lab carbohydrate metabolism by sohail sarwar from university of lhh
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L.A.B.: Carbohydrate Metabolism
Transport of sugars across cell membrane
Catabolism of sugars for energy
Synthesis of polysaccharides
Homolactic fermentationMixed acid fermentation Heterolactic fermentation
• Pathways for glucose fermentation
• Pathways for other hexoses
• Pathways for pentoses• Pathway for citrate
• Disaccharides
Selected references
• Salminen, S. and A. vonWright. 1998. Lactic acid bacteria. Marcel Dekker, Inc., New York, NY. pp. 51-56 (sugar transport), pp. 22-43 (degradation of sugars for energy).
• Marth, E.H. and J.L. Steele (eds.). 1998. Applied Dairy Microbiology. Marcel Dekker, Inc., New York, NY. Pp. 178-187.
Sugar Transport in L.A.B.ce
llm
embr
ane
OUT
IN
ConcentrationGradient
(S1 & S2) PEP
Sugar
Sugar
H+
H+
Symport
Permease
Sugar
Sugar-PO4
PEP-PTSAntiport
Sugar1
Sugar1
Sugar2
Sugar2
Permease
PEP Pyruvate
EIIBCS
EIIAS
HPr
EI
ATP
H+
H+
ADP
F1F0 ATPase
PMF(pH + )
Energyfrom:
PEP-PTS
Sugar Transport Systems are Sugar-Specific ce
llm
embr
ane
OUT
IN
Lactose
Lactose
H+
H+
LactosePermease
Glucose
Glucose
H+
H+
GlucosePermease
Galactose
Galactose
H+
H+
GalactosePermease
Also sugar specific…… Antiport permease PEP-PTS – EIIA and EIIBC
Example: Lactose Transport
Type of Transport System for a Sugar is Species Specific
PEP-PTS:LactococcusLactobacillus casei
Antiport (lactose/galactose):S. thermophilusLactobacillus delbrueckii subsp. bulgaricus
SymportLactobacillus helveticusLactobacillus acidophilusLeuconostocPediococcus
Lactose
Lactose
H+
H+
Lactose
Lactose
Galactose
Galactose
Lactose
Lactose-PO4
Homolactic Fermentation of Glucose (Embden-Meyerhof Pathway)
Glucose
Fructose-1,6-diphosphate
ATP
ADP
(2) 3-Phosphoglycerate(2) ATP
(2) ADP(2) 1,3-Diphosphoglycerate
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) 2-Phosphoglycerate
(2) Phosphoenolpyruvate (PEP)
(2) H2O
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
Products:2 ATP2 Lactate
Key enzymes:FDP aldolaseLactate dehydrogenase
(2) Lactate(2) NAD+
(2) NADH + (2) H+
Lactate dehydrogenase (LDH)
cofa
ctor
reg
ener
atio
n
O-P-OO
O
phosphate groupGlucose-6-phosphate
ATP
ADP O-P-OO
O
Fructose-6-phosphate
Pyruvate = key intermediate
(Glycolysis + LDH)
Two roles for PEP
Transport (PEP-PTS) or ATP generation
Mixed Acid Fermentation: Alternative endproducts for pyruvate
Glucose
(2) ATP
(2) ADP
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
2 ATP
2 ADP
(2) Lactate
(2) NAD+ (2) NADH + (2) H+
(LDH)Homolactic
cofa
ctor
reg
ener
atio
n
ATP
ADP
Acetate
Acetyl-phosphate
(2) Acetyl-CoA
Acetaldehyde
Ethanol
NAD+
NADH + H+
(2) Formate(2) CoA
NAD+
NADH + H+
CoAH2PO4
-
CoA
Products:3 ATP 2 Formate1 Ethanol1 Acetate
Key enzymes:FDP aldolasePyruvate formate lyase (PFL)
Homolactic vs. Mixed Acid Fermentation
Homolactic fermentation prevails when glucose is abundant.
Mixed acid fermentation prevails when sugars are limited – “semi-starvation”.
Substrate availability and the nature of the substrate determine which pathway is used…..
Mixed acid fermentation prevails during growth on galactose as the primary sugar source.
Homolactic fermentation prevails under aerobic conditions because the pyruvate formate lyase (PFL) enzyme is oxygen sensitive.
Glucose
Glucose-6-phosphate
ATP
ADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATP
ADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATP
ADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose (Pentose Phosphate Pathway)
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 CO2
1 Lactate1 Acetate
Key enzymes:PhosphoketolaseLactate dehydrogenaseNADH oxidase
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphate
Phosphoketolase
ATP
ADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
--- Aerobic conditions
Glucose
Glucose-6-phosphate
6-phospho-gluconate
Ribulose-5-phosphate
Glyceraldehyde-3-phosphate
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATP
ADP
ATP
ADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATP
ADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose --- Anaerobic conditions
NAD+
NADH + H+
Xylulose-5-phosphate
H2PO4-
NAD+
NADH + H+CO2
Acetyl-phosphate
Acetyl-CoA
Acetaldehyde
Ethanol
NAD+
NADH + H+
H2PO4-
CoA
NAD+
NADH + H+
CoA
Products:1 ATP1 CO2
1 Lactate1 Ethanol
Key enzymes:PhosphoketolaseLactate dehydrogenase
Phosphoketolase
Differentiation of L.A.B. Genera
Hexoses other than glucose
Fructose, mannose and galactose enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization and phosphorylation steps
Glucose
Glucose-6-phosphate
Fructose-6-phosphate
HOMOLACTIC &
MIXED ACID
Glucose
Glucose-6-phosphate
6-phospho-gluconate
HETEROLACTIC
Galactose
Galactose-1-P
Glucose-1-P
Fructose
(when galactose is transported by permease – Leloir – next slide)
Galactose metabolism pathway depends on transport system used
GalactoseH+
H+
GalactosePermease
Galactose
Galactose
Galactose-6-PO4
PEP
PyruvateEIEI
LeloirPathway
Galactose-1-PO4
Glucose-1-PO4
Glucose-6-PO4
ATP
ADP
Homolactic, *mixed acid, or heterolactic pathway
Tagatose-1,6-diPO4
ATP
ADP
(2) Glyceraldehyde- 3-phosphate
Dihydroxyacetone-phosphate
TagatosePathway
Tagatose-6-PO4
Homolactic or *mixed acid pathway
* Mixed acid fermentation dominates if galactose is the most abundant sugar available
Some L.A.B. cannot metabolize galactose
Lactose
Lactose
Galactose
Galactose
Permeasecell
mem
bran
e
OUT
IN
Example: S. thermophilus and Lb. delbrueckii subsp. bulgaricus
Galactose is exported via antiport system
Glucose
Homolactic, mixed acid or heterolactic
pathway
Disaccharides: broken into monosaccharides before metabolized
sucrose maltoselactose
galactose glucose
glucose fructose glucoseglucose
Lactose Breakdown: depends on transport system
+
+
PO4 PO4
-galactosidase
Phospho--galactosidase
Homolactic, mixed acid, or heterolactic
pathway
Homolactic, mixed acid, or heterolactic
pathway
Tagatose pathway
Leloir pathway
Summary of Lactose Metabolism in L.A.B.
How many ATPs from one lactose?
LactococcusLb. casei
*S. thermophilus, *Lb. delbrueckii, Lb. Helveticus, Lb. lactis
* S. thermophilus, and Lb. delbrueckii do not metabolize the galactose part of lactose. They export galactose from the cell.
LeuconostocGroup III Lactobacillus
(Figure from Fox et al. 1990. Critical Reviews in Food Science and Nutrition. 29:237-253.)
Tagatose Pathway Homolactic Pathway
Leloir Pathway
Heterolactic Pathway
-galactosidasephospho--galactosidase
CO2
Pentoses
Pentose
Pentose
Pentose-PO4
Xylulose-PO4
orRibulose-PO4
Heterolactic fermentationpathway
ATP
ADP
isomerization
Pentoses cannot enter the homolactic or mixed acid pathways
Glucose
Glucose-6-phosphate
ATP
ADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATP
ADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATP
ADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation: Pentose-PO4 entry
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 Lactate1 Acetate
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphate
Phosphoketolase
ATP
ADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
L.A.B. groups based on fermentation pathways
• Obligately homofermentative
• Obligately heterofermentative
• Facultatively heterofermentative
Obligately homofermentative
Group I Lactobacillus species and a few other species
• Hexoses are fermented by homolactic fermentation pathway (glycolysis + LDH)
• Can do mixed acid fermentation of hexoses under certain conditions
• Do not ferment pentoses
• Have FDP aldolase enzyme
• Do not have phosphoketolase enzyme
Homolactic Fermentation of Glucose (Glycolysis + LDH)
Glucose
Fructose-1,6-diphosphate
ATP
ADP
(2) 3-Phosphoglycerate(2) ATP
(2) ADP(2) 1,3-Diphosphoglycerate
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) 2-Phosphoglycerate
(2) Phosphoenolpyruvate (PEP)
(2) H2O
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
Products:2 ATP2 Lactate
Key enzymes:FDP aldolaseLactate dehydrogenase
(2) Lactate(2) NAD+
(2) NADH + (2) H+
Lactate dehydrogenase (LDH)
cofa
ctor
reg
ener
atio
n
O-P-OO
O
phosphate groupGlucose-6-phosphate
ATP
ADP O-P-OO
O
Fructose-6-phosphate
Glucose
Glucose-6-phosphate
ATP
ADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATP
ADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATP
ADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose --- Aerobic conditions
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 CO2
1 Lactate1 Acetate
Key enzymes:PhosphoketolaseLactate dehydrogenaseNADH oxidase
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphate
Phosphoketolase
ATP
ADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
Obligately heterofermentative
Group III Lactobacillus species, Leuconostoc, Oenococcus
• Hexoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Pentoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Have phosphoketolase enzyme
• Do not have FDP aldolase enzyme
Facultatively heterofermentative
Group II Lactobacillus species, Lactococcus, Pediococcus, Streptococcus thermophilus
• Hexoses are fermented by homolactic fermentation pathway (glycolysis + LDH)
• Can do mixed acid fermentation of hexoses under certain conditions
• Pentoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Have both FDP aldolase and phosphoketolase enzymes
Lb. rhamnosus
Lactobacillus
Homolactic and facultatively heterolactic – no CO2 from glucose, FDP aldolase presentObligately heterolactic – CO2 from glucose, phosphoketolase present
Lb. sanfranciscoLb. kefir
Citrate Transport
CitrateH+
CitrateH+
CitratePermease
Citrate permease is pH dependent – only functions pH 5 – 6. Optimum = pH 5.2
CH2 - COOH
CH2 - COOH
HO - C - COOH
~1.5 mg/ml citrate in milk
Citrate Metabolism
NAD+
NADH + H+
NAD+
NADH + H+
AcetateCO2
Leuc. mesenteroides subsp. cremorisLc. lactis subsp. lactis biovar. diacetylactis
citrate lyase
CH2 - COOH
CH2 - COOH
HO - C - COOH
Pathway does not generate ATP, but regenerates NAD+.
citrate
CH3 – C – C – CH3
O O
Lactate Export
Sugar Lactate-
Malate-
Antiport
Symport
Lactate-2H+
2H+
(Not common)
Exopolysaccharide Synthesis
Some strains of Lc. lactis, S. thermophilus, Leuconostoc, Lactobacillus
Exopolysaccharide types and applications
• Capsular: yogurts (thickener), cheeses (increase moisture)
• Ropy: some fermented milks such as viili and långfil
(Perry et al. 1997. Journal of Dairy Science. 80:799-805.)
L.A.B.: Carbohydrate Metabolism
Transport of sugars across cell membrane
Catabolism of sugars for energy
Synthesis of polysaccharides
Homolactic fermentationMixed acid fermentation Heterolactic fermentation
• Pathways for glucose fermentation
• Pathways for other hexoses
• Pathways for pentoses• Pathway for citrate
• Disaccharides
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