AMINO ACIDS

I. COMMERCIAL APPLICATIONS

II. PRODUCTION METHODS/TOOLS

III. PRODUCTION OF INDIVIDUAL

AMINO ACIDS
I. COMMERCIAL APPLICATIONS

 Flavor enhancer.

 Sweetener.

Special dietary foods.

 Feed additives (methionine and

lysine to soybean meal).
I. COMMERCIAL APPLICATIONS
I. COMMERCIAL APPLICATIONS
I. COMMERCIAL APPLICATIONS

 Market has grown 5-10%/year.

 Efficient fermentation technology

lowers price.

 Competes with other food

additives.

 Amino acids with largest market are

cheapest.
I. COMMERCIAL APPLICATIONS
I. COMMERCIAL APPLICATIONS
II. PRODUCTION METHODS/TOOLS

 Chemical synthesis.

 Isolation from acid hydrolysates of

proteins.

 Large volume fermentation.

II. PRODUCTION METHODS & TOOLS
 Strain development.
 Classical.
 Mutagenesis.
 Phenotype selection.
 Amino acid accumulation.
 Lysine overproducers =
resistant to chemical analogues.
 Disadvantages = reduced growth
rate and low pathway conversion
rate.
II. PRODUCTION METHODS/TOOLS
II. PRODUCTION METHODS/TOOLS
 Genomic techniques.
Rebuilding = introduction of
only necessary mutations
using genomic sequence.
Rebuilding ensures high
productivity of the mutant isolated.
 Intracellular flux analysis.
 NMR spectroscopy.
 13
C-labeled substrate.
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Glutamate.
 120,000 tons/year.
 Fermentation.
 Biochemistry.
 Glutamate dehydrogenase
(α-ketoglutarate to glutamate,
49.1kDa, multimer, high
specific activity).
 high glutamate levels in
cells.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-Glutamate.
Biochemistry.
Glutamate dehydrogenase.
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Glutamate.
 Overproduction relies on
anaplerotic reactions in C.
glutamicum for α-ketoglutarate
formation.
 Production strains.
 Treatments include
limiting biotin, adding
penicillin/ surfactant.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
L-Glutamate.
 Production strains.
 Grow oleic acid auxotroph on
oleic acid/glycerol.
 Treatments affect cell wall,
membrane
composition/ carrier or
lowers α-
ketoglutarate dihydrogenase
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
L-Glutamate.
 Production process.
 NH4+, dissolved
+
O 2, pH.
 Low NH4 & high
O2.
 Large volume fermentor
surfactant induces L-
glutamate synthesis with 60-
70% yield.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
L-Lysine
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Lysine.
 Biochemistry.
 First 2 steps of lysine
synthesis shared with
threonine, isoleucine and
methionine.
 Aspartate kinase.
o First reaction = aspartate
semialdehyde.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Lysine.
 Biochemistry.
 Aspartate kinase
o Feedback inhibited by L-
lysine & L-threonine
together.
o 2 α & β subunits with β
subunit regulated -
isolated
β subunit feedback
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Lysine.
 Biochemistry.
 Dihydropicolinate synthase
(dapA).
o Controls pathway flux =
competes with homoserine
dehydrogenase for aspartate
semialdehyde.
o Low homoserine dehydrogen-
ase activity mutants =
increased lysine production.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Lysine.
Biochemistry.
Lysine synthesis in C.
glutamicum
split for cell wall formation.
o Succinylase variant =
produces DAP for cell wall
peptidoglycan.
oDehydrogenase variant = DAP
III. PRODUCTION OF INDIVIDUALAMINO ACIDS
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Lysine.
 Biochemistry.
Export of lysine – lysE encodes
lysine carrier in C. glutamicum
o Small dimeric membrane protein
(24.5kDa).
o Catalyzes translocation
mechanism driven by change in
membrane potential.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Lysine.
 Production strains.
 Resistant to analogues –
S-(2-aminoethyl)-L-cysteine,
methyllysine and α-chlorocapro-
lactam.
 Excrete >170 g/L lysine.
Point mutations in homoserine
dehydrogenase, pyruvate
carboxylase, aspartate kinase =
overproducer.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Lysine.
 Production process.
 Carbon sources = molasses,
sucrose and starch
hydrolysates.
 Nitrogen sources = ammonium
sulfate & ammonia.
Growth factors = plant protein
hydrolysates, corn steep
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Lysine.
 Production process.
 Fermentation = limiting carbon
initiates lysine synthesis.
 Up to 170 g/L lysine.
 Recovery = HCl salt – ion
exchange, evaporation &
crystallization; L-lysine –
evaporation & filtration; sulfate
salt – spray drying & granulation.
III. PRODUCTION OF INDIVIDUAL AMINO
ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Lysine.
 Production process.
 60,000 tons/year.
 Feed additive.
 L-Threonine.
 Biochemistry.
 E. coli thrABC operon = thrA-
homoserine dehase, thrB-
homoserine kinase, thrC-threonine
synthase.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-THREONINE
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-Threonine.
Biochemistry.
 thrABC operon regulated
by transcriptional attenuation =
10- fold increase in
transcription without
attenuation.
 Producer strains.
 2 targets - prevent
isoleucine formation & high
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
L-Threonine.
 Producer strains.
 Threonine deaminase with low
isoleucine affinity.
 Strain with cloned feedback-
resistant aspartate kinase &
homoserine dehydrogenase =
high thrABC levels.
 Substrate uptake – clone scr or
csc regulons into E. coli K12 to
use sucrose.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-Threonine.
Production process.
 Fermentation using
overproducer strain.
 Mineral salts medium, glucose/
sucrose, yeast extract, NH4OH –
100 g/L, 77 h with up to 60% yield.
 Recovery- treat cells with pH/heat,
filtration & concentration,
crystallization (>90%
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
L-PHENYLALANINE.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-Phenylalanine.
Biochemistry.
E. coli or C. glutamicum.
 aroFGH encodes 3-
deoxy-D- arabino-
heptulosonate-7- phosphate
(DAHP) synthase.
Synthase controls flux of
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-Phenylalanine.
Production strains.
 Feedback-resistant DAHP
synthase (aroF or aroG) or
chorismate mutase-
prephenate dehydratase
(pheA).
 L-Tyrosine auxotrophic
III. PRODUCTION OF INDIVIDUAL
AMINO ACIDS
 L-Phenylalanine.
 Production process.
 4 stage fermentor process (2.5
days, 50.8 g/L, 27.5% yield).
o 1 = High glucose/O 2 .
o 2 = Feed glucose.
o 3 = Tyrosine consumed,
level of biomass constant,
increase in phenylalanine
produced.
o 4 =Acetic acid 
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF
INDIVIDUAL AMINO
ACIDS
L-
Tryptophan.
III. P R O D U C T I O N O F INDIVIDUAL
A M I N O ACIDS
 L-Tryptophan.
 Biochemistry.
 Fermentation with E. coli, C.
glutamicum or B.
subtilis mutants.
 Tryptophan synthase = indole-
3-glycerol phosphate + L-
serine.
  subunit of synthase
forms L- tryptophan from
indole + L- serine.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Tryptophan.
 Production process.
 E. coli trpEDCBA mutants
with deleted repressor
and attenuation region.
 10% Protein = synthase.
 Substrates = indole (petroleum
byproduct) + L-serine (sugar
beet molasses).
 75 g/L/day, high price feed
additive.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
L-ASPARTIC ACID.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Aspartic acid.
 Biochemistry.
Aspartase = converts
fumarate + ammonia (NH3)
to aspartic acid.
oTetramer (196 kDa).
oRequires divalent metal ions
for activity.
oNot thermostable.
III. PRODUCTION OF
INDIVIDUAL AMINO ACIDS
 L-Aspartic acid.
 Production process.
 Immobilized E. coli cells (1000
L columns).
o Polyacrylamide gel.
o Carrageenan
(polysaccharide matrix).
o Fumarase inactivated.
 3.4 tons/day and purified by
crystallization.
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS
III. PRODUCTION OF INDIVIDUAL AMINO ACIDS