Metabolic engineering of Vibrio natriegens for anaerobic succinate production

The biotechnological production of succinate bears serious potential to fully replace existing petrochemical approaches in the future. In order to establish an economically viable bioprocess, obtaining high titre, yield and productivity is of central importance. In this study, we present a straightforward engineering approach for anaerobic succinate production with Vibrio natriegens, consisting of essential metabolic engineering and optimization of process conditions. The final producer strain V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pyc_Cg (Succ1) yielded 1.46 mol of succinate per mol of glucose under anaerobic conditions (85% of the theoretical maximum) and revealed a particularly high biomass-specific succinate production rate of 1.33 g_Succ g_CDW ^-1  h^-1 compared with well-established production systems. By applying carbon and redox balancing, we determined the intracellular flux distribution and show that under the tested conditions the reductive TCA as well as the oxidative TCA/glyoxylate pathway contributed to succinate formation. In a zero-growth bioprocess using minimal medium devoid of complex additives and expensive supplements, we obtained a final titre of 60.4 g_Succ l^-1 with a maximum productivity of 20.8 g_Succ  l^-1  h^-1 and an overall volumetric productivity of 8.6 g_Succ l^-1  h^-1 during the 7 h fermentation. The key performance indicators (titre, yield and productivity) of this first engineering approach in V. natriegens are encouraging and compete with costly tailored microbial production systems.