Adaptively evolved Methylorubrum extorquens with enhanced formate tolerance and its application in 3-hydroxypropionic...

Methylorubrum extorquens AM1, a native formate-utilizing bacterium, has exhibited limited capacity to tolerate formate. In this study, we employed an adaptive laboratory evolution (ALE) strategy to develop an evolved strain FT3 derived from M. extorquens AM1, with enhanced formate tolerance. When cultivated with a mixture of carbon sources containing 90 mM formate and 30 mM methanol, the FT3 strain exhibited 5.3 times higher optical density (OD₆₀₀) compared to the parental strain. FT3 strain was shown to efficiently utilize both methanol and formate in experiments using ¹³C-labeled carbon sources. Furthermore, the mechanism underlying the enhanced formate tolerance in FT3 strain was investigated through a combination of DNA re-sequencing, transcriptome analysis, and ALE-inspired gene manipulation experiments. The FT3 strain was identified as a hypermutant, and its enhanced formate tolerance was attributed to increased formate transport, an improved methanol oxidation pathway, and enhanced formate oxidation and assimilation pathways. In addition, gene overexpression experiments indicated the involvement of genes META1_0287*, META1_3027, META1_3028, META1_3029, META1_1261, META1_1418, and META1_2965 in formate tolerance. Notably, the addition of formate resulted in a significant improvement in the generation of NADH and NADPH in the FT3 strain. Moreover, using the FT3 strain as a chassis, an improved 3-hydroxypropionic acid (3-HP) production of 2.47 g/L through fed-batch fermentation was achieved. This study provides an important foundation for further engineering of the evolved M. extorquens strain as an efficient platform for the co-utilization of methanol and formate in the production of reduced chemicals.

In the present study, we successfully obtained an evolved strain FT3 derived from M. extorquens AM1 with high formate tolerance using the ALE strategy. The FT3 strain was identified as a hypermutant, with its enhanced formate tolerance attributed to increased formate transport, an improved methanol oxidation pathway, and enhanced formate oxidation and assimilation pathways. Through transcriptome analysis and ALE-inspired gene manipulation experiments, we identified several genes that contribute to the FT3 strain's tolerance to formate. The enhanced levels of reducing equivalents and the increased tolerance to 3-HP make FT3 a suitable chassis for 3-HP production, achieving an improved yield of 2.47 g/L through fed-batch fermentation. This study provides an important foundation for further engineering of the evolved M. extorquens strain as an efficient platform for the co-utilization of methanol and formate in the production of reduced chemicals.