Willians de Oliveira Santos

As the world recognizes the need for more sustainable practices, greater effort has been put into improving the economic viability of bioprocesses. Fields like metabolic engineering explore strategies to improve the production of bioproducts of industrial relevance, such as the use of alternative carbon sources. Regarding the performance assessment of a bioprocess, the product yield can be helpful, but other parameters like product titer and productivity (rate of production) are just as important. Furthermore, the highest performance may not come from a scenario with the highest yield, titer or productivity, but from a titer, rate, yield (TRY) set that maximizes profitability when the production costs are considered. Our previous work introduced the DySEEP approach, which uses an economic metric based on TRY parameters for evaluation of the potential of bioprocesses. This work expands the DySEEP program in key areas such as adding the possibility of exploring fed-batch processes and using strain design algorithms. Taking batch and fed-batch poly-3-hydroxybutyrate (PHB) production with E. coli as the case study, the scenarios that would lead to economic viability and the theoretical maximum performance for three different carbon sources, glucose, glycerol, and xylose, were identified. The results were then set in a strain design algorithm using Minimal Cut Sets (MCS) to find genetic interventions that could achieve the desired performance. The genetic interventions found with the MCS algorithm successfully led to strains that achieve the economic viability scenario, based on in silico simulations. This work illustrates how the expanded approach can be used to guide metabolic engineering strategies to improve the production of important bioproducts.

Concerns with sustainability have led to increasing interest in bioprocesses in the last decades. In particular, environmental problems with plastic disposal have been a major issue. Bioplastics such as poly-3-hydroxybutyrate (PHB) are potential substitutes since they are biodegradable and less toxic. However, their production costs are high and optimization is required. Many works have therefore aimed to build strains capable of higher product yields. But in the case of products that share a precursor with biomass and in the case of intracellular metabolites, a trade-off may occur. Not only yield but also final biomass, titer, and productivity have to be considered. Therefore, this work presents an approach named Dynamic Simulations for Evaluation of Economic Potential (DySEEP), which uses dynamic flux balance analysis (DFBA) and an economic metric as a function of the bioprocess parameters for evaluation of the production of bioproducts of industrial interest. As a case study, the PHB production potential of recombinant Cupriavidus necator, Escherichia coli, and Saccharomyces cerevisiae was analyzed. While some key polymer properties cannot be predicted due to the nature of DFBA simulations, the PHB production is a good case study to highlight the trade-off between biomass and product formation. It was identified that for growth-associated production with recombinant E. coli and the NADPH-dependent PHB synthesis pathway, the scenario that starts positive cash flows is when a yield of 0.37 g/g and its respective final biomass, titer, and productivity is achieved, and the maximum theoretical profit would be achieved when a yield of 0.50 g/g and its respective parameters is reached. Furthermore, comparison between the internal flux distribution of the best scenario identified and the flux distributions of a simulation constrained with experimental data from the literature allowed the suggestion of genetic modifications that could enhance PHB production, such as knockouts for interruption of the oxidative phase of the pentose phosphate pathway, of the acetate production reaction, and of the reaction catalyzed by the 2-oxoglutarate dehydrogenase enzyme or downregulation of the TCA cycle, setting therefore potential targets for metabolic engineering strategies. For nongrowth-associated production with both recombinant E. coli and C. necator, the scenario at which cash flow starts to become positive is when 40% (mol) of the available glucose is used in the growth phase and the remaining 60% is used in the production phase, and the scenario that leads to the maximum theoretical profit, within a realistic maximum PHB content, is when 20% (mol) is used in the growth phase and the remaining 80% in the production phase, information that sets targets for bioreactor operation strategies such as defining the moments for nutrient limitation and for synthetic biology by showing when to activate genetic toggle-switches.