Decoding metabolic trade-offs in Halomonas elongata: Chemostat-based flux remodeling for industrial ectoine biosynthesis

Abstract

Halomonas elongata, a moderately halophilic γ-proteobacterium of industrial interest, serves as a microbial cell factory for ectoine-a high-value compatible solute extensively utilized in biopharmaceuticals and cosmetics. While its ectoine biosynthesis potential is well-documented, the systemic metabolic adaptations underlying osmoadaptation remain poorly characterized, limiting rational engineering strategies for optimized production. To address this gap, we employed chemostat cultivation coupled with multi-omics integration (physiological profiling, metabolomics, and metabolic flux analysis) to dissect salt-dependent metabolic network rewiring in the model strain DSM 2581^T under moderate (6.0 % NaCl) and high salinity (13.0 % NaCl). Results demonstrated that, under moderate salt conditions, a specific growth rate (μ) of 0.20 h^-1 significantly enhanced the ectoine-specific production rate (q _p), intracellular ectoine content (p _ectoine), and yield coefficient (Y _p/s), concurrent with redirection of carbon flux toward the Entner-Doudoroff (ED) pathway and ectoine biosynthesis. Under high salt conditions, flux through both the ED pathway and ectoine biosynthesis was further upregulated, whereas fluxes through the pentose phosphate (PP) pathway, tricarboxylic acid (TCA) cycle, and CO₂ generation were downregulated. Simultaneously, suppression of the flux from malate to pyruvate enhanced oxaloacetate synthesis, thereby increasing the supply of key precursors including glutamate, aspartate, and NADPH to fuel ectoine biosynthesis. Stepwise salt reduction experiments revealed bidirectional metabolic flexibility: elevated salinity prioritized carbon investment into ED-driven ectoine production, whereas hypo-osmotic conditions reactivate respiratory activity and the TCA cycle to fuel energy metabolism. These findings establish H. elongata as a paradigm of dynamic flux rewiring, where carbon economy is strategically reallocated between stress-protective solute biosynthesis and energy homeostasis. This study bridges the knowledge gap in understanding the physiological characteristics of H. elongata and provides a foundation for improving ectoine production and engineering strains through metabolic optimization.

Keywords: Chemostat culture; Ectoine biosynthesis; Halomonas elongata; Metabolic flux analysis; Salt adaptation.

Fig. 1

Physiological parameters of H. elongata grown under different dilution conditions at 6.0 % salt concentration. (A) C_x, biomass. (B) C_p, ectoine content. (C) C_s, residual sugar concentration. (D) q_co2, specific carbon dioxide consumption rate. (E) q_p, specific product formation rate. (F) q_s, specific substrate consumption rate; (G) Y_x/s, biomass yield on substrate; (H) p_ectoine, the content of ectoine per unit biomass; (I) Y_co2/S, carbon dioxide yield on glucose.

Fig. 2

The chemostat cultivation kinetics of H. elongata. (A) and (E), linear relationship between q_p and μ; (B) and (F), linear relationship between 1/μ and 1/C_s; (C) and (G), linear relationship between q_s and μ; (D) and (H), linear relationship between q_o2 and μ. Here, (A–D) and (D–H) correspond to the data under 6 % and 13 % salt concentration conditions, respectively.

Fig. 3

The relative flux changes within H. elongata grown under the μ of 0.10, 0.20, and 0.30 h⁻¹. The negative values in the figure represent that the directionality of the net flux was the contrary to that in the figure. The flux from metabolites to biomass is not indicated.

Fig. 4

(A) Heatmaps of intracellular metabolic flux in H. elongata grown under 13 % and 6 % salt concentrations. (B) Comparison of metabolic flux magnitudes between 13 % and 6 % salt concentrations. Red font indicates reactions with higher flux under 13 % salt conditions compared to 6 % salt conditions, while green font indicates the opposite.

Fig. 5

Intracellular organic acids and phosphoric sugar substances under different dilution conditions at 6 % NaCl. The vertical axis of each graph represents μmol/g DCW, and the horizontal axis represents the corresponding dilution rate.

Fig. 6

Changes in macroscopic physiological metabolism and intracellular fluxes of H. elongata during dynamic salt concentration reduction. (A) Variations in macroscopic physiological parameters of H. elongata. (B–I) Corresponding to: C_x, biomass; C_ectoine, ectoine content; p_ectoine, the content of ectoine per unit biomass; q_p, specific product formation rate; q_co2, specific carbon dioxide consumption rate; q_s, specific substrate consumption rate; Y_co2/S, carbon dioxide yield on glucose; Y_x/s, biomass yield on substrate. (J) Heatmap of intracellular metabolic flux changes, where color coding corresponds to the associated metabolic pathways.