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

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 FadR, significantly boosted R-MBFA titers. While an engineered strain carrying the Mus musculus TE domain (TE) 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 ('TesA) and FadR 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.