Exploring the versatility of fatty acid biosynthesis in Escherichia coli: Production of random methyl branched fatty acids

Abstract

Microbial fatty acids (FAs) hold significant potential as alternatives for the oleochemical industry. However, expanding the functional and structural diversity of microbial FA-derived products is essential to fully leverage this potential. Methyl-branched-chain FAs (MBFAs) are of particular interest as high-performance industrial compounds. This study examines the ability of the Escherichia coli FA biosynthesis pathway to produce a diverse mixture of random MBFAs (R-MBFAs) by utilizing both the natural malonyl-ACP substrate and the branched-chain methylmalonyl-ACP (mm-ACP) as an unnatural elongation unit. First, E. coli was engineered to accumulate methylmalonyl-CoA (mm-CoA) through a methylmalonate or a propionate-dependent pathway, and the capacity of E. coli FASII enzymes to synthesize mm-ACP and utilize it as a substrate was confirmed by the production of R-MBFAs. However, low R-MBFA accumulation and propionate-induced growth inhibition was observed. To improve R-MBFA yields, various malonyl-/mm-CoA acyltransferase (AT) enzymes were expressed, and their efficacy in generating mm-ACP was indirectly assessed through R-MBFA production levels. When expressing selected ATs, including native malonyl CoA-acyl carrier protein transacylase FabD, propionate-induced growth inhibition was alleviated and R-MBFA titers ranged from 5.9% to 7.7% of total FAs. Further strain optimization, analyzing two thioesterase (TE) activities and overexpression of the E. coli transciptional regulator ^EcFadR, significantly boosted R-MBFA titers. While an engineered strain carrying the Mus musculus TE domain (^MmTE) produced 55.2 mg/L of R-MBFAs, representing an 11.8% of total FAs, another strain combining the overexpression of the cytosolic version of the TE TesA from E. coli (^Ec‘TesA) and ^EcFadR produced approximately 1.1 g/L of total FAs, with an R-MBFA fraction of 6.7% (70.5 mg/L), marking the highest yield recorded in shake-flask cultures. Finally, these two recombinant E. coli strains were grown in laboratory-scale fed-batch fermentations, and produced approximately 10 g/L of total FAs and over 1–1.2 g/L of R-MBFAs, underscoring the potential for large-scale production of these valuable FA-derived compounds.