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Fig. 8. Relative use of NADH over NADPH (or vice versa) by the acetoacetyl-CoA reductases from Ca. A. phosphatis and C. necator.

Figure 8 Relative use of NADH over NADPH (or vice versa) by the acetoacetyl-CoA reductases from Ca. A. phosphatis and C. necator.

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Fig. 2. Specific initial rates of reactions catalyzed by AAR‘AP at different concentrations of acetoacetyl-CoA. Concen- tration of the cofactor NADH was fixed at 300 mM. The reaction buffer was 50 mM Tris (pH 8), 5 mM MgCl, 5 mM NaCl. Temperature was set at 30°C. Best fit curve was obtained with a simple Michaelian model.
Fig. 2. Specific initial rates of reactions catalyzed by AAR‘AP at different concentrations of acetoacetyl-CoA. Concen- tration of the cofactor NADH was fixed at 300 mM. The reaction buffer was 50 mM Tris (pH 8), 5 mM MgCl, 5 mM NaCl. Temperature was set at 30°C. Best fit curve was obtained with a simple Michaelian model.
Fig. 3. Progress curves of reactions catalyzed by AAR“? at different acetoacetyl-CoA concentrations. The NADPH concen- tration was fixed at 300 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of acetoacetyl-CoA and enzyme were: (1) AcAcCoA = 20 mM, Enzyme = 0.07 mM. (2) AcAcCoA = 38 mM, Enzyme = 0.07 mM. (3) AcAcCoA = 48 mM, Enzyme = 0.07 mM. (4) AcAcCoA = 80 mM, Enzyme = 0.08 mM. (5) AcAcCoA = 120 mM, Enzyme = 0.09 mM. (6) AcAcCoA = 150 mM, Enzyme = 0.1 mM. (7) AcAcCoA = 165 mM, Enzyme = 0.1 mM. (8) AcAcCoA = 190 mM, Enzyme = 0.1 mM. (9) AcAcCoA = 70 mM, Enzyme = 0.08 mM. (10) AcAcCoA = 115 mM, Enzyme = 0.09 mM.
Fig. 3. Progress curves of reactions catalyzed by AAR“? at different acetoacetyl-CoA concentrations. The NADPH concen- tration was fixed at 300 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of acetoacetyl-CoA and enzyme were: (1) AcAcCoA = 20 mM, Enzyme = 0.07 mM. (2) AcAcCoA = 38 mM, Enzyme = 0.07 mM. (3) AcAcCoA = 48 mM, Enzyme = 0.07 mM. (4) AcAcCoA = 80 mM, Enzyme = 0.08 mM. (5) AcAcCoA = 120 mM, Enzyme = 0.09 mM. (6) AcAcCoA = 150 mM, Enzyme = 0.1 mM. (7) AcAcCoA = 165 mM, Enzyme = 0.1 mM. (8) AcAcCoA = 190 mM, Enzyme = 0.1 mM. (9) AcAcCoA = 70 mM, Enzyme = 0.08 mM. (10) AcAcCoA = 115 mM, Enzyme = 0.09 mM.
Fig. 4. Progress curves of reactions catalyzed by AAR“? at different NADH concentrations. The acetoacetyl-CoA concen- tration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a competitive product inhibition model. The initial concentrations of NADH and enzyme were: (1) NADH = 12 mM, Enzyme = 0.05 mM. (2) NADH = 30 mM, Enzyme = 0.05 mM. (3) NADH = 46 mM, Enzyme = 0.05 mM. (4) NADH = 77 mM, Enzyme = 0.05 mM. (5) NADH = 78 mM, Enzyme = 0.05 mM. (6) NADH = 94 mM, Enzyme = 0.05 mM. (7) NADH = 111 mM, Enzyme = 0.05 mM. (8) NADH = 136 mM, Enzyme = 0.05 mM. (9) NADH = 150 mM, Enzyme = 0.05 mM. (10) NADH = 168 mM, Enzyme = 0.05 mM. (11) NADH = 9 mM, Enzyme = 0.1 mM. (12) NADH = 20 mM, Enzyme = 0.1mM. (13) NADH = 35 mM, Enzyme = 0.1 mM. (14) NADH = 47 mM, Enzyme = 0.1 mM. (15) NADH = 78 mM, Enzyme = 0.1 mM.
Fig. 4. Progress curves of reactions catalyzed by AAR“? at different NADH concentrations. The acetoacetyl-CoA concen- tration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a competitive product inhibition model. The initial concentrations of NADH and enzyme were: (1) NADH = 12 mM, Enzyme = 0.05 mM. (2) NADH = 30 mM, Enzyme = 0.05 mM. (3) NADH = 46 mM, Enzyme = 0.05 mM. (4) NADH = 77 mM, Enzyme = 0.05 mM. (5) NADH = 78 mM, Enzyme = 0.05 mM. (6) NADH = 94 mM, Enzyme = 0.05 mM. (7) NADH = 111 mM, Enzyme = 0.05 mM. (8) NADH = 136 mM, Enzyme = 0.05 mM. (9) NADH = 150 mM, Enzyme = 0.05 mM. (10) NADH = 168 mM, Enzyme = 0.05 mM. (11) NADH = 9 mM, Enzyme = 0.1 mM. (12) NADH = 20 mM, Enzyme = 0.1mM. (13) NADH = 35 mM, Enzyme = 0.1 mM. (14) NADH = 47 mM, Enzyme = 0.1 mM. (15) NADH = 78 mM, Enzyme = 0.1 mM.
Fig. 5. Progress curves of reactions catalyzed by AAR“? at different NADPH concentrations. The acetoacetyl-CoA concen- tration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a competitive product inhibition model. The initial concentrations of NADPH and enzyme were: (1) NADPH = 18 mM, Enzyme = 0.05 mM. (2) NADPH = 33 mM, Enzyme = 0.05 mM. (3) NADPH = 40 mM, Enzyme = 0.05 mM. (4) NADPH = 66 mM, Enzyme = 0.05 mM. (5) NADPH = 96 mM, Enzyme = 0.05 mM. (6) NADPH = 30 mM, Enzyme = 0.5 mM. (7) NADPH = 46 mM, Enzyme = 0.5 mM. (8) NADPH = 60 mM, Enzyme = 0.5 mM. (9) NADPH = 76 mM, Enzyme = 0.5 mM. (10) NADPH = 89 mM, Enzyme = 0.5 mM. (11) NADPH = 103 mM, Enzyme = 0.5 mM. (12) NADPH = 119 mM, Enzyme = 0.5 mM. (13) NADPH = 134 mM, Enzyme = 0.5 mM. (14) NADPH = 149 mM, Enzyme = 0.5 mM. (15) NADPH = 162 mM, Enzyme = 0.5 mM. (16) NADPH = 26 mM, Enzyme = 1.5 mM. (17) NADPH = 56 mM, Enzyme = 1.5 mM. (18) NADPH = 61 mM, Enzyme = 1.5 mM. (19) NADPH = 78 mM, Enzyme = 1.5 mM. (20) NADPH = 90 mM, Enzyme = 1.5 mM. (21) NADPH = 104 mM, Enzyme = 1.5 mM. (22) NADPH = 121 mM, Enzyme = 1.5 mM. (23) NADPH = 155 mM, Enzyme = 1.5 mM. (24) NADPH = 151 mM, Enzyme = 1.5 mM. (25) NADPH = 175 mM, Enzyme = 1.5 mM.
Fig. 5. Progress curves of reactions catalyzed by AAR“? at different NADPH concentrations. The acetoacetyl-CoA concen- tration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a competitive product inhibition model. The initial concentrations of NADPH and enzyme were: (1) NADPH = 18 mM, Enzyme = 0.05 mM. (2) NADPH = 33 mM, Enzyme = 0.05 mM. (3) NADPH = 40 mM, Enzyme = 0.05 mM. (4) NADPH = 66 mM, Enzyme = 0.05 mM. (5) NADPH = 96 mM, Enzyme = 0.05 mM. (6) NADPH = 30 mM, Enzyme = 0.5 mM. (7) NADPH = 46 mM, Enzyme = 0.5 mM. (8) NADPH = 60 mM, Enzyme = 0.5 mM. (9) NADPH = 76 mM, Enzyme = 0.5 mM. (10) NADPH = 89 mM, Enzyme = 0.5 mM. (11) NADPH = 103 mM, Enzyme = 0.5 mM. (12) NADPH = 119 mM, Enzyme = 0.5 mM. (13) NADPH = 134 mM, Enzyme = 0.5 mM. (14) NADPH = 149 mM, Enzyme = 0.5 mM. (15) NADPH = 162 mM, Enzyme = 0.5 mM. (16) NADPH = 26 mM, Enzyme = 1.5 mM. (17) NADPH = 56 mM, Enzyme = 1.5 mM. (18) NADPH = 61 mM, Enzyme = 1.5 mM. (19) NADPH = 78 mM, Enzyme = 1.5 mM. (20) NADPH = 90 mM, Enzyme = 1.5 mM. (21) NADPH = 104 mM, Enzyme = 1.5 mM. (22) NADPH = 121 mM, Enzyme = 1.5 mM. (23) NADPH = 155 mM, Enzyme = 1.5 mM. (24) NADPH = 151 mM, Enzyme = 1.5 mM. (25) NADPH = 175 mM, Enzyme = 1.5 mM.
Fig. 6. Progress curves of reactions catalyzed by AAR“mera at different NADH concentrations. The acetoacetyl-CoA con- centration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of NADH and enzyme were: (1) NADH = 19.6 mM, Enzyme = 0.002 mM. (2) NADH = 19.4 mM, Enzyme = 0.002 mM. (3) NADH = 18.6 mM, Enzyme = 0.002 mM. (4) NADH = 37.2 mM, Enzyme = 0.002 mM. (5) NADH = 34.6 mM, Enzyme = 0.002 mM. (6) NADH = 36.1 mM, Enzyme = 0.002 mM. (7) NADH = 54 mM, Enzyme = 0.002 mM. (8) NADH = 54 mM, Enzyme = 0.002 mM. (9) NADH = 92.5 mM, Enzyme = 0.002 mM. (10) NADH = 102.5 mM, Enzyme = 0.002 mM. (11) NADH = 102.5 mM, Enzyme = 0.002 mM. (12) NADH = 143 mM, Enzyme = 0.002 mM. (13) NADH = 142 mM, Enzyme = 0.002 mM. (14) NADH = 314 mM, Enzyme = 0.002 mM. (15) NADH = 326 mM, Enzyme = 0.002 mM. (16) NADH = 322 mM, Enzyme = 0.002 mM. (17) NADH = 534 mM, Enzyme = 0.002 mM. (18) NADH = 540 mM, Enzyme = 0.002 mM. (19) NADH = 532 mM, Enzyme = 0.002 mM.
Fig. 6. Progress curves of reactions catalyzed by AAR“mera at different NADH concentrations. The acetoacetyl-CoA con- centration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of NADH and enzyme were: (1) NADH = 19.6 mM, Enzyme = 0.002 mM. (2) NADH = 19.4 mM, Enzyme = 0.002 mM. (3) NADH = 18.6 mM, Enzyme = 0.002 mM. (4) NADH = 37.2 mM, Enzyme = 0.002 mM. (5) NADH = 34.6 mM, Enzyme = 0.002 mM. (6) NADH = 36.1 mM, Enzyme = 0.002 mM. (7) NADH = 54 mM, Enzyme = 0.002 mM. (8) NADH = 54 mM, Enzyme = 0.002 mM. (9) NADH = 92.5 mM, Enzyme = 0.002 mM. (10) NADH = 102.5 mM, Enzyme = 0.002 mM. (11) NADH = 102.5 mM, Enzyme = 0.002 mM. (12) NADH = 143 mM, Enzyme = 0.002 mM. (13) NADH = 142 mM, Enzyme = 0.002 mM. (14) NADH = 314 mM, Enzyme = 0.002 mM. (15) NADH = 326 mM, Enzyme = 0.002 mM. (16) NADH = 322 mM, Enzyme = 0.002 mM. (17) NADH = 534 mM, Enzyme = 0.002 mM. (18) NADH = 540 mM, Enzyme = 0.002 mM. (19) NADH = 532 mM, Enzyme = 0.002 mM.
Fig. 7. Progress curves of reaction catalyzed by AAR“ !™e" at different NADPH concentrations. The acetoacetyl-CoA con- centration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of NADPH and enzyme were: (1) NADPH = 38 mM, Enzyme = 0.0015 mM. (2) NADPH = 56 mM, Enzyme = 0.0018 mM. (3) NADPH = 56 mM, Enzyme = 0.0015 mM. (4) NADPH = 56 mM, Enzyme = 0.0015 mM. (5) NADPH = 110 mM, Enzyme = 0.0015 mM. (6) NADPH = 112 mM, Enzyme = 0.0016 mM. (7) NADPH = 112 mM, Enzyme = 0.0015 mM. (8) NADPH = 155 mM, Enzyme = 0.0016 mM. (9) NADPH = 155 mM, Enzyme = 0.0015 mM. (10) NADPH = 155 mM, Enzyme = 0.0015 mM. (11) NADPH = 325 mM, Enzyme = 0.0018 mM. (12) NADPH = 320 mM, Enzyme = 0.0018 mM. (13) NADPH = 327 mM, Enzyme = 0.002 mM. (14) NADPH = 532 mM, Enzyme = 0.0021 mM. (15) NADPH = 533 mM, Enzyme = 0.0022 mM. (16) NADPH = 531 mM, Enzyme = 0.0022 mM.
Fig. 7. Progress curves of reaction catalyzed by AAR“ !™e" at different NADPH concentrations. The acetoacetyl-CoA con- centration was fixed at 400 mM. Time scale is in seconds. The thick lines represent the global fitting, while thinner lines represent the experimental data. Best fit curves were obtained with a simple Michaelian model. The initial concentra- tions of NADPH and enzyme were: (1) NADPH = 38 mM, Enzyme = 0.0015 mM. (2) NADPH = 56 mM, Enzyme = 0.0018 mM. (3) NADPH = 56 mM, Enzyme = 0.0015 mM. (4) NADPH = 56 mM, Enzyme = 0.0015 mM. (5) NADPH = 110 mM, Enzyme = 0.0015 mM. (6) NADPH = 112 mM, Enzyme = 0.0016 mM. (7) NADPH = 112 mM, Enzyme = 0.0015 mM. (8) NADPH = 155 mM, Enzyme = 0.0016 mM. (9) NADPH = 155 mM, Enzyme = 0.0015 mM. (10) NADPH = 155 mM, Enzyme = 0.0015 mM. (11) NADPH = 325 mM, Enzyme = 0.0018 mM. (12) NADPH = 320 mM, Enzyme = 0.0018 mM. (13) NADPH = 327 mM, Enzyme = 0.002 mM. (14) NADPH = 532 mM, Enzyme = 0.0021 mM. (15) NADPH = 533 mM, Enzyme = 0.0022 mM. (16) NADPH = 531 mM, Enzyme = 0.0022 mM.
Fig. 9. Flux capacity of the reaction catalyzed by AAR‘? at different NADH/NAD+ and NADPH/NADP* concentration ratios.
Fig. 9. Flux capacity of the reaction catalyzed by AAR‘? at different NADH/NAD+ and NADPH/NADP* concentration ratios.
Biomass composition of the engineered strain after considering plasmid and heterologous protein contributions. Relative elemental composition for each component is below the columns identified with the symbol of the elements carbon, hydrogen, nitrogen, oxygen, sulfur and phosphorous. After considering recombinant DNA and protein burdens, 1 carbon- mol of biomass (CmolX) has a “molecular” weight of 23.1840 gCDW/CmolX, and its reduction degree is y=4.2920. DNA amplification, restriction and ligation as well as bacterial transformation, selection and identification were executed according to the standard procedures described elsewhere [6]. When amplifying DNA for cloning purposes, Q5 High-Fidelity DNA Polymerase (New England Biolabs) was used. GoTaq Green Master Mix (Promega) was used for colony PCR. Plasmid pu- rification from cultures was done using the QlAprep Spin Miniprep Kit (Qiagen). For plasmid propagation and long-term storage of constructions, introduction of the plasmids in E. coli One Shot® TOP10 (ThermoFisher Scientific) was implemented. All DNA modifying enzymes employed in this study were purchased from New England Biolabs. BADCAD *= AneAdad tw CHA ABSDCADE zane "TRA READCADE cand: x:7ae HeAlstad) feArS 4 Mmateca.
Biomass composition of the engineered strain after considering plasmid and heterologous protein contributions. Relative elemental composition for each component is below the columns identified with the symbol of the elements carbon, hydrogen, nitrogen, oxygen, sulfur and phosphorous. After considering recombinant DNA and protein burdens, 1 carbon- mol of biomass (CmolX) has a “molecular” weight of 23.1840 gCDW/CmolX, and its reduction degree is y=4.2920. DNA amplification, restriction and ligation as well as bacterial transformation, selection and identification were executed according to the standard procedures described elsewhere [6]. When amplifying DNA for cloning purposes, Q5 High-Fidelity DNA Polymerase (New England Biolabs) was used. GoTaq Green Master Mix (Promega) was used for colony PCR. Plasmid pu- rification from cultures was done using the QlAprep Spin Miniprep Kit (Qiagen). For plasmid propagation and long-term storage of constructions, introduction of the plasmids in E. coli One Shot® TOP10 (ThermoFisher Scientific) was implemented. All DNA modifying enzymes employed in this study were purchased from New England Biolabs. BADCAD *= AneAdad tw CHA ABSDCADE zane "TRA READCADE cand: x:7ae HeAlstad) feArS 4 Mmateca.
b.d.l.: below detection level. Table 2
b.d.l.: below detection level. Table 2
Metabolic fluxes distributions for the engineered strain during the two studied steady-states. The feasible minimal and maximal fluxes for each reaction is also reported. Metabolit labelled with the symbol [e] are considered extracellular while the symbol [c] signal the intracellular metabolites.
Metabolic fluxes distributions for the engineered strain during the two studied steady-states. The feasible minimal and maximal fluxes for each reaction is also reported. Metabolit labelled with the symbol [e] are considered extracellular while the symbol [c] signal the intracellular metabolites.
Contribution of the heterologous proteins to the biomass composition.
Contribution of the heterologous proteins to the biomass composition.
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