High-Throughput Screening and Directed Evolution of β-1,3-<i>N</i>-Acetylglucosaminyltransferase for Enhanced LNnT Production in Engineered <i>Saccharomyces cerevisiae</i>

Lacto-<i>N</i>-triose II (LNTII), lacto-<i>N</i>-tetraose (LNT), and lacto-<i>N</i>-neotetraose (LNnT) are key neutral human milk oligosaccharides (HMOs) with significant physiological functions. In cell factory-based synthesis, glycosyltransferases are rate-limiting enzymes, enhancing the activity of which is, therefore, crucial for efficient synthesis of HMOs. To address this issue, we developed a high-throughput screening method based on a UDP-glucose regeneration-coupled colorimetric reaction. As a case study, we applied this screening method for the directed evolution of β-1,3-<i>N</i>-acetylglucosamine transferase in <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>), resulting in a mutant strain that doubled LNnT production. Additionally, by enhancing the metabolic flux of the heterologous pathway, we further elevated the total LNnT titer to 1.29 g/L, setting a new record for LNnT production in <i>S. cerevisiae.</i> Our study demonstrates the effectiveness of the high-throughput screening method based on UDP-glucose regeneration-coupled disaccharide transferase colorimetric assay and provides a new strategy for improving the biosynthetic efficiency of HMOs that release UDP during the synthesis process.