Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophicbacteria like Methylobacterium extorquensAM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanollimits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanolvia adaptive directed evolutionusing the GM3 technology of automated continuous culture. Turbidostatand conditional medium swap regimes were employed for the parallel evolutionof the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanolthan the ancestor strains. Genome sequencing identified the gene metYcoding for an O-acetyl- L-homoserine-sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanolas substrate at elevated concentrations. This side reaction would produce methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metYalleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl- L-homoserinesulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanolexposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanollike formate dehydrogenasesand ATP synthaseswas boosted in the evolved cells upon a short-term methanolstress. D-lactate production from methanolby adapted cells overexpressing the native D-lactate dehydrogenasewas quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanolto assimilate this one carbon feedstock more efficiently.