Synthetic biosensor of natural products - design, construction and application

• Applicant: Professor Dr. Andriy Luzhetskyy
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Microbial Ecology and Applied Microbiology
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 326726704

Actinobacteria are Gram-positive bacteria producing a large number of compounds (secondary metabolites) used in medicine and agriculture. Typically their production level is far from being sufficient for biological activity testing and other applications. Moreover, many genes for secondary metabolite production are silent under laboratory conditions. The optimization of producing strains and awakening the cryptic pathways require availability of simple high-throughput screening procedures allowing identification of clones with the desired features. For small molecule production the most promising solution of the screening problem is the use of the biosensor technique. Typical biosensors are built from a regulatory protein (signal input) controlling transcription of a reporter gene (signal output) in response to the presence of a certain ligand. Such constructs specific to mevalonate, succinate and amino acids were successfully used for the optimization of E. coli and Corynebacterium strains.With the project proposed here we will establish the basic design rules for the construction of secondary metabolite sensors in actinobacteria based on regulatory proteins. Pamamycins are a group of closely related polyketides produced by Streptomyces alboniger DSM40043 with remarkable activity against multi-drug resistant Mycobacterium tuberculosis. The export of pamamycins in S. alboniger is tightly controlled by TetR-like transcriptional regulator PamR2 in response to accumulation of the compound. We successfully used PamR2 to construct the prototype of an in vivo pamamycin detection system. Subsequently we determined the atomic resolution structure of PamR2 in the presence and absence of pamamycin 607. This structural information will be used to significantly improve our working prototype, which suffered from high read-out noise and broad specificity to different pamamycins. We accomplish this goal by employing two complimentary approaches: (1) We will tune the pamamycin binding pocket of PamR2 (signal input part of the sensor) by rational protein engineering in order to shift its specificity towards particular pamamycins. (2) In order to tune the signal output part of the pamamycin biosensor PamR2, operator sequences will be identified and combined with the synthetic actinobacterial promoters. In the second stage of the proposed work we will use the newly developed pamamycin sensors as a platform to construct artificial biological sensors with new specificities to other secondary metabolites.

• DFG Programme: Research Grants