Maurice Hädrich (Straubing / DE), Clarissa Schulze (Straubing / DE), Felix Thoma (Straubing / DE), Bastian Blombach (Straubing / DE)
Most industrial production processes must operate at a high final biomass concentration to reach a high volumetric productivity (QP), 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 (qS) [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 cx,max of 6.6 ± 0.4 g L-1. Moreover, the qs of the non-growing cells increased to 3.5 g glucose g-1CDW h-1, close to the qS 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.