A microbial platform for selective biosynthesis of monoterpene esters

Area of Science:

• Biotechnology and Synthetic Biology
• Metabolic Engineering
• Natural Product Biosynthesis

Background:

• Synthesizing complex natural esters like monoterpene esters in microbes is challenging due to limited enzyme selectivity and pathway incompatibility.
• Existing methods struggle with the precise reconstruction of diverse monoterpene ester biosynthetic pathways.

Purpose of the Study:

• To establish a dual-substrate microbial platform for profiling alcohol acyltransferase (AAT) activity.
• To enable the synthesis of three distinct classes of monoterpene esters: monoterpenyl esters, monoterpenoate esters, and monoterpenyl monoterpenoate esters.
• To provide access to both natural and non-natural monoterpene ester biosynthetic pathways.

Main Methods:

• Development of a dual-substrate microbial platform to profile alcohol acyltransferase (AAT) activities.
• Structure-guided engineering of AATs and tuning of dual-substrate molar ratios for selective ester biosynthesis.
• Coculture engineering to optimize metabolic flux towards acyl-CoA and alcohol precursors.

Main Results:

• Achieved selective biosynthesis of >C2 acyl-CoA-derived monoterpene esters, overcoming competition from endogenous acetyl-CoA.
• Demonstrated the synthesis of monoterpenyl esters, monoterpenoate esters, and monoterpenyl monoterpenoate esters.
• Obtained high yields of 11.50 g/L linalyl acetate and 3.16 g/L geranyl butyrate in a 1-L bioreactor through coculture engineering.

Conclusions:

• Expanded the biosynthetic potential for monoterpene esters using engineered microbes.
• Provided a versatile strategy for controlling AAT selectivity in ester biosynthesis.
• Established a plug-and-play, scalable framework for industrial ester biomanufacturing.