Construction and Optimization of a BgaR-Based Lactose Biosensor for High-Throughput Selection of Human Milk Oligosaccharide Producers

Biosensors are crucial for high-throughput screening (HTS) of metabolite overproducers, but human milk oligosaccharide (HMO) biosensors are rarely reported. The concentrations of HMOs were evaluated by detecting the residual lactose, since lactose served as a universal substrate. In this study, a lactose biosensor was developed using the lactose-sensing transcription factor BgaR, correlating lactose concentration with strain growth. The biosensor was optimized, expanding the detection and dynamic ranges from 5-10 g/L and 1.47-fold to 0-12 g/L and 5.19-fold, respectively. To convert growth coupling with lactose to that with HMOs, LacI was introduced. LacI expression was controlled by BgaR and fine-tuned to optimize the biosensor. Lacto-<i>N</i>-triose II and 2'-fucosyllactose (2'-FL) overproducers were obtained through HTS. A <i>secY</i>^T1124C mutation was identified as a new mechanism for high production of 2'-FL, which reached 78.21 ± 2.10 g/L. This study provided an approach to construct and optimize universal biosensors for HMOs and is beneficial for developing HMO overproducers.