OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG

Area of Science:

• Synthetic biology
• Optogenetics
• Molecular engineering

Background:

• Optogenetics offers tunable, reversible, and low-toxicity control of biological processes.
• The lactose operon system in E. coli is regulated by the lactose repressor protein (LacI) and chemical inducers like IPTG.
• Existing induction systems lack the fine-tuned control offered by light-based methods.

Purpose of the Study:

• To engineer light-responsive variants of the LacI repressor for optogenetic control of gene expression in E. coli.
• To develop novel light-controlled gene expression systems (OptoE.coliLight and OptoE.coliDark) that eliminate the need for chemical inducers.
• To demonstrate the efficacy of these systems in protein production and metabolic flux control.

Main Methods:

• Harnessing the light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1.
• Engineering LacI into light-responsive variants (OptoLacIL and OptoLacID).
• Constructing and applying OptoE.coliLight and OptoE.coliDark systems for gene expression regulation.

Main Results:

• OptoLacIL and OptoLacID variants respond directly to light and darkness, replacing IPTG.
• Developed bifunctional gene expression systems (OptoE.coliLight and OptoE.coliDark) with superior controllability.
• Dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded IPTG-induced levels by over 110% and 60%, respectively.

Conclusions:

• OptoLacI variants provide a novel platform for light-based control of gene expression in E. coli.
• The developed optogenetic systems offer enhanced control and efficiency for protein production and metabolic engineering.
• This advancement holds significant potential for optogenetic protein engineering and diverse biotechnological applications.