Development of a Biosensor-Guided Evolution Platform for Engineering Synthetic Methylotrophic Escherichia coli Towards L-Lactate Bioproduction

Abstract

Current strategies for microbial evolution aimed at enhancing bioproduction remain limited, primarily relying on growth-coupled selection methods. These approaches often fail to identify strains with high production potential. Decoupling growth from product formation using biosensors offers a promising alternative to effectively screen for high-performing production strains. This project aims to develop a methodological standard for biosensor-guided evolutionary platform, specifically based on genetically encoded biosensors consisting of a fluorescent protein linked to a ligand-binding domain. The proof of concept is focused on engineering synthetic methylotrophic Escherichia coli strains for efficient L-lactate production from methanol. The experimental approach integrates three main modules: methanol assimilation in industrially relevant M9 medium, chromosome-based L-lactate biosynthesis, and intracellular lactate detection using single-fluorescent protein based biosensors. Key results of this work include the identification and genomic integration of an effective heterologous L-lactate dehydrogenase, resulting in stable, plasmid-free production; the functional validation of biosensors responsiveness within engineered strains; and critical insights into genetic stability, redox balance, and modular expression systems. More work remains to bring together the progress achieved in different streams – improving growth rate, producing L-lactate, tuning detection – and ultimately derive a reproducible cell factory development methodology, applicable to broader metabolite portfolios, leveraging the modularity of single fluorescent protein based biosensors. Ultimately, this work bridges biosensing, synthetic biology, and evolution engineering.