GFP Scaffold-Based Engineering for the Production of Unbranched Very Long Chain Fatty Acids in Escherichia coli With Oleic Acid and Cerulenin Supplementation.

Elias Kassab,Thomas Brueck,Norbert Mehlmer

doi:10.3389/fbioe.2019.00408

Abstract

Currently, very long chain fatty acids (VLCFAs) for oleochemical, pharmaceutical, cosmetic, or food applications are extracted from plant or marine organism resources, which is associated with a negative environmental impact. Therefore, there is an industrial demand to develop sustainable, microbial resources. Due to its ease of genetic modification and well-characterized metabolism, Escherichia coli has established itself as a model organism to study and tailor microbial fatty acid biosynthesis using a concerted genetic engineering approach. In this study, we systematically implemented a plant-derived (Arabidopsis thaliana) enzymatic cascade in Escherichia coli to enable unbranched VLCFA biosynthesis. The four Arabidopsis thaliana membrane-bound VLCFA enzymes were expressed using a synthetic expression cassette. To facilitate enzyme solubilization and interaction of the synthetic VLCFA synthase complex, we applied a self-assembly GFP scaffold. In order to initiate VLCFA biosynthesis, external oleic acid and cerulenin were supplemented to cultures. In this context, we detected the generation of arachidic (20:0), cis-11-eicosenoic (20:1) and cis-13-eicosenoic acid (20:1).

Highlights

Microbial oils have been recently designated as a sustainable alternative to plant- and animal-based lipids
In order to enable the recombinant production of very long chain fatty acids (VLCFAs) in E. coli BL21 (DE3), we constructed and expressed the characterized Arabidopsis thaliana VLCFA multi-enzyme elongase system using a synthetic expression cassette
After induction with 0.05 mM IPTG, we performed a lipid analysis where we found that the individual expression of three 3-ketoacyl-CoA synthases (KCS1, KCS6, and KCS18) in E. coli BL21 (DE3) did not result in detectable amounts of VLCFA (Supplementary Material)

Summary

Introduction

Microbial oils have been recently designated as a sustainable alternative to plant- and animal-based lipids. E. coli has established itself as a model organism to study and tailor microbial fatty acid biosynthesis using a concerted genetic engineering approach (Janssen and Steinbuchel, 2014). While E. coli is not an oleaginous organism per se the option for extensive genetic alternation has allowed the generation of respectable product titers (Janssen and Steinbuchel, 2014). Despite giant strides in increasing the intracellular fatty acid pool and diversifying the profile of natural fatty acids by genetic engineering, currently the ability to generate plant-like fatty acids with industrial demand is very limited (Handke et al, 2011; Janssen and Steinbuchel, 2014; Pfleger et al, 2015). There are a Engineering VLCFA Production in E. coli few studies that describe the generation of unbranched VLCFAs (C20–C28) that could be applied as food additives or in specialized high value chemical applications, such as performance lubricants (Handke et al, 2011; Pfleger et al, 2015)

Methods

Results

Conclusion