Demonstrating and Engineering the <i>De Novo</i> Synthesis of Two Aromatic Chemicals Estragole and Anethole in <i>Escherichia coli</i>

Estragole and anethole are the major flavoring and aroma components of the essential oils extracted from basil (Ocimum basilicum L.), anise (Pimpinella anisum), star anise (Illicium verum), and fennel (Foeniculum vulgare). They also exhibit bioactivities, such as antioxidation, antimicrobial, immunomodulation, anti-inflammation, and antidiabetes. These properties make them suitable for substantial applications in the industries of food, cosmetics, pharmaceutics, and agriculture. Commercial estragole and anethole are currently obtained from the extraction of their original plants, which suffer from unstable quality and resources. Microbial synthesis is a promising alternative approach to sustainably obtain estragole and anethole, but has not been achieved. In this study, we modularly redesigned the synthetic pathways based on Escherichia coli chassis. First, a p-coumaryl alcohol overaccumulating strain (436.28 mg/L) was engineered to facilitate the validation of the undemonstrated downstream pathway. De novo biosynthesis of estragole and anethole was achieved by stepwise analysis and the BioBricks assembly. The intracellular availability of S-adenosyl-l-methionine (SAM) was revealed to be the key limiting factor and was enhanced by deregulating the synthesis. Finally, the production of estragole and anethole was improved by 246.88 and 202.44% compared with the initial strains. This study represents the first synthesis of estragole and anethole in microbial chassis and the demonstration of microbial synthesis of allyl and propenyl phenols. Besides, it provided an alternative, sustainable approach to obtain flavoring and aroma chemicals.