The in silico reconstruction of the genome-scale metabolic networks of two human-derived L. reuteri strains was performed by implementing the AUTOGRAPH method [73] (link). This semi-automatic method combines orthology predictions with available curated metabolic networks to infer gene-reaction associations. Using this same methodology, a metabolic model was recently constructed for the type strain L. reuteri JCM1112 [56] , based on the networks of L. plantarum[74] (link), Lactococcus lactis[75] (link), Bacillus subtilis[76] (link), and E. coli[77] (link). Due to the obvious close proximity between all human-derived L. reuteri strains relative to members of different taxa, the manually curated metabolic network of JCM1112 was used as a template for the development of the genome-scale models for L. reuteri ATCC PTA 6475 and ATCC 55730. Pair-wise orthologous relationships between the query species and JCM1112 were established by comparing their genome sequences (retrieved in May 2009 from GenBank), resorting to the stand-alone version of Inparanoid (version 3.0) using BLOSUM80 as the substitution matrix [78] (link). The original gene-reaction association of the genes considered to be orthologous between the two strains was then transferred to the corresponding genes of the query species.
The fully automated version of the model was further curated by manual inspection of the list of gene-reaction associations, incorporating experimental evidence regarding carbohydrate utilization. With this purpose, the growth of L. reuteri 55730 and 6475 on different carbohydrates was measured for 24 h in LDMIII at 600 nm (OD600 nm) using commercially available sugars and well established prebiotics as previously described [79] (link). Simple carbohydrates tested consist of glucose, sucrose, lactose, raffinose, fructose, arabinose, maltose, mannose, arabinogalactan, starch and 1,2 propanediol (Sigma, St Louis, MO). Growth on following prebiotics as the sole carbon source were also tested: fructooligosaccharides (FOS, Beneo™ P95, Orafti, Belgium, 5% glucose, fructose and sucrose, degree of polymerization [DP] = 2–10), short-chain fructooligosaccharides (ScFOS, Actilight 950P, Beighin-Meiji, France, 5% glucose, fructose and sucrose, DP = 2–5), high-molecular weight inulin (Beneo™ HP, Orafti, 100% inulin, average DP = 23), galactooligosaccharides (Vivinal GOS, Friesland Food, partially dried by evaporation to form a syrup containing approximately 45% galactooligosaccharides, DP = 3–8, 15% lactose, 14% glucose, and 1% galactose).
The comparison of the newly obtained genome-scale metabolic models for L. reuteri ATCC PTA 6475 and ATCC 55730, along with the visualization of experimental data was carried out within the SimPheny™ software platform (Genomatica, Inc., San Diego, CA).
The fully automated version of the model was further curated by manual inspection of the list of gene-reaction associations, incorporating experimental evidence regarding carbohydrate utilization. With this purpose, the growth of L. reuteri 55730 and 6475 on different carbohydrates was measured for 24 h in LDMIII at 600 nm (OD600 nm) using commercially available sugars and well established prebiotics as previously described [79] (link). Simple carbohydrates tested consist of glucose, sucrose, lactose, raffinose, fructose, arabinose, maltose, mannose, arabinogalactan, starch and 1,2 propanediol (Sigma, St Louis, MO). Growth on following prebiotics as the sole carbon source were also tested: fructooligosaccharides (FOS, Beneo™ P95, Orafti, Belgium, 5% glucose, fructose and sucrose, degree of polymerization [DP] = 2–10), short-chain fructooligosaccharides (ScFOS, Actilight 950P, Beighin-Meiji, France, 5% glucose, fructose and sucrose, DP = 2–5), high-molecular weight inulin (Beneo™ HP, Orafti, 100% inulin, average DP = 23), galactooligosaccharides (Vivinal GOS, Friesland Food, partially dried by evaporation to form a syrup containing approximately 45% galactooligosaccharides, DP = 3–8, 15% lactose, 14% glucose, and 1% galactose).
The comparison of the newly obtained genome-scale metabolic models for L. reuteri ATCC PTA 6475 and ATCC 55730, along with the visualization of experimental data was carried out within the SimPheny™ software platform (Genomatica, Inc., San Diego, CA).
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