Engineering diverse fatty acid compositions of phospholipids in Escherichia coli

Bacterial fatty acids (FAs) are an essential component of the cellular membrane and are an important source of renewable chemicals as they can be converted to fatty alcohols, esters, ketones, and alkanes, and used as biofuels, detergents, lubricants, and commodity chemicals. Most prior FA bioconversions have been performed on the carboxylic acid group. Modification of the FA hydrocarbon chain could substantially expand the structural and functional diversity of FA-derived products. Additionally, the effects of such modified FAs on the growth and metabolic state of their producing cells are not well understood. Here we engineer novel Escherichia coli phospholipid biosynthetic pathways, creating strains with distinct FA profiles enriched in ω7-unsaturated FAs (ω7-UFAs, 75%), Δ5-unsaturated FAs (Δ5-UFAs, 60%), cyclopropane FAs (CFAs, 55%), internally-branched FAs (IBFAs, 40%), and Δ5,ω7-double unsaturated FAs (DUFAs, 46%). Although bearing drastically different FA profiles in phospholipids, UFA, CFA, and IBFA enriched strains display wild-type-like phenotypic profiling and growth. Transcriptomic analysis reveals DUFA production drives increased differential expression and the induction of the fur iron starvation transcriptional cascade, but higher TCA cycle activation compared to the UFA producing strain. This likely reflects a slight cost imparted for DUFA production, which resulted in lower maximum growth in some, but not all, environmental conditions. The IBFA-enriched strain was further engineered to produce free IBFAs, releasing 96 mg/L free IBFAs from 154 mg/L of the total cellular IBFA pool. This work has resulted in significantly altered FA profiles of membrane lipids in E. coli, greatly increasing our understanding of the effects of FA structure diversity on the transcriptome, growth, and ability to react to stress.