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Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1,3-propanediol synthesis at high glycerol loading.

Gungormusler-Yilmaz M, Shamshurin D, Grigoryan M, Taillefer M, Spicer V, Krokhin OV, Sparling R, Levin DB - AMB Express (2014)

Bottom Line: The analyses revealed that high glycerol concentrations reduced cell growth.Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures.The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosystems Engineering, Faculty of Engineering, University of Manitoba, Manitoba, Winnipeg R3T 5 V6, MB, Canada ; Department of Bioengineering, Ege University, Izmir, Turkey.

ABSTRACT
Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

No MeSH data available.


Growth curves, pH profiles, glycerol utilization, and end-product synthesis profiles ofC. butyricumcultures with low (170 mM) and high (620 mM) initial glycerol concentrations. a) OD of cells in log scale versus pH values, b) residual glycerol concentrations versus 1,3-PDO concentrations, c) H2 and CO2 concentrations, d) lactate, acetate, butyrate and ethanol concentrations.
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Figure 1: Growth curves, pH profiles, glycerol utilization, and end-product synthesis profiles ofC. butyricumcultures with low (170 mM) and high (620 mM) initial glycerol concentrations. a) OD of cells in log scale versus pH values, b) residual glycerol concentrations versus 1,3-PDO concentrations, c) H2 and CO2 concentrations, d) lactate, acetate, butyrate and ethanol concentrations.

Mentions: The target enzymes were selected using in-house pathway tools and the NCBI C. butyricum 5521 genome sequence. The end-product synthesis patterns were then correlated with the proteomic analyses (Figure 1b, c and d). The enzymes selected for analysis (and their corresponding locus tags) included: glycerol dehydratase (CBY_0501 and 0502), 1,3-PDO dehydrogenase (oxidoreductase) (CBY_0500), glycerol dehydrogenase (CBY_3235 and 0508), dihydroxyacetone kinase (dhaK) (CBY_0505, 0506, 3691 and 3690), pyruvate-ferredoxin oxidoreductase (CBY_0233, CBY_1889 and 3642), FeFe hydrogenase (CBY_2300), acetyl CoA acetyltransferase (CBY_1290), 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045), 3-hydroxybutyryl-coa dehydratase (CBY_3041), acyl-CoA dehydrogenase (CBY_3258 and 3042), phosphate butyryltransferase (CBY_2919), butyrate kinase (CBY_2920), butanol dehydrogenase (CBY_3747 and 3751), phosphate acetyltransferase (CBY_0205), acetate kinase (CBY_0206), lactate dehydrogenase (CBY_0742, 2341 and 2757), and ethanol dehydrogenase (CBY_3753) (Figure 2 and Table 2).


Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1,3-propanediol synthesis at high glycerol loading.

Gungormusler-Yilmaz M, Shamshurin D, Grigoryan M, Taillefer M, Spicer V, Krokhin OV, Sparling R, Levin DB - AMB Express (2014)

Growth curves, pH profiles, glycerol utilization, and end-product synthesis profiles ofC. butyricumcultures with low (170 mM) and high (620 mM) initial glycerol concentrations. a) OD of cells in log scale versus pH values, b) residual glycerol concentrations versus 1,3-PDO concentrations, c) H2 and CO2 concentrations, d) lactate, acetate, butyrate and ethanol concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230902&req=5

Figure 1: Growth curves, pH profiles, glycerol utilization, and end-product synthesis profiles ofC. butyricumcultures with low (170 mM) and high (620 mM) initial glycerol concentrations. a) OD of cells in log scale versus pH values, b) residual glycerol concentrations versus 1,3-PDO concentrations, c) H2 and CO2 concentrations, d) lactate, acetate, butyrate and ethanol concentrations.
Mentions: The target enzymes were selected using in-house pathway tools and the NCBI C. butyricum 5521 genome sequence. The end-product synthesis patterns were then correlated with the proteomic analyses (Figure 1b, c and d). The enzymes selected for analysis (and their corresponding locus tags) included: glycerol dehydratase (CBY_0501 and 0502), 1,3-PDO dehydrogenase (oxidoreductase) (CBY_0500), glycerol dehydrogenase (CBY_3235 and 0508), dihydroxyacetone kinase (dhaK) (CBY_0505, 0506, 3691 and 3690), pyruvate-ferredoxin oxidoreductase (CBY_0233, CBY_1889 and 3642), FeFe hydrogenase (CBY_2300), acetyl CoA acetyltransferase (CBY_1290), 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045), 3-hydroxybutyryl-coa dehydratase (CBY_3041), acyl-CoA dehydrogenase (CBY_3258 and 3042), phosphate butyryltransferase (CBY_2919), butyrate kinase (CBY_2920), butanol dehydrogenase (CBY_3747 and 3751), phosphate acetyltransferase (CBY_0205), acetate kinase (CBY_0206), lactate dehydrogenase (CBY_0742, 2341 and 2757), and ethanol dehydrogenase (CBY_3753) (Figure 2 and Table 2).

Bottom Line: The analyses revealed that high glycerol concentrations reduced cell growth.Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures.The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosystems Engineering, Faculty of Engineering, University of Manitoba, Manitoba, Winnipeg R3T 5 V6, MB, Canada ; Department of Bioengineering, Ege University, Izmir, Turkey.

ABSTRACT
Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

No MeSH data available.