Structurally Tunable Cell-Free System: to Expand the Synthetic Biology Toolkit for Studies on Extracellular...

Cell-free systems not only offer an efficient strategy for producing proteins that are hard to express but also serve as an innovative platform for studying biosynthetic modules of complex microbial metabolites and reconstructing metabolic pathways in vitro. As microbial extracellular polysaccharides (EPS) hold significant industrial potential, the biosynthetic pathway is highly complex, involving numerous key enzymes and membrane-associated biochemical reactions that are challenging to analyze. In this research, a flexible cell-free (membrane) protein synthesis system (CF(M)PS) was developed and applied to glycobiology associated with membrane proteins (MPs) by incorporating nanoliposomes as membrane mimics to facilitate in vitro transcription-translation coupling and promote MPs solubilization. The membrane-bound phosphoglycosyl transferase from Bacillus licheniformis CGMCC 2876, which initiates the assembly of sugar repeat units, was successfully expressed with CF(M)PS, and subsequent catalytic reaction was coupled to produce glycoconjugate. This initiation of EPS synthesis in a cell-free platform also facilitated the identification of targets for component-oriented regulation of extracellular polymers. Furthermore, CF(M)PS enabled efficient Levan biosynthesis, revealing the application potential of a newly identified levansucrase. The successful application of CF(M)PS provides an efficient and versatile platform for glycans synthesis. CF(M)PS expands the synthetic glycobiology toolbox and is expected to accelerate the study of complex metabolites.