Low-biomass pyruvate production with engineered Vibrio natriegens is accompanied by parapyruvate formation

Background: Pyruvate is a precursor for various compounds in the chemical, drug, and food industries and is therefore an attractive target molecule for microbial production processes. The fast-growing bacterium Vibrio natriegens excels with its specific substrate uptake rate as an unconventional chassis for industrial biotechnology. Here, we aim to exploit the traits of V. natriegens for pyruvate production in fermentations with low biomass concentrations. Results: We inactivated the pyruvate dehydrogenase complex in V. natriegens Δvnp12, which harbors deletions of the prophage regions vnp12. The resulting strain V. natriegens Δvnp12 ΔaceE was unable to grow in minimal medium with glucose unless supplemented with acetate. In shaking flasks, the strain showed a growth rate of 1.16 ± 0.03 h^{− 1} and produced 4.0 ± 0.3 g_Pyr L^{− 1} within 5 h. We optimized the parameters in an aerobic fermentation process and applied a constant maintenance feed of 0.24 g_Ac h^{− 1} which resulted in a maximal biomass concentration of only 6.6 ± 0.4 g_CDW L^{− 1} and yielded highly active resting cells with a glucose uptake rate (q_S) of 3.5 ± 0.2 g_Glc g_CDW⁻¹ h^{− 1}. V. natriegens Δvnp12 ΔaceE produced 41.0 ± 1.8 g_Pyr L^{− 1} with a volumetric productivity of 4.1 ± 0.2 g_Pyr L^{− 1} h^{− 1}. Carbon balancing disclosed a gap of 30%, which we identified partly as parapyruvate. Deletion of ligK encoding the HMG/CHA aldolase in V. natriegens Δvnp12 ΔaceE did not impact biomass formation but plasmid-based overexpression of ligK negatively affected growth and led to a 3-fold higher parapyruvate concentration in the culture broth. Notably, we also identified parapyruvate in supernatants of a pyruvate-producing Corynebacterium glutamicum strain. Cell-free bioreactor experiments mimicking the biological process also resulted in parapyruvate formation, pointing to a chemical reaction contributing to its synthesis. Conclusions: We engineered metabolically highly active resting cells of V. natriegens producing pyruvate with high productivity at a low biomass concentration. However, we also found that pyruvate production is accompanied by parapyruvate formation in V. natriegens as well as in a pyruvate producing C. glutamicum strain. Parapyruvate formation seems to be a result of chemical pyruvate conversion and might be supported biochemically by an aldolase reaction.