Tools and Strategies for Engineering Bacillus methanolicus: A Versatile Thermophilic Platform for Sustainable Bioproduction from Methanol and Alternative Feedstocks

ABSTRACT: Bacillus methanolicus MGA3 is a methylotrophic bacterium with a high potential as a production host in the bioeconomy, particularly with methanol as a feedstock. This review presents the recent acceleration in strain engineering technologies through advances in transformation efficiency, the development of CRISPR/Cas9-based genome editing, and the application of genome-scale models (GSMs) for strain design. The generation of novel genetic tools broadens the biotechnological potential of this thermophilic methylotroph. B. methanolicus is a facultative methylotroph and apart from methanol it can grow on mannitol, arabitol and glucose, and was engineered for starch and xylose utilisation. Here, the central carbon metabolism of B. methanolicus for various native and non-native carbon sources is described, with an emphasis on methanol metabolism. With its expanding product portfolio, B. methanolicus demonstrates its potential as a microbial cell factory for the production of tricarboxylic acid(TCA) cycle and ribulose monophosphate (RuMP) cycle intermediates and their derivatives. Beyond small chemicals, B. methanolicus is both a valuable source of novel thermostable proteins and a host for the production of heterologous proteins, enabled by advances in genetic tools and cultivation methods. Continued progress in understanding its physiology and refining its genetic toolbox will be decisive in transforming B. methanolicus from a promising candidate into a fully established industrial workhorse for sustainable methanol-based biomanufacturing.