Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high‐throughput microbial screening platform
| Autor: | Caleb Young, Cong T. Trinh, Jong-Won Lee, Hyeongmin Seo |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities Protein design High selectivity Bioengineering Alcohol Saccharomyces cerevisiae Protein Engineering Saccharomyces Applied Microbiology and Biotechnology Chloramphenicol acetyltransferase chemistry.chemical_compound Biosynthesis Escherichia coli biology Isobutyl acetate Proteins Substrate (chemistry) Esters biology.organism_classification Recombinant Proteins High-Throughput Screening Assays Molecular Docking Simulation Rapid identification chemistry Biochemistry Acyltransferases Fermentation Colorimetry Butyl acetate Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 118:4655-4667 |
| ISSN: | 1097-0290 0006-3592 |
| Popis: | 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 (CATSa) 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 confirm CATSa F97W with enhanced isobutyl acetate synthesis but also identify three ATF1Sc (P348M, P348A, and P348S) variants, derived from Saccharomyces cerevisiae’s AAT and engineered by model-guided protein design, for enhanced butyl acetate production. We anticipate the high-throughput microbial screening platform is a useful tool to identify novel AATs that have important roles in nature and industrial biocatalysis for designer bioester production. |
| Databáze: | OpenAIRE |
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