A curcumin direct protein biosensor for cell-free prototyping.

Area of Science:

• Synthetic biology
• Biochemistry
• Molecular engineering

Background:

• Biosensors are crucial tools in synthetic biology for detecting molecules and signals.
• Naturally fluorescent protein-ligand complexes offer potential for various applications.

Purpose of the Study:

• To develop a direct protein (DiPro) biosensor utilizing the *Escherichia coli* double bond reductase (EcCurA) and its substrate curcumin.
• To optimize cell-free curcumin biosynthesis using the developed biosensor and acoustic liquid handling robotics.

Main Methods:

• A novel DiPro biosensor was created based on the fluorescent interaction between EcCurA and curcumin.
• Cell-free reactions were employed to optimize 10 reaction parameters, including cofactor, substrate, and enzyme levels.
• Acoustic liquid handling robotics were utilized for high-throughput parameter screening.

Main Results:

• The EcCurA-curcumin DiPro biosensor demonstrated a 78-fold increase in fluorescence within cell-free reactions.
• Optimization of reaction parameters significantly enhanced the efficiency of cell-free curcumin biosynthesis detection.
• The study successfully fine-tuned multiple parameters for improved biosensor performance.

Conclusions:

• The EcCurA-curcumin DiPro biosensor represents a novel, naturally fluorescent detection unit for synthetic biology applications.
• This approach facilitates the engineering of high-value chemicals and holds potential for medical imaging.
• The findings contribute to the expanding repertoire of protein-ligand complexes for biotechnological innovation.