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Calculation of metabolic flow of xylose in Lactococcus lactis
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 103, No. 1, 92–94. 2007 DOI: 10.1263/jbb.103.92
© 2007, The Society for Biotechnology, Japan
Calculation of Metabolic Flow of Xylose in Lactococcus lactis Hitomi Ohara,1* Michiko Owaki,2 and Kenji Sonomoto2,3 R&D Center for Bio-based Materials, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan,1 Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,2 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 3 Received 20 June 2006/Accepted 4 August 2006
A circuit diagram is proposed on the basis of an analysis of metabolic pathways of lactic acid bacteria, namely, a phosphoketolase pathway and a pentose phosphate/glycolic pathway. An augmented matrix was derived from carbon balances and stoichiometries from the circuit diagram, and solved by Gaussian elimination. Results indicated that 70% of the amount of xylose was metabolized through the pentose phosphate/glycolic pathway and that the yield of lactic acid from xylose ranged from 0.34 to 0.52 (mol/mol), whereas that of pyruvic acid was more than 1.4 (mol/mol). [Key words: Lactococcus lactis, pentose phosphate/glycolic pathway, phosphoketolase pathway, xylose]
HClO4; and flow rate, 1.0 ml/min. Dry cell weight was determined from the optical density at 562 nm using a spectrophotometer (BioSpec-1600; Shimadzu, Kyoto) and a calibration curve for the relationship between dry cell weight and optical density. Cells of L. lactis IO-1 grew well with xylose as the carbon source, and produced lactic acid, formic acid, ethanol, and acetic acid (Fig. 1). The total carbon yields of lactic acid, formic acid, ethanol, and acetic acid relative to the amount of consumed xylose were 5.0 at 3 h and 1.0 at 6, 10, 12, 19, and 24 h. In other words, all the consumed xylose was metabolized to lactic acid, formic acid, ethanol, and acetic acid, except at 3 h. The metabolic pathways of L. lactis IO-1 are shown in Fig. 2 (3). This figure is not suitable for the calculation of
The technologies used to produce fuel and plastics from biomass have been developed because of diminishing oil reserves and global warming caused by the greenhouse effect brought about by carbon dioxide emission. The efficient utilization of nonedible wooden biomass is one of the most important areas of research in biotechnology. Poly(lactic acid) is highlighted because of its environmentally benign nature. About 30% of the amount of xylose is derived from wooden biomass; therefore, it is an important sugar in the production of lactic acid (1). Lactococcus lactis IO-1 (JCM7638) (2) possesses a phosphoketolase pathway and a pentose phosphate (PP)/glycolic pathway, and metabolizes xylose to L-lactic acid (3). In the PP/glycolic pathway, 3 mol of xylose yields 5 mol of lactic acid. Therefore, it is important to analyze the metabolic pathway of this microorganism, because in its pathway, there is no loss of carbon from xylose to lactic acid. The strain L. lactis IO-1 was used in this study. The medium used contained 10 g/l Bacto peptone (Difco, Detroit, MI, USA), 8 g/l beef extract (Difco), 4 g/l yeast extract (Difco), 1 ml/l Tween 80, 2 g/l K2HPO4, 5 g/l sodium acetate ⋅3H2O, 2 g/l triammonium citrate, 0.2 g/l MgSO4 ⋅7H2O, 0.05 g/l MnSO4 ⋅4H2O, and 20 g/l xylose. A 360-ml aliquot of inoculated medium was cultivated in a 1-l stirred fermentor at 37°C and an agitation speed of 400 rpm. pH was controlled at 6.7 with 5 M NaOH during cultivation. The amounts of lactic acid, formic acid, ethanol, acetic acid, and xylose were determined using an HPLC system (US-HPLC 1210; JASCO, Tokyo) equipped with a refractometer (RI-2031; JASCO). The analytical conditions used were as follows: column, Shodex (Sugar SH1011; Showa Denko, Tokyo); column temperature, 50°C; solvent used for elution, 3 mM
FIG. 1. Fermentation profile of L. lactis IO-1 with xylose as carbon source. Symbols: open circles, lactic acid; open triangles, formic acid; open squares, ethanol; closed circles, acetic acid; closed triangles, xylose; closed squares, dry cell weight.
* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)75-724-7689 92
VOL. 103, 2007
NOTES
93
and outflow at each junction is not preserved, the number of carbons is preserved. Here, fn expresses the number of carbons in each pathway and is equal to the number of molecules in the flow multiplied by the number of carbons in the molecules. For example, the following equation applies to junction A in Fig. 3: f1 = f2 + f9 + f10
(1)
Similarly, the equations for other junctions can be formed. The junctions can be classified into the following two types: “and” and “or”. The molecular rate of inflow or outflow is constant at junction “and”, whereas it is not at junction “or”. B, F, G, H, and J are “and” junctions. Concerning the inflow of molecules to G (Gin) and the outflow of molecules from G (Gout), the following equation can be formed. The number of molecules was calculated by dividing fn by the number of carbons in the molecules.
FIG. 2. Pathway of xylose metabolism in L. lactis IO-1. Junctions A–J correspond to those in Fig. 3. Abbreviations: GAP, glyceraldehyde 3-phosphate; FBP, fructose 1,6-diphosphate.
metabolites, because glyceraldehyde 3-phosphate and D-xylulose 5-phosphate have been placed at several points. Hence, the junctions A–K were set, the same letters were organized in a circuit diagram, and each pathway was named f1–f22 (Fig. 3). Although the number of molecules in the inflow
Gin: f11/7 = f13/3
(2)
Gout: f14/4 = f15/6
(3)
Similar equations for other “and” junctions can be formed in the same manner. These equations were expressed in an augmented matrix (Fig. 4). The number of carbons at each time point, calculated from Fig. 1, was subscribed to the augmented matrix (Fig. 4), and fn was derived by Gaussian elimination (Table 1). Strain IO-1 has two metabolic pathways, namely, the phosphoketolase and PP/glycolic pathways. In the phosphoketolase pathway, the yield coefficient of lactic acid per consumed xylose is less than 1.0 mol/mol, whereas the PP/glycolic pathway produces 5 mol of lactic acid from 3 mol of xylose. The numbers of carbons metabolized through the phosphoketolase and pentose phosphate/glycolic pathways were estimated by dividing f2 and f9 + f10 by dry cell weight (Fig. 5). From the slope of each line, it is observed that 70% of the amount of xylose was metabolized through the PP/glycolic pathway. The yield of lactic acid from xylose ranged from 0.34 to
FIG. 3. Modified metabolic pathway in L. lactic IO-1. fn (n = 1–22) expresses the number of carbons in each pathway and is equal to the number of molecules in the flow multiplied by the number of carbons in the molecules. CL, CF, CE, and CA are the carbon masses contained in the measured lactic acid, formic acid, ethanol, and acetic acid, respectively.
94
J. BIOSCI. BIOENG.,
OHARA ET AL.
FIG. 4. Augmented matrix derived from carbon balance and stoichiometry of flow shown in Fig. 2. A–K are the junctions shown in Fig. 2. Gin and Hin are the stoichiometric inflows at each point, and Bout, Fout, Gout, Hout, and Jout are the outflows at each point. TABLE 1. Calculation results for augmented matrix at each fermentation time Time (h) 0 3 6 10 12 19 24
f1
f2
f3
0 0 0 16 9 5 75 37 22 211 99 59 357 208 125 348 191 114 378 228 137
f4 0 4 15 39 83 76 91
f5
f6
f7
0 0 0 26 −3 16 136 −40 28 396 −87 138 574 −99 217 587 −114 209 587 −108 209
f8
f9
f10
f11
0 0 0 0 7 7 14 9 55 38 76 53 127 112 225 157 182 150 300 210 190 158 315 221 199 150 300 210
(mM carbon)
f12
f13
f14
f15
f16
f17
f18
f19
0 4 23 67 90 95 90
0 4 23 67 90 95 90
0 5 30 90 120 126 120
0 8 46 135 180 189 180
0 8 46 135 180 189 180
0 16 91 270 360 378 360
0 4 23 67 90 95 90
0 10 108 259 357 378 378
f20
f21
f22
0 0 0 7 3 3 72 36 32 172 86 85 238 119 139 252 126 138 252 126 144
tion is regulated by the NADH produced from glyceraldehyde 3-phosphate, which is converted to pyruvic acid, and is used in the reaction converting acetyl-CoA to ethanol. Note that the yield of lactic acid from xylose could be increased up to 1.4 mol/mol by increasing the NADH concentration in the cell. In this study, a method of calculating metabolic flow in L. lactis IO-1, which has a phosphoketolase pathway and a PP/glycolic pathway, and that of achieving an efficient lactic acid production from xylose were shown. REFERENCES FIG. 5. Distributions of carbon in xylose to phosphoketolase pathway ( f2) and pentose phosphate/glycolic pathway ( f9 + f10) per dry cell weight. Symbols: open circles, f2; closed circles, f9 + f10.
0.52 (mol/mol) at each cultivation time (Fig. 1), whereas that of pyruvic acid calculated as f5/3 was more than 1.4 (mol/mol). The reaction proceeded from acetyl-CoA to acetyl phosphate at 0–19 h because of the decreasing negative values of f6 (Table 1). This result indicates that the reac-
1. Ohara, H., Owaki, M., and Sonomoto, K.: Xylooligosaccharide fermentation with Leuconostoc lactis, J. Biosci. Bioeng., 101, 415–420 (2006). 2. Ishizaki, A. and Ohta, T.: Batch culture kinetics of L-lactate fermentation employing Streptococcus sp. IO-1. J. Ferment. Bioeng., 67, 46–51 (1989). 3. Tanaka, K., Komiyama, A., Sonomoto, K., Ishizaki, A., Hall, S. J., and Stanbury, P. F.: Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1. Appl. Microbiol. Biotechnol., 60, 160–167 (2002).
© 2007, The Society for Biotechnology, Japan
Calculation of Metabolic Flow of Xylose in Lactococcus lactis Hitomi Ohara,1* Michiko Owaki,2 and Kenji Sonomoto2,3 R&D Center for Bio-based Materials, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan,1 Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,2 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 3 Received 20 June 2006/Accepted 4 August 2006
A circuit diagram is proposed on the basis of an analysis of metabolic pathways of lactic acid bacteria, namely, a phosphoketolase pathway and a pentose phosphate/glycolic pathway. An augmented matrix was derived from carbon balances and stoichiometries from the circuit diagram, and solved by Gaussian elimination. Results indicated that 70% of the amount of xylose was metabolized through the pentose phosphate/glycolic pathway and that the yield of lactic acid from xylose ranged from 0.34 to 0.52 (mol/mol), whereas that of pyruvic acid was more than 1.4 (mol/mol). [Key words: Lactococcus lactis, pentose phosphate/glycolic pathway, phosphoketolase pathway, xylose]
HClO4; and flow rate, 1.0 ml/min. Dry cell weight was determined from the optical density at 562 nm using a spectrophotometer (BioSpec-1600; Shimadzu, Kyoto) and a calibration curve for the relationship between dry cell weight and optical density. Cells of L. lactis IO-1 grew well with xylose as the carbon source, and produced lactic acid, formic acid, ethanol, and acetic acid (Fig. 1). The total carbon yields of lactic acid, formic acid, ethanol, and acetic acid relative to the amount of consumed xylose were 5.0 at 3 h and 1.0 at 6, 10, 12, 19, and 24 h. In other words, all the consumed xylose was metabolized to lactic acid, formic acid, ethanol, and acetic acid, except at 3 h. The metabolic pathways of L. lactis IO-1 are shown in Fig. 2 (3). This figure is not suitable for the calculation of
The technologies used to produce fuel and plastics from biomass have been developed because of diminishing oil reserves and global warming caused by the greenhouse effect brought about by carbon dioxide emission. The efficient utilization of nonedible wooden biomass is one of the most important areas of research in biotechnology. Poly(lactic acid) is highlighted because of its environmentally benign nature. About 30% of the amount of xylose is derived from wooden biomass; therefore, it is an important sugar in the production of lactic acid (1). Lactococcus lactis IO-1 (JCM7638) (2) possesses a phosphoketolase pathway and a pentose phosphate (PP)/glycolic pathway, and metabolizes xylose to L-lactic acid (3). In the PP/glycolic pathway, 3 mol of xylose yields 5 mol of lactic acid. Therefore, it is important to analyze the metabolic pathway of this microorganism, because in its pathway, there is no loss of carbon from xylose to lactic acid. The strain L. lactis IO-1 was used in this study. The medium used contained 10 g/l Bacto peptone (Difco, Detroit, MI, USA), 8 g/l beef extract (Difco), 4 g/l yeast extract (Difco), 1 ml/l Tween 80, 2 g/l K2HPO4, 5 g/l sodium acetate ⋅3H2O, 2 g/l triammonium citrate, 0.2 g/l MgSO4 ⋅7H2O, 0.05 g/l MnSO4 ⋅4H2O, and 20 g/l xylose. A 360-ml aliquot of inoculated medium was cultivated in a 1-l stirred fermentor at 37°C and an agitation speed of 400 rpm. pH was controlled at 6.7 with 5 M NaOH during cultivation. The amounts of lactic acid, formic acid, ethanol, acetic acid, and xylose were determined using an HPLC system (US-HPLC 1210; JASCO, Tokyo) equipped with a refractometer (RI-2031; JASCO). The analytical conditions used were as follows: column, Shodex (Sugar SH1011; Showa Denko, Tokyo); column temperature, 50°C; solvent used for elution, 3 mM
FIG. 1. Fermentation profile of L. lactis IO-1 with xylose as carbon source. Symbols: open circles, lactic acid; open triangles, formic acid; open squares, ethanol; closed circles, acetic acid; closed triangles, xylose; closed squares, dry cell weight.
* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)75-724-7689 92
VOL. 103, 2007
NOTES
93
and outflow at each junction is not preserved, the number of carbons is preserved. Here, fn expresses the number of carbons in each pathway and is equal to the number of molecules in the flow multiplied by the number of carbons in the molecules. For example, the following equation applies to junction A in Fig. 3: f1 = f2 + f9 + f10
(1)
Similarly, the equations for other junctions can be formed. The junctions can be classified into the following two types: “and” and “or”. The molecular rate of inflow or outflow is constant at junction “and”, whereas it is not at junction “or”. B, F, G, H, and J are “and” junctions. Concerning the inflow of molecules to G (Gin) and the outflow of molecules from G (Gout), the following equation can be formed. The number of molecules was calculated by dividing fn by the number of carbons in the molecules.
FIG. 2. Pathway of xylose metabolism in L. lactis IO-1. Junctions A–J correspond to those in Fig. 3. Abbreviations: GAP, glyceraldehyde 3-phosphate; FBP, fructose 1,6-diphosphate.
metabolites, because glyceraldehyde 3-phosphate and D-xylulose 5-phosphate have been placed at several points. Hence, the junctions A–K were set, the same letters were organized in a circuit diagram, and each pathway was named f1–f22 (Fig. 3). Although the number of molecules in the inflow
Gin: f11/7 = f13/3
(2)
Gout: f14/4 = f15/6
(3)
Similar equations for other “and” junctions can be formed in the same manner. These equations were expressed in an augmented matrix (Fig. 4). The number of carbons at each time point, calculated from Fig. 1, was subscribed to the augmented matrix (Fig. 4), and fn was derived by Gaussian elimination (Table 1). Strain IO-1 has two metabolic pathways, namely, the phosphoketolase and PP/glycolic pathways. In the phosphoketolase pathway, the yield coefficient of lactic acid per consumed xylose is less than 1.0 mol/mol, whereas the PP/glycolic pathway produces 5 mol of lactic acid from 3 mol of xylose. The numbers of carbons metabolized through the phosphoketolase and pentose phosphate/glycolic pathways were estimated by dividing f2 and f9 + f10 by dry cell weight (Fig. 5). From the slope of each line, it is observed that 70% of the amount of xylose was metabolized through the PP/glycolic pathway. The yield of lactic acid from xylose ranged from 0.34 to
FIG. 3. Modified metabolic pathway in L. lactic IO-1. fn (n = 1–22) expresses the number of carbons in each pathway and is equal to the number of molecules in the flow multiplied by the number of carbons in the molecules. CL, CF, CE, and CA are the carbon masses contained in the measured lactic acid, formic acid, ethanol, and acetic acid, respectively.
94
J. BIOSCI. BIOENG.,
OHARA ET AL.
FIG. 4. Augmented matrix derived from carbon balance and stoichiometry of flow shown in Fig. 2. A–K are the junctions shown in Fig. 2. Gin and Hin are the stoichiometric inflows at each point, and Bout, Fout, Gout, Hout, and Jout are the outflows at each point. TABLE 1. Calculation results for augmented matrix at each fermentation time Time (h) 0 3 6 10 12 19 24
f1
f2
f3
0 0 0 16 9 5 75 37 22 211 99 59 357 208 125 348 191 114 378 228 137
f4 0 4 15 39 83 76 91
f5
f6
f7
0 0 0 26 −3 16 136 −40 28 396 −87 138 574 −99 217 587 −114 209 587 −108 209
f8
f9
f10
f11
0 0 0 0 7 7 14 9 55 38 76 53 127 112 225 157 182 150 300 210 190 158 315 221 199 150 300 210
(mM carbon)
f12
f13
f14
f15
f16
f17
f18
f19
0 4 23 67 90 95 90
0 4 23 67 90 95 90
0 5 30 90 120 126 120
0 8 46 135 180 189 180
0 8 46 135 180 189 180
0 16 91 270 360 378 360
0 4 23 67 90 95 90
0 10 108 259 357 378 378
f20
f21
f22
0 0 0 7 3 3 72 36 32 172 86 85 238 119 139 252 126 138 252 126 144
tion is regulated by the NADH produced from glyceraldehyde 3-phosphate, which is converted to pyruvic acid, and is used in the reaction converting acetyl-CoA to ethanol. Note that the yield of lactic acid from xylose could be increased up to 1.4 mol/mol by increasing the NADH concentration in the cell. In this study, a method of calculating metabolic flow in L. lactis IO-1, which has a phosphoketolase pathway and a PP/glycolic pathway, and that of achieving an efficient lactic acid production from xylose were shown. REFERENCES FIG. 5. Distributions of carbon in xylose to phosphoketolase pathway ( f2) and pentose phosphate/glycolic pathway ( f9 + f10) per dry cell weight. Symbols: open circles, f2; closed circles, f9 + f10.
0.52 (mol/mol) at each cultivation time (Fig. 1), whereas that of pyruvic acid calculated as f5/3 was more than 1.4 (mol/mol). The reaction proceeded from acetyl-CoA to acetyl phosphate at 0–19 h because of the decreasing negative values of f6 (Table 1). This result indicates that the reac-
1. Ohara, H., Owaki, M., and Sonomoto, K.: Xylooligosaccharide fermentation with Leuconostoc lactis, J. Biosci. Bioeng., 101, 415–420 (2006). 2. Ishizaki, A. and Ohta, T.: Batch culture kinetics of L-lactate fermentation employing Streptococcus sp. IO-1. J. Ferment. Bioeng., 67, 46–51 (1989). 3. Tanaka, K., Komiyama, A., Sonomoto, K., Ishizaki, A., Hall, S. J., and Stanbury, P. F.: Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1. Appl. Microbiol. Biotechnol., 60, 160–167 (2002).