Engineering Escherichia coli for the production of butyl octanoate from endogenous octanoyl-CoA.

Micaela G Chacón,Emanuele G Kendrick,David J Leak

doi:10.7717/peerj.6971

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.

Highlights

Volatile short and medium chain esters (C4–C14) are found naturally in the flowering and vegetative tissue of plants where they can function either to attract pollinators, or as anti-microbial or anti-herbivore agents (Dudareva, Pichersky & Gershenzon, 2004; Beekwilder et al, 2004; Rodriguez, Tashiro & Atsumi, 2014)
While butyl octanoate is not the preferred end-product for any of these three enzymes, AAT16 from Actinidia chinensis showed the highest activity for its production, and so was chosen as the candidate alcohol acyl transferase (AAT) for this work (Günther et al, 2011)
Expression cultures of E. coli C43 (DE3) harbouring the pET21a::AAT16 plasmid were supplemented with 10 mM butanol and 5 mM octanoic acid to provide an excess of substrate to the AAT16 enzyme

Summary

Introduction

Volatile short and medium chain esters (C4–C14) are found naturally in the flowering and vegetative tissue of plants where they can function either to attract pollinators, or as anti-microbial or anti-herbivore agents (Dudareva, Pichersky & Gershenzon, 2004; Beekwilder et al, 2004; Rodriguez, Tashiro & Atsumi, 2014). Short and medium chain esters occur naturally as fermentation products from certain species of yeast and lactic acid bacteria Those produced by the former have garnered particular interest as they strongly influence both the aroma and flavour profiles of beers and wines (Verstrepen et al, 2003; Costello et al, 2013). The majority of short and medium chain esters possess fruity and floral aromas—with their chemical structure influencing their characteristic scent As such, they have been widely adopted as valuable flavour and fragrance compounds in perfumes, cosmetics, food, and beverages (Sose, Bansode & Rathod, 2017; Rodriguez, Tashiro & Atsumi, 2014; Schrader et al, 2004), representing a key product group in the. C4–C14 esters have a number of potential applications in the pharmaceutical, industrial solvent, and biofuel industries (Sose, Bansode & Rathod, 2017; Tai, Xiong & Zhang, 2015; Layton & Trinh, 2014; Jin et al, 2012)

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Results

Conclusion