Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform

Lee, Jong‐Won; Seo, Hyeongmin; Young, Caleb; Trinh, Cong T.

doi:10.1002/bit.27926

Title: Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform

Journal Article · 25 August 2021 · Biotechnology and Bioengineering

DOI: https://doi.org/10.1002/bit.27926 · OSTI ID:1864484

Lee, Jong‐Won ^[1]; Seo, Hyeongmin ^[2]; Young, Caleb ^[3];

^[4]

Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); University of Tennessee, Knoxville
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Univ. of Tennessee, Knoxville, TN (United States)
Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

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. Here, 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.

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Research Organization:: Univ. of Tennessee, Knoxville, TN (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; SC0019412

OSTI ID:: 1864484

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 12 Vol. 118; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
AAT
CAT
Escherichia coli
alcohol acetyltransferase
chloramphenicol acetyltransferase
colorimetric assay
esters
ethyl acetate
high-throughput microbial screening
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model-guided protein design
n-butyl acetate
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Title: Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform

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