A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• Metabolic flux redirection is key for improving microbial production yields.
• Identifying target genes for repression that enhance yield without impacting cell growth remains challenging.
• Existing methods lack dynamic control and scalability for complex metabolic pathways.

Purpose of the Study:

• To develop a novel strategy for dynamic, cell-density-dependent gene repression in metabolic engineering.
• To enable precise control over metabolic fluxes to optimize the production of valuable compounds.
• To demonstrate the broad applicability of this system across different target molecules.

Main Methods:

• Implementation of a quorum-sensing system to control small RNA transcription for gene repression.
• Characterization of repression dynamics using parameters like AHL concentrations (A_i, A_m, R_A).
• Identification of LuxR_I58N as a tuning factor and dynamic overexpression of the Hfq chaperone for combinatorial repression.

Main Results:

• The developed strategy allows convenient, dynamic, and simultaneous regulation of multiple target genes.
• LuxR_I58N variant and Hfq chaperone dynamic overexpression enable effective combinatorial repression without growth impairment.
• Significant improvements in production titers: 365.3% for pinene, 79.5% for pentalenene, and 302.9% for psilocybin.

Conclusions:

• The quorum-sensing-based small RNA system offers a robust platform for dynamic metabolic regulation.
• This approach overcomes limitations of static gene repression, enabling enhanced microbial production.
• The strategy demonstrates broad applicability and significant yield improvements for diverse bioproducts.