Journal article
Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis
Metabolic engineering, Vol.66(C), pp.179-190
07/01/2021
DOI: 10.1016/j.ymben.2021.04.005
PMID: 33872779
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 degrees 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 degrees 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.
Details
- Title: Subtitle
- Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis
- Creators
- Hyeongmin Seo - Oak Ridge National LaboratoryJong-Won Lee - University of Tennessee at KnoxvilleRichard J. Giannone - Oak Ridge National LaboratoryNoah J. Dunlap - University of Tennessee at KnoxvilleCong T. Trinh - Oak Ridge National Laboratory
- Resource Type
- Journal article
- Publication Details
- Metabolic engineering, Vol.66(C), pp.179-190
- Publisher
- Elsevier
- DOI
- 10.1016/j.ymben.2021.04.005
- PMID
- 33872779
- ISSN
- 1096-7176
- eISSN
- 1096-7184
- Number of pages
- 12
- Language
- English
- Date published
- 07/01/2021
- Academic Unit
- Chemical and Biochemical Engineering
- Record Identifier
- 9984696723002771
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