Systems-level analysis provides insights on methanol-based production of l-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus

Bacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain MGA3 of B. methanolicus naturally overproduces l-glutamate in methanol-based fed-batch fermentations. Here we generated, by directed evolution, a B. methanolicus mutant strain exhibiting enhanced l-glutamate production capability (> 150%). To showcase the potential of the evolved strain, further metabolic engineering enabled the production of γ-aminobutyric acid (GABA) directly from l-glutamate, with a yield of >13 g/L from methanol during fed-batch fermentations. By using a system level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards α-ketoglutarate and its derivative, l-glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on l-glutamate production and explained it by an upregulation of various transaminases involved in the l-glutamate biosynthesis from α-ketoglutarate. These insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of l-glutamate and its derivatives. Transcriptomic data was conducted for the following Bacillus methanolicus strains and conditions: MGA3gad (wild-type with vector pBV2xp-gadST), ABBM4307 (mutant), ABBM4307gad (mutant with vector pBV2xp-gadST)) and ABBM4307gad NI (mutant with uninduced vector pBV2xp-gadST) The processed data comprise the same transcriptome reads mapped onto the reference's chromosome (Chr_*) and native plasmids (pBM19_* and pBM69_*).