TolC Is Required for a Mixed-Linkage β-Glucan (MLG) Biosynthesis: Engineering Bacteria for MLG Overproduction.

Mixed-linkage β-glucans (MLGs) are emerging as promising biopolymers with significant biotechnological potential due to their unique structural and rheological properties. In rhizobia, MLG biosynthesis is controlled by the second messenger cyclic di-GMP (c-di-GMP) and mediated by the bicistronic operon bgsBA. However, the full composition of the biosynthetic machinery and strategies for enhanced production remain incompletely understood. In this study, we demonstrate that the outer membrane protein TolC is essential for MLG production in Sinorhizobium meliloti. Genetic disruption of tolC abolished MLG synthesis, while its complementation restored production. We propose that TolC functions as part of a tripartite complex with BgsA and BgsB, enabling efficient polymer synthesis and/or export. Furthermore, co-overexpression of tolC, bgsBA, and a constitutively active diguanylate cyclase (pleD*) yielded an 8-fold increase of MLG over a control plasmid without tolC, reaching up to ~10 g/L under bioreactor conditions. Additionally, this genetic module enabled de novo MLG production in otherwise non-producer rhizobial hosts (e.g., Mesorhizobium japonicum), allowing bacterial chassis exchanges and highlighting its portability and potential for synthetic biology applications. Overall, our findings identify TolC as a key component of the MLG biosynthetic machinery and provide a robust platform for the scalable production of this valuable biopolymer.