In Vivo Reduction of Medium- to Long-Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

Abstract

Fatty aldehyde production by chemical synthesis causes an immense burden to the environment. Within this study, we explored a sustainable, aldehyde-selective and mild alternative approach by utilizing carboxylic acid reductases (CARs). CARs from Neurospora crassa (NcCAR), Thermothelomyces thermophila (TtCAR), Nocardia iowensis (NiCAR), Mycobacterium marinum (MmCAR) and Trametes versicolor (TvCAR) were overexpressed in E. coli K-12 MG1655 RARE (DE3) and screened for medium- to long-chain fatty acid (C6–C18) reduction. MmCAR showed the broadest tolerance towards all carbon-chain lengths and was selected for further investigations of fatty aldehyde synthesis in whole cells. To yield relevant product concentrations, different limitations of CAR whole-cell conversions were elucidated and compensated. We coupled an in vitro cofactor recycling system to a whole-cell biocatalyst to support cofactor supply and achieved 12.36 g L−1 of octanal (STY 0.458 g L−1 h−1) with less than 1.5 % of 1-octanol.