Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis

• Published in: Journal of agricultural and food chemistry 2024-07, Vol.72 (27), p.15293-15300
• Main Authors: Liu, Wen-Kai, Su, Bing-Mei, Xu, Xin-Qi, Xu, Lian, Lin, Juan
• Format: Article
• Language: English
• Subjects: Biotechnology and Biological Transformations
• ISSN: 0021-8561; 1520-5118; 1520-5118
• DOI: 10.1021/acs.jafc.4c04228

Hydroxytyrosol, a naturally occurring compound with antioxidant and antiviral activity, is widely applied in the cosmetic, food, and nutraceutical industries. The development of a biocatalytic approach for producing hydroxytyrosol from simple and readily accessible substrates remains a challenge. Here, we designed and implemented an effective biocatalytic cascade to obtain hydroxytyrosol from 3,4-dihydroxybenzaldehyde and l-threonine via a four-step enzymatic cascade composed of seven enzymes. To prevent cross-reactions and protein expression burden caused by multiple enzymes expressed in a single cell, the designed enzymatic cascade was divided into two modules and catalyzed in a stepwise manner. The first module (FM) assisted the assembly of 3,4-dihydroxybenzaldehyde and l-threonine into (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and the second module (SM) entailed converting (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid into hydroxytyrosol. Each module was cloned into Escherichia coli BL21 (DE3) and engineered in parallel by fine-tuning enzyme expression, resulting in two engineered whole-cell catalyst modules, BL21­(FM01) and BL21­(SM13), capable of converting 30 mM 3,4-dihydroxybenzaldehyde to 28.7 mM hydroxytyrosol with a high space–time yield (0.88 g/L/h). To summarize, the current study proposes a simple and effective approach for biosynthesizing hydroxytyrosol from low-cost substrates and thus has great potential for industrial applications.