Libro de resumenes SAMIGE - RANDOM METHYL-BRANCHED CHAIN FATTY ACIDS PRODUCTION IN ENGINEERED Escherichia coli STRAINS

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Microbial fatty acids (FA) and derived molecules have emerged as promising alternatives to petroleum-based chemicals aimed at reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often provide a narrow variety of linear long-chain saturated and unsaturated hydrocarbons, yielding end products with limited structural diversity. Methyl-branched chain fatty acids (MBFA) have lower melting points and better cold flow properties than their corresponding linear-chain analogs and exhibit better oxidative stability than linear unsaturated FA of the same chain length. These attributes make them attractive for multiple environmentally friendly applications, such as biodegradable lubricant additives, coating agents, and personal care products.Thereby, we aimed to produce a novel type of MBFA in the commonly used biotechnological host Escherichia coli. First, we supplemented an E. coli BL21(DE3) ΔfadE::kan strain carrying the Vibrio harveyi Acyl-ACP Synthetase with 4-methyloctanoic acid. After total FA extraction and analysis by GC-MS, we found the elongation products: 6-methyldecanoic, 8-methyldodecanoic, 10-methyltetradecanoic, and 12-methylhexadecanoic acids. Proven that native E. coli Fatty Acid Synthase (FAS) was able to elongate methyl-branched intermediates, we aimed to design a metabolic pathway for de novo production of MBFA. We hypothesized that if the FAS system was in the presence of methylmalonyl-ACP (MM-ACP, an unnatural elongation unit), FA with methyl-branches in even carbon number may be produced. Therefore, to achieve the biosynthesis of MM-ACP, different acyltransferases (AT) domains/enzymes capable of catalyzing the transacylation of the methylmalonyl moiety from methylmalonyl-CoA (MM-CoA) to MM-ACP have to be explored. To begin with, we analyzed total FA production of the E. coli BAP1/pBF01 strain, which was designed and constructed to in vivo synthesize high-levels of the precursor MM-CoA after feeding with propionate, by deletion of the propionate catabolism prpBCDE operon and overexpression of native Propionyl-CoA Synthetase (PrpE) and heterologous Streptomyces coelicolor Propionyl-CoA Carboxilase (pBF01). Effectively, a wide variety of MBFA were found, ranging from 11 to 16 carbon-length, with branches located in random even-number positions varying from 2 to 12. This result suggested that the native E. coli FabD transacylase, which typically transfers malonyl-CoA to ACP, was indeed able to synthesize MM-ACP in vivo as an unspecific reaction, as it was already reported in vitro. Finally, by overexpression of E. coli TesA’ thioesterase in the BAP1/pBF01 strain, MBFA titers were improved, representing a 2.2% of total FA production. Selected heterologous AT with specificity to MM-CoA were cloned and are planned to be introduced into this promising strain. In conclusion, a metabolic pathway was established in E. coli to produce MBFA, although further efforts are needed to improve the final titers. Información suministrada por el agente en SIGEVA