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

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.

Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars

“…While the coculture strategy substantially improved titers and selectivity of isobutyl butyrate through the pathway and resource compartmentalization which is generally applicable to other designer ester biosynthesis, further improvement in catalytic efficiency and substrate specificity of AATs by protein engineering is needed for an efficient and scalable production of designer bioesters. This future research is highly feasible based on the recent advances in the model-guided protein engineering of AATs , and high-throughput screening method …”

“…This future research is highly feasible based on the recent advances in the model-guided protein engineering of AATs 38,42 and high-throughput screening method. 11 The compartmentalization strategy to engineer the complex diverging−converging metabolic pathways can offer practical benefits for rapid identification of pathway bottlenecks and optimization of pathways and strains for designer ester biosynthesis. However, careful analysis must be taken upon implementation of the compartmentalization strategy.…”

“…Microbial biosynthesis of esters uses an alcohol acyltransferase (AAT) that can perform a thermodynamically favorable condensation reaction of an acyl-CoA and an alcohol in an aqueous phase . The de novo ester biosynthetic pathways for converting fermentable sugars to esters can be modularized into submodules, including the sugar-utilizing submodules, acyl-CoA production submodule, alcohol production submodule, and/or ester synthesis submodule (AAT). ,, Harnessing the modular ester biosynthetic pathways enables combinatorial biosynthesis of a large number of unique esters. ,,− However, due to the complex nature of diverging–converging ester biosynthetic pathways, it requires optimal metabolic flux balance and control to achieve selective ester biosynthesis. , …”

“…6,7,8−10 However, due to the complex nature of diverging−converging ester biosynthetic pathways, it requires optimal metabolic flux balance and control to achieve selective ester biosynthesis. 6,11 This article is licensed under CC-BY-NC-ND 4 Isobutyl butyrate is a hydrophobic and sweet-scented medium carbon chain length (C 8 ) ester that is naturally found in flowers. 12 A de novo isobutyl butyrate biosynthetic pathway is derived from the converging sugar-utilizing pathway submodules to generate pyruvate, then the isobutanol and butyryl-CoA pathway submodules that diverge from pyruvate to make the precursors isobutanol and butyryl-CoA, and finally the ester biosynthesis submodule that converges to synthesize isobutyl butyrate by the AAT-dependent condensation of isobutanol and butyryl-CoA 7 (Figure 1A).…”

The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters

“…Using the same method, Lee et al developed a high throughput screening approach for alcohol acyltransferases (AATs) identification. This platform could investigate the alcohol substrate specificity of AATs and recognize the beneficial mutations in the engineered AATs for enhanced ester synthesis ( Lee et al, 2021 ). Lin et al developed a novel assay method for AATase activity.…”