Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens

Abstract

Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed-batch processes are applied to obtain high space-time yields. In this study, we established an aerobic glucose-limited fed-batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 g_X L^-1 . However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L^-1 . Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two-fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pyc_Cg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five- to six-fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L^-1 with a volumetric productivity of 8.5 and 7.7 g_Suc L^-1 h^-1 , respectively. Both strains showed a product yield of about 1.4 mol_Suc mol_Glc ^-1 , which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.

FIGURE 1

Glucose‐limited fed‐batch process with Vibrio natriegens wt. The courses of cell dry weight (hollow circle) and dissolved oxygen (dark blue line) are shown in (A). The concentrations of acetate (circles), pyruvate (squares), and viscosity (green line) measured at a shear rate of 0.1 s⁻¹ are depicted in (B). Results shown are means and standard deviations (error bars for cell dry weight and metabolites; grey line for dissolved oxygen) of three independent experiments. The arrow indicates a deviation from the growth rate μ_set.

FIGURE 2

Genetic regions and genes potentially related to polysaccharide biosynthesis in Vibrio natriegens. Genes are represented by arrows in scale. Positions are ordered by their locations on the chromosomes and labelled with their respective gene identifiers. A detailed overview of the gene names and the corresponding locus tags is provided in Tables S2 and S3. Similarities of the syp and cps regions to published clusters of V. fischeri (VF_A1020‐VF_A1037) (Yip et al., 2005) and V. parahaemolyticus (VPBB_A1276‐VPBB_A1286) (Güvener & McCarter, 2003) are shown by matching colours for genes displaying over 60% sequence identity by BLASTN (Tables S2 and S3).

FIGURE 3

Glucose‐limited fed‐batch processes with Vibrio natriegens wt (black), V. natriegens ΔcpsR (orange), V. natriegens ΔwbfF (dark blue), V. natriegens Δcps (light blue), and V. natriegens Δsyp Δcps (green). The courses of cell dry weight (hollow circle) and glucose concentration (cross) are shown in (A). In (B), the oxygen transfer rate (bar) over time is depicted. The course of the viscosity (diamond) measured at a shear rate of 0.1 s⁻¹ is shown in (C) and the EPS concentrations are pictured in (D). Results given are means and standard deviations of three independent biological replicates.

FIGURE 4

Product yield (dark blue) and final viscosity (orange) of an anaerobic resting cell approach for succinate production. Fermentations were performed with Vibrio natriegens Succ1, Succ1 ΔcpsR and Succ1 Δcps (n = 1). The black dashed line represents the maximum theoretical yield on glucose (1.71 mol_Suc mol_Glc ⁻¹). The viscosity was measured as a technical triplicate at a shear rate of 0.1 s⁻¹.