Massively parallel barcode sequencing revealed the interchangeability of capsule transporters in .

Multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) family transporters are essential in glycan synthesis, flipping lipid-linked precursors across cell membranes. Yet, how they select their substrates remains enigmatic. Here, we investigate the substrate specificity of the MOP transporters in the capsular polysaccharide (CPS) synthesis pathway in . These capsule flippases collectively transport more than 100 types of capsule precursors. To determine whether they can substitute for one another, we developed a high-throughput approach to systematically examine nearly 6000 combinations of flippases and substrates. CPS flippases fall into three groups: relaxed, type-specific, and strictly specific. Cargo size and CPS acetylation affect transport, and we isolated additional gain-of-function flippase variants that can substitute for the peptidoglycan flippase YtgP (MurJ). We also showed that combining flippase variants in a single cassette allows various CPS precursors to be flipped, which may aid glycoengineering. This study reveals that MOP flippases exhibit broad specificity, shaping the evolution of glycan synthesis.