Sequence Redesign of Glycosyltransferases for Enhanced Heterologous Expression and Glycosylation Efficiency in Escherichia coli

Heterologous expression of enzymes from higher organisms remains a major bottleneck in the construction of microbial cell factory. To address the problem, we developed a sequence redesign strategy guided by essential structural features and evolutionary conservation. This strategy integrates ProteinMPNN with several scorers to comprehensively redesign enzymes that inherently exhibit poor heterologous expression. We applied this approach to two glycosyltransferases, TOGT and UGT84A56, for esculetin glycosylation. The redesigned enzymes featured alterations in more than 20% of their sequences, resulting in significant improvements in soluble expression in Escherichia coli. Furthermore, both in vitro and in vivo assays demonstrated notable enhancements in the catalytic activity of the redesigned enzymes. Specifically, the best-performing TOGT variant exhibited a 6.4-fold increase in soluble expression and an 11.8-fold improvement in catalytic efficiency, enabling the production of cichoriin at a titer of 299 mg/L in whole-cell catalysis. Similarly, the top-performing UGT84A56 variant achieved 402 mg/L aesculin, representing a 13.9-fold improvement over the native enzyme. Fed-batch fermentation using these redesigned enzymes achieved record-high product titers of 2.11 g/L for cichoriin and 4.05 g/L for aesculin, respectively. This study demonstrates a powerful strategy for enzyme redesign, paving the way for efficient microbial cell factories to synthesize high-value natural products.