Systematically Engineering Escherichia coli for Efficient and Complete Hydroxytyrosol Biosynthesis.

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Hydroxytyrosol (HT) is a potent antioxidant found in olive oil with significant food and nutraceutical value.
• Current methods for HT production face challenges in sustainability and scalability.

Purpose of the Study:

• To establish an efficient and scalable de novo biosynthesis pathway for hydroxytyrosol (HT) in *Escherichia coli*.
• To overcome limitations in HT production through metabolic engineering and enzyme optimization.

Main Methods:

• Engineered *Escherichia coli* using a glucose-glycerol dual carbon source for de novo HT biosynthesis.
• Eliminated feedback inhibition and competing metabolic pathways.
• Rationally mutated the rate-limiting enzyme HpaBC (S462A/M293Y) to enhance catalytic efficiency, guided by molecular dynamics simulations.
• Optimized global cofactors and dissolved oxygen levels for increased HT production.

Main Results:

• Achieved an HT titer of 4.90 g/L in shake flasks through metabolic engineering and cofactor optimization.
• Reached a record-high HT production of 13.25 g/L in a 5 L bioreactor under optimized conditions.
• Demonstrated enhanced catalytic efficiency of the mutated HpaBC enzyme.

Conclusions:

• Developed a robust and sustainable biomanufacturing strategy for hydroxytyrosol (HT) production in *Escherichia coli*.
• The engineered microbial strain and optimized bioprocess represent a significant advancement for industrial HT synthesis.
• This work provides a scalable solution for meeting the growing demand for HT in various industries.