Sustainable l-tryptophan biosynthesis by engineered Bacillus methanolicus utilizing methanol and mannitol

Abstract Background Bacillus methanolicus is a thermophilic, methylotrophic bacterium with well-established methanol-based bioproduction of value-added chemicals. It is also capable of utilizing mannitol as sole carbon source, a characteristic of relevance given that mannitol constitutes a carbohydrate fraction of waste and residual biomass. Results In this study, we evaluated B. methanolicus as a production host of L-tryptophan (Trp) by overexpressing its native trp operon ( trpEGDCFBA ) using plasmid-based systems. Strains carrying the overexpressed operon exhibited a titer of 150 mg L⁻¹ Trp upon growth on methanol, as opposed to approximately 6 mg L⁻¹ achieved by the wild-type strain. Co-expression of the anthranilate synthase-phosphoribosyl transferase complex genes trpEGD further enhanced production to 295 mg L − 1 , highlighting the enzyme complex as a dosage-sensitive control point in Trp biosynthesis in B. methanolicus . Here, the carbon yield on methanol reached by engineered strains was approximately 0.1 g g − 1 in small-scale shake flask conditions, a performance similar to that seen in traditional glucose-based fermentations. Transcriptomic analysis revealed differential expression of 91 genes under Trp overproduction, including downregulation of central carbon metabolism, stress response, and transport genes, suggesting an energy-saving reallocation of cellular resources toward Trp synthesis. Upregulation of purine and pyrimidine metabolism indicated a tight interplay between Trp biosynthesis and nucleotide metabolism. Flux balance analysis corroborated these transcriptomic data, suggesting similar metabolic reprogramming during Trp production. Operon analysis revealed two distinct transcriptional units within the trp cluster and suggested that B. methanolicus possesses T-box regulatory elements in the 5’-untranslated regions. Medium optimization using methanol and mannitol demonstrated Trp titers up to 861 mg L⁻¹ in flask-scale conditions. Moreover, a methanol fed-batch fermentation resulted in a Trp titer of 6.8 g L − 1 , representing the first demonstration of such levels in methanol-based production. Conclusions The results in this study underscore the potential of methanol, as well as mannitol, as sustainable substrates for Trp production. The titers and transcriptional insights obtained provide a foundation for future metabolic engineering strategies and the scaled-up production of Trp in B. methanolicus using next-generation feedstocks.