Figure 1. Canonical pathway for the synthesis of branched-chain fatty acids (BCFA) via the BKDH complex. The branched-chain amino acids isoleucine (Ile), leucine (Leu), and valine (Val) are converted into their respective α-ketoacids by the transaminase IlvE; α-KMV (α-keto-β-methylvalerate), α-KIC (α-ketoisocaproate), and α-KIV (α-ketoisovalerate) undergo oxidative decarboxylation catalyzed by the α-keto acid dehydrogenase (BKDH) complex and the respective acyl-CoA primers, once condensed with malonyl CoA via 3-ketoacyl-ACP synthase III (FabH), are elongated into their respective BCFAs through the type II fatty acid synthase (FASII).
Figure 2. The putative acyl-CoA synthetase gene mbcScontributes to BCFAs synthesis in the absence of an active BKDH complex . Wild-type (WT), lpdA mutant, lpdA suppressor mutant with a modified mbcS promoter (lpdA mbcS1) andlpdA mbcS double mutant cells were grown in (A) TSB,(B) TSB supplemented with a mixture of 0.5 mM BCCAs i C4,i C5, and a C5 or (C) TSB supplemented with 0.5 mMa 15:0 fatty acid, and growth behavior was monitored over time as an increase in optical density at 600 nm (OD600). Data are plotted as mean ± SD of three biological replicates. ****p<0.0001, using two-way ANOVA with Tukey’s multiple comparison test at 5-8 h. In panel A, asterisks indicate thatlpdA single mutant and lpdA mbcS double mutant are significantly different compared to WT and lpdA mbcS1 . In panels B and C, asterisks indicate that lpdA mbcS double mutant is significantly different compared to WT, lpdA single mutant, andlpdA mbcS1 .
Figure 3. Mutations in the mbcS promoter increase its activity and result in synthesis of i14:0 BCFAs. (A)Selected alleles identified during suppressor analysis are compared to the WT allele of the mbcS promoter region. Changes are highlighted in magenta, and -10 and -35 boxs are indicated by the gray boxes. (B) WT cells harboring plasmids with either WT or mutantmbcS promoter regions  fused translationally to gfp(PmbcS-gfp ) were grown to stationary phase in TSB, at which time cells were pelleted and washed with PBS. Promoter activity was then measured (relative fluorescence units [RFUs]; GFP/OD600). **** p<0.000, one-way ANOVA with Tukey’s multiple comparison test. (C) Suppressor mutants with the indicated mbcS alleles were grown to exponential phase in TSB. Cells were washed with PBS, and membrane fatty acid content was analyzed by GC-FAME. Data are presented as mean ± SD from three biological replicates. **** p<0.0001, ***p<0.001, two-way ANOVA with Tukey’s multiple comparison test; ns, not significant.