Citrate metabolism produces various end products including acetate, diacetyl, acetoin, and butanediol. Citrate is transported into bacteria via citrate permeases and metabolized through pathways involving citrate lyase and other enzymes. This metabolism generates a proton motive force through citrate/lactate antiport and consumption of protons during decarboxylation reactions. Regulating enzymes in the pathway and combining citrate fermenters with acid producers can enhance diacetyl formation, an important flavor component in dairy products. Genetic modifications can also be used to modify citrate metabolism and increase desirable end products.

1. Citrate metabolism
and transport
Upma
Phd 2nd yr

2. Introduction
The level of citrate in milk is low (8mM).
It is an important property of some mesophilic cultures.
The products of citrate metabolism are acetate, diacetyl, acetoin, 2,3- butanediol
and co2.
Production of variety of metabolic products depend on the genus of bacteria and
growth conditions.
Ability of strains to ferment citrate and form diacetyl is important property in
many dairy products.
It contributes buttery aroma and flavour attributes in cultured butter milk, sour
cream and cheeses.

3. Metabolism

4. Citrate utilization pathway in bacteria and its end products
T = specific membrane transporter
CL= citrate lyase activity
OAD = oxaloacetate decarboxylase activity

5. Associated with
Leuconostoc sp. And
selected strains of
Lactococcus sp.
Lactococcus lactis
subsp. lactis biovar
diacetylactis is linked
with an 8kb plasmid
encode citrate
pemease (CitP).
Leuconostoc
associated with 22kb
plasmid encode CitP
and citrate lyase
Diacetyl synthesis

7. LAB could conceivably reduce all excess pyruvate to
lactate via lactate dehydrogenase, this does not
occur normally.
• Because pyruvate reduction requires NADH, which is made
during glycolysis, but not formed in citrate fermentation
pathway.
Using NADH to reduce citrate generated pyruvate
would quickly deprive cells of the NADH pool
necessary to reduce pyruvate produced during
glycolysis.
• Instead, excess pyruvate is decarboxylated by pyruvate
decarboxylase in a thiamine pyrophosphate (TPP)- dependent
reaction, and acetaldehyde-TPP formed

8. Citrate transport in lactic acid bacteria

9. Transport is mediated by
homologous citrate/lactate
antiporter citrate permease
(CitP) which is encoded by
the plasmid citP gene.
CitP have optimum activity
between pH 4.5-5.5.
It has been found that CitP
does not function at pH 7.0.
This transport leads to the
generation of a proton
motive force.
The PMF generated by
citrate metabolism enables
the LAB to withstand energy
depletion.

10. The PMF is an electrochemical gradient across the membrane involving
two components,
A membrane potential ΔΨ (inside negative) and a pH gradient
(inside alkaline for LAB).
The ΔΨ is produced during the antiport transport system
catalyzed by CitP in which one molecule of divalent citrate enters
into the cell, and one molecule of monovalent lactate is excreted.
The result of this process is the cellular uptake of one negative net
charge.

11. The ΔpH arises because of the consumption of protons during
citrate degradation.
Therefore citrate metabolism cause alkalinzation of the
cytoplasm by consumption of intracellular protons, and by
excretion of less acidic compounds.
There is also consumption of protons during decarboxylation
reactions. contributes to the cytoplasm alkalization.
Hence, the citrate metabolism plays an important role in the
regulation of the intracellular pH homeostasis necessary for the
maintenance of metabolic activities and growth.

12. • Specific transporters facilitate citrate entrance into the cells.
• The bacterial citrate transporters occur in four different families
which have been named as
Metabolite : proton
symporters (MHS)
Citrate : Cation
symporters (CCS)
2hydroxycarboxylate
transporters (2HCT)
Citrate : metal
symporters (Cit MHS)
Citrate transporters

13. It includes permeases utilized by Enterobacteriaceae during
aerobic citrate fermentations.
• These transporters are the CitH of Klebsiella pneumoniae
and the CitAs of Escherichia coli and Salmonella
typhimurium.
They are symporters, which transport one molecule of
citrate and three protons.
Metabolite : proton symporters (MHS)

14. Citrate : cation symporters (CCS)
Includes CitT of E. coli,
transports citrate under
anaerobic conditions
Involves an antiport of
citrate and succinate
(end-product
metabolism)

15. Include CitS permease of K.pneumoniae
• CitS is a symporter which uses a Na2+-gradient
to transport citrate
while Lactococcus, Leuconostoc include CitP
permeases
• CitP is responsible for the antiport of H-citrate2-
and lactate1- generating a membrane potential
2-Hydroxycarboxylate transporters (2-HCT)

16. They are associated with the aerobic metabolism of citrate.
Includes the transporters CitM and CitH of B. subtilis
Both permeases transport citrate complexed with
metal ions
CitM takes up citrate in complex with Mg2+
CitH transports the complex of citrate and Ca2+
This family also includes CitM of S. mutans , where citrate is co-
transported with Ca2+ (also Mn2+ but not Fe2+or Fe3+)
CitH of E. faecalis citrate is co-transported with Fe3+ (also Mn2+ but not
Ca2+ or Mg2+)
Citrate : metal symporters (Cit MHS)

18. Regulation of key enzyme in citrate metabolism
Citrate lyase
Lactate dehydrogenase
Pyruvate formate lyase
Pyruvate dehydrogenase
Acetolacate synthase
Acetoin / diacetyl reductase

19. • 20-fold increase in specific activity of citrate lyase when
Leuconostoc was grown in citrate-containing growth
media
• In Lactococcus lactis no regulation of citrate lyase found in
all strains
Citrate lyase
• Lactate dehydrogenase: pyruvate + NADH -~L-lactate +
NAD
• Higher the glycolysis higher will be the LDH activity and
rapid production of LA.
• During metabolism of citrate resulting in low activity of
this enzyme when citrate is present as only growth
substrate.
Lactate dehydrogenase

20. • Responsible for mixed acid fermentation at pH value near to
neutrality
• This enzyme inactivate at low oxygen levels which leads to complete
disappearance of formate and ethanol
• When pH drops below 6 no more formate produced for citrate or
lactose
Pyruvate formate lyase
• Found in aerobic and facultative anaerobic microorganism
• Consist three enzymes
Enzyme 1 for decarboxylation of pyruvate
Enzyme 2 for regeneration of cofactor
Enzyme 3 for oxidation reaction
• The activity of PDC dependent on the presence of cofactors
coA and TPP
NADH has a strong inhibitory effect on enzyme activity, in
particular enzyme 3.
Pyruvate dehydrogenase

21. • It catalyse the TPP-dependent condensation reaction of two pyruvate
molecules to the C5 component a-acetolactate with the release of carbon
dioxide.
• One pyruvate molecule is decarboxylated with TPP acting as the coenzyme,
resulting in the formation of hydroxyethyl-TPP, also called 'active
acetaldehyde'.
• This is an important regulating factor in the production of acetoin and
diacetyl, a- Acetolactate will only be produced by the lactic acid bacteria
when pyruvate is accumulated inside the cells.
Acetolactate synthase
• In lactobacilli and Lactococci the enzyme uses NADH as reducing cofactor
while in most Leuconostoc spp. NADPH is used for reduction
• When both acetoin and diacetyl are present in the reaction mixture, both
substrates compete for reduction with acetoin being the preferred substrate.
• At concentrations above 1 mM, acetoin has a inhibitory effect on enzyme
activity.
Acetoin / diacetyl reductase

22. Combination of citrate fermenting
strains with acid producing strain.
Oxygen stimulates diacetyl
production as 30-fold
Oxygen also Reduce activity of lactate
dehydrogenase.
It also increase the oxidative
decarboxylation reaction
Oxidize NADH prevent diacetyl
reduction to acetoin or 2,3 butanediol
Enhancing diacetyl formation in dairy products

23. Genetic modification
Inactivation of
lactate
dehydrogenase.
Enhanced
expression of
plasmid-born
copies of genes
coding for alpha-
acetolactate
synthase or
NADH oxidase
Inactivation of
the genes coding
for alpha-
acetolactate
dehydrogenase
which form
acetoin