Periplasmic Engineering Enhances Terminal Hydroxylation for Efficient Caffeic Acid Biosynthesis in Escherichia coli

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Caffeic acid (CA) is a valuable phenylpropanoid with diverse industrial applications.
• Microbial CA production is often hindered by the low efficiency of the terminal hydroxylation step catalyzed by 4-hydroxyphenylacetate 3-monooxygenase (HpaBC).

Purpose of the Study:

• To develop a de novo biosynthetic pathway for CA in Escherichia coli.
• To enhance CA production through metabolic and spatial engineering strategies.

Main Methods:

• Constructed a de novo CA biosynthetic pathway in E. coli.
• Optimized l-tyrosine supply and HpaBC expression.
• Engineered HpaBC periplasmic localization using the Tat pathway.
• Performed periplasmic remodeling and fermentation optimization.
• Conducted fed-batch fermentation in a bioreactor.

Main Results:

• Initial optimization increased CA production to 61.0 mg/L.
• Periplasmic relocation of HpaBC resulted in a 4.9-fold increase in CA production to 299.1 mg/L, with reduced byproducts.
• Shake-flask production reached 463.7 mg/L after further engineering and optimization.
• Fed-batch fermentation achieved a final CA concentration of 5.1 g/L in a 3 L bioreactor.

Conclusions:

• Periplasmic engineering is a highly effective strategy for enhancing oxidation-dependent phenylpropanoid biosynthesis.
• This study presents a robust microbial platform for high-yield caffeic acid production.