A multi-layer genome mining and phylogenomic analysis to construct efficient and autonomous efflux system for medium chain fatty acids

Medium-chain fatty acids (MCFAs) are important components for food, pharmaceutical and fuel industries. Nevertheless, engineering microorganisms to produce MCFAs often induces toxicity and stresses towards host strains, which could be alleviated via accelerating the export of MCFAs from cells. However, current secretory systems are inefficient and require inducible promoters. Here, a multi-layer genome mining and phylogenomic analysis was developed to identify efficient efflux transporters. Firstly, based on the genomic mining of 397 strains throughout various representative species, the evolutionary history of efflux transporters was recapitulated, and further experimental analysis revealed that acrE from Citrobacter exhibited the best performance. Secondly, according to the further mining of 797 Citrobacter genomes and 1084 Escherichia genomes, a detailed phylogenomic analysis of efflux transporter-centric genomic vicinities was performed. This led to the identification of efficient efflux pump combination acrE and acrF. These efflux pumps were then combined with the quorum-sensing circuit from Enterococcus faecalis to regulate MCFA efflux in an autonomous manner, which achieved a 4.9-fold boost in MCFA production and firstly demonstrated the efficient and autonomous efflux pump specially for MCFAs. The integrative omics technologies described here are enabling the utilization of the increasingly large database and the effective mining of target gene diversities.