Chapter 6. Development of a mathematical model to understand the
6.2 Materials and methods
6.2.3 Reconstruction of metabolic network
Reconstruction of metabolic network forC. acetobutylicumwas carried out using C. acetobutylicum ATCC 824 genome annotated in KEGG database as the primary source along with other databases such as MetaCyc (Caspi et al., 2012) and SEED (Overbeek et al., 2005). For the purpose of gap filing, enzymes which were not found in any of the aforementioned databases were added to model based on prior literature. Fig. 6.1 shows the schematic representation of the central metabolic pathways in C. acetobutylicum. All the manually added reactions are enlisted in Table 6.1.In the present study, FBA was performed with glucose as the sole carbon source. Glucose metabolism inC. acetobutylicumis initiated through the glycolytic pathway where glucose is catabolized to pyruvate and further into acetylCoA and CO2. Conversion of pyruvate to acetyl-CoA is catalyzed by the enzyme pyruvate:
Fig. 6.1. Major metabolic pathways in C. acetobutylicum. Genes whose products were identified are labeled with the corresponding gene locus while genes with no locus are the ones whose products were not identified.
ferredoxin oxidoreductase (Jones and Woods, 1986) and is also linked with hydrogen production.
Production of hydrogen plays an important role in maintaining redox balance in clostridial spp. This involves the oxidation of ferredoxin coupled with reduction of NAD(P)+ and hydrogen production which are catalyzed by ferredoxin: NAD(P)H oxidoreductase and hydrogenase enzyme, respectively (Jones and Woods, 1986).
Till date, ferredoxin: NADH oxidoreductase is not found in any of the clostridial genomes, still it was added to the model on the basis of literature where it was proposed to play a key role as part of the redox balancing pathway (Yoo et al., 2015).
This reaction was considered reversible (Fig. 6.1), as evinced from experiments carried out by Gheshlaghi et al. (2009). Significant enzymatic activity of both NADH: ferredoxin oxidoreductase and ferredoxin: NAD reductase was observed in C. acetobutylicum. Former enzyme performs the function of ferredoxin reduction by NADH, while the latter catalyzes reduction of NAD by ferredoxin. However, in case of ferredoxin: NADPH oxidoreductase, specific activity of NADPH: ferredoxin oxidoreductase enzyme was found to be low (Gheshlaghi et al., 2009). Hence, the enzyme was considered irreversible with the main role being assumed as the production of NADPH (Jungermann et al., 1973).
Solventogenic phase in C. acetobutylicum involves acetone, butanol, and ethanol production along with acid reutilization (Jones and Woods, 1986). The enzyme acetoacetyl-CoA: acetate/butyrate: CoA transferase couples acetic acid and butyric acid reutilization with acetone formation (Jones and Woods, 1986).
However, acetone decoupled butyric acid reutilization has been reported by Desai et al. (1999) and hence based on the model developed by Shinto et al. (2007), directionality of the enzymes phosphotransacetylase-acetate kinase (PTA-AK) and phosphotransbutyrylase-butyrate kinase (PTB-BK) was considered reversible for alternate acid reutilization (Fig. 6.1). The end reactions of butanol and ethanol synthesis pathway are catalyzed by alcohol dehydrogenase, an enzyme known to have NADH and/or NADPH cofactor dependence (Gheshlaghi et al., 2009). Since the substrate requirement for generation of both the cofactors is same, NADH was considered as the common redox representative for the purpose of simplification (Villadsen et al., 2011).
Clostridial spp. generate essential precursors for nucleotide and amino acid biosynthesis via pentose phosphate pathway (PPP). These precursors can be produced via two routes, either oxidative route from glucose-6-phosphate (G6P) or non- oxidative route from fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GAP). Oxidative route of PPP was found to be absent according to13C based studies carried out inC. acetobutylicum(Crown et al., 2011) and hence the oxidative reactions were excluded from the metabolic network.
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Table 6.1. Enzymes manually added in the different biosynthesis pathways S. No Missing EC
no. Enzyme name Reaction Pathway Reference
1 1.3.5.4 Fumarate reductase Fum+RFRD=⇒OFRD+Suc' TCA Amador-Noguez
et al., 2011
2 6.2.1.5 Succinyl-coenzyme A
synthetase 'SCOA+ADP+Pi→ Suc+CoA+ATP' TCA Dash et al., 2014 3 2.7.4.6 Nucleoside-diphosphate
kinase
ATP+UDP⇔ADP+UTP’
CDP+ATP⇔CTP+ADP’
Nucleic acid
biosynthesis Dash et al., 2014
4 2.3.1.38
Acetyl coenzyme A-acyl-carrier-protein
transacylase
Acetyl-CoA+Acyl-carrier protein⇔CoA+ Acetyl-[acyl-carrier protein]
Fatty acid biosynthesis
Senger and Papoutsakis,
2008a 5 3.1.3.27 phosphatidylglycerophos-
phatase
Phosphatidylglycerophosphate+H2O⇔ Phosphatidylglycerol+Orthophosphate
Fatty acid biosynthesis
Senger and Papoutsakis,
2008a 6 1.18.1.3 NAD+-ferredoxin reductase 2 Reduced ferredoxin+NAD+ +H+ ⇔2
Oxidized ferredoxin+NADH
Hydrogen
production Yoo et al., 2015 7 1.18.1.2 ferredoxin-NADP+ reductase Reduced ferredoxin+NADP+ ⇔Oxidized
ferredoxin+NADPH+H+
Ferredoxin reductase
Senger and Papoutsakis,
2008a 8 2.7.7.39 glycerol-3-phosphate
cytidylyltransferase
CTP+sn-Glycerol 3-phosphate⇔ Diphosphate+CDP-glycerol
Glycerophospho- lipid
metabolism
Yoo et al., 2015
9 4.2.1.91 arogenate dehydratase L-Arogenate⇔L-Phenylalanine+H2O+ CO2
Amino acid biosynthesis
Senger and Papoutsakis,
2008a
Catalysis of reactions involved in non-oxidative route is carried out by transketolase and transaldolase, therefore these reactions were incorporated in the model. Lack of Rnf complex rendersC. acetobutylicumunable to derive energy during electron transfer from reduced ferredoxin to NAD and undertake substrate level phosphorylation for energy generation (Millat and Winzer, 2017). In the absence of oxidative phosphorylation, the key role of tricarboxylic acid (TCA) cycle is assumed to be the generation of essential precursor for amino acid synthesis.
13C experiments conducted forC. acetobutylicumhave shown that the TCA cycle is bifurcated from oxaloacetate towards oxidative and reductive route culminating in secretion of succinate (Amador-Noguez et al., 2010; Crown et al., 2011). With the exception of succinyl-CoA synthetase and fumarate reductase, enzymes catalyzing all the other reactions of TCA cycle are annotated in the genome (Fig. 6.1). These two genes are yet to be identified in any of the clostridial spp. (Au et al., 2014).
However, the reactions describing conversion of fumarate to succinate and succinyl- CoA to succinate were manually added in the metabolic network (Fig. 6.1) and the directionality was decided on the basis of published literature (Crown et al. 2011;
Senger and Papoutsakis, 2008a; Au et al., 2014). Certain other reactions apart from the TCA cycle, for which the genes were not found, were added manually to the network are listed in Table 6.1. Detailed list of all the reactions is provided in the appendix.