A Genetically Encoded Biosensor for the Detection of Levulinic Acid.

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Levulinic acid (LA) is a versatile platform chemical with applications in fuels, fragrances, and polymers.
• Developing efficient biosynthetic pathways for LA is crucial for sustainable chemical production.

Purpose of the Study:

• To propose biosynthetic pathways for levulinic acid (LA) production from lignin and poly(ethylene terephthalate).
• To engineer a genetically encoded biosensor for LA detection and screening of biosynthesis pathway genes.

Main Methods:

• Utilized the LvaR transcriptional regulator from *Pseudomonas putida* KT2440 to create an LA-responsive fluorescent biosensor.
• Tested the biosensor in *Escherichia coli* and *P. putida* KT2440, optimizing its function in the latter.
• Determined the linear correlation range (0.156-10 mM) and detection limit (0.156 mM) of the biosensor in *P. putida*.

Main Results:

• The LA biosensor was non-functional in *E. coli* but functional in *P. putida* KT2440.
• Demonstrated a linear relationship between fluorescence intensity and LA concentration (0.156-10 mM) in *P. putida*.
• Achieved a 12.3-fold maximal fluorescence increase in the presence of 10 mM LA, with a detection limit of 0.156 mM.

Conclusions:

• The developed LA biosensor in *P. putida* KT2440 enables effective high-throughput screening for LA biosynthesis pathway engineering.
• The biosensor facilitates the evolution of enzymes and metabolic pathways for sustainable LA production in engineered microbes.