Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform
Abstract
Abstract Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl‐CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high‐throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate‐based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CAT Sa ) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate‐based culturing technique with a previously established colorimetric assay, we developed a high‐throughput microbial screening platform for AATs. We demonstrated that this platform could not only probe the alcohol substrate specificity of both native and engineered AATs but also identify the beneficial mutations in engineered AATs for enhanced ester synthesis. We anticipate the high‐throughput microbial screening platform provides a useful tool to identify novel wildtype and engineered AATs that have important roles in nature and industrial biocatalysis for designer bioester production.
- Authors:
-
[1]
- Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
- Univ. of Tennessee, Knoxville, TN (United States)
- Publication Date:
- Research Org.:
- Univ. of Tennessee, Knoxville, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1864484
- Alternate Identifier(s):
- OSTI ID: 1818592
- Grant/Contract Number:
- SC0019412; AC02-05CH11231; AC05-000R22725; DE‐AC05‐000R22725; DE‐SC0019412
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biotechnology and Bioengineering
- Additional Journal Information:
- Journal Volume: 118; Journal Issue: 12; Journal ID: ISSN 0006-3592
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; high-throughput microbial screening; esters; isobutyl acetate; n-butyl acetate; ethyl acetate; 2-phenhylethyl acetate; alcohol acetyltransferase; AAT; chloramphenicol acetyltransferase; CAT; Escherichia coli; solvent overlays; colorimetric assay; model-guided protein design
Citation Formats
Lee, Jong‐Won, Seo, Hyeongmin, Young, Caleb, and Trinh, Cong T. Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform. United States: N. p., 2021.
Web. doi:10.1002/bit.27926.
Lee, Jong‐Won, Seo, Hyeongmin, Young, Caleb, & Trinh, Cong T. Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform. United States. https://doi.org/10.1002/bit.27926
Lee, Jong‐Won, Seo, Hyeongmin, Young, Caleb, and Trinh, Cong T. Thu .
"Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform". United States. https://doi.org/10.1002/bit.27926. https://www.osti.gov/servlets/purl/1864484.
@article{osti_1864484,
title = {Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform},
author = {Lee, Jong‐Won and Seo, Hyeongmin and Young, Caleb and Trinh, Cong T.},
abstractNote = {Abstract Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl‐CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high‐throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate‐based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CAT Sa ) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate‐based culturing technique with a previously established colorimetric assay, we developed a high‐throughput microbial screening platform for AATs. We demonstrated that this platform could not only probe the alcohol substrate specificity of both native and engineered AATs but also identify the beneficial mutations in engineered AATs for enhanced ester synthesis. We anticipate the high‐throughput microbial screening platform provides a useful tool to identify novel wildtype and engineered AATs that have important roles in nature and industrial biocatalysis for designer bioester production.},
doi = {10.1002/bit.27926},
journal = {Biotechnology and Bioengineering},
number = 12,
volume = 118,
place = {United States},
year = {Thu Aug 26 00:00:00 EDT 2021},
month = {Thu Aug 26 00:00:00 EDT 2021}
}
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