(448d) Exploring the Potential of NON-Growth Associated POLY-3-Hydroxybutyrate Production with Recombinant Escherichia coli through Dynamic Flux Balance Analysis

For the past decades there is a push for the development of sustainable processes due to increasing concerns with environmental impacts. Plastic disposal, for instance, has become a major problem due to how long conventional oil-derived plastics take to degrade when disposed, and because of problems with toxicity and bioaccumulation (ANDREESSEN; TAYLOR; STEINBÜCHEL, 2014; BRADFORD, 2017; PARKER, 2018). In this scenario, bioplastics such as poly-3-hydroxybutyrate (PHB) are seem as a possible and more environmentally friendly alternative for certain applications (ANDREESSEN; TAYLOR; STEINBÜCHEL, 2014; CHEN, 2009; KESHAVARZ; ROY, 2010; ZHENG et al., 2020). But PHB production costs remain a barrier for its broader commercialization (GODINHO, 2005; ZHANG et al., 2014; ZHENG et al., 2020). Many researches therefore were focused on trying to improve the PHB production processes through the use of metabolic engineering strategies. Recombinant microorganisms such as Escherichia coli harboring the PHB synthesis pathway from Cupriavidus necator can often offer advantages over naturally producing microorganisms such as a higher growth rate and easier polymer extraction (GODINHO, 2005). While the literature points that recombinant E. coli produces PHB even during growth (CARLSON; WLASCHIN; SRIENC, 2005), a dedicated growth phase followed by a dedicated production phase can lead to higher productivities, which theoretically can be achieved with strategies such as genetic toggle-switches, where after reaching a predetermined cell concentration, a trigger shifts carbon flux from biomass to product formation (ANESIADIS; CLUETT; MAHADEVAN, 2008; ZHUANG et al., 2013). This scenario leads to an optimization problem in order to find how much of a given total glucose should be used for the growth phase, with the remaining glucose being used for the PHB production phase. With the use of Dynamic Flux Balance Analyses simulations and an estimation of the monthly gross profit for each scenario, this work explores the two-phase PHB production potential of recombinant E. coli for both aerobic and anaerobic batch cultures. The best result for the two-phase simulations under aerobic conditions is achieved when using 20%(mol) of the available glucose on the growth phase and the remaining 80% in the PHB production phase, leading to a final yield of 0.47 g PHB/g glu, titer of 11.73 g PHB/L, and productivity of 1.51 g PHB/L.h, which results in a monthly gross profit of USD 5460.09, in the conditions simulated. For the anaerobic two-phase simulations, the best performance within a realistic PHB content is achieved when 30%(mol) of the available glucose is used on the growth phase and the remaining 70% in the PHB production phase, leading to a final yield of 0.32 g PHB/g glu, titer of 7.99 g PHB/L, and productivity of 0.85 g PHB/L.h, which results in a monthly gross profit of USD 758.02. These results show how DFBA can be used to identify the approaches that lead to the most profitable scenarios, setting therefore more precise targets for future metabolic engineering strategies.

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Acknowledgments

We gratefully acknowledge support of the RCGI – Research Centre for Greenhouse Gas Innovation, hosted by the University of São Paulo (USP) and sponsored by FAPESP – São Paulo Research Foundation (2014/50279-4 and 2020/15230-5) and Shell Brasil, and the strategic importance of the support given by ANP (Brazil’s National Oil, Natural Gas and Biofuels Agency) through the R&D levy regulation.