Engineering of a Biosensor in Response to Malate in Bacillus licheniformis .

Malate is an essential intermediate in the tricarboxylic acid (TCA) cycle; it also has valuable uses in medicine and food. The production of malate with a microbial synthesis method is still in its early stages. One of the key problems in metabolic engineering is that the dynamic and subtle changes in malate are difficult to detect. It remains critical to develop techniques with direct and precise detection of malate in microbial metabolism, which facilitates high-throughput screening of the engineered strains. In this study, a genetically encoded biosensor in response to malate was constructed in B. licheniformis. Key regulator MalR and the action site of the biosensor were first identified. Then, the output of the reporter gene expression was amplified by introducing a strong constitutive promoter and iteratively tuning the action sites. The engineered biosensor can respond to malate from 5 to 15 g/L; within this range, it shows a linear correlation between eGFP fluorescence and malate concentration. This biosensor enrich our toolbox of synthetic biology in pathway engineering for malate production in microorganisms.