Adaptive laboratory evolution of Cupriavidus necator to sodium salts as a whole cell engineering strategy for enhanced trehalose production

Trehalose is an important compatible solute with diverse applications in the pharmaceutical, food, and cosmetic industries. In Cupriavidus necator, trehalose biosynthesis is naturally associated with osmotic stress, but typically retained intracellularly as an osmo-protectant. In this study, adaptive laboratory evolution (ALE) was used as a whole cell engineering strategy to adapt C. necator H16 to increasing osmotic stress. ALE was performed with independent lineages and gradually increasing concentrations of NaCl and Na₂SO₄, reaching stable growth at up to 500 mM NaCl or 400 mM Na₂SO₄. In selected single colonies of the adapted cultures, the SetA sugar efflux transporter from Escherichia coli was expressed to enable trehalose secretion. Moderate concentrations of Na₂SO₄ were identified as most effective osmotic trigger, balancing growth and trehalose production. Under the selected production conditions the best-performing adapted isolate produced 11.5 g/L trehalose from 20 g/L fructose, corresponding to 60.5% of the theoretical maximum yield, compared to 7.4 g/L and 42.1% in the parental strain. Genome sequencing revealed mutations affecting transporters, membrane structure, metabolism and regulators. These mutations are considered contributors to the observed osmoadaptive phenotype, while individual effects on trehalose production were not resolved. Overall, the results demonstrate the potential of ALE as a whole cell engineering strategy for generating C. necator isolates with improved extracellular trehalose production under defined conditions. Cupriavidus necator H16 was adapted to increasing NaCl and Na2SO4 concentrations by ALE with final concentrations of 500 mM NaCl and 400 mM Na2SO4. ALE was used as a whole cell engineering strategy with subsequent selection for osmotic-stress resistance. In the adapted isolate, the trehalose exporter SetA was expressed using a plasmid. Moderate Na2SO4 concentrations of 150 mM provided an effective osmotic trigger for enhanced trehalose production. The selected NaCl-adapted isolate produced 11.5 g/L trehalose from 20 g/L fructose. Genome sequencing revealed mutations in genes potentially associated with osmoadaptation.