Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• P-coumaric acid (p-CA) is a plant metabolite with antioxidant and anti-inflammatory properties, widely used in biomedicine, food, and cosmetics.
• Existing methods for p-CA production face limitations due to enzyme expression and activity bottlenecks.

Purpose of the Study:

• To enhance the production of p-coumaric acid (p-CA) in an engineered Escherichia coli strain.
• To overcome the limitations of low soluble expression and activity of the AtC4H enzyme in the p-CA synthetic pathway.
• To increase intracellular NADPH levels for improved p-CA biosynthesis.

Main Methods:

• Designed a synthetic pathway (PAL) integrating AtPAL2, AtC4H, and AtATR2 enzymes into Escherichia coli PHE05.
• Enhanced soluble expression and activity of AtC4H through N-terminal modifications, developing an optimal mutant AtC4H^L373T/G211H.
• Employed metabolic engineering strategies, including ppnk overexpression, to increase intracellular NADPH pool and the NADPH/NADP⁺ ratio.

Main Results:

• Developed an optimal AtC4H mutant with a 4.3-fold higher k_cat/K_m value.
• Overexpression of ppnk in engineered E. coli PHCA20 resulted in a 13.9-fold increase in NADPH content and a 1.3-fold increase in the NADPH/NADP⁺ ratio.
• Achieved a 29.1% increase in p-CA titer, reaching 3.09 g/L with a yield of 20.01 mg/g glucose and productivity of 49.05 mg/L/h in a 5-L fermenter.

Conclusions:

• N-terminal modification of AtC4H and metabolic engineering to boost NADPH are effective strategies for enhancing p-CA production.
• The engineered E. coli strain PHCA20 demonstrates significant potential for efficient industrial-scale production of p-coumaric acid.
• This study presents a novel approach for the efficient biosynthesis of plant metabolites using industrial microbial strains.