Figures (8)  Tables (0)

    • Figure 1. 

      The evolutionary relationships of AcrE homology hits. When using AcrE as a query, the evolutionary relationships of 287 homology hits were analyzed and each homolog information was confirmed with BLASTp. It was found that the homology hits of AcrE mainly included AcrE families, AcrA families, MdtE families, and other efflux RND transporter periplasmic adaptor subunits. The violet and red indicated the selected predicted efflux pumps for further analysis.

    • Figure 2. 

      The evolutionary relationships of MdtE homology hits. When using MdtE as a query, the evolutionary relationships of 284 homology hits were analyzed and each homolog information was confirmed with BLASTp. It was found that the homology hits of MdtE also mainly comprised AcrE families, AcrA families, MdtE families, and other efflux RND transporter periplasmic adaptor subunits. The colored areas indicate the relationships between Citrobacteria and E. coli species.

    • Figure 3. 

      The evolutionary relationships of MdtC homology hits. (a) When using MdtC as a query, the evolutionary relationships of 1,446 homology hits were analyzed and each homolog information was confirmed with BLASTp. These homology hits could be divided into seven different enzyme families such as MdtC, MdtB, AcrD, AcrF, MdtF, AcrB, and CusA families. (b) The evolutionary history of MdtC families was further recapitulated. The colored areas indicate the selected predicted efflux pumps for further analysis.

    • Figure 4. 

      The taxonomic relationship of each species used for general evolutionary recapitulation. The taxonomic relationship of each species was defined via constructing a species tree with the amino acid sequences of their RNA polymerase beta subunits (RpoB). It was found that Salmonella species exhibited closer evolutionary relationship with Escherichia species than Citrobacteria species, which was different when using AcrE, MdtE, or MdtC as queries.

    • Figure 5. 

      Construction of MCFA efflux pump library. (a) Microbial production of MCFAs from D-glucose via the reversal of β-oxidation cycle and transporter engineering. (b) Illustration of the test system. This test system consisted of two individual plasmids. The first plasmid pCDFD-T7-bktB-T7-fadB-T7-ter-T7-ydiI-t7-acs was responsible for MCFA production, whereas the other pETDuet-1 derived plasmid was utilized for the expression of various bacterial transporters. (c) Effect of predicted efflux pump engineering on extracellular, intracellular and total MCFA production. Each experiment in this study was conducted in triplicate and error bars signify standard deviation (SD) with 95% confidence interval (CI).

    • Figure 6. 

      Detailed evolutionary divergence of MCFA transporter families in Citrobacter species. (a) Analysis of this AcrE-centric phylogenetic tree based on genomes from Citrobacter species. This revealed that EnvR homologues, a predicted AcrEF/EnvCD operon regulator, were present in most Citrobacteria species. (b) Effect of transcriptional regulator EnvR engineering on MCFA production. CT_EnvR indicated envR of Citrobacter tructae. Experiments in this study were conducted in triplicate and error bars signify SD with 95% CI.

    • Figure 7. 

      Detailed evolutionary divergence of MCFA transporter families in Escherichia species. (a) Analysis of the AcrE-centric phylogenetic tree based on genomes of Escherichia species. This phylogenetic tree also manifested that EnvR homologues were existing in most Escherichia species. (b) Effect of transcriptional regulator EnvR engineering on MCFA production. EC_EnvR indicated envR of E. coli; CT_EnvR indicated envR of Citrobacter tructae; CT_AcrE indicated acrE of Citrobacter tructae; CT_AcrF indicated acrF of Citrobacter tructae. Experiments in this study were conducted in triplicate and error bars signify SD with 95% CI.

    • Figure 8. 

      Construction of autonomous MCFA secreting systems. (a) Schematic of QEX circuity. (b) The effect of replacing T7 promoter with QEX circuity on MCFA production. The signal synthase CcfA was driven by constitutive promoters with different strength ranging from high strength P1 to low strength P6, to trigger QEX circuity at various times. (c) The evaluation of the performance of this autonomous MCFA secreting system in scaled-up bioreactors. Experiments in this study were conducted in triplicate and error bars signify SD with 95% CI.