Development of N-acetylneuraminic acid responsive biosensors based on the transcriptional regulator NanR.

Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high-throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences without the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental. In contrast, a bottom-up approach to design tailor-made biosensors was pursued here. Novel biosensors were created that respond to N-acetylneuraminic acid (Neu5Ac), an important sugar moiety with various biological functions, by employing native and engineered promoters that interact with the TF NanR. This bottom-up approach, whereby various tuned modules, e.g., the ribosome binding site (RBS) controlling NanR translation can be combined, enabled the reliable engineering of various response curve characteristics. The latter was validated by testing these biosensors in combination with various Neu5Ac-producing pathways, which allowed to produce up to 1.4 ± 0.4 g/L extracellular Neu5Ac. In this way, the repertoire of biosensors was expanded with seven novel functional Neu5Ac-responsive biosensors.