Cryptic Antibiotic Discovery Using Elicitor-Activated BGC Screening
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Solution Overview
Problem
Existing methods for discovering cryptic metabolites in bacteria require genetic manipulations and are slow, and do not directly link bioactivity to the metabolites, missing a significant portion of potentially bioactive molecules.
Innovation Solution
A method that involves growing bacteria in the presence of elicitors in a high-throughput format, screening the supernatant for bioactivity, and identifying cryptic metabolites with desired properties without genetic manipulations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If genetic manipulations are used to discover cryptic metabolites, then the ability to activate silent biosynthetic gene clusters is improved, but the discovery process becomes slow and complex
Solution Approach 1:
The patent extracts and removes the requirement for genetic manipulations from the cryptic metabolite discovery process. Instead of modifying the organism's genome, the method uses small molecule elicitors to activate silent BGCs in wild-type strains, thereby eliminating the time-consuming steps of genetic engineering while maintaining the ability to induce metabolite production
Solution Approach 2:
The patent introduces small molecule elicitors as intermediary substances that mediate between the researcher and the silent BGCs. These elicitors naturally activate the cryptic biosynthetic pathways without requiring direct genetic manipulation, serving as a bridge that triggers metabolite production through chemical signaling rather than genetic engineering
2Ease of operation
If traditional screening methods are used, then the process is simple, but bioactivity cannot be linked to specific cryptic metabolites before isolation
Solution Approach 1:
The patent performs preliminary bioactivity screening on the crude supernatant from elicitor-treated cultures before metabolite isolation. This allows the identification of bioactive fractions and preliminary linkage to specific metabolites early in the process, guiding subsequent purification efforts and preventing the loss of bioactivity information that would occur with traditional post-isolation screening
Solution Approach 2:
The patent segments the discovery process into distinct stages: elicitor treatment, supernatant collection, bioactivity screening, and metabolite isolation. This segmentation allows bioactivity assessment to occur at an intermediate stage, providing information about which metabolites are likely responsible for observed effects before the time-consuming isolation step
3Ease of manufacture
If wild-type organisms are screened without elicitors, then no genetic manipulation is needed, but cryptic metabolites remain undetected
Solution Approach 1:
The patent changes the chemical environment parameters by adding small molecule elicitors to the culture medium. This parameter change triggers a physiological response in the wild-type organisms, activating silent BGCs and increasing the quantity of detectable metabolites without requiring any modification to the organism's genetic makeup or complex preparation procedures
Data Source
AI summary
Bacteria harbor an immense reservoir of potentially new and therapeutic small molecules in the form of “silent” biosynthetic gene clusters. These clusters can be identified bioinformatically but are at best sparingly expressed under normal laboratory growth conditions; their products are therefore not interrogated during bioactivity screening exercises. An estimated 80-90% of biosynthetic loci are silent, meaning that routine bioactivity screens miss the majority of microbial biosynthetic potential. Disclosed herein is a method that allows access to this vast hidden metabolome, thereby allowing researchers to screen the complete metabolomes of microorganisms in the search of new therapeutic leads. The disclosed approach, “Bioactivity-HiTES”, broadly activates the secondary metabolomes of bacteria and links the cryptic metabolites produced to a desired biological activity. Using the disclosed method, induction of cryptic antibiotics was detected in all four actinomycete bacterial strains that were tested as proof-of-concept. Follow-up in two cases demonstrated the production of two new antibiotics: In one case, the taylorflavins, pyrimidine antibiotics that harbor selective growth-inhibitory activity toward Gram-negative bacteria, were identified. For example, taylorflavin B shows potent minimal inhibitory concentration toward Neisseria gonorrhoeae and Acinetobacter baumanii, but not against a panel of Gram-positive bacteria. In the second case, the lanthipeptide cebulantin, which is specific toward Gram-negative Vibrio pathogens, but does not affect the growth of Gram-positive bacteria tested, was identified. These compounds, taylorflavins and cebulantin, may serve as useful leads in the future. At the same time, Bioactivity-HiTES may be applied broadly to identify cryptic metabolites with the desired biological properties.