active reactions for a target point in the feasible solution space (envelope) using a mixed-integer linear programming formula. The method initially finds the reduced desirable solution space envelope in the product versus biomass plot by removing all inactive reactions. Then, with reinsertion of the deleted reactions, OptEnvelope attempts to reduce the number of knockouts so that the desirable production envelope is preserved. Additionally, OptEnvelope searches for envelopes with higher minimum production rates or fewer knockouts by evaluating different target points within the desired solution space. It is possible to limit the maximal number of knockouts. The method was implemented on metabolic models of E. coli and S. cerevisiae to test the method benchmarking the capability of these industrial microbes for overproduction of acetate and glycerol under aerobic conditions and succinate and ethanol under anaerobic conditions. The results illustrate that OptEnvelope is capable to find multiple strong coupled envelopes located in the desired solution space because of its novel target point oriented strategy of envelope search. The results indicate that E. coli is more appropriate to produce acetate and succinate while S. cerevisiae is a better host for glycerol production. Gene deletions for some of the proposed reaction knockouts have been previously reported to increase the production of these metabolites in experiments. Both organisms are suitable for ethanol production, however, more knockouts for the adaptation of E. coli are required. OptEnvelope is available at https://github.com/lv-csbg/optEnvelope.

Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by Crypthecodinium cohnii from Glycerol, Glucose and Ethanol.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.