Advances on medium-chain fatty acids synthesis in Escherichia coli

Medium-chain fatty acids (MCFAs), straight-chain fatty acids with 6-12 carbons, can be transformed into aliphatic hydrocarbons, triglyccrides, and other olcochemicals. MCFAs and their derivatives show strong potential as useful materials in the energy, medicine, food and chemical industries. Compared with the conventional extraction methods based on petroleum or plant biomass, microbial fermentation is a green and sustainable alternative for MCFA production. Escherichia coli are frequently used as model microorganism for the analyses of metabolic mechanisms and industrial processes owing to the factors: rapid replication, clear genetics, case of rearing in the laboratory, simple process for amplification and abundance of gene manipulation tools. In E. coli, MCFAs can be synthesised through the fatty acid biosynthesis (FAB) pathw and the reverse beta-oxidation (RBO) pathway. However, there are still many challenges that severely limit MCFAs biosynthesis in E. coli, such as chain-length control, synthesis efficiency and cytotoxicity. With the development and improvement of metabolic engineering, synthetic biology and high-throughput sequencing technology, great progress has been made in recent years in the engineering of E. coli for MCFAs overproduction. In this review, we first introduce the MCFAs synthetic pathways in E. coil, including the FAB and RBO pathways, and compare these two pathways in terms of extension unit, rate-limiting step and cofactors. We then summarise the challenges, advances and representative studies in MCFAS production. with a focus on three aspects: chain-length control, synthetic pathway optimisation and tolerance improvement. Chain-length control is the key to the directed synthesis of MCFAs, as multiple rounds of cycles in the synthetic pathway tend to generate mixtures of various chain lengths. We discuss the catalysis mechanism of functional protein in the synthetic pathways to achieving chain-length control, including thioesterase, thiolase, and ketoacyl synthase. Protein and metabolic engineering of these key enzymes for targeted synthesis of MCFAs are also summarised in this review. Furthermore, synthetic pathway optimisation is an effective strategy for the enhancement of MCFAs biosynthesis. The "Pull-Push" of the FAB and RBO pathways, a process frequently used to increase precursor supply and improve product release. is reviewed here. Tolerance engineering is extremely critical for MCFAs overproduction, owing to the cell toxicity of MCFAs. We discuss in this review the injury mechanisms associated with MCFAs production and the strategies for improving cellular tolerance. Specifically, MCFAs production reduces intracellular pH and changes the composition of the cell membrane. leading to disruption of cytoplasmic homeostasis and destruction of membrane structure and properties. Membrane engineering, stress response regulation and adaptive evolution are generally employed for improving cellular tolerance of MCFAs. In summary, we cover in this review the recent advancements in the bio-engineering of E. coil for MCFAs production, based on the FAB and RBO biosynthetic pathways. Protein, metabolic and membrane engineering are generally applied to control the chain length, optimise the synthetic pathway and improve cellular tolerance. We also provide some perspectives for further strengthening MCFAs biosynthesis. including improvement of computer technology for protein assessment, indepth and full perspective analysis of MCFCs toxicity and tolerance mechanisms and the mining of useful targets from exogenous and endogenous species using high-throughput screening.