Fig. 1 Glutarate biosynthetic pathway optimization results inEscherichia coli. Tfu_0875 : α-ketothiolase,Tfu_2399 : 3-hydroxyacyl-CoA dehydrogenase, Tfu_0067 : 3-hydroxyadipyl-CoA dehydrogenase, Tfu_1647 : 5-carboxy-2-pentenoyl-CoA reductase, Tfu_2576-7 : adipyl-CoA synthetase, malonic acid synthetase: matB , malonic acid carrier protein: matC . The ArcA , ldhA , atoB ,pflB , ackA and poxB genes were deleted using the CRISPR/Cas9 system. A rough arrow represents genes or enzymes subject to overexpression, X represents those deleted. ldhA , L-lactate dehydrogenase; atoB , acetyl-CoA C-acetyltransferase; arcA , aerobic respiration control protein; pflB , formate C-acetyltransferase 1; ackA , acetate kinase; poxB , pyruvate dehydrogenase; pta , phosphate acetyltransferase;acs , acetyl-CoA synthetase.
Fig. 2 Glutarate tolerance and degradation test. A. Time profiles of Bgl4146 treated with various glutarate concentrations. Cells were grown at 37 °C in the SOB medium (pH 7.0) and exposed at exponential growth phase (OD600~0.3). B. Growth of Bgl4146 on glutarate. Growth (blue line) and the consumption of glutarate (orange line) was measured in M9 supplemented with 5 g/L glutarate as the single carbon source. Bgl4146: Bgl4 carrying pAD1, pAD4 and pAD6; Bgl4: BL21(DE3) ΔarcA ΔldhA ΔatoBΔpflB ; pAD1: pRSFDuet-1 carrying Tfu_0875 andTfu_2399 ; pAD4: pTrc99a carrying Tfu_0067 andTfu_1647 ; pAD6: pCDFDuet-1 carrying Tfu_2576-7 . Data shown are mean ± s.d. (n = 3 independent experiments).
Fig. 3 Different recombinant strains for glutarate production.A. Aerobic Condition. B. Microaerobic Condition. C. Anaerobic Condition. The all strains were fermented in SOB with 4 g/L glucose initially. Error bars represent the s.d. from three independent assays. Bgl4146: Bgl4 carrying pAD1, pAD4 and pAD6; Bgl4: BL21(DE3) ΔarcA ΔldhA ΔatoB ΔpflB ; pAD1: pRSFDuet-1 carryin9g Tfu_0875 and Tfu_2399 ; pAD4: pTrc99a carryingTfu_0067 and Tfu_1647 ; pAD6: pCDFDuet-1 carryingTfu_2576-7 ; Bgl41461: Bgl4146 carrying pACC1 (pACYCDuet-1 carrying accABCD from E. coli ); Bgl41462: Bgl4146 carrying pACC2 (pACYCDuet-1 carrying accBC , dtsR1 fromCorynebacterium glutamicum ); Bgl41463: Bgl4146 carrying pACC3 (pACYCDuet-1 carrying acs from E. coli ); Bgl41464: Bgl4146 carrying pACC4 (pACYCDuet-1 carrying acs , accBC ,dtsR1 ).
Fig. 4 Enhanced production of glutarate at different acetate concentrations in E. coli Bgl41464, Bgl51464, Bgl61464. A. The glutarate production at different acetate concentrations under 4 g/L glucose and microaerobic condition. B. The glutarate production at different acetate concentrations under microaerobic condition. Bgl4: BL21(DE3) ΔarcA ΔldhA ΔatoB ΔpflB ; Bgl5: BL21(DE3) ΔarcA ΔldhA ΔatoB ΔpflBΔackA ; Bgl6: BL21(DE3) ΔarcA ΔldhA ΔatoBΔpflB ΔackA ΔpoxB ; Bgl41464: Bgl4 carrying pAD1, pAD4, pAD6 and pACC4; Bgl51464: Bgl5 carrying pAD1, pAD4, pAD6 and pACC4; Bgl61464: Bgl6 carrying pAD1, pAD4, pAD6 and pACC4; Bgl4: BL21(DE3) ΔarcA ΔldhA ΔatoB ΔpflB ; Bgl5: BL21(DE3) ΔarcA ΔldhA ΔatoB ΔpflBΔackA ; Bgl6: BL21(DE3) ΔarcA ΔldhA ΔatoBΔpflB ΔackA ΔpoxB ; pAD1: pRSFDuet-1 carryingTfu_0875 and Tfu_2399 ; pAD4: pTrc99a carryingTfu_0067 and Tfu_1647 ; pAD6: pCDFDuet-1 carryingTfu_2576-7 ; pACC4: pACYCDuet-1 carrying acs ,accBC , dtsR1 .
Fig. 5 Fed-batch production of glutarate in 5 L bioreactors.Fermentation process of Bgl51464. The medium initially contained 6 g/L acetate. Substrates consumptions, metabolites, and cell growth during fed-batch fermentation with 8 g/L glucose initially, 0.5 vvm aeration rate and 400 rpm agitation rate in SOB medium. Error bars represent the s.d. from three independent assays. Bgl51464: Bgl5 carrying pAD1, pAD4, pAD6 and pACC4; Bgl5: BL21(DE3) ΔarcA ΔldhA ΔatoBΔpflB ΔackA ; pAD1: pRSFDuet-1 carrying Tfu_0875and Tfu_2399 ; pAD4: pTrc99a carrying Tfu_0067 andTfu_1647 ; pAD6: pCDFDuet-1 carrying Tfu_2576-7 ; pACC4: pACYCDuet-1 carrying acs , accBC , dtsR1 .