Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico

Abstract

Background: Genome-scale metabolic networks and flux models are an effective platform for linking an organism genotype to its phenotype. However, few modeling approaches offer predictive capabilities to evaluate potential metabolic engineering strategies in silico.

Results: A new method called "flux balance analysis with flux ratios (FBrAtio)" was developed in this research and applied to a new genome-scale model of Clostridium acetobutylicum ATCC 824 (iCAC490) that contains 707 metabolites and 794 reactions. FBrAtio was used to model wild-type metabolism and metabolically engineered strains of C. acetobutylicum where only flux ratio constraints and thermodynamic reversibility of reactions were required. The FBrAtio approach allowed solutions to be found through standard linear programming. Five flux ratio constraints were required to achieve a qualitative picture of wild-type metabolism for C. acetobutylicum for the production of: (i) acetate, (ii) lactate, (iii) butyrate, (iv) acetone, (v) butanol, (vi) ethanol, (vii) CO2 and (viii) H2. Results of this simulation study coincide with published experimental results and show the knockdown of the acetoacetyl-CoA transferase increases butanol to acetone selectivity, while the simultaneous over-expression of the aldehyde/alcohol dehydrogenase greatly increases ethanol production.

Conclusions: FBrAtio is a promising new method for constraining genome-scale models using internal flux ratios. The method was effective for modeling wild-type and engineered strains of C. acetobutylicum.

Figure 1

Primary central carbon metabolism of C. acetobutylicum. Cofactors consumed by each reaction are listed as (−) and cofactors produced (+) (H⁺ ions are not shown). The following enzymes are shown in bold: (LDH) lactate dehydrogenase, (PFO) pyruvate ferredoxin oxidoreductase, (FNO) ferredoxin NAD⁺ oxidoreductase, (FNPO) ferredoxin NADP⁺ oxidoreductase, (HYDA) hydrogenase, (AAD) acetaldehyde/alcohol dehydrogenase, (PTA) phosphotransacetylase, (AK) acetate kinase, (THL) thiolase, (CoAT) acetoacetyl-CoA transferase (for acetate and butyrate), (AADC) acetoacetate decarboxylase, (BHBD) β-hydroxybutyryl-CoA dehydrogenase, (CRO) crotonase, (BCD) butyryl-CoA dehydrogenase, (PTB) phosphotransbutyrylase, (BK) butyrate kinase, (BDHA) butanol dehydrogenase A, and (BDHB) butanol dehydrogenase B. The CoAT can function with either acetate or butyrate substrate; it does not require both. The AAD can catalyze three reactions in the model. These are listed as (i) AAD_1, (ii) AAD_2, and (iii) AAD_3.

Figure 2

FBA results of wild-type metabolism using iCAC490. The model was simulated given (i) a glucose uptake rate of 10 $\frac{mmol}{hr·gDCW}$, (ii) varied SPF, and (iii) constraints listed in Table 1. These results do not coincide with experimental observation.

Figure 3

FBrAtio results of wild-type metabolism using iCAC490. The model was simulated model given (i) a glucose uptake rate of 10 $\frac{mmol}{hr·gDCW}$, (ii) varied SPF, (iii) constraints listed in Table 1, (iv) unidirectional acetate and butyrate secretion by diffusion, and (v) f (rTHL) f (rPTA) = 2, (vi) f (rTHL): f (rAAD_1) = 10, (vii) f (CO₂export): f (CO₂conversion) = 5, (viii) f (rPFO): f (rLDH) = 10, (ix) f (rCoAT, butyrate):f (rCoAT, acetate) = 0.63. Results qualitatively fit wild-type metabolism.

Figure 4

The wild-type f(rCoAT):f(rBHBD) flux ratio. Knockdowns of the CoAT of (i) 25%, (ii) 50%, and (iii) 75% are shown.

Figure 5

FBrAtio predictions of solvent production using the iCAC490 model and ratios of Figure4. The model was simulated given (i) a glucose uptake rate of $10\frac{mmol}{hr·gDCW}$, (ii) varied SPF, (iii) constraints listed in Table 1, (iv) unidirectional acetate and butyrate secretion by diffusion, (v) f (rTHL): f (rPTA) = 2, (vi) f (rTHL): f (rAAD_1) = 10, (vii) f (CO₂export): f (CO₂conversion) = 5, (viii) f (rPFO): f (rLDH) = 10, (ix) f (rCoAT, butrate): f (rCoAT, acetate) = 0.63, and (x) f (rBHBD) flux ratios defined in Figure 4 as a function of the SPF. The following curves are shown: acetone (cyan), butanol (red), and ethanol (green).

Figure 6

FBrAtio predictions of solvent production using the iCAC490 model and fixed ratios. The model was simulated given (i) a glucose uptake rate of $10\frac{mmol}{hr·gDCW}$, (ii) varied SPF, (iii) constraints listed in Table 1, (iv) unidirectional acetate and butyrate secretion by diffusion, (v) f (rTHL): f (rPTA) = 2, (vi) f (rTHL): f (rAAD_1) = 10, (vii) f (CO₂export): f (CO₂ conversion) = 5, (viii) f (rPFO): f (rLDH) = 10, (ix) f (rCoAT, butrate): f (rCoAT, acetate) = 0.63, and (x) constant f (rCoAT): f (rBHBD) flux ratios of 1, 0.5, 0.1, and 0.01. The following curves are shown: acetone (cyan), butanol (red), and ethanol (green).

Figure 7

FBrAtio predictions using the iCAC490 model given various levels of CoAT and AAD knockdown. Shown are predictions of growth (x10) (blue), acetone (cyan), butanol (red), and ethanol (green) during solventogenesis (SPF = 5). The following flux ratio constraints were applied: (i) f(rTHL):f(rAAD_1), (ii) f(rCoAT):f(rBHBD), (iii) f(rPTB):f(rAAD_2), and (iv) f(rAAD_3):f(rAAD_1). The following were held constant: (i) glucose uptake rate of $10\frac{mml}{hr·gDCW}$, (ii) constraints listed in Table 1, (iii) f(rTHL):f(rPTA) = 2, (iv) f(CO₂export):f(CO₂conversion) = 5, (v) f(rPFO):f(rLDH) = 10, and (vi) f(rCoAT, butyrate):f(rCoAT, acetate) = 0.63.

Figure 8

FBrAtio predictions using the iCAC490 model at various levels of CoAT knockdown and AAD over-expression. Shown are predictions of growth (x10) (blue), acetone (cyan), butanol (red), and ethanol (green) production during solventogenesis (SPF = 5). The following flux ratios were applied: (i) f(rTHL):f(rAAD_1), (ii) f(rCoAT):f(rBHBD), (iii) f(rPTB): f (rAAD_2), and (iv) f(rADD_3):f(rAAD_1). The following were held constant: (i) glucose uptake rate of $10\frac{mmol}{hr·gDCW}$, (ii) constraints listed in Table 1, (iii) f(rTHL):f(rPTA) = 2, (iv) f(CO₂export):f(CO₂conversion) = 5, (v) f(rPFO):f(rLDH) = 10, and (vi) f(rCoAT, butyrate):f(rCoAT, acetate) = 0.63.