Adaptive laboratory evolution of Methylorubrum extorquens for lanthanide-dependent robust growth under high concentrations of methanol.

Methylorubrum extorquens AM1 has been extensively studied as a platform for biomanufacturing using a methanol feedstock. Given that lanthanide (Ln) ions are recognized as important metals related to methylotrophy of methylotrophic bacteria, this study applied adaptive laboratory evolution to M. extorquens under Ln-supplemented conditions to obtain strains with robust growth at high concentrations of methanol. The cultivation results indicated a possible Ln-dependent mechanism for methanol tolerance in this methylotroph. The parent strain was unable to grow at 5% (v/v) methanol under Ln-free conditions, but exhibited slight yet definite growth with La3+. Repeated passaging under high methanol concentrations resulted in isolation of evolved strains that acquired additional Ln-independent methanol tolerance, thereby could grow at high concentrations of methanol up to 7% with La3+. The identification of the mutations by whole-genome resequencing and reconstitution of the identified mutations in the parent strain suggested an association between impaired function of proton-transport protein and high methanol tolerance of M. extorquens. At 5% methanol, the evolved strain exhibited a cell yield comparable to that of the parent strain at 0.5% methanol, while its PHA content was approximately 2.5-fold higher, resulting in enhanced production of PHA from methanol. No increase in PHA production was observed between the parent and the evolved strain at 0.5% methanol, indicating that the acquired ability to grow at high methanol concentrations contributed to the enhanced PHA production. This study demonstrates the importance of Ln for methanol tolerance of M. extorquens and the advantage of high-methanol conditions for bioproduction using methylotrophs.