Use of Microorganisms

for the Production of

Amino Acids
By:
Mubasshira
MSc-2 Food Processing and Preservation
Subject: Food Biotechnology
Subject Teacher: Ms. Trisha Ganguly
 INDEX
 Introduction
 Amino acid production: A short history
 Amino acid production process
 Amino acid producing bacteria
 L- glutamic acid
 L- lysine
 L- tryptophan
 INTRODUCTION
 What are amino Acids? (11)
 APPLICATIONS OF AMINO ACIDS (4,5,6)
 animal feed additives, flavor enhancers in human
nutrition or as ingredients in cosmetic and medical
products. (1)
 involved in the regulation of key metabolic pathways
(4).
 maximizing the efficiency of food utilization
 reducing the adiposity
 regulating the muscle protein metabolism
 controlling the growth and immunity of the
organism(5)
LIST OF ESSENTIAL AND NON- ESSENTIAL
AMINO ACIDS
Amino acid production: a Short history (2)

 In 1909, the major umami component of

‘kombu’, a kind of seaweed, is sodium
glutamate
 In 1957, direct fermentation method using
Corynebacterium glutamicum
 The discovery of l-glutamate accumulation
by a wild-type bacteria was a
breakthrough of the amino acid industry.
AMINO ACID PRODUCTION PROCESS

 Extraction from protein hydrolysates (1)

 Chemical synthesis (1)
 Enzymatic and fermentation pathways
with the aid of microorganisms (The most
common industrially used amino acid
producing bacteria Corynebacterium
glutamicum and Escherichia coli) (1)
 Process Principle Advantages Disadvantages

Extraction An amino acid can be  Large scale industrial  Few kinds of amino acids
from protein separated from the others production  Depend on the
hydrolysates present in the protein-  Can use industrial by-products availability of natural
hydrolysates if its or waste protein rich resources
properties are different  Common reagents such as  Possible protein
from the others hydrochloric acid and sodium degradation
hydroxide  By-products
 Wastewater generation

Chemical Amino acids obtained Produce achiral-amino acids  Production of racemic

Synthesis through a chemical mixtures additional
reaction optical resolution step is
necessary to obtain only
the L-forms
 Price of the catalyst
 Hazardous sources

Enzymatic Application of  Produce optically pure D and L-  Price and instability of

synthesis proteases to peptide amino acids the enzyme
hydrolysis  Very few amount of by-product  Not favorable for
 Simple process downstream production of L-amino
acids at industrial scale
Fermentation Microorganisms  Large scale industrial  Sterility has to be
convert the sugars production of most of L- amino ensured
present in a acids  Energy required for
substrate into amino  Economic method oxygen transfer and
acids  Production of L- amino acids mixing
form  High operational costs
 Mild conditions
 Low plant maintenance costs
 FERMENTATION(13,14,15)
 Fed batch fermentation
 Batch fermentation
 Continuous fermentation
 Submerged fermentation
 AMINO ACID PRODUCING BACTERIA

 The most common bacteria used for amino

acid production via fermentation, are C.
glutamicum and E. coli (1)
 Genetically modified C. glutamicum is used
to produce lysine or glutamic acid with high
yields (up to 50% w w−1), while E. coli has
been modified to enable the production of
the extremely interesting aromatic amino
acids such as L-tryptophan, L-phenylalanine
and L-tyrosine. (1)
 CENTRAL CARBON
METABOLISM(1)
 C. glutamicum  E.coli
 3 pathways: EMP  3 pathways
pathway, TCA  PPP is responsible
pathways and for the breakdown of
Pentose phosphate the carbon sources
pathway. that E. coli utilizes
 Different enzymes for the biosynthesis
are involved in the of amino acids
conversion of
carbon
STRAIN IMPROVEMENT(1)
PROCESS MONITORING OF AMINO ACID PRODUCTION
(1)

 Downstream separation
 Important parameters
and purification
 Quality of inoculum, pH,  Crucial to reduce the cost
feed rate, aeration  Separation- centrifugation
intensity and process or filtration
temperature is required  chromatographic
 Inoculum preparation is techniques (solubility,
a key step isoelectric point and
affinity to adsorbent).
-Significant loss occurs
 Membrane based
processes (good
alternative)
 Nano filtration is an
innovative technique
L- glutamic acid
 L-glutamic acid

 Important amino acid. (7)

 The turning point in the history of glutamic acid
production was the discovery of Micrococcus
glutamicus (later identified as C. glutamicum in
1957), which produces 30 g/L of L-glutamic acid
in glucose medium through fermentation. (7)
 Glutamic acid and its salt MSG represent the
largest product segment within the amino acid
market (7)
Biosynthesis of L-glutamic acid in C. glutamicum using glucose as a
carbon source.
BIOTECHNOLOGICAL APPROACH AND
PRODUCTION (7)

 The most popular Coryneform species include C.

glutamicum, Corynebacterium lilium,
Corynebacterium herculis, Brevibacterium flavum,
Brevibacterium lactofermentum, Brevibacterium
divarticum, Brevibacterium ammoniagenes,
Brevibacterium thio­genetalis, Brevibacterium
saccharoliticum, and Brevibacterium roseum
 Other glutamic acid-producing organisms include
Escherichia coli, Bacillus megaterium, Bacillus
circulans, Bacillus cereus, and Sarcina lutea
 Typical Example of submerged fermentation on
a lab scale for the production of L-glutamic acid
utilizing a mutant strain of C.glutamicum.
 Fermentation conditions are designed properly.
 Yields have increased to 100g/L with genetically
modified organisms as compared to the wild
which gives only 10g/L.
 Other methods of increasing yields- by growing
biotin auxotrophs on biotin deficient medium
L- glutamic acid production can be achieved
through immobilized cells of C.glutamicum.
(7)
Batch fermentation gives good yield(93g/L)
with low productivity (3.8g/L/h) (7)
In case of continuous fermentation, 73g/L
was recovered with high productivity
(29.1g/L/h) (7)
 APPLICATION (7)

 Flavour enhancer
 Serves as a unique brain fuel and
performs some other important function-
detoxification of ammonia, aid in peptic
ulcer healing etc.
 As a neurotransmitter
 Research paper- Effects of proteases on L-
glutamic acid fermentation (12)
●
During fermentation, some proteins can not be decomposed and utilized by

Introduction
the bacteria.
●
Causes problems of increased foam problem- low fermentation efficiency
●
Main sources of fermentation nutrients include corn syrup, soybean meal
hydrolysate. 

●
Brevibacterium flavum GDK-168 (suboptimal biotin high-yield strains

Methodology
●
Providing proper culture condition
●
10.0 g (total volume of fermentation volume of 20.0 L) of alkaline protease,
neutral protease, complex flavor protease, and trypsin were weighed separately.
●
Determination-TPC , total DO , organic acids , amino acids , glutamic acid

●
Efficiency of dissolved oxygen and improve the fermentation state. 

Conclusion
●
0.5 g/L trypsin at the initial fermentation stage was the best choice
●
the amount of ventilation and the amount of defoamer were also reduced
●
The final L-glutamic acid production reached 177.0 g/L, which was 14.9% higher than the
control fermentation (154.0 g/L), and the glucose conversion rate was 68.3%, which was
4.0% higher than the control fermentation (65.6%). 
L-Lysine
 L-Lysine
 The introduction of L-glutamic acid
fermentation using C. glutamicum had a
great economic effect in the field of L-
lysine fermentation (7)
 Efficient mutant of C.glutamicum for the
production of L-Lysine. (7)
Fig: Production of l-lysine through the conversion of its immediate
precursor DAP (two-step process) (7)
Biotechnological Approach and
Production
 Mutagenesis by protoplast fusion (8)
 Mutagenesis by Recombinant DNA
Technology (8)
 Intracellular activity (8)
 Enhancement or Overexpression of important genes for L-Lysine
formation (8)
Attenuation or switch off of genes reducing L-Lysine formation
(8)
 Homoserine auxotroph or resistant
strains (e.g. to homoserine
dehydrogenase or S-(2-aminoethyl)-
cysteine) of Coryneform bacteria (7)
Fig: 2 Regulatory pathways of l-lysine biosynthetic pathways in C. glutamicum. (7)
 Production(7)

 Organisms maintained at low temperature,

 Fermentation
 Carbon sources- Molasses and glucose
 Balance between nitrogen and carbon
source
 The production of l-lysine can be enhanced
by using the leucine fermentation liquor
(0.2%–15%) in the production medium
 Optimum Ph(7.2) and optimum temperature:
30 0 C.
Downstream processing of L-
Lysine (8)
 Ion- exchange resins
 Direct crystallization of L-lysine from
culture medium
 Evaporation and spray drying
biolys production scheme (Patent EP 533039) compared to lysine
hydrochloride production scheme (6)
INDUSTRIAL
APPLICATION(7)
 In food industry, l-lysine is used in a
number of dietary or nutritional
supplements that are popularly used by
athletes, weight lifters, bodybuilders.
 Nutritional supplements- syrups, film-
coated tablets, capsules, and powder for
instant drinks
Application of
lysine
L-Tryptophan
 L-Tryptophan
 Chemical method to produce tryptophan
was the first method used for industrial-
scale manufacturing (7)
 Bacillus subtilis, Pseudomonas
aeruginosa, and others containing
tryptophan synthase (TSase; EC 4.2.1.20)
or tryptophanase (TPase; EC 4.1.pp.1) can
be used for enzymatic reaction.
 strains of Corynebacterium and E. coli(7)
BIOTECHNOLOGICAL
APPROACH AND PRODUCTION

Tryptophan biosynthetic pathway in E. coli.(7)

 Overproduction strains-overexpressed
genes- yield increases (7)
 Produce 40.2 g/L of tryptophan on suitable
medium containing glucose as a carbon
source in 40 h (7)
 PRODUCTION (7)
 Organisms maintained in monthly transfer
schedule on NA slant supplemented with 0.5%
NaCl.
 Optimum pH. (6.8)
 30 ml of sterilized medium+ inoculum
 Flask should be kept on shaker at 200rpm for 24
hr/ 30 0 C
 Fermentation medium should contain C-source
and N-source.
 Antifoam agent- 1ml of 20% silicon RD in
deionized water.
Case study: L-Tryptophan Production
in Escherichia coli Improved by Weakening the
Pta-AckA Pathway (10)
 Use of E.coli in order to increase the yield of
tryptophan.
 Acetate- by-product of fermentation process.
 2 metabolic pathways are involved in acetate
formation-
• PoxB oxidase-catalyzes the formation
of acetate
• Pta-AckA pathway- formation of acetate
 Recombinant E. coli FB-04
 In order to reduce the acetate formation,
phosphate acetyltransferase gene (pta)
from E. coli FB-04 was deleted.
 The new strain is called FB-04(Δpta)
 Growth defect.
 Other strain- pta was replaced with
a pta variant (pta1) from E. coli CCTCC M
2016009, forming strain FB-04(pta1).
 FB-04(pta1) reached 44.0 g/L, representing a
15% increase over that of FB-04.
 APPLICATION
 Precursor for a number of
neurotransmitters (7)
 Help in the treatment of Schizophrenia. (7)
 Have potential to contribute to the therapy
of autism, CVD, cognitive function,
depression etc. (10)
 REFERENCES
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Microbial Production of Food Ingredients, Enzymes and Nutraceuticals, 385–412. doi:10.1533/9780857093547.2.385
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