Adaptive Laboratory Evolution of Halomonas bluephagenesis Enhances Acetate Tolerance and Utilization to Produce Poly(3-hydroxybutyrate)

Acetate is a promising economical and sustainable carbon source for bioproduction, but it is also a known cell-growth inhibitor. In this study, adaptive laboratory evolution (ALE) with acetate as selective pressure was applied to Halomonas bluephagenesis TD1.0, a fast-growing and contamination-resistant halophilic bacterium that naturally accumulates poly(3-hydroxybutyrate) (PHB). After 71 transfers, the evolved strain, B71, was isolated, which not only showed better fitness (in terms of tolerance and utilization rate) to high concentrations of acetate but also produced a higher PHB titer compared with the parental strain TD1.0. Subsequently, overexpression of acetyl-CoA synthetase (ACS) in B71 resulted in a further increase in acetate utilization but a decrease in PHB production. Through whole-genome resequencing, it was speculated that genetic mutations (single-nucleotide variation (SNV) in phaB, mdh, and the upstream of OmpA, and insertion of TolA) in B71 might contribute to its improved acetate adaptability and PHB production. Finally, in a 5 L bioreactor with intermittent feeding of acetic acid, B71 was able to produce 49.79 g/L PHB and 70.01 g/L dry cell mass, which were 147.2% and 82.32% higher than those of TD1.0, respectively. These results highlight that ALE provides a reliable method to harness H. bluephagenesis to metabolize acetate for the production of PHB or other high-value chemicals more efficiently.

Automated summary

In this study, adaptive laboratory evolution (ALE) was used to improve the acetate adaptability and poly(3-hydroxybutyrate) (PHB) production of Halomonas bluephagenesis. The evolved strain B71 showed better fitness to high concentrations of acetate and produced a higher PHB titer compared to the parental strain. Genetic mutations were identified as possible contributors to the improved acetate adaptability and PHB production. The results highlight the efficient utilization of H. bluephagenesis for the production of PHB or other high-value chemicals using ALE.