Elsevier

Metabolic Engineering

Volume 66, July 2021, Pages 179-190
Metabolic Engineering

Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis

https://doi.org/10.1016/j.ymben.2021.04.005Get rights and content
Under a Creative Commons license
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Highlights

  • Developed a highly thermostable and promiscuous ester biosynthesis platform.

  • Engineered promiscuity of chloramphenicol acetyltransferases (CATs).

  • Demonstrated efficiency of engineered CATs towards various alcohols and acyl-CoAs.

  • Demonstrated whole-cell bioconversion of various alcohols into designer esters.

  • CAT thermostability is critical for efficient ester production in Clostridium thermocellum.

  • Engineered C. thermocellum produced up to 1 g/L of isobutyl esters from cellulose.

Abstract

Robust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as interchangeable metabolic modules for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and engineered promiscuity of chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacterium Escherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinant E. coli was also capable of simulating the ester profile of roses with high conversion (>97%) and titer (>1 g/L) from fermentable sugars at 37 °C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacterium Clostridium thermocellum harboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50 °C) due to the engineered enzyme's remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.

Keywords

Chloramphenicol acetyltransferase
Alcohol acyltransferase
Ester biosynthesis
Enzyme thermostability
Escherichia coli
Clostridium thermocellum

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