EngineeringEscherichia colifor the production of butyl octanoate from endogenous octanoyl-CoA

Abstract

Medium chain esters produced from fruits and flowering plants have a number of commercial applications including use as flavour and fragrance ingredients, biofuels, and in pharmaceutical formulations. These esters are typically made via the activity of an alcohol acyl transferase (AAT) enzyme which catalyses the condensation of an alcohol and an acyl-CoA. Developing a microbial platform for medium chain ester production using AAT activity presents several obstacles, including the low product specificity of these enzymes for the desired ester and/or low endogenous substrate availability. In this study, we engineered Escherichia coli for the production of butyl octanoate from endogenously produced octanoyl-CoA. This was achieved through rational protein engineering of an AAT enzyme from Actinidia chinensis for improved octanoyl-CoA substrate specificity and metabolic engineering of E. coli fatty acid metabolism for increased endogenous octanoyl-CoA availability. This resulted in accumulation of 3.3 + 0.1 mg/L butyl octanoate as the sole product from E. coli after 48 h. This study represents a preliminary examination of the feasibility of developing E. coli platforms for the synthesis single medium chain esters from endogenous fatty acids.

Microbial production of butyl butyrate: from single strain to cognate consortium

“…AATs have also been known to contribute to ester biosynthesis in various microorganisms, for instance, Saccharomyces cerevisiae (Kruis et al 2018 ; Saerens et al 2010 ), Yarrowia lipolytica (Gao et al 2018 ; Yu et al 2020 ), C . acetobutylicum (Noh et al 2018 ), and E. coli (Chacon et al 2019 ). Another contribution of AAT on esters synthesis, particularly in E. coli , has been demonstrated by chloramphenicol O -acetyltransferases (CATs, EC 2.3.1.28), an AAT homolog isolated from Streptococcus aureus (Alonso-Gutierrez et al 2013 ; Rodriguez et al 2014 ).…”

Exploring a Streptomyces wax synthase using acyl-SNACs as donor substrates

“…21 Analogous studies have also described how the selection of the mutation site was driven by its proximity to the conserved HHXXXDG motif of wax ester synthases, and how altering its surrounding could modify the enzyme substrate affinity. 22,23…”

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

“…Bioprospecting and protein engineering are promising strategies to find novel AATs with high specificity and activity towards a target ester. For instance, AAT of Actinidia chinensis (AAT Ac ) was engineered to create a AAT Ac S99G variant that enhanced n ‐butyl octanoate production in Escherichia coli about 4.5‐fold higher than the wildtype (Chacon et al, 2019). Similarly, Seo et al reported that a single F97W mutation in CAT of the mesophilic Staphylococcus aureus (CAT Sa ), identified by a model‐guided protein design, achieved ~3.5‐fold increase in isobutyl acetate (IBA) production in a thermophilic, cellulolytic bacterium Clostridium thermocellum (Seo et al, 2019).…”