Metabolic engineering of Vibrio natriegens for anaerobic succinate production

Abstract

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.

Fig. 1

Central metabolic pathways of V. natriegens. Enzymatic reactions (shown in blue boxes) are indicated as annotated in the KEGG database. Redox and energetic co‐factors of the reactions are highlighted in orange and green respectively. Prominent products of mixed acid fermentation are boxed. Colour coding: glycolysis, grey; acetate pathway, ochre; pyruvate‐derived fermentation products, red; TCA cycle, blue; glyoxylate pathway, turquoise and anaplerotic reactions, purple. Multiple enzymatic steps are indicated by a dashed line. Inactivated enzymes are framed in red. Abbreviations for metabolites: AC – acetate, AC‐CoA – acetyl‐coenzyme A, ACN – aconitate, AC‐P – acetylphosphate, aKG – α‐ketoglutarate, ALA – alanine, 1,3‐BPG – 1,3‐bisphosphoglycerate, CIT – citrate, DHAP – dihydroxyacetone phosphate, FOR – formate, FRC‐1,6‐BP – fructose‐1,6‐bisphosphate, FUM – fumarate, GLC – glucose, GLC‐6P ‐ glucose‐6‐phosphate, GLX – glyoxylate, ICT – isocitrate, LAC – lactate, MAL – malate, OAA – oxaloacetate, PEP – phosphoenolpyruvate, PYR – pyruvate, Q^ox/red – quinone/quinol, SUCC – succinate and SUCC‐CoA – succinyl‐coenzyme A; Abbreviations for enzymes: AckA – acetate kinase, Acn – aconitase, Acs – Acetyl‐CoA synthetase, aKGDHC – α‐ketoglutarate dehydrogenase complex, CS – citrate synthase, FH – fumarate hydratase, Frd – fumarate reductase, ICD – isocitrate dehydrogenase, Icl – isocitrate lyase, Mae – malic enzyme, Mdh – malate dehydrogenase, Mqo – malate:quinone oxidoreductase, MS – malate synthase, Odc – oxaloacetate decarboxylase, Pck – PEP carboxykinase, Pcx – PEP carboxylase, PDHC – pyruvate dehydrogenase complex, Pps – PEP synthase, Pta – phosphate acetyltransferase, Pts – phosphotransferase system, Pyk – pyruvate kinase, Sdh – succinate dehydrogenase and Suc – succinyl‐CoA synthetase.

Fig. 2

Relevant pathways and overall reaction equations of the different modules (regarding substrate, product, NADH equivalents and CO₂) for succinate production from glucose in V. natriegens. (A) Glucose dissimilation to pyruvate and possible NADH regeneration through acetate formation. Required reactions for succinate production from pyruvate following (B) the reductive branch, (C) the oxidative branch of the TCA cycle and (D) the glyoxylate pathway are highlighted in magenta, green and orange respectively. Abbreviations as in Fig. 1.

Fig. 3

Anaerobic succinate production in test tubes containing 50 ml VN minimal medium with 27.5 mM glucose. Succinate yields on glucose affected (A) by supplementation of 100 mM KHCO₃ to cultures that were inoculated from stationary phase and growing precultures, respectively, and (B) by metabolic engineering of V. natriegens. The red dashed line indicates the theoretical maximum succinate yield on glucose (1.71 mol_Succ mol_Glc ⁻¹). (C) Carbon balance to access the anaerobic product spectrum and unaccounted by‐products of engineered strains from (B). To avoid an overestimation of the carbon proportion, we calculated only three glucose‐derived carbon atoms for succinate. (D) Succinate production kinetics of V. natriegens Succ1 (∆lldh ∆dldh ∆pfl ∆ald ∆dns::pyc _Cg). All data represent mean values with error bars indicating the standard deviation from at least three independent biological replicates. (C) Standard deviations were calculated by error propagation.

Fig. 4

Metabolic flux distribution (A) and refined carbon balance (B) of V. natriegens Succ1 (∆lldh ∆dldh ∆pfl ∆ald ∆dns::pyc _Cg). As outlined above, we could not distinguish between carbon flux via the oxidative branch of the TCA cycle and glyoxylate pathway (A). For simplicity, only the oxTCA route is highlighted.

Fig. 5

Anaerobic zero‐growth succinate production with V. natriegens Succ1 (∆lldh ∆dldh ∆pfl ∆ald ∆dns::pyc _Cg). The bioreactor system contained initially 400 ml VN minimal medium with 555 mM glucose and 150 mM KHCO₃. 100 mM KHCO₃ h⁻¹ was continuously fed into the bioreactor. All data represent mean values with error bars indicating the standard deviation of three independent biological replicates.