Most industrial production processes must operate at a high final biomass concentration to reach a high volumetric productivity (Q_P), wasting significant amounts of carbon for biocatalyst generation. Therefore, we are using Vibrio natriegens due to its very fast growth and associated high specific substrate uptake rate (q_S) [1] in a low-biomass processes with non-growing but metabolically highly active catalysts.

We engineered the prophage-free strain V. natriegens Δvnp12 [2] by deleting the aceE gene encoding the E1 subunit of the pyruvate dehydrogenase complex (PDHC). This PDHC-deficient strain is acetate auxotroph, allowing adjustment of the biomass formation by changing the amount of acetate in the process. Through this, the whole process is split into a growth and a production phase in which pyruvate is secreted into the medium.

Shaking flask experiments in minimal medium with 7.5 g glucose L^-1 and 1 g acetate L^-1 achieve up to 4.0 ± 0.3 g pyruvate L^-1 in the supernatant. Therefore, batch fermentations were performed and up to 22.2 ± 0.8 g pyruvate L^-1 were produced after 10 hours with a yield of 0.59 ± 0.04 g pyruvate g^-1 glucose. An additional acetate feed of 8 mM h^-1 increased the titer to 41 ± 2 g pyruvate L^-1 with a c_x,max of 6.6 ± 0.4 g L^-1. Moreover, the qs of the non-growing cells increased to 3.5 g glucose g^-1_CDW h^-1, close to the q_S of exponentially growing cells [1] and therefore over twice as high as the qs of growing E. coli.

Following gaps in the carbon balance, significant amounts of parapyruvate (4-Hydroxy-4-methyl-2-oxoglutarate, HMG) were found in the reactor samples. The ligase LigK was investigated as a possible candidate for biological parapyruvate formation using overexpression and deletion strains, showing significant phenotypes. Moreover, chemical parapyruvate formation was explored in cell-free reactor experiments.

[1] Hoffart, E., Applied and environmental microbiology, 2017. 83(22): e01614-17.

[2] Pfeifer, E., et al., Applied and environmental microbiology, 2019. 85(17): e00853-19.