Survey Report on the Systematization of Amino Acid
Fermentation Technology
Shigeru Nakamori
Abstract
Amino acids are the building blocks of proteins. There are 20 kinds of amino acids; these have been found to have various biological andchemical functions. They are used many useful applications, including seasoning, medicines, supplement, feed-additives, chemicals, andcosmetics. While there are three methods currently used to produce amino acids, namely hydrolysis from protein, chemical synthesis, andfermentation, the dominant method is fermentation. Amino acid fermentation, in which microbes are used to produce amino acids, originated anddeveloped in Japan; this technology is now used to supply over half of the world’s amino acid market. This report provides a systematic survey ofamino acid fermentation technology, tracing its history and outlining the prospects for future technological developments.
Monosodium glutamate (MSG) was the first amino acid to be commercialized. In 1908, Kikunae Ikeda invented a method of producing thisamino acid from gluten, wheat protein. It was released as a seasoning by Saburōsuke Suzuki under the brand name of “Aji-no-moto.” While thisenterprise succeeded despite much difficulty, further technological improvements were needed due to issues with securing raw materials andworking with hot hydrochloric acid. These improvements were taken from two approaches: chemical synthesis and fermentation. Although ameans of producing glutamic acid by chemical synthesis was established to the point of factory production, chemically synthesized glutamic acidwas not well received by consumers. Meanwhile, researchers at Kyowa Hakko discovered a new glutamic-acid-producing bacterium,
Corynebacterium glutamicum, resulting in the first successful fermentative production of glutamic acid. Amino acids produced by fermentation
were characterized in that a natural form of amino acid could be produced from a low-cost raw material such as glucose through a reaction inmild conditions. Glutamic acid was followed by over 15 more amino acid products produced by fermentation, including lysine, aspartic acid, andthreonine. Amino acid fermentation technology spread throughout the world, with an increase in amino acid production volume. MSG production
reached 1.7 million tons worldwide in 2005, with an expected annual increase of 3-4 %.
While there are four elements to amino acid fermentation technology, namely breeding of producing strains, large-scale cultivation methods,
separating/refining, and engineering, the fundamental element is the breeding producing strains. Microbe cells have a mechanism or metabolic
regulation operating within them that governs the production of metabolites like amino acids. Accordingly, breeding amino-acid-producing
strains involves applying science and technology from biochemistry, genetics and new genetic engineering techniques to overcome this metabolic
regulation.
The following specific methods have been achieved to break down or bypass the metabolic regulation. Glutamic acid fermentation was
accomplished by culturing C. glutamicum under the optimum conditions for its characteristics. Lysine and ornithine fermentation was developed
by deriving auxotrophic mutants from C. glutamicum to reduce the concentration of threonine or arginine, which have a metabolic regulation
effect. Many strains producing threonine, lysine, tryptophan, and other amino acids were developed by selecting amino acid analog resistantstrains. Other methods developed include bioreactor and enzymatic production using microbes with enzymes that convert to amino acids fromother cheaply available materials. The application of new genetic engineering technology has resulted in improvements in producing strains, forboth fermentation and enzymatic approaches, and new fermentation production.
Large-scale cultivation for amino acid production was established based on cultivation methods for antibiotics production. Isolating and purifying
the amino acids from the fermentation fluid is an important part of the process, not only because it determines the quality of the product, but also
because the yield at this stage has a significant impact on the cost. Computer-controlled systems have been developed to handle the engineeringof large-scale cultivation and large-scale purification.
Amino acid production plants using Japanese technology have spread throughout the world. The raw materials used include starch, raw sugar, or
molasses. Therefore, amino acid production is strongly linked to the availability of raw materials. Most amino acids that are consumed overseas,such as glutamic acid, lysine, and threonine, are produced at overseas factories.
Amino acid fermentation processes are natural processes, such as those used at brewing plants for sake or soy sauce. Environmentally-friendly
systems are being put in place. Grains and other livestock fodder are lacking in certain amino acids; adding these amino acids to livestock fodder
is useful in not only improving the amino acid balance of foods, but also reducing ammonia emissions from animal waste and improving theenvironment.
The 1970s saw consumer action over the safety of amino acids, but it was proven scientifically that this was not an issue. Nevertheless, the“aftereffects” of this action still linger today, with deep-seated concern over the safety of amino acids.
Future developments can be expected in the field of amino acids, as there are no alternative products to them. A significant issue will be the rising
price of raw materials; technology and price competition against overseas manufacturers with a secure supply of raw materials will be a major
challenge for Japanese engineers, although they invented the technology. To meet this challenge, it will become all the more important to breedstrains for improved production using genetic information and genetic engineering, to develop new raw materials and to cultivate strains that canuse these raw materials, and to develop environmentally-focused processes. Technological developments can be expected in these areas.
Technology has recently been developed to synthesize peptides from amino acids using microbial enzymes. Use of this technology can be
expected to extend to pharmaceuticals and other bioactive compounds.

Profile
Shigeru Nakamori

Contents

Born September 1940
March 1963:
April 1963:
January 1975:
March 1993:
April 1993:
April 2006:

1
2
3
4

Introduction ........................................................................... 2
About Amino Acids ............................................................... 3
Origins of Amino Acid Fermentation ..................................... 8
Origins of Amino Acid Fermentation:Amino-Acid-Producing
Bacteria Screening and Strain Breeding ............................... 11
Graduated from Kyoto University Faculty of
5 Development of Cultivation Technology and Equipment..... 23
Agriculture, Department of Agricultural Chemistry 6 Development of Amino Acid Separation and Purification
Employed by Ajinomoto Co., Inc.
Technology .......................................................................... 25
PhD in Agriculture
7 Amino Acid Standards ......................................................... 27
Retired from Ajinomoto Co., Inc.
8 Main Raw Materials for Amino Acid Fermentation ............. 28
Professor, Fukui Prefectural University Faculty of 9 Overseas Expansion of Amino Acid Fermentation Technology
Biotechnology............................................................................................. 29
Professor Emeritus, Fukui Prefectural University
10 Amino Acids and Environmental Issues .............................. 31
11 Safety of Amino Acids ......................................................... 32
12 Systematization of Amino Acid Fermentation Technology .. 34
13 Future Developments ........................................................... 36
14 Acknowledgements .............................................................. 38

Chief Survey Officer, Center of the History of Japanese Industrial
Technology, National Museum of Nature and Science1 Introduction
Amino acids are the building blocks of proteins. Evenas simple substances, these have many useful functions
and are widely used to benefit human welfare
throughout the world, as flavor enhancers or as
nutritional components in pharmaceutical products oranimal feed. While amino acids have gained recent
media attention and come into the awareness of thegeneral public, it is not widely known that amino acidtechnology, particularly the fermentation process usedat the core of the production technology, originated inJapan or that it was Japanese technology that grew it
into a world-leading industry, accounting for over halfof the world’s production output. No other substancecan replace the functions of amino acids. Accordingly,
the industry can be expected to develop further, withfurther increase in demand inevitable. In light of thedevelopment that has taken place in the 50 years sinceamino acid fermentation began in Japan, it is verysignificant to anticipate the prospects of future2

development.
Chapter 2 of this report discusses what kind of
substance amino acids are, what functions they have,
how they are used, the progress in production methods,
and changes in production output, as well as providing
an overview of amino acid fermentation technology,
the main topic of this report. Chapter 3 outlines thefactors leading up to the emergence of amino acidfermentation in Japan. Chapter 4 and onwards
discusses the history and current state of the four basic
technologies for amino acid fermentation, namely
screening and breeding producing strains, large-scalecultivation, separation/purification, and engineering, as
well as systematizing the technology and examining
the future and prospects.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

2 About Amino Acids
protein (= peptides)

2.1

What are Amino Acids?

Amino acid is a general term for chemical compoundswith an amino group (-NH2) and a carboxyl group(-COOH) in the same molecule. While there aredifferent amino acids such as α, β, γ, etc., with thecarbon in different positions in the amino and carboxylgroup (Fig. 2.1), the α-amino acids are the mostcommon and act as a structural component in theprotein in all organisms. This report discusses α-aminoacids (little mention is made of β-, γ- or other aminoacids, as little substantive development has been madeon them at this stage).
α carbon(a) α-amino acid

β carbon(b) β-amino acid

(hydrolysis)Fig. 2.2. Formation of Amino Acids due to Hydrolysis of Protein(= Peptides)
The position of the arrows represents the severing of the peptide bond.

Since there are theoretically 20 kinds of amino acids
contained in the hydrolyzed solution obtained byprocessing proteins with hydrochloric acid or enzymes
in a reaction vessel, each amino acid could be obtainedby separating and purifying these. While these would
appear difficult to differentiate from external
appearance due to being in white crystal (or powder)
form, the crystals have distinctive characteristics whenviewed under a microscope. Glutamic acid crystals andlysine crystals are shown here as examples (Fig. 2.3).

α carbon(c) γ-amino acid

Fig. 2.1.α, β and γ-Amino Acid Structural Formulas
These structures differ according to the different amino group binding
positions; R is a chemical side chain and represents an atomic group orhydrogen atom.

α-Amino acids are structural components in protein;
their chemical formula can be represented as shown in
Fig. 2.1 (a). There are 20 kinds of amino acids inordinary protein, depending on differences in R. The
names, abbreviations, single-letter designations,structural formulas, molecular formulas, and molecularweights are given in Table 2.1. Of these, isoleucine,
threonine, tryptophan, methionine, phenylalanine,
valine, lysine, and leucine cannot be synthesized denovo by humans, and so are called essential aminoacids.
Amino acids rarely exist in an isolated state in an
organism, instead existing as high molecular protein inwhich the amino group in one amino acid links to thecarboxyl group in another amino acid throughdehydrated and condensed peptide bonds. As iswell-known, proteins are an essential component of
biological functions, forming the muscles, blood, bones,
skin, nails, and other tissue in an organism, as well as
the hormones, or regulatory substances, and enzymes,
which act as biocatalysts. If a protein is processed withhydrochloric acid or a proteolytic enzyme, the peptide
bond hydrolyzes, resulting in amino acid (Fig. 2.2). In
practice, proteins ingested as part of the diet are broken
down through the action of pepsin, trypsin,chymotrypsin, and other proteolytic enzymes in thestomach, small intestine, and other parts of an animal’s
alimentary canal. The various amino acids produced asa result re-form into specific proteins needed by the
animal.

Fig. 2.3 (1) Photograph of Glutamic Acid (α) Crystals (suppliedby Ajinomoto Co., Inc.)

Fig. 2.3 (2) Photograph of Lysine Hydrochloride (supplied by
Ajinomoto Co., Inc.)

Survey Report on the Systematization of Amino Acid Fermentation Technology

3Table 2.1 Classification of Proteinous Amino Acid, Notation, Chemical Structure, Molecular Formula, andMolecular WeightClassification

Name

SymbolChemical Structure

Molecular Formula

Glycine
AlanineValineAliphatic amino
acids
LeucineIsoleucine

SerineHydroxyamino
acids
Threonine
Cysteine

Sulfur amino
acids

Neutralamino acids

Cystine

Methionine

Phenyl-alanine

Aromatic amino
acids

Tyrosine

Tryptophan

ProlineImino acids
Hydroxyproline

Asparagine
Acidic amino acidamidesGlutamine

Aspartic acid
Acidic amino acids
Glutamic acid

LysineBasic amino acids

Histidine

Arginine

4

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

Molecular WeightCarbon atoms that attach to four different atoms or
atom groups, such as the amino group, the carboxylgroup, and R (atom group or hydrogen atom) in theamino acid, are called asymmetric carbon atoms. When
exposed to polarized light, chemical compounds withasymmetric carbons, such as amino acids, rotate theplane of polarized light at a constant angle to the left or
to the right. Some compounds are L-compounds andothers are D-compounds; all the natural amino acids inprotein are L-compounds.
All amino acids produced by fermentation, the main
topic of this report, are L-compounds. While this paper
focuses on L-amino acids in principle, D-amino acids
will sometimes be mentioned in a limited context. By
contrast, amino acids obtained by chemical synthesis
are DL-compounds (or racemic compounds)
comprising an equal mixture of enantiomers D- andL-compounds. To obtain a biologically-active
L-compound from a DL-compound requires an opticalresolution process. While a small quantity of D-aminoacid has recently been found in the brain and otherorgans, its purpose and function are still in the processof being identified, so it is not touched on in this report.

2.2

Functions and Uses of Amino Acids

While amino acids play an important role in organisms
as the building blocks of proteins, what function dothey serve? In other words, what utility value do they
have? This utility value can be represented as thecommodity value of amino acids.
The first amino acid to be developed into a commercial
product was monosodium glutamate, or MSG. Kikunae
Ikeda, a professor of the Tokyo Imperial University
College of Science, discovered MSG as the flavor
component of kombu and invented a method of
producing it from wheat gluten.(1) It was released by
Ajinomoto Co., Inc. under the name of “Ajinomoto”
and continues to be a major product used around theworld. The other amino acids produced as byproducts
to glutamic acid initially received no particularattention, but their importance was later recognizedwhen they were discovered to have various functions in
nutrition and medicine as those fields advanced. They
have continued to expand in use as the below
applications for them have developed. To
accommodate these applications, there has been a needto find a cheap production method for these aminoacids as well as glutamic acid.
The uses for amino acids can be broadly divided into
the following four categories according to function.(2)1) Use as a Flavor Component
Amino acids each have a specific flavor. The mostrepresentative example of these is MSG, which is
produced on a large scale as a flavor enhancer. Glycine

and alanine are sweet, while valine and isoleucine arebitter. Mixtures of amino acids are also used forseasoning. Aspartame, a dipeptide made up of aspartic
acid and phenylalanine methyl ester, has also beendeveloped as a low-calorie sweetener.
2) Use as a Nutritional Component
Amino acids are used as a nutritional component.
Amino acid infusion and enteral nutrition involve
dispensing a pharmaceutical product made up of a
combination of effective amino acids to match theamino acid composition of a protein such as bloodplasma, ovum, or milk to perioperative patients fornutritional support, fatigue recovery or to improvestrength. Amino acids have also been used as
nutritional fortifiers or food additives in health foods,
sports nutrition products, and supplements, as well as
in cosmetics. Many other applications have beendeveloped, such as for improving the nutritional qualityof the proteins in feed grain for pigs and chickens or asa medium component for culturing zooblasts or
producing microbial enzymes. Combinations ofeffective amino acids are used in these applications
rather than amino acids on their own.
3) Use in Pharmacological Functions
Amino acids also have particular pharmacological
functions and are used in pharmaceutical products,
including liver disease treatments (isoleucine, valine,
leucine, arginine, etc.), digestive organ ulcer treatments(glutamine), Parkinson’s disease treatments(dioxyphenylalanine), and mucolytic agents (cysteine).
4) Uses for Amino Acid Reactivity
Amino acids also have a particular reactivity as
chemical compounds and are used in the following
applications: synthetic pharmaceutical materials, suchas diabetes medicines (D-phenylalanine),anti-hypertensive agents (hydroxyproline, valine),
antiviral agents (valine), and antibiotics(D-phenylglycine, D-p-hydroxyphenylglycine), as wellas production materials, such as reaction flavors(cysteine), bread fermentation agents (cysteine),surfactants (N-acylglutamic acid), and perm solutions(cysteine). They have also been used in moisturizing
products (proline). Amino acids also constitute the raw
material for producing newly-developed peptides.

2.3

How are Amino Acids Produced?

The MSG production method invented by Kikunae
Ikeda, mentioned in Section 2.2, was a proteinhydrolysis method involving hydrolyzing wheat gluten
or soy protein in hydrochloric acid and then separating
and purifying the MSG from the resulting solution.
While this method of production continued for around50 years until about 1960, when the fermentation
Survey Report on the Systematization of Amino Acid Fermentation Technology

5method was established, it had a few technical areasthat needed improvement, as shall be mentioned in
Section 3.1. These obstacles were surmounted in a newchemical synthesis method that was developed, as wellas in the fermentation method, which is the main topic
of this report. While the fermentation method is themain method currently used to produce glutamic acidand many other amino acids, each method has its own
advantages and disadvantages and all three methods arestill in use today. The main factors for selecting a
production method are cost and the image that method
projects to consumers, since amino acids are mainly
used in food and other internal applications. Aminoacids produced by chemical synthesis include glycine,which has no enantiomers, and methionine, which has
both D- and L-compounds and can be used in animal
fodder, while tyrosine, leucine, cysteine, andasparagine are produced by protein hydrolysis.
The main raw materials for protein hydrolysis are soyprotein and casein; for chemical synthesis, the main
raw materials are ethylene nitrile compounds andammonia; the main raw materials used for thefermentation method are starch solutions (saccharified
starch solutions), molasses, or raw sugar.

2.4

Amino Acid Production Quantities

The Japan Essential Amino Acids Association (nowabolished) has published worldwide amino acidproduction quantity estimates every few years. Fig. 2.4(3)shows the figures from this data for 1979 and 1996.
As indicated by the graphs, there is a clear significant
increase in production quantities of all amino acids
except for DL-alanine. Glutamic acid has a particularly
notable increase in quantity, with 1 million tons in
1996 rising to 1.7 million tons by 2005, visibly
increasing by a few per cent per annum. As a single
chemical compound, it is an unprecedented productthat has had continued growth since its inception 100
years before. While other epochal chemical compounds
have emerged and been commercialized around theworld, such as ammonium sulfate, urea, penicillin, andnylon, none of these have demonstrated such stable
growth for such a long period of time. The significant
value of glutamic acid is worth noting.
Other amino acids that have undergone significant
growth in production quantity include lysine, used inanimal fodder, with around 300,000 tons produced in
1996 rising to 800,000 tons in 2005, threonine, also
used in animal fodder, which increased from onlyaround 4,000 tons in 1996 to 80,000 tons in 2005, andtryptophan, which is estimated to have increased from500 tons to 1,800 tons. Phenylalanine, used as a main
ingredient in the low-calorie sweetener aspartame, andaspartic acid have also shown remarkable growth.
Other amino acids have also increased in production
quantities as pharmaceutical products and supplements.

6

1979

1996

Fig. 2.4 Global Amino Acid Production Quantity Estimates (3)2.5Amino Acid Safety

While amino acids are the building blocks of proteins
and were not considered to have any safety issues, inthe 1970s there was a worldwide wave of concern overthe safety of chemical compounds used in food andmedicine and amino acids were caught up in thesesafety concerns as well. For example, there werereports of brain disorders in juvenile mice resulting
from being force-fed MSG and other reports of theso-called “Chinese restaurant syndrome” occurring as aresult of ingesting MSG added to Chinese cuisine.
Nevertheless, earnest endeavors by mainly Japanesemanufacturers established scientific evidence that theseconcerns were unfounded. Open and impartial
discussions resulted in the USFDA and other agencies
concluding that there were no safety issues and issuinga scientific “safety declaration.” However, the reality
remains that there are many people around the world
who do not believe that these are safe. In the 1980s,
there were fatalities in the United States believed to be
due to a tryptophan degradation product contained in

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

health foods, demonstrating the importance of checking
the ingredients in an amino acid product.

2.6

What is the Amino Acid Fermentation
Process?

The development of new applications and the decreasein costs due to improved production technology havemade a significant contribution to the increase in aminoacid production quantities shown in Section 2.4. Whileamino acids are essential substances, they are not a
product that will always have a demand regardless ofthe price. Rather, the skillful combination of two
factors – cost reduction due to improved production
technology and the development of new applications –has resulted in increased demand.
Amino acid production technology includes thetechnology used for protein hydrolysis, fermentation,and chemical synthesis, mentioned in Section 2.3, as
well as related technologies for separating, purifying,and engineering. Currently, the predominant production
method is fermentation, the main topic of this report.
Fig. 2.5 shows an overview of the amino acidfermentation process. The main technologies involved
are for strain breeding, large-scale cultivation,
separation/purification, and engineering. Of these,strain breeding is the fundamental technology thatplays the greatest role in characterizing the amino acidfermentation.
producingstrainstrainbreeding

large-scalecultivation

production
mediumculturing
methodseparation andpurification

finishedproduct

separation andpurificationengineering

Fig. 2.5 Overview of Amino Acid Fermentation and Main
Related Technologies

Strain breeding is the art of manipulating the cells
involved in controlling the course of metabolism within
a tiny cell, several micrometers in size, which takes thesubstrate (raw material) glucose and forms it into theresulting product amino acid, and secreting the aminoacid outside of the cell. Specifically, this involves
making repeated improvements to increase amino acidproduction by identifying the metabolic pathway forthe amino acid and using cell mutation and genemanipulation to prevent metabolic regulation andthereby enable more effective production properties.
There is usually no outwardly discernable difference
between newly-obtained strains and parent strains.
Large-scale cultivation technology involves placingthese resulting microbes into the optimum conditions
where they will multiply at a rate of 1016-17 and produce
the maximum quantity of amino acid.
Separation/purification technology is the process of
separating out the amino acid accumulated in the
microbe culture fluid and refining it to crystals with apurity of almost 100 %. Engineering is the
management of the processes involved in large-scalecultivation and large-scale purification, as well as
managing the equipment. Computers are used forprocess control. Engineering also includes a lot oftechnological development challenges that can only beworked out at the stage of upscaling from a small-scalelaboratory experiment.
<References>(1) K. Ikeda: “Shin Chōmiryō ni tsuite [On a New
Seasoning]”, Journal of the Tokyo Chemical
Society, 30, 820, 1909.
(2) T. Tsunoda: “Aminosan Riyō no Genjō [Current
State of Amino Acid Utilization]”, Japan
Bioindustry Association, Amino Acid/Nucleic
Acid Group, ed., Hakkō kara Nyūbaiotekunorojī e[From Fermentation to New Biotechnology], p. 49,
1987.
(3) Japan Essential Amino Acids Association, ed.:Aminosan Shiryōshū [Collected Works on Amino
Acid], 1979, 1996.

Survey Report on the Systematization of Amino Acid Fermentation Technology

7

3 Origins of Amino Acid Fermentation
Amino acid fermentation is a technology that originatedin Japan around 50 years ago. An underlying factor
leading up to the emergence of this technology is that asa humid rice-farming area situated in Asia’s monsoon
zone, Japan had long put microbes to use with its
exceptional fermentation and brewing technology,
typified by its sake, miso, and soy sauce. More directly,
the Japanese already had MSG, the specific target ofinterest, and the scientific knowledge in biochemistry,
microbiology, and molecular biology to enable its
production by fermentation, as well as the necessary
screening and cultivation technology used for
antibiotic-producing strains.

3.1

The Success of “Ajinomoto” (MSG) and

Fig. 3.2 Sample Bottle Containing Kikunae Ikeda’s First
Successful Extract of Glutamic Acid (supplied by AjinomotoCo., Inc.)

Technological Issues
The first target for amino acid fermentation was“Ajinomoto” or MSG. As mentioned in Section 2.2,
MSG had been discovered as the flavor component ofkombu by Kikunae Ikeda in 1908 and patented as aseasoning. Later, a method of producing it byhydrolyzing gluten, the protein found in wheat, or soy
protein using hydrochloric acid was established and thefamous “Ajinomoto” was developed through a joint
venture between Prof. Ikeda and Saburōsuke Suzuki,
founder of Suzuki Pharmaceutical Company, later
Ajinomoto Co., Inc. Fig. 3.1 shows photographs ofKikunae Ikeda and Saburōsuke Suzuki II. Fig. 3.2
shows a photograph of the first glutamic acid sample(labelled “gurutami acid”) successfully extracted fromkombu and crystallized. This invention has been lauded
as one of the three greatest inventions of the Meiji era,along with Kōkichi Mikimoto’s cultured pearl andSakichi Toyoda’s automatic power loom.

Fig. 3.1 Prof. Kikunae Ikeda (left) and Saburōsuke Suzuki II(right), Second President of Ajinomoto Co., Inc. (supplied by
Ajinomoto Co., Inc.)

8

Since “Ajinomoto” was a completely new product,
there were many challenges in making it into a viablebusiness, in terms of both technology and marketing;however, these were overcome and the business was
successfully established.(1)Kikunae Ikeda majored in physical chemistry and so theinvention of “Ajinomoto” was a successful achievement
outside of his area of expertise. This success can be
attributed to his strong entrepreneurial aptitude and his
belief in advocating the idea that “taste promotes
digestion” to the Japanese people, who had poor
nutrition at the time. He had a strong desire to make foodtaste better and believed that he himself and all Japanese
had more refined palates than people in the West. He
sensed that a seasoning product would be accepted.
Ten years after its founding, Ajinomoto Co., Inc. wasperforming stably and the business expanded its
operations overseas.(1) Ikeda’s patent lapsed in 1923, but
a six-year extension was granted. When it expired in
1929, many companies started producing MSG. By 1930,
these companies totaled 28, excluding Ajinomoto Co.,
Inc. (2)However, a number of problems were identified withthe production method that had been developed(proteolysis). One issue was with raw materials: as
wheat and soy supplies were heavily reliant on imports,
they were subject to quantitative limitations dependingon circumstances overseas. There were also importing
restrictions in place to protect domestic agriculture.
Prices were high and it was difficult to keep costs down.
Another issue was that hot hydrochloric acid was used
for protein hydrolysis, which presented major problems
with equipment corrosion. Various workarounds weredevised, resulting in the development of a system using
fine ceramic “Dōmyōji-game” pots as acid-proof
containers. However this alone did not constitute any
radical solution and there were still unresolved issues
remaining, such as the difficulty of sequencing andautomating operations and the need to consider
workers’ health and public pollution. A new method of

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

production was therefore needed to overcome theseissues; investigations were carried out from two
different approaches – chemical synthesis andfermentation. Ajinomoto Co., Inc. started working on
this research around 1950.
Several methods of chemical synthesis were devised
and trialed. A method was perfected using a Strecker
reaction to combine carbon monoxide and hydrogencyanide with acrylonitrile and Ajinomoto Co., Inc.
started production at its plant in Tokai. This technology
was lauded as epoch-making technology that could
produce glutamic acid, a foodstuff, from petroleum
products, initially thought to be inexhaustible. It wasawarded the Chemical Society of Japan Award forTechnology in 1964 and the Okouchi Memorial
Production Award in 1965. However, consumers had ahuge change of heart soon afterwards and the concept
of “making food by chemosynthesis” was no longer
accepted. Progress had also been made withfermentation, which had begun around the same time,and so the MSG chemical synthesizing plant closed in
1973.(3)
Meanwhile, the fermentation method had certain
scientific and technological prerequisites, such as
biochemical expertise on microbial amino acidmetabolism, microbial genetics expertise on applied
microbiology screening methods, mutation andharvesting mutants, and methods for culturing
microbes. One key point of success was that theserequirements happened to coincide with a sudden boomin this science and technology, which was quickly
applied to the establishment of amino acid fermentation
technology.

3.2

Advances in Biochemistry and Microbiology

From the 1930s to the 1950s, significant progress had
been made in research on metabolisms withinorganisms, including the discovery of the glycolytic
pathway, which breaks down glucose, the tri-carboxylic
acid (TCA) cycle, and other major reaction pathways
and identifying the biochemical significance of these.
Naturally, this also meant an increase of knowledge
about amino acid metabolism, which is closely linked
to these areas. Research from the 1960s onwards
clarified the regulation of enzyme synthesis, typified in
Jacob & Monod’s operon theory on the metabolic
control of amino acids and other substances, and theexistence and mechanisms of negative feedback control
to inhibit feedback. In other words, amino acidsynthesis in an organism is strongly linked to metabolic
regulation, in which the amino acid itself regulates the
production and activity of the enzymes that catalyze
the synthesis reaction (see Section 4.3). Accordingly, it
became clear that overproduction or fermentation ofamino acid could not be achieved unless the control
effect in the amino acid itself could be eliminated. Thisknowledge was a prerequisite strategy in making aminoacid fermentation possible.

3.3

Advances in Molecular Biology andMicrobial Genetics

The transformation of Pneumococcus by O. Avery et al.
demonstrated that DNA is the main substance in genes.
In 1953, Watson & Crick clarified the mechanisms in
the central dogma, in which DNA has a double helix
structure and that the genetic information in DNA istranscribed to RNA and then translated to protein. This
opened up a new major research field, molecularbiology. Major research avenues were opened relating
to the collection and utilization of mutant strains,
including the idea that mutation occurs due to changes
in the base sequence of the DNA and the idea that
mutations occur in greater frequency when treated withultra-violet rays, X-rays, or chemical substances. These
discoveries provided the tools required to develop
amino acid fermentation technology using mutant
microbe strains. The use of mutant strains was notdirectly related to glutamic acid fermentation, but it
was used in lysine fermentation and subsequent
compounds.

3.4

Microbe Screening Technology / Discoveryof Antibiotics and Development of
Production Processes

In 1929, A. Fleming discovered that blue moldproduces penicillin. Around 1940, H. W. Florey et al.
improved penicillin production to practicalimplementation level and it proved to be remarkably
effective in treating infections contracted by woundedsoldiers in the Second World War. This was followedby the discoveries of a number of antibiotics, such as
streptomycin by S. A. Waksman, chloramphenicol andaureomycin. The golden age of antibiotics continued
from around 1940 to around 1980. These discoveries
were the major dream of microbe researchers.
Professor Kinichiro Sakaguchi of the University ofTokyo, an authority on applied microbiology with along record of achievements, is quoted as saying, “I put
my hope in microbes and have never been let down.”
There are a great variety of microbes and countless
numbers of them; it is certain that there wereresearchers that believed in the existence of microbesthat produce amino acids. There was a lot to be learnt
about microbe screening from the discovery ofantibiotics.

Survey Report on the Systematization of Amino Acid Fermentation Technology

9

3.5

Advances in Large-Scale Cultivation
Technology

As discussed in Section 3.4, antibiotics were being
produced using blue mold and actinomycetes. Jar
fermenters and fermentation tanks were developed for
aeration-agitation cultivating. Many companies inJapan started working on developing technology forpenicillin fermentation in the late 1940s; thisexperience proved to be a major advantage inestablishing amino acid fermentation.

10<References>(1) Ajinomoto Co., Inc.: Aji o Tagayasu: Ajinomoto
Hachijūnen-shi [Cultivating Taste: 80-Year
History of Ajinomoto]”, pp. 48-102, July 1990.
(2) S. Ota: Umami Chōmiryō no Chishiki[Knowledgeof Flavor Seasoning], p. 4, Saiwai Shobo, June
1992.
(3) Ajinomoto Co., Inc.: Kenkyūjo 50-nen-shi: ‘Ashita
no Moto’ o Motomete [A 50-Year History of theInstitute: Searching for the ‘Substance ofTomorrow’], p. 100, September 2006.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

4 Origins of Amino Acid Fermentation:

Amino-Acid-Producing

Bacteria Screening and Strain Breeding

4.1(6) succinate dehydrogenase complex; (7) fumarate hydratase;(8) malate dehydrogenase; (9) glutamate dehydrogenase

Glutamic Acid Production through Two-Step
Fermentation

Glutamic acid was known to be produced by introducing
an amino group to 2-oxo-glutaric acid (2OG,
α-ketoglutarate) produced by a reaction in the TCA cycle(Fig. 4.1) in an organism. It was also known that 2OG
could be produced by fermentation. It was thus believedthat glutamic acid production was “almost within reach”
from the perspective of biochemical metabolic pathways
and appears to have come to the attention of many
researchers. Initial studies by companies including
Ajinomoto Co., Inc. and Kyowa Hakko Kogyo Co., Ltd.
investigated two-stage fermentation to increase andaminate the 2OG output. The need for Ajinomoto Co., Inc.
to develop technology as the prior specialist manufacturer
is mentioned above. The fact that Kyowa Hakko was a
newcomer to glutamic acid did not mean that it had any
trouble producing MSG. It already had fermentation
technology, as it had intended to use it in a new research
project producing protein to address the nutrition
problems in postwar Japan. Instead, it changed its aim
from protein to glutamic acid and started its research.(1)glucosefatty acidpyruvic acidacetyl-CoA

oxaloacetate

citric acidmalic acidcis-aconitic acidfumaric acidisocitric acidsuccinic acidsuccinyl-CoA

reversible reaction
irreversible reaction
2-oxoglutarate

oxalosuccinic acidglutamic acid

Fig. 4.1 Positional Relationship of Glutamic Acid in the TCA Cycle(1) citrate synthase; (2) aconitate hydratase; (3) isocitrate dehydrogenase;(4) 2-oxoglutarate dehydrogenase complex; (5) succinyl-CoA synthetase;

While the production quantities of 2OG increased
considerably, there are no reports of its amination. Thisis probably because the direct fermentation method
proved to be successful in the meantime, as shall be
discussed next, and this method was discontinued.

4.2

Glutamic Acid Production through Direct
Fermentation

4.2.1

Discovery of Glutamic-Acid-Producing
Microbes
As well as working on the two-stage fermentation
process mentioned above, Kyowa Hakko also
successfully screened a strain of bacterium that directly
produced glutamic acid and succeeded in establishing a
new fermentation method, namely glutamic acidfermentation.(2)It was not common knowledge at the time amino acids
such as glutamic acid could be produced directly fromglucose. Accordingly, although this experiment literally
defied common sense, perhaps some researchers
believed that they could achieve a glutamic acidmicrobe. The keys to this success were in the accurate
method of strain screening and in the clever applicationof a bioassay method to quantify the amino acid. In
other words, some lactic acid bacteria require aminoacids for growth; the bioassay determined the quantityof the amino acid using the proportional relationshipbetween the quantity of the required amino acid and thegrowth amount. Specifically, the lactic acid bacteriathat require several types of amino acids for growth
were pre-coated onto nutrient agar with the glutamic
acid removed and the naturally-selected microbes werethen cultivated. This system made it possible to easily
detect the microbes that produced glutamic acid, as
they had an observable ring of lactic acid bacteria
growth around them. Within a short time, the ones witha ring of growth around them were collected andconfirmed to be producing glutamic acid in a liquid
culture.(3)Microscope observation revealed that this strain
exhibited a spherical shape. It was categorized as a newstrain under the name Micrococcus glutamicus; thisname was later changed to Corynebacterium
glutamicum as it was observed to change in shape
under different cultivation conditions.
Cultivation was carried out using several combinationsof culture mediums to alter the amount of required
nutrients. The amount of added biotin, a type ofvitamin, was found to be a significant factor. In otherwords, while biotin had to be added to the culture

Survey Report on the Systematization of Amino Acid Fermentation Technology

11medium because the microbe required biotin to grow,the concentration was extremely important. Anythingover 10 μg/L resulted in no glutamic acid production atall, while cultivation at 3 μg/L resulted in 40-50 g ofglutamic acid per liter from 100 g of glucose.(4)Once Kyowa Hakko had reproduced this experiment at
its pilot plant at flask level, it issued a press release on
September 21, 1956 announcing its successful glutamic
acid fermentation and its intention to enter the MSG
industry. This successful glutamic acid fermentation isregarded as the start of amino acid fermentation. The
discovery of a glutamic acid microbe had hugesignificance in that it opened up an entirely newtechnological field, that of producing not only glutamic
acid but also many other amino acids by fermentation.
This announcement by Kyowa Hakko shocked
Ajinomoto Co., Inc. and other existing MSG
manufacturers. Having taken over the patent rights to a
“Method for Cultivating α-Ketoglutaric-acid-producing
strains in a culture medium containing a large amountof nitrogen” (the “Tada patent”) granted to Yasuji Tada
and Oki Nakayama in 1954, Ajinomoto Co., Inc. andSanraku Shuzo handled the matter at patent level. At
the same time, the various companies and universities
actively engaged in developing new microbe strains.
This resulted in a succession of strain discoveries,
including Brevibacterium flavum and Brevibacterium
lactofermentum by Ajinomoto Co., Inc. and Sanraku
Shuzo, Microbacterium ammoniaphilum by
Asahi-Kasei, Brevibacterium thiogenitalis by Takeda
Pharmaceutical Co., Ltd., and Brevibacterium
divaricatum by the University of Tokyo. These strains
shared similar taxonomical characteristics, such as (1)producing glutamic acid under aerobic conditions; (2)requiring biotin for growth; (3) showing positive in a
Gram stain test, a classification indicator for bacteria;(4) forming no spores; (5) having no motility; (6) being
short-rod-shaped or spherical. Due to these factors, arecommendation was made in the 1980s to unite themunder the name Corynebacterium glutamicum; thisname is now popularly used.

Fig. 4.2 (2) Photograph of Corynebacterium glutamicum Colony(supplied by Kyowa Hakko Kogyo Co., Ltd.)

These strains were not only used to produce glutamic
acid, but also as the parent strains for mutations
producing many amino acids, including lysine,
threonine, and phenylalanine, as shall be discussed in
Sections 4.5 and 4.7.
Kyowa Hakko received the Okouchi Memorial Awardin 1958 and the Japan Science and Technology Agency
Director-General’s Award in 1960 for the invention of
glutamic acid fermentation, as well as the Japan
Academy Prize in 1966 for the establishment of aminoacid fermentation including ornithine and lysine
fermentation, as shall be discussed in Section 4.5.
In 1957, an agreement was made that all glutamic acidproduced using Kyowa Hakko’s fermentation method
was to be supplied to Ajinomoto Co., Inc. for the next
five years. However, there was trouble with thepurification process and the product was not supplied
as planned. In 1961, Ajinomoto Co., Inc. cancelled theagreement, having developed a method that did not
infringe on Kyowa Hakko’s patent. Kyowa Hakko later
supplied MSG to Takeda Pharmaceutical for its nucleic
acid seasoning “Inoichiban,” but Takeda
Pharmaceutical discontinued the supply in 1966,
having also started producing MSG from strains it had
developed on its own.(5) These developments werefraught with microbe-strain-related patent disputes.
4.2.2

Fig. 4.2 (1) Electron Micrograph of Corynebacterium
glutamicum (supplied by Kyowa Hakko Kogyo Co., Ltd.)

12

Glutamic-Acid-Producing Strain Cultivation
Technology
Large-scale cultivation methods for producing glutamic
acid involved using flasks and jar fermenters developed
for antibiotic fermentation. It was clear that the key toachieving the maximum production quantity was
limiting the biotin concentration to around 3 μg/L,(4) as
mentioned in Section 4.2.1, but there was a major issue
in that molasses, a cheap raw material, contains highconcentrations of biotin and could not be used to
produce glutamic acid. Faced with this practicalproblem, Merck, which had adopted Kyowa Hakko’s
technology, successfully developed the technology thatenabled the use of molasses, having proven that

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

glutamic acid could be produced from molasses with ahigh biotin concentration by adding a low
concentration of penicillin to the molasses culture.(6)Meanwhile, Ajinomoto Co., Inc. developed a method
involving the adding of a surfactant.(7)4.2.3
Glutamic Acid Production Mechanism
There was a lot of practical and academic interest as towhy such high concentrations of glutamic acidaccumulated and much research was devoted to clarify
the matter. As mentioned in Section 4.2.2, there were
two main cultivation methods for producing large
quantities of glutamic acid: (1) suppressing theconcentration of biotin; (2) adding penicillin or asurfactant to high concentrations of biotin. Later
research demonstrated the following.
1) Wild-type strains inhibit enzyme activity related to
glutamic acid biosynthesis or repress the formation of
those enzymes. In other words, they have a metabolism
controlling function, mentioned in Section 4.3, as seenin B. flavum.
2) Under glutamic-acid-producing conditions, as theglutamic acid produced within the microbe is secreted,the metabolic flow predominantly progresses towards
glutamic acid.
In the second point above, since biotin is a factor in thesynthesis of the fatty acids that form the cellular
membrane, limiting the biotin means limiting thesynthesis of fatty acids and limiting the synthesis of thecellular membrane. Adding penicillin or a surfactant
has a similar effect. While the cellular membrane thus
plays an important role,(12) a recent joint study between
Ajinomoto Co., Inc. and Tokyo Institute of Technology
on the secretion function of the cellular membrane has
found that when glutamic acid is produced, mutations
occur in the genes believed to correspond tomechanosensitive channels. Mechanosensitive
channels are channels (pathways) that have thefunction of sensing changes in the osmotic pressure on
the microbe’s cellular membrane and secreting acorresponding solute (in this case glutamic acid)outside of the cell. This has been explained as thechannel always being “on” in this mutation, while inwild-type strains the channel only turns “on” andsecretes glutamic acid in conditions that induce theproduction of glutamic acid ((1) and (2) above).(8)4.2.4

Academic Research on
Glutamic-Acid-Producing Bacteria
As mentioned in Section 4.2.1, glutamic acid bacteriasuch as C. glutamicum and B. flavum were just used for
producing glutamic acid. In later studies, these
extremely important strains served as the parent stock
for the mutant strains that produced lysine and manyother amino acids. Extensive academic research was
carried out on these microbes, mainly in Japan,

covering not only the breeding ofamino-acid-producing strains, but also taxonomy,
amino acid metabolism, gene manipulation systems,
and genome analyses. The results of these studies are
summarized in the literature.(9), (10), (11)4.2.5

Overseas Expansion of Glutamic Acid
Fermentation Technology
Kyowa Hakko’s technology is said to have been
licensed to Merck, a major American chemicalcompany, and to companies in Taiwan.
Meanwhile, Ajinomoto Co., Inc. started production at
its factories in Japan and as early as 1960 started
actively expanding its operations overseas in the formof MSG plants in Thailand, Malaysia, the Philippines,
Italy, and other places. Since the price of MSG heavily
depends on the raw materials, there are hardly any
MSG factories in Japan any more, having spreadinstead to Southeast Asia, Brazil, the United States,
Taiwan, and China that produce molasses and starch.
As mentioned in Section 2.4, glutamic acidfermentation developed into a major global industry,
with an estimated MSG production of 1.7 million tons
in 2005.
4.2.6

Industry-University Collaboration on Amino
Acid Fermentation Research
Given the success of glutamic acid fermentation,
Kinichiro Sakaguchi of the University of Tokyo
proposed that Japan develop the technology for aminoacid production by fermentation into a uniquely
Japanese industry. In 1958, the Ministry of Education,
Culture, Sports, Science and Technology started anamino acid research group that included the leading
universities across the country. The “Amino Acid
Group” was launched as a forum for presenting
research and many members of the industry actively
took part as well, carrying out vigorous research anddiscussions. Although the group later changed its nameto the “Amino Acid/Nucleic Acid Group” and now
exists as the “Technical Committee on Fermentationand Metabolism” within the Japan Bioindustry
Association, it still continues its activities as the main
driving force behind amino acid fermentation researchin Japan.

4.3

What is Metabolic Regulation to Prevent
Overproduction of Amino Acid?

Since amino acid production is closely connected tometabolic regulation (Section 3.2), let us provide an
outline of that here.
Microbes have a regulating mechanism to prevent
excessive production of amino acids and othermetabolites necessary to their biological activity. Thisis metabolic regulation.

Survey Report on the Systematization of Amino Acid Fermentation Technology

13

Alcohol fermentation is similar to glutamic
fermentation in that it produces a substance that isuseful to humans through the fermentation process.
While the reaction from the action of the yeast
produces alcohol as well as carbon dioxide gas fromglucose, the alcohol is produced as a byproduct of theyeast growth reaction. Consequently, no metabolic
regulation operates in this reaction. By contrast,
glutamic acid is an essential substance as proteinmaterial, so it would be irrational for the microbe to
produce it in excess and secrete it. The mechanism
preventing this from happening is metabolic regulation.
Thus, while alcohol fermentation and amino acidfermentation are similar in that they both use microbes
to produce a useful substance, they differ in the details.
Due to this metabolic regulation effect, ordinary
wild-type microbes cultivated through normal methods
do not over-produce amino acid (an exception is
wild-type C. glutamicum, which sometimes stores up
small amounts of alanine and valine with a low
metabolic regulation effect).
An overview of the amino acid biosynthesis pathways in
microbes and the regulation of these was determined in
1955-1960s (Fig. 4.3), with research in molecular biology
also shedding light on these mechanisms. The regulating
functions mentioned here are feedback inhibition andrepression of enzyme formation by end product, or aminoacid. For example, in a metabolic pathway thatsynthesizes end product E from start substance A, as
shown in Fig. 4.4, if the concentration of E increases due
to over-production, feedback inhibition inhibits the directbinding to the A→E reaction-catalyzing enzyme (a) while
repression suppresses the production of the enzymes (a-d)
that catalyze the reactions from A to E. Thus, if there is ahigh concentration of E, the A→E reaction stops, but once
consumption of E eventually reduces the concentration,
the control is lifted and synthesis resumes.
glucoseGenerally, enzyme (a), the starting enzyme involved in
the A→E reaction is called a key enzyme. This enzyme
has an active site, where A binds, and a control site,where end product E binds. If E binds, the spatial
structure of the enzyme protein changes and A cannot
bind, thereby inhibiting the reaction. These enzymes
are called allosteric enzymes. While feedback
inhibition directly binds E to the enzyme, repression ismore indirect, suppressing the production of enzymes
through mRNA transcription. E. coli and othergram-negative bacteria also have anamino-acid-decomposition reaction; this is also
metabolic regulation.
Thus, breaking down or bypassing metabolic regulation
is a necessary condition for the over-production ofamino acid. The following methods can be used to
bypass the specific types of metabolic regulation
discussed thus far.

(transcription)(translation)
enzymesFig. 4.4 Schematic Representation of Feedback Inhibition and
Repression (Suppression of Enzyme Production)
Feedback inhibition is the phenomenon of inhibiting the enzyme a thatcatalyzes the A→B reaction in E in the series of biosynthetic reactions that turnstarting product A into end product E (amino acid). Repression is thephenomenon of suppressing the production of proteins (= enzymes) a-d, acting
at the stage in which E transcribes the genetic information in the DNA tomRNA.
biosynthetic
reaction

4.4

Feedback inhibition

Overcoming Metabolic Regulation (1)Promoting Glutamic Acid Secretion

As discussed in Section 4.2.3, increasing the secretionof glutamic acid, in other words, increasing thepermeability of the cell membrane of the glutamic acidmicrobe has been shown to cause it to produce large
quantities of glutamic acid even with the metabolic
regulation found in the glutamic acid biosynthesis, asmentioned in Section 4.3.(12) A molecular-level analysisof glutamic acid secretion suggests the existence ofsecretion channels.(8)pentosephosphate
cycle(glycolytic
pathway)(pyruvate)

TCA cycle

Fig. 4.3 Schematic Representation of Amino Acid Biosynthesis
and Regulation in Microbes
amino acidintermediatebiosynthesis pathway

Main Feedback Control* 1: glucose-6-phosphate, * 2: 3-phosphoglycerate, * 3: phosphoenolpyruvate,* 4: oxaloacetate, * 5: 2-oxoglutarate, * 6: erythrose-4-phosphate, * 7:
ribose-5-phosphate, * 8: homoserine

14

repression

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

4.5

Overcoming Metabolic Regulation (2)Production Using Auxotrophic Mutants

While mutants are strains with hereditarycharacteristics that differ from those of the parent stock,auxotrophic mutants are mutants that genetically losethe enzymes related to synthesizing amino acids orother metabolites. Since they cannot biosynthesize
these metabolites, they require them as nutritive
materials in order to grow. These mutants have been
induced in order to block specific reactions. The
method discussed here uses auxotrophic mutants toeliminate feedback inhibition by reducing theconcentration of amino acid, the feedback inhibitory
factor, thereby producing an excess of amino acid.
4.5.1
Ornithine
Having achieved the capability to discoverglutamic-acid-producing microbes, Kyowa Hakko
attempted to screen other amino-acid-producing
microbes from soil and other natural elements, but it
appears that these attempts were not successful. As
mentioned in Section 4.3, there were no further reportsof wild-type microbes producing an excess of aminoacids other than glutamic acid, alanine, and valine.
Given the knowledge of metabolic regulation
mentioned above, the obvious conclusion is that there
are no producing strains for these other amino acids.
Meanwhile, there have been reports of auxotrophic
mutants of E. coli with the amino acid biosynthetic
pathway blocked secreting an intermediate before theblocking reaction. Kyowa Hakko used these clues toselect various auxotrophic mutants of C. glutamicum
and examining what they produce. Strains requiring
arginine were found to produce ornithine; strainsrequiring homoserine were found to produce lysine;strains requiring threonine were found to produce
homoserine; strains requiring tyrosine were found to
produce phenylalanine. Of these, the highest quantities
produced were of ornithine (13) and lysine.(14) These
were put to practical use, resulting in the creation ofornithine and lysine fermentation. Rather than simply
blocking biosynthesis and storing up the intermediate,
over-production was achieved by eliminating the
feedback inhibition involved in amino acidbiosynthesis.
As shown in Fig. 4.5, biosynthesis of wild-typearginine is achieved by the starting enzyme
N-acetylglutamate synthase being subject to feedback
inhibition from the arginine, as well as regulation byrepressing the synthesis of a series of enzymes related
to biosynthesis. Arginine-requiring strains lacking
ornithine carbamoyl transferase (OTC), whichcatalyzes the next arginine reaction, are cultivated witha limited low concentration of arginine, which
eliminates feedback inhibition and repression. While
this also results in a relative increase inN-acetylglutamate synthase activity, theexcessively-produced ornithine is stored due to the lackof OTC. The quantity of ornithine produced using
arginine-requiring strains indicated a 36 % higher

molar yield than glucose.(13)
While ornithine is an amino acid not found in protein,
it is used as an ingredient in amino acid transfusions, aswell as in a salt form with aspartic acid as an agent toimprove liver function.
glutamic acidglutamic acidornithineornithineargininearginine

wild-type strain

arginine auxotroph

Accumulated

Fig. 4.5 Regulation of Arginine Biosynthesis in C. glutamicumand Ornithine Production in Arginine Auxotrophs (10)biosynthetic reaction

feedback inhibition

AGS : N-acetylglutamate synthase、
OCT : ornithin carbamoyl transferase

Since arginine auxotrophs lack OCT (represented by) and can be cultivated with alimited low concentration of arginine, ACS feedback inhibition and the production of aseries of enzymes can be regulated. Enzyme activity increases (represented by) andexcessively-produced ornithine is accumulated.

4.5.2
LysineAs shown in Fig. 4.6, there is concerted feedback
inhibition in wild-type C. glutamicum whenaspartokinase, the starting enzyme for lysine, threonine,and methionine synthesis, coexists with both lysine andthreonine. If homoserine auxotrophs lacking
homoserine dehydrogenase are cultivated with a low
concentration of homoserine (or threonine plus
methionine), there will be no feedback inhibition of
aspartokinase due to the low threonine concentration
and an excess of the intermediate
aspartate-semialdehyde will be produced. Since there isa lack of homoserine dehydrogenase, the intermediate
will not convert to threonine, but flow into lysine,causing an excess to be produced and accumulated(14).
aspartic acidaspartic acidaspartyl phosphate

aspartyl phosphate

aspartate-semialdehyde

aspartate-semialdehydelysinelysinehomoserine

homoserine
Accumulated

threoninemethionine

wild-type strain

threoninemethionine

homoserine auxotroph

Fig. 4.6 Regulation of Lysine, Threonine, and Methionine
Biosynthesis in C. glutamicum and Lysine Production in
Homoserine Auxotrophs (11)biosynthetic
feedback inhibition
reaction
regulation of enzyme synthesis, AK: aspartokinase; HD: homoserine dehydrogenase
Homoserine auxotrophs lack HD (represented by); if they are cultivated with alimited low concentration of threonine and methionine, there is no AK feedback inhibition
and AK activity increases (represented by). Excessively-produced lysine isaccumulated via aspartate-semialdehyde.

Survey Report on the Systematization of Amino Acid Fermentation Technology

15

4.5.3 Other Amino Acids
Based on these successes, various other auxotrophic
mutants have been selected, including
proline-producing strains derived from isoleucine
auxotrophs (15) and citrulline-producing strains fromarginine auxotrophs.(16)Production using these auxotrophic mutants has been
adopted as a historically-significant discovery.
However, when using these methods of using
auxotrophic mutants it is difficult to control the
optimum concentration of the auxotrophic substance toobtain the maximum production quantity. There arealso inherent limitations; in other words, amino acidauxotrophy cannot be achieved in these producing
strains because they block amino acid synthesis. The
use of analog-resistant strains, as shown in Fig. 4.7,
solved these issues all at once.

4.6

4.7

Overcoming Metabolic Regulation (4)Production using Analog-Resistant Strains

Analogs are substances with a similar structure tonatural amino acids or nucleic acid bases and aremainly used as ingredients for pharmaceutical products
or metabolism research. Some example analogs foramino acids include ethionine for methionine,α-amino-β-hydroxyvaleric acid (AHV) for threonine,and S-(2-aminoethylcysteine) (AEC) for lysine. Fig.
4.7 shows a comparison of structural formulas between
these and their natural amino acid counterparts.

Overcoming Metabolic Regulation (3)Production by Converting Intermediate
Metabolites

methionine

ethionine

threoninelysineFig. 4.7 Example Amino Acid Analog Structural Formulas

Amino acid metabolic regulation is mainly achieved
through inhibition of the starting enzyme in thesynthesis reaction (Section 4.3). Accordingly, since
there is essentially no inhibition of reactions from thestarting enzyme onwards or of formation reactions
from side substances, it was believed possible toconvert amino acid precursors or intermediates in thisposition into amino acids. Examples include turning
threonine precursor homoserine into threonine,tryptophan precursors anthranilic acid or indole into
tryptophan, fumaric acid into aspartic acid, glycine into
serine, and chemically-synthesized α- aminobutyrate
into isoleucine. The method involves screening strains
capable of converting to amino acids using theseprecursors and intermediates as the substrate (rawmaterial), thereby proceeding with the reaction while
ingeniously avoiding the key enzymes subject tometabolic regulation. Although, such methods made it
possible to achieve the target strain, many suchmethods were never implemented. The reasons for thiswere that the precursors and intermediates used as raw
material were expensive and that a means of directly
fermenting amino acids using analog-resistant strains,mentioned in Section 4.7, was established not longafterwards. Methods that successfully made it topractical implementation were production methods forincluded those that used relatively inexpensive raw
materials such as fumaric acid to produce aspartic acidand alanine and those that could not be adopted using
analog-resistant strains, such as D-phydroxyphenylglycine and cysteine. These were
implemented as fermentation production methods, withmore details given in Section 4.8. Studies on
analog-resistant strains of serine made slow progress,
and it was produced from glycine for a while.

16*AHV: α-amino-β-hydroxyvaleric acid** AEC: S-(2-aminoethyl) cysteine

While wild-type microbes cannot grow in minimal
media (media containing only the minimum
components necessary for the growth of the
microorganism) with the analog added, they can berevived if the corresponding amino acid is
supplemented. Since these analogs are “counterfeits” of
the real amino acids, they can antagonistically interferewith the real amino acids in reactions that captureamino acids to build them into protein (aminoacyltRNA synthetase) and reactions for key enzyme
feedback inhibition. Accordingly, an analog-resistantstrain that can grow in a medium that includes ananalog is a mutant strain thought to be able towithstand interference and produce an excess of realamino acid. Thus, in the early 1960s there were reportsof E. coli and other amino acid analog-resistant strains
overproducing and secreting various types of aminoacids. These secretions were literally minisculeamounts and these reports did not include any
quantitative descriptions. Examples include secretionsof methionine from ethionine-resistant strains,
threonine from AHV-resistant strains, and tryptophan
from 5-methyl-tryptophan-resistant strains (Later, when
the author visited Dr. G. N. Cohen at the Pasteur
Institute, who had reported the first threonine secretionfrom an AHV-resistant strain of E. coli, Dr. Cohen said
that he “never dreamed that such research would haveany potential commercial interest”).

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

Ajinomoto Co., Inc. induced an AHV-resistant mutant
strain from Brevibacterium flavum (C. glutamicum)
parent stock and achieved a strain that could produce
large quantities of threonine. This was far from the“secretions” previously reported on. The company
immediately industrialized threonine fermentation
using this producing strain. This was the firstsuccessful industrialization of amino acid fermentation
using an analog-resistant strain. The reason this mutant
strain produced threonine was that it genetically
excluded feedback inhibition of homoserine
dehydrogenase through threonine, as shown in Fig. 4.6,
and that its sensitivity to inhibition was around 1,300
times lower than the parent stock, showing a clear
correlation between production and enzyme activity.
Later research showed that this mutation occurred due
to permutation of an amino acid residue in thehomoserine dehydrogenase control area amino acidsequence. A threonine-producing strain derived at thesame time from E. coli K-12 was used for
improvement through genetic engineering 15 years
later.(17)As the pioneers in successful threonine fermentation,
Ajinomoto Co., Inc., Kyowa Hakko, and Tanabe
Pharmaceuticals Co., Ltd. (now Mitsubishi Tanabe
Pharma Corporation) reported production of various
amino acids from analog-resistant strains. While thesereports are many in number, the main products thought
to have immediately gone on to industrialization are
given in Table 4.1.
Thus, the use of analog-resistant strains resulted in theachievement of a number of amino acids being
produced by fermentation. It is increasingly evident
that for many of these the activity of the starting
enzyme for amino acid synthesis was excluded throughamino acid feedback inhibition or regulating enzyme

production, such as using lysine + threonine to inhibit
the feedback of aspartokinase, the key enzyme inlysine-producing strains from B. flavum, or using
histidine to eliminate the inhibition of
ATP-phosphoribo-syltransferase, likewise the key
enzyme in histidine-producing strains from C.
glutamicum. The research findings by Tanabe
Pharmaceuticals are characterized by the use of entericbacteria Serratia marcescens as a parent stock. This
strain resembles E. coli and is distinctive in that it cantransduce by phage. Some mutant strains have beencreated by using this distinctive trait to inhibit feedback
and eliminate repression of enzyme production.

4.8

Enzymatic Method and Production using
Bioreactors

The enzymatic method is a technology developed
through application of the methods outlined in Section
4.6, producing amino acids by converting raw materials
other than glucose, such as intermediates on themetabolic pathway or chemically synthesizedintermediates, using microbial enzymes or microbesthat contain enzymes as the catalyst. This differs
slightly from the fermentation method in that while thefermentation method produces amino acids as the
microbes multiply, the enzymatic method uses themultiplying enzymes as the catalyst. In this report, theenzymatic method is treated as a fermentation methodin the broader sense. The main research findings using
this method are as follows.

Table 4.1 Amino-Acid-Producing Strains Obtained through Analog-Resistant Mutations
Amino Acid

Analog Used

StrainLiterature

threonineα-amino-β-hydroxyvale
ric acid

E. coli
B. flavum
C. glutamicum
S. marcescens*

I. Shiio and S. Nakamori(18)I. Shiio and S. Nakamori (19)
H. Kase et al.(20)S, Komatsubara, et al.(21)lysineS-(2-aminoethylcysteine)B. flavum
B. lactofermentum

K. Sano and I. Shiio (22)
O. Tosaka et al.(23)isoleucine

AHV+o-methyl
threonine
AHV+ethionine
isoleucine hydroxamate

B. flavum

I. Shiio et al.(24)

B. flavum
S. marcescens *S. Ikeda et al. (25)
M. Kisumi, et al.(26)

histidine

1,2,4-triazole alanine
2-thiazole alanine

C. glutamicum
B. flavum

K. Araki et al. (27)
H. Kamijyo et al (28)tryptophan

5-fluoro tryptophan, etc. B. subtilis *5-fluoro tryptophan, etc. B. flavum

I. Shiio et al. (29)
I. Shiio et al. (30)phenylalanine

p-fluorophenylalanine,etc.

B. lactofermentum

T. Tsuchida et al.(31)prolinearginine

3,4-dehydropoline
D-Ser, D-Arg, etc.
2-thiazole alanine

B. flavum
C. glutamicum
B. flavum

S. Nakamori el al(32)
N. Nakayama et al.(33)K. Kubota el at.(34)

glutamine

sulfur guanidine

B. flavum

T. Tsuchida et al.(35)

valineα-amino butyric acid
2-thiazole alanine

S. marcescens *B. lactofermentum

M. Kisumi et al.(36)
T. Tsuchida et al.(37)leucineα- amino butyric acidS. marcescens *M. Kisumi et al.(38)

serineazaserine

B. flavum

W. Hibino el al.(39)* Thought to be no longer used
While B. flavum and B. lactofermentum are now recommended to be designated as C.
glutamicum, the former designations are used here in accordance with the original report.

Survey Report on the Systematization of Amino Acid Fermentation Technology

174.8.1

Production of L-Amino Acids from
DL-Amino Acids with Aminoacylase
Before the regularization of amino acid production byfermentation, the method of production that had madethe most progress in research was chemical synthesis.
The amino acids produced using this method were
DL-compounds (racemic compounds). While it isrelatively simple to get to the DL-compound stage, theoptical resolution to convert a DL-compound into anatural L-compound is quite a difficult challenge andhardly ever made it to industrialization. A method
developed by Tanabe Pharmaceuticals achieved thisthrough asymmetric hydrolysis using aminoacylase, an
enzyme that derives N-acyl-DL-amino acid, easily
synthesized from DL-amino acid, from Aspergillus
oryzae. This enzyme catalyzes a reaction that detaches
the acyl group from the N-acyl-DL-amino acid and ischaracterized in that it only acts on L-compounds, noton D-compounds. The remaining D-compounds thathave not decomposed are racemized by heating to form
DL-compounds. Repeating this reaction eventuallyconverts everything into L-compounds (Fig. 4.8).
Examples of early L-amino acids successfully
produced by the enzymatic method include
L-methionine, phenylalanine and valine, mainly usedin amino acid infusion requiring L-compounds.

aminoacylase

DL-amino acidN-acyl-DL-amino acidN-acyl-D-amino acidL-amino acid(racemization)

control panel

flow
acetyl-DL-ami
no acidcontinuous
concentrator

heat

crystallizationtankacetyl-D-aminoacid

flowmeter
heat exchanger

racemizationtank

enzymecolumnseparator

hot
water
tank
pump

filterpump

automatic
reaction rate
recorderThe entrapping method targets not only the enzyme but
also the microbe containing the enzyme.
In 1969, Tanabe Pharmaceuticals succeeded in thecontinuous production of L-amino acid using
aminoacylase with an immobilized bioreactor reaction,
having immobilized the enzymes by ionically bonding
them to a DEAE-Sephadex ion-exchange resin.(40)4.8.2
Aspartic Acid and Alanine
Aspartic acid is used as a component in amino acidinfusions and in oral, enteral, and other clinicalnutritional supplements, as well as in pharmaceuticalapplications in salt form together with arginine andornithine for fatigue recovery and improving liverfunction. It is also used as a seasoning to boost
sourness and heartiness and is in high demand as theraw material used in the sweetener aspartame. Alanine
is used in amino acid infusions and in oral, enteral, andother clinical nutritional supplements.
In 1959, both Tanabe Pharmaceuticals and Kakuo
Kitahara et al. of Tokyo University reported a methodof producing aspartic acid from fumaric acid using E.
coli. The enzyme catalyzing this reaction was aspartase.
In 1973, Tanabe Pharmaceuticals successfully
industrialized the continuous production of aspartic
acid using immobilized aspartase (41) (Fig. 4.9).
Tanabe Pharmaceuticals also discovered Pseudomonas
dacunhae, which produces aspartic acid decarboxylase,that converts aspartic acid into alanine, andsuccessfully developed a process for producing alaninefrom aspartic acid (42)(Fig. 4.9).
Since production of aspartic acid from glucose byfermentation offers low yields and production ofalanine results in a DL-compound, both aspartic acidand alanine are produced industrially using the
enzymatic method.
Tanabe Pharmaceuticals immobilized microbes
containing aspartase and aspartic acid decarboxylaseby entrapping them in polyacrylamide (and later inκ-carrageenan) and succeeded in long-term, stable,
continuous production of aspartic acid and alaninefrom fumaric acid.(43) This reaction system is shown in
Fig. 4.9.

L-amino acid

Fig. 4.8 Reaction Formula for Producing L-Amino Acids from
DL-Amino Acids using Aminoacylase and Flow Diagram of
Production Equipment(40)fumaric acid

A point of difficulty lies in that since the enzymes areused in an aqueous solution state, they have low
stability and cannot be used for repeated continuous
use like solid catalysts. Enzyme immobilization
technology solved this issue by making it possible totreat these enzymes like solid catalysts. Reactions
using these immobilized enzymes are called bioreactor.
Enzyme immobilization is achieved by binding the
enzyme to a high-molecular carrier such as an
ion-exchange resin, linking the enzyme using
glutaraldehyde or other binding agent or entrapping the
enzyme in a bilayer membrane such as a liposome or ahigh polymer such as carrageenan or polyacrylamide.

18

aspartic acidimmobilized microbes(aspartase)alanineimmobilized microbes(aspartic aciddecarboxylase)

Fig. 4.9 Production of Aspartic Acid and Alanine from Fumaric
Acid using Immobilized Microbes containing Enzymes

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

4.8.3
Dioxyphenylalanine
Dioxyphenylalanine (DOPA) is a precursor amino acidof a series of chemical compounds such as dopamine,
which governs the neural functions in the brain. It is apharmaceutical product shown to be very effective intreating Parkinson’s disease and other cranial nerve
disorders in the elderly. Since the amino acid isunrelated to microbial biological activity, it cannot be
produced through direct fermentation, but a method has
been developed to synthesize it using microbial
enzymes. Hideaki Yamada et al. of Kyoto University
discovered that microbes such as Citrobacterintermedius and Erwinia herbicola produce
β-tyrosinase, the enzyme that catalyzes the reactions
below which decompose tyrosine at β position (44) (Fig.
4.10). While the reverse reaction to this reaction showsthat tyrosine is composed of phenol, pyruvate, andammonia within the same group, the enzyme has broad
substrate specificity and DOPA can be synthesized byreplacing phenol with pyrocatechol as the raw material(Fig. 4.11). This process was industrialized by
Ajinomoto Co., Inc. in 1992.(45)phenol, pyruvate, ammonia

tyrosineβ-tyrosinase

Fig. 4.10 Tyrosine Decomposition Reaction with β-Tyrosinasepyrocatechol

pyruvateβ-tyrosinase

Fig. 4.11 Synthesis of DOPA using β-Tyrosinase Reverse
Reaction

4.8.4
Cysteine
Cysteine has many uses as a raw material for infusions,liver function improvement agents, pigmentation
improvement agents, and bread fermentation agent,
with its acetyl derivative used as a raw material for
expectorants. Cysteine is produced by hydrolyzing hairor keratin with a high cysteine content in hydrochloric
acid. As yet there has been no method established for
producing it by fermentation. Ajinomoto Co., Inc.
developed a method of obtaining L-cysteine byasymmetrically hydrolyzing
DL-2-aminothiazoline-4-carboxylic acid (DL-ATC), anintermediate in cysteine chemical synthesis, using
microbial enzymes.(46) The microbe Pseudomonas
thiazolinophilum has been screened as one that can becultivated using DL-ATC as the sole nitrogen source
and is presumed to contain ATC racemase andhydrolase, enzymes involved in subsequent reactions(Fig. 4.12). This production method was industrialized
by Ajinomoto Co., Inc. in 1982.

hydrolase

racemase

S-carbamoyl cysteinehydrolase
L-cysteine

Fig. 4.12 Enzymatic Production of L-Cysteine from DL-ATC
ATC: 2-aminothiazoline-4-carboxylic acid

4.8.5
D-p-Hydroxyphenylglycine
D-p-hydroxyphenylglycine (D-HPG) is a D-amino aciddeveloped as a component in semi-synthetic penicillin
amoxicillin, rather than a natural amino acid.
Amoxicillin is synthesized by introducing D-HPG tothe amino group at position 6 in 6-aminopenicillanic
acid, the penicillin structure.
While there have been several reports of methods for
obtaining L-amino acids by enzymatic asymmetrichydrolysis of 5-substituted hydantoins, intermediates inamino acid chemical synthesis, Hideaki Yamada et al.
of Kyoto University screened microbes that produce
hydantoinase, the enzyme that specifically produces
D-amino acids.(47) Of these, Pseudomonas putida wasselected as being the most active in convertingDL-5-(p-hydroxyphenyl) hydantoin into
D-N-carbamoyl-p-hydroxyphenylglycine. The
decarbamoyl reaction to convert
D-N-carbamoyl-p-hydroxyphenylglycine into D-HPG
was performed using a method of nitrite treatment
under acidic conditions. Kaneka achieved industrial
production of D-HPG and runs the manufacturing
process mentioned as follows at its Singapore plant(Fig 4.13).
chemical
synthesis

phenolglyoxylic acidurea

DL-5-(p-hydroxyphenyl) hydantoin

asymmetric hydrolysis (hydantoinase)

D-N-carbamoyl-phydroxyphenylglycine

D-p-hydroxyphenylglycine
decarbamoyl reaction

Fig. 4.13 Synthesis Reaction of D-p-Hydroxyphenylglycine

Survey Report on the Systematization of Amino Acid Fermentation Technology

19

The decarbamoyl reaction at the final stage in thisprocess was initially carried out by nitrite treatment
under acidic conditions, as mentioned above. However,this method produced a low yield and a lot of
byproduct. It also had significant environmental issues
due to heavy use of alkalis. Kaneka Corporation
screened Agrobacterium sp., a microbe containing the
D-carbamylase enzyme that catalyzes this reaction andsuccessfully cloned and expressed the D-carbamylase
gene in E. coli. This made it possible to establish a
production method using a bioreactor system andimmobilizing hydantoinase-containing Pseudomonas
putida and carbamylase-containing E. coli.(48)
4.8.6
LysineToray Industries, Inc. developed a system of producing
L-lysine from DL-α-amino-ε- caprolactam (ACL), theraw material for nylon, using the enzymatic method.(49)As shown in Fig. 4.14, the reaction involves selecting
Achromobacter obae, a microbe with an ACL racemase
activity, and Cryptococcus laurentii, a yeast containing
a hydrolyase that hydrolyzes ACL, combining theseand converting the DL-ACL almost quantitatively toL-lysine. Although this method was temporarily
industrialized in partnership with Takara Shuzo, it is
not practiced today.

L-lysine
ACL racemase

hydrolase

Fig. 4.14 Production of L-Lysine from
DL-α-Amino-ε-Caprolactam using the Enzymatic Method
ACL: α-amino-ε-caprolactam

4.9

Improving Producing Strains by Applying
Genetic Engineering (Recombinant DNA)
Technology

Genetic engineering technology, established around the1970s-1980s, has enabled various applications, such as
the isolating of specific genes and analyzing,
modifying or amplifying these genes. Since
overproduction of amino acids requires eliminating themetabolic regulation for each amino acid andincreasing the enzyme activity related to amino acidbiosynthesis as mentioned in Section 3.3, the ability toalter enzymes by gene-specific cloning or to increase
enzyme production meant increased amino acidbiosynthesis. This technology has an extremely wide
range of applications in breeding and improving
strains.

20

When genetic engineering technology was firstestablished, there were concerns about the safety oforganisms with heterologous genes and the impact on
the ecosystem. Experimentation guidelines wereenacted for gene recombination experiments and thenlegislated in 2004 as the Act on the Conservation andSustainable Use of Biological Diversity throughRegulations on the Use of Living Modified Organisms(commonly known to the Japanese as the “Cartagena
Protocol Hypothecation Act”). Since there are no issues
with safety where the origin of the microbe or gene isclear, appropriate experimentation is permitted andprogress has been made on practical implementation.
The E. coli K-12 strain has been demonstrated to besafe and is widely used throughout the world. The
wealth of information on relevant materials andmethods make it the easiest strain to apply genemanipulation technology. Improvements have been
made using this strain and it has even been used to
produce new amino acids.
By contrast, genome analysis on C. glutamicum, themain strain used in amino acid fermentation, has cometo an end. Although vector construction, gene transfer
methods, and other gene manipulation systems havebeen developed for it and Brevibacterium and thesehave been shown to increase production of threonine
and phenylalanine, they have not reached the point ofpractical implementation.
4.9.1
Threonine
Ajinomoto Co., Inc. obtained threonine-producing
strains from E. coli K-12 as an analog AHV-resistantmutant.(18) As mentioned in Section 4.7, homoserine
dehydrogenase, the key enzyme for threonine synthesis,
was desensitized in this mutation to make it insensitive
to threonine feedback inhibition. Based on thisinformation, Genetika, the Russian State ResearchInstitute for Genetics and Selection of Industrial
Microorganisms, selected its own threonine-producing
strains and applied gene manipulation technology toamplify the desensitized homoserine dehydrogenase
gene to increase threonine production to three times
that of the parent stock, reporting a yield of 20 g /L bycultivating it in a medium containing 6.2 % glucose.(50)
Ajinomoto Co., Inc. likewise increased threonine
production by three times that of the parent stock byamplified the E. coli K-12 threonine-producing gene
and went on to achieve a yield of 55 g/L by cultivating
it in a jar fermenter.(51)Ajinomoto Co., Inc. later improved this by introducing
microbes from Genetika. In other words, the HD gene wasinserted into the plasmid used as a vector, but the plasmid
was often unstable and the threonine production titerwould decrease. This was successfully stabilized bycloning by chromosomal recombination using Mu
phage.(52) Fig. 4.15 shows a schematic outline of strain
breeding. This strain was industrialized in 1991 by French

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

company Eurolysine, a joint venture of Ajinomoto Co.,
Inc., with the approval of the French government, having
been presented with the data on its safety. This is thought
to be the first instance of industrialization of a microbe
strain achieved by gene manipulation.
4.9.2
Lysine and Tryptophan
While Ajinomoto Co., Inc. made no specific externalannouncements regarding this, the company is believed
to have worked on improving lysine- andtryptophan-producing strains by applying genemanipulation technology to the E. coli K-12 strain withthe approval of the Ministry of Agriculture, Forestry
and Fisheries Agricultural Materials Council Feed
Committee and confirmation of adherence to Ministryof Economy, Trade and Industry guidelines.(53)threonine-producing strain
HD with desensitized chromosomal regulation
Mutant with gene ()(HD: homoserine dehydrogenase)

DNA extractionhydrolysate. Since this amino acid is unrelated tomicrobial biological activity, it cannot be produced bydirect fermentation using microbes. However,technology developed by Kyowa Hakko has made thispossible by using recombination technology to
combine the functions of different microbes.
In addition to being D- or L-compounds, the chemical
structure of hydroxyproline also has eight isomers,
depending on if the hydroxyl group is in position 3 or 4,
or if the hydroxyl and carboxyl groups are cis or trans.
The biologically active isomer istrans-4-hydroxyproline.
The method developed by Kyowa Hakko involved
screening Dactytylosporangium sp. producing proline
4-hydroxylase, the enzyme that introduces the hydroxyl
group at carbon 4 in the proline, cloning this gene andinducing it in a proline-producing strain bred from adifferent E. coli, thereby adding a hydroxyl group to
the proline. With a conversion yield of 100 % from theproline in this strain and a fermentation performance of
40 g/L, this method conforms to the industrialization
guidelines for genetically-modified organisms and isbeing implemented in production.(54)plasmidtarget enzymes not cut
degradation by restriction
enzymes(linking (ligation))transformation

selection of plasmids thatcan complement the
mutation lacking the HD
gene
miniMu phagetransformation

HD gene recombined into multiple
plasmids; increased HD activity
corresponding to the gene andimproved threonine production

multiple HD genes chromosomally
recombined and stabilized;
threonine production also stabilized

Fig. 4.15 Schematic Diagram of Improvements toThreonine-Producing Strains by Recombinant DNA Technology

4.10 Production of Novel Amino Acids throughApplication of Genetic Engineering
Technology

4.10.1
Hydroxyproline
This amino acid is the main component in animal
collagen. Used as an ingredient in pharmaceuticalproducts, hundreds of tons of it have been produced
around the world to date by extracting it from collagen4.10.2D-p-Hydroxyphenylglycine
The decarbamoyl reaction in the final stage of the
D-HPG synthesis process developed by Kaneka, as
mentioned in Section 4.8.5, has been established in a
production process that involves taking the
D-carbamylase gene that catalyzes the reaction from
Agrobacterium sp., fermenting it with E. coli and using
these cells as the enzyme source.
<References>(1) S. Kinoshita: “Amino-san Hakkō no Hakken to
Shinpo [Discovery of and Progress in Amino
Acid Fermentation]”, Japan Bioindustry
Association Amino Acid/Nucleic Acid Group(ed.), Hakkō kara Nyūbaiotekunorojī e [FromFermentation to New Biotechnology], p. 7, 1987.
(2) S. Kinosita et al.: J. Gen. Appl. Microbiol., 3, 193,
1957.
(3) S. Udaka: J. Bacteriol., 79, 754, 1960.
(4) K. Tanaka et al.: J. Agric. Chem. Soc., Japan, 34,593, 1960.
(5) History of Kyowa Hakko Co., Ltd., “Sore kara
Sore e [From There to There]”, Kyōwahakkō
50-nen no Kiseki to Shinseiki e no Ishizue[Kyowa Hakko’s 50-Year Trajectory andFoundation for a New Century], p. 67, September
2000.
(6) N. L. Sommerson and T. Phillips: Jpn. Examined
Patent Application Publication No. Sho 37-1965.
(7) K. Takinami et al.: Agric. Biol. Chem., 27, 858,
1963.
(8) J. Nakamura, et al.: Appl. Environ. Microbiol., 73,
4491, 2007.
(9) Amino Acid/Nucleic Acid Group (ed.):Amino-san Hakkō [Amino Acid Fermentation] 1& 2, Kyoritsu Shuppan, 1972.
(10) Aida, Takinami, Chibata, Nakayama and Yamada(eds.): Amino-san Hakkō [Amino Acid
Survey Report on the Systematization of Amino Acid Fermentation Technology

21Fermentation], Japan Scientific Societies Press,
1986.
(11) K. Aida, I. Chibata, K. Nakayama, K. Takinami,and H. Yamada (eds.): Biotechnology of Amino
Acid Production, Kodansha, Tokyo, and Elsevier,
Amsterdam-Oxford-New York-Tokyo, 1986.
(12) I. Shiio, et al.: J. Biochem., 53, 333, 1963.
(13) S. Udaka, et al.: J. Gen. Appl. Microbiol, 4, 272,
1958.
(14) K. Nakayama, et al.: J. Gen. Appl. Microbiol., 7,
145, 1961.
(15) F. Yoshinaga, et al.: J. Gen. Appl. Microbiol., 12,219, 1966.
(16) S. Okumura, et al.: Agric. Biol. Chem., 28, 742,
1964.
(17) S. Nakamori, H. Aida (ed.): Amino-san Hakkō[Amino Acid Fermentation], p. 307, Japan
Scientific Societies Press, 1986.
(18) I. Shiio, et al.: Agric. Biol. Chem., 33, 1152,
1969.
(19) I. Shiio, et al.: Agric. Biol. Chem., 34, 448, 1970.
(20) H. Kase, et al.: Agric. Biol. Chem., 36, 1611,
1972.
(21) S. Komatsubara, et al.: Appl. Environ. Microbiol.,35, 834, 1978.
(22) K. Sano, et al.: J. Gen. Appl. Microbiol., 16, 373,
1970.
(23) O. Tosaka, et al.: Agric. Biol. Chem., 42, 1773,
1978.
(24) I. Shiio, et al.: Agric. Biol. Chem., 37, 2053,
1973.
(25) S. Ikeda, et al.: Agric. Biol. Chem., 40, 511, 1976.
(26) M. Kisumi, et al.: J. Bacteriol., 110, 761, 1972.
(27) K. Araki, et al.: Agric. Biol. Chem., 35, 2081,
1971.
(28) H. Kamijo et al.: Abstracts from the Japan
Society for Bioscience, Biotechnology, andAgrochemistry, p. 112, 1973.
(29) I. Shiio, et al.: Agric. Biol. Chem., 36, 2315,
1972.
(30) I. Shiio, et al.: Agric. Biol. Chem., 37, 1991, 1973(31) T. Tsuchida et al.: Jpn. Unexamined Patent
Application Publication No. Sho 49-116294.
(32) S. Nakamori, et al.: Agric. Biol. Chem., 46, 487,
1982.

22

(33) K. Nakayama, et al.: Agric. Biol. Chem., 36, 1675,
1972.
(34) K. Kubota, et al.: J. Gen. Appl. Microbiol., 19,339, 1973.
(35) T. Tsuchida, et al.: Agric. Biol. Chem., 51, 2089,
1987.
(36) M. Kisumi, et al.: Amino Acid and Nucleic Acid
Fermentation and Metabolism, 19, 1, 1969.
(37) T. Tsuchida, et al.: Agric. Biol. Chem., 39, 1319,
1975.
(38) M. Kisumi, et al.: J. Biochem., 73, 107, 1973.
(39) Hibino et al.: Abstracts from the Japan Society
for Bioscience, Biotechnology, andAgrochemistry, p.247, 2000.
(40) T. Tosa: “Baioriakutā no Kōgyō-ka[Industrialization of the Bioreactor]”, Frontiers in
Chemistry, p. 50, September 1992.
(41) T. Tosa, et al.: Biotechnol. Bioeng., 19, 1973.
(42) K. Yamamoto, et al.: Biotechnol. Bioeng., 22,2945, 1980.
(43) T. Sato, et al.: Enzyme Engineering, 6, 271, 1982.
(44) H. Kumagai, et al.: Biochem. Biophys. Res.
Commun., 34, 266, 1969.
(45) H. Enei et al.: Bioscience and Industry, 54, 11,1996.
(46) K. Sano et al.: Appl. Environ. Microbiol., 34, 806,
1977.
(47) H. Yamada et al.: J. Ferment. Technol., 56, 484,
1978.
(48) S. Takahashi: J. Agric. Chem. Soc., Japan, 74,961, 2000.
(49) T. Fukumura: Agric. Biol. Chem., 41, 1327, 1977.
(50) V. G. Debabov et al.: Proceedings of IVthInternational Symposium on GIM (Kyoto), p. 254,
1982.
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1983.
(52) O. Kurahashi et al.: Proceedings of VIthInternational Symposium on GIM (Strasbourg), p.
178, 1988.
(53) Nikkei-Bio Yearbook 2004, p. 575.
(54) Shibasaki et al: Biosci. Biotech. Biochem., 64,
746, 2000.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

5 Development of Cultivation Technology andEquipment

Large-scale cultivation involves the scaling up andreproduction of test results for screened or bredmicrobes obtained at test tube or flask level tolarge-scale jar fermenter or tank cultivation, as well as
the development of cultivation technology that cannot
be achieved at test tube or flask level.

Fig. 5.1 shows an outline of the large-scale cultivationequipment commonly currently used in amino acidfermentation. This technology has been establishedfundamentally based on a submerged cultivation
system developed for producing antibiotics.

1. Air Sterilization
2. Medium Sterilization
air inlet
medium flow control
preparation tanktemperature
controlpreheaterheaterdust removal
devicesteam

coolant

dehumidifier

heaterfilterflow control
pump
pre-filter
gas-liquid
separatorpressure control

air supply linepressure control

high-pressure
steam

exhaust5. Entire System Sterilization

pH electrodecoolerfermentation tank

3. Temperature Control

temperature
controloxygenelectrodeliquid ammonia
4. pH Control through Ammonia Supply

Fig. 5.1 Schematic Diagram of Large-Scale Cultivation Equipment (from (1))

There are five main elements to the system: (1) air
sterilization; (2) medium sterilization; (3) temperature
control; (4) pH control; (5) entire system sterilization.
Together, these form the system that controls theaeration and agitation culture taking place in thecentrally-positioned fermentation tank. This is all
controlled by computer. The following technologies
have been developed for this system.

5.1

Oxygen Supply

Oxygen is not only required for energy acquisition
through microbe respiration, but it is also thesecond-highest consumed raw material in thecomposition of amino acid next to sugar, which
provides the carbon source. If there is not enoughoxygen in glutamic acid fermentation, succinic acidand lactic acid will be produced and the amino acidyield will decrease significantly. Extensive
investigation has been carried out on the impact ofoxygen concentration on amino acid production byamino-acid-producing strains derived from
Brevibacterium flavum and B. lactofermentum(C.
glutamicum). High oxygen concentrations produced thegreatest yield of glutamic acid and its synthesis
products glutamine, alanine, and proline, while low
oxygen concentrations produced the greatest yield ofvaline and phenylalanine.(2)Since oxygen has a low solubility, the oxygen supply
can form a bottleneck in fermentation productivity if ahigh oxygen concentration is required. This technicalissue was successfully resolved by using large turbine
blades for culture agitation and increasing the oxygen
supply capacity.(3) There are ongoing investigations
into improving the oxygen supply by using liquid
oxygen to increase the oxygen partial pressure in the
air or by using oxygen enrichment devices.
Survey Report on the Systematization of Amino Acid Fermentation Technology

23

5.2

Fed-Batch Cultivation

This method is an improved batch culture method inwhich the target product concentration and batch-wiseproductivity is increased by adding a special reserve ofsugar to continue cultivation once the culture has
consumed all of the sugar content in the main raw
material. While this method was developed for
antibiotic fermentation, Ajinomoto Co., Inc. applied
this technology to the production of glutamic acid andother amino acids, increasing productivity by
extracting liquid in proportion to the amount of liquid
added.(3)

5.3

Regulating the Osmotic Pressure of the
Culture Fluid

Ajinomoto Co., Inc. also discovered that the amount ofglutamic acid or other amino acid accumulated reaches
a plateau after a certain time. While this is due to a
decline in microbial production activity, one cause wasshown to be an increase in the osmotic pressure of theculture fluid. Countermeasures to increase production
speed included gradually adding to the sugarconcentration or adding glycine betaine.

5.4

Controlling Foaming

Large amounts of foaming can occur, depending on thetype of fermentation. The causes of foaming arecomplex and have not been identified, but depend on a
combination of factors, including medium composition,
cell composition, amino acid or other product,temperature, and pH. Therefore, it is not uncommon for

24

foaming to occur unexpectedly due to differences in thetype of amino acid or microbe. Where there is a lot of
foaming, the fermentation liquid can spurt out of thefermentation tank, causing the microbes to becomecontaminated and requiring the volume of fermentation
liquid to be reduced. Reducing the volume of liquidmeans reducing the productivity. To solve this issue,
technology has been developed that treats the foamwith an anti-foaming agent or mechanically breaks it
down by sending it to the cyclone separator along withthe exhaust emissions.(3)As mentioned previously, these technologies haveessentially taken the knowledge gained from majoradvances in the industrialization of penicillin
fermentation and applied it to amino acid fermentation.
Although developed mainly for glutamic acidfermentation, they have of course also been widelyadopted into fermentation processes for other aminoacids as well.
Large-scale cultivation and separation/purification
processes require a lot of equipment. This is all
controlled by computer.(4)<References>(1) S. Nakamori and H. Shibai: “R&D Trend
Analysis”, Biotechnology in Japan, p. 84, KRI, Jan.
2001.
(2) K. Akashi et al., H. Aida (ed.): Amino-san Hakkō[Amino Acid Fermentation], p. 127, Japan
Scientific Societies Press, 1986.
(3) Ajinomoto Co., Inc.: Kenkyūjo 50-nen-shi: ‘Ashita
no Moto’ o Motomete [A 50-Year History of theInstitute: Searching for the ‘Substance ofTomorrow’], pp. 135-136, September 2006.
(4) Y. Goto, H. Aida (ed.): Amino-san Hakkō [Amino
Acid Fermentation], p. 145, Japan Scientific
Societies Press, 1986.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

6 Development of Amino Acid Separation and
Purification Technology

The so-called “downstream” technology of amino acidfermentation is the process of separating, purifying,
and collecting the amino acid from the culture fluid andbioreactor reaction solution. This includes decoloring
and crystalizing the amino acid, as well as labor-saving
technologies. This is an important process that
determines the quality of the product, while the qualityof the yield significantly impacts the cost. While there
are very few academic reports on this technology, there
is a lot of expertise in the field that can be drawn fromresources found in company histories and similarsources. The following sections outline
separating/purifying glutamic acid and lysine fromfermentation liquid and separating/purifying aspartic
acid from fumaric acid using the fermentation method.
Processes for other amino acids are generally the sameas these, with only minor differences.

6.1

Monosodium Glutamate

neutralizing
solution(crystallization)(separation)

L-glutamic acid crude crystals

mother liquor(neutralizing)(separation)

Activated carbon

L-glutamic acidhydrochloride crude
crystals(decolorization)

Fig. 6.1 shows an overview of this separation/purification
process. This method has been used since the early daysof glutamic acid fermentation to collect the glutamic acidas a salt from the fermentation mother liquor. Later,technology was developed to improve the elimination of
impurities by transferring the glutamic acid α-form
crystals into β-form crystals. Technology was also
developed to collect the α-form crystals from the motherliquor by concentrated crystallization. These technologies
have increased the recovery rate of glutamic acid and also
have the advantage of suppressing dispersal of theammonia formed in the concentration of the culture fluid.

6.2

L-glutamic acid fermentation
liquid(concentration)(crystallization)(separation)mother liquor(drying)
MSGcrystals

Fig. 6.1 L-Monosodium Glutamate Purification and Separation
Process (1)L-lysine fermentation
liquidLysine Hydrochloride(microbe separation)

Fig. 6.2 shows an overview of theseparation/purification process for lysine hydrochloride.
This process also faced major technical development
issues in terms of increasing production, conserving
energy and minimizing environmental impact. This
process centers around ion exchange resin treatment. In
the early days, this involved filling a large resin column
with resin and draining out the culture fluid, eluent, andregenerating solution in turn by switching valves. As
this method was time-consuming and produced low
yields, new technology was developed to allow
continuous operation by having the culture fluid, eluent,and regenerating solution drain quickly, with smallercolumns for each fluid switched in turn.
The resin operation was further simplified when E. coli
was introduced as a producing strain andmicrofiltration membranes started being used for thefiltration of this strain.(3)(neutralization)(ion exchange resin)aqueous ammonia
eluenteluate(concentration)(crystallization)(separation)mother liquor(drying)
L-lysine hydrochloride crystals

Fig. 6.2 L-Lysine Hydrochloride Purification and
Separation Process (2)

25

6.3

Aspartic Acid

Since aspartic acid is produced from fumaric acid in abioreactor using immobilized E. coli with aspartase
activity, there are no microbial cells, fermentation
medium, or byproducts secreted from the microbes
such as those found in the glutamic acid or lysine
culture fluid. Accordingly, the reaction solution is far
simpler in composition than the culture fluid and it iseasier to process. Fig. 6.3 shows an overview of theaspartic acid separation/purification process.
<References>(1) H. Ito: Society for Chemical Engineers, Japan(ed.) Baiopurosesu Binran [Bioprocess
Handbook], p. 236, 1996.
(2) T. Kawakita: Society for Chemical Engineers,
Japan (ed.) Baiopurosesu Binran [Bioprocess
Handbook], p. 289, 1996.
(3) Ajinomoto Co., Inc.: Kenkyūjo 50-nen-shi:‘Ashita no Moto’ o Motomete [A 50-Year Historyof the Institute: Searching for the ‘Substance ofTomorrow’], pp. 135-136, September 2006.
(4) I. Umemura: Society for Chemical Engineers,
Japan (ed.) Baiopurosesu Binran [Bioprocess
Handbook], p. 71, 1996.

26

reactionsolution
activated carbon(decolorization, 80-90 °C)(ultrafiltration)
sulfuric acid(crystallization, <90 °C)(cooling, >15 °C)(crystal separation)(drying)

L-aspartic acid crystals

Fig. 6.3 L-Aspartic Acid Purification and Separation Process (4)National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

7 Amino Acid Standards
Amino acid standards have been listed in all kinds of
documents (including the Japanese Pharmacopoeia, theJapanese Pharmaceutical Codex, the Japan Chemical
Additives Specifications, and the Japanese Standards ofFood Additives). Companies have also determined theirown standards. Here, we shall outline the amino acidtest standards adopted by Ajinomoto Co., Inc. The
main items and details are as follows.(1)Specific optical rotation [α]A means of determining the optical purity of aminoacids, measured using a polarimeter. Optical rotation
measurements are carried out at a temperature of 20 ˚C,a layer length of 100mm and the light used is thesodium D line.
Dissolved state
A limit test of the turbidity and color of the sample
solution. Test methods include visually checking theturbidity and color against a white background or using
a spectrophotometer to measure the transmittance at ameasurement wavelength of 430mm using a 10mm cellwith a solvent for contrast.
Chloride (Cl)A limit test of the chloride (Cl) mixed in with thesample or a quantitative test of the chloride present in
the sample composition. Silver nitrate is used for thelimit test, while the Volhard method is used for thequantitative test.
Ammonium (NH4)
A limit test of the ammonium (NH4+) mixed in with thesample. After the sample solution has been distilled,
the indophenol method is used with sodium ferrous
phenol pentacyanonitrosyl and a mixture of sodium
hypochlorite and sodium hydroxide.
Sulfate (SO4)
A limit test of the sulfate (SO4) mixed in with thesample, using barium chloride.
Iron (Fe)A limit test of the iron (Fe) mixed in with the sample,
using 1,10-phenanthroline.
Heavy metals (Pb)
A limit test of the heavy metals mixed in with thesample. These heavy metals are metallic contaminants
that are colored by acidic sodium sulfide solution; thelimit is expressed as the amount of lead.
Arsenic (AS2O3)
A limit test of the arsenic mixed in with the sample.
The limit is expressed as the amount of arsenic trioxide(AS2O3).

Other amino acids
A method of separating and detecting other aminoacids mixed in with the sample. The test method usedis thin-layer chromatography, in which a layer of silica
gel is thinly spread onto a glass plate is used as thestationary phase. The sample is spotted onto this anddried and the lower part dipped into an eluting solvent
solution. The sample then separates and moves in thespace between it and the solvent. The piece is sprayedwith a ninhydrin reagent and the ratio between the
distance travelled by the colored amino acids and the
distance travelled by the solvent is used to identify theamino acids contained in the sample.
Drying loss
Measuring the amount of moisture content, part or allof the crystallization water and volatile substances lost
during drying.
Moisture content
A method of measuring the moisture content in thesample using titration (Karl Fischer method).
Ignition residue (sulfate)Measuring the amount of residual substance that did
not volatilize when ignited to constant weight at
550-650 ˚C as a sulfate.
Content
Non-aqueous titration: Titrating an amino aciddissolved in a non-aqueous solvent (usually acetic acid)as a base in perchloric acid and measuring the change
in potential difference.
Neutralization titration: Titrating an acidic amino acid(aspartic acid, glutamic acid) dissolved in water as anacid in a sodium hydroxide solution and quantifying itwith the indicator phenolphthalein or measuring thechange in potential difference.
Iodometric titration: Titration of excess iodine insodium thiosulfate using the reaction of cysteine withiodine.
pH
Measured using a glass-electrode pH meter.
Endotoxins (injected)
A method used for detecting endotoxins from
Gram-negative microbes using a lysate reagent
prepared from components extracted from horseshoe
crab blood cells.
<References>(1) Ajinomoto Co., Inc. Amino Acid Division (ed.):Ajinomoto’s Amino Acids Handbook, pp. 71-73,
January 2003.

Survey Report on the Systematization of Amino Acid Fermentation Technology

27

8 Main Raw Materials for Amino Acid Fermentation
Besides the carbon source, which is the main raw
material for amino acid fermentation, other raw
materials include ammonia and amino acid mixtures as
nitrogen sources, phosphoric acid, potassium,magnesium, iron, or manganese as mineral components
and biotin, thiamine, or other vitamins. Surfactants andβ-lactam antibiotics are also used in glutamic acidfermentation. The carbon source occupies the largest
proportion of the raw materials. Since the other raw
materials are substantially lower in quantity than the
carbon source, they are omitted from the present
description. While this report includes the enzyme
method of amino acid production as production byfermentation in the broader sense, it does not cover the
raw materials for the enzyme method, as these are
wide-ranging, depending on the type of amino acid.
Accordingly, the main raw material for the direct
fermentation method refers to the main carbon source.
Table 8.1 shows the current situation and points ofissue related to the main carbon source materials andpotential raw materials. The main raw material for thefermentation process is glucose, either starch-based or
sugar-based. Starch-based glucose is converted to sugarwith an enzyme (amylase). Sugar-based glucose can beeither raw sugar from sugarcane or molasses. Molasses
is the crystallization mother liquor that is left after the
raw sugar crystals have been collected. It can be maderelatively cheaply from sugarcane or sugar beet; theformer is called cane molasses, while the latter is called
beet molasses. Both are composed of sucrose, glucose,and fructose. Of the materials shown in Table 8.1,
acetic acid and ethanol are able to be used (assimilated)by the glutamic acid bacteria. Special screening has
been carried out on microbes able to assimilate
methanol and n-paraffin and producing strains arebeing bred from these.

A major current issue is a sudden price rise for starch,sugar, and molasses due to a growing demand for theseas a source of bioethanol due to rising oil prices
worldwide. Accordingly, a future issue will be
developing biomass, methanol, carbon dioxide, andother underused source materials to avoid acompetition between food and energy.
Table 8.1 Current Situation and Points of Issue
Regarding Currently Used and Potential Main Carbon
Sources for Amino Acid Fermentation
Carbon Source
Starch-basedCorn, cassava,
sago palm, etc.

Sugar-based
Cane molasses
Beet molasses
Raw sugar(sucrose)

Assimilated by enzymes (amylase); the most widelyused in current amino acid fermentation. Priceincrease due to competition with bioethanol asignificant point of issue.
Molasses is the crystallization mother liquor fromsugar and is made up of sucrose, glucose, andfructose. Used in glutamic acid and lysine
fermentation. Price increase a significant point ofissue.

Other raw materials

Acetic acid

Methanol

Ethanoln-paraffin(n-alkane)

Cellulose-basedmaterials(biomass)
Carbon dioxide

28

Current Situation and Points of Issue

A superior raw material for usability and amino acid
purification than colored mixtures such as molasses
due to its solubility and purity. Used briefly forfermentation of glutamic acid, lysine, and threonine,
but its use was discontinued after it rose in pricefollowing the oil crisis (1980).
Superior for usability and amino acid purification
due to its purity. Currently receiving some attention
due to rising prices of starch-based and sugar-basedsources, but present productivity is low. For further
study.
Superior for usability and amino acid purification
due to its purity. Studied briefly for practicalimplementation but this is currently discontinued.
Received attention as a potential source material
prior to the oil crisis (1980), but was discontinued
beyond the research stage due to price increase andconsumer aversion.
Receiving attention as a renewable resource fromrice straw, chaff, bagasse, scrap wood, etc.
Bottlenecked at collection and sugar conversion.
Thought to be the ultimate ideal raw material, but
has low production yield in its present state. For
further study.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

9 Overseas Expansion of Amino Acid Fermentation
Technology

Kyowa Hakko exported its glutamic acid fermentation
technology developed in 1957 to US company Merck in 1958
and to Taiwan company Ve Wong around the same time. The
company also exported its lysine fermentation technology to
Merck in 1958.
Meanwhile, Ajinomoto Co., Inc. used a strategy ofestablishing factories to expand its glutamic acidfermentation technology overseas. This expansion madeactive progress, with plants successively established in
Thailand in 1961, Malaysia, the Philippines, and Italy in 1962,
Peru in 1968, Indonesia in 1969, Brazil in 1974, and then into
the United States, China, Vietnam, and other countries.
Ajinomoto Co., Inc. also established subsidiary company
Eurolysine in France in 1974 and began fermentation
production of lysine for use in animal fodder. The company
also established Heartland Lysine Inc. in the United States(Iowa) in 1984, followed by further expansion into Brazil,
Thailand, Italy, and China. In France and the United States,
production began on threonine and tryptophan for animal
fodder in addition to lysine. This successful overseas
expansion of amino acid fermentation technology led to anincrease in amino acid production volume worldwide, as
mentioned in Section 2.4.
Meanwhile, although Kyowa Hakko advanced its lysine
technology into Hungary and its lysine, threonine, andtryptophan technology into the United States and Mexico,
according to the Nikkei-Bio Yearbook,(1) it closed down its
operations in Mexico in March 2004, sold all of its stock in
Hungary, including its intellectual property related to lysine,
threonine, and tryptophan, to Degussa and faced thechallenge of restructuring its BioKyowa facilities in the
United States. This shows a stark contrast between the twocompanies.
Ajinomoto Co., Inc. started increasing its production ofamino acids for pharmaceutical products at its plant in the
United States (North Carolina), followed by China and Brazil.
By contrast, although Kyowa Hakko faced a slump in the
United States, as mentioned above, it was increasing its
production in China (Shanghai Kyowa).
Table 9.1 shows an outline of the distribution, product lines
and production capacity (including estimates) of the mainamino acid plants around the world.
Most of the processes used at these factories are based onJapanese technology; in many cases, the overseas
manufacturer has access to a cheap supply of raw materials
and is using Japanese technology that has come off patent.
China is a major player in MSG production and consumption,
consuming around one third of the MSG produced around theworld. Production volumes, around 30,000 tons in 1980,
increased by around 20 times to around 600,000 tons in 1997.
While further increases in demand can be expected as living
standards rise and eating habits improve, there are around
100 manufacturers in the industry in addition to the major
ones listed in Table 9.1, meaning intense competition inwhich only the strong will survive. There is a growing
demand to respond to environmental issues and the major
players are beginning to consolidate.(2)In contrast to theflourishing overseas expansion, there is very little amino acidbeing produced within Japan at present (2007). This is anatural consequence of the dependence of amino acidfermentation on raw materials, as well as the large market
and low cost of labor overseas.

Table 9.1 Distribution, Product Lines and Production
Capacity of the Main Amino Acid Plants Worldwide
Country

BrazilChina

FranceGermany
Hungary
Italy
Indonesia

Japan

Company(conglomerate)

RegionAjinomotoSao PauloInteramericana
Ajinomoto Biolatina “Laranjal
Paulista
Valparaiso
Pederneiras
Linghua MSG
Jining, ShandongGroup
Lianhua MSG
Xiangcheng,
Group
Henan
Hongmei MSG
Shenyang,Group
Liaoning
Guangzhou Orsan
Guangzhou,
Gourmet Powder
Guangdong
JiangsuZhangjiagang,
Chrysanthemum
Jiangsu
Gourmet Powder
Group
GBT
GBT Technology
ChangchunGroup
BBCA Biochemical Anhui
Juneng Group
Shandong
Sichuan Chuanhua Chengdu, Sichuan
Ajinomoto
AjinomotoShanghai
Kyowa Hakko“
Ajinomoto
AmiensEurolysine““
Ajinomoto Foods
Nales
EuropeDegussaFrankfurt
Kyowa Hakko
Kaba
Ajinomoto Bioitalia Bottorighe
Cheil Sugar
Pasruan““
Ajinomoto
Slavaya
SASA
Miwon Indnesia
Ajinomoto“

Kawasaki
Saga

Kyowa Hakko
YamaguchiSouth Korea Cheil Sugar
Desan
BASF
Malaysia
Malaysia Ajinomoto Kuala Lumpur
MexicoKyowa Hakko
Philippines
Peru
Slovakia

ThailandTaiwanUnitedStatesVietnam

Ajinomoto
AjinomotoDegussa

ManilaLima
Slovenska Lupca““
Thailand Ajinomoto Bangkok
Kamphaengphet
Patontani
Vedan
USA Ajinomoto
Iowa
North Carolina
Ajinomoto
Iowa
Heartland“
BioKyowa
Missouri“
ADM
Illinois“
Vietnam Ajinomoto
Vedan
Miwon
-

Products

Glu

Production Capacity(tons/year) (including
estimates)
90,000various
Glu
Lys
Thr
Lys
Glu

2,000
80,00060,00013,00060,000140,000“

120,000“

50,000“40,000“40,000Lys“

240,000
120,000“““

20,00020,00030,000

Various
“
Lys

800
800
70,000

Thr
Trp
Glu

35,0003,000
65,000

Met
Lys
Lys
Glu
Lys
Thr
Glu
Glu
Glu

222,000(withdrawn)
30,00050,000100,000
10,00050,00065,00080,000variousPhe, Arg, Asp,
Cysvarious
Glu
Phe
Lys
Glu
Lys
Thr
Glu
Glu
Lys
Thr
Trp
Glu
Glu
Lys
Glu
Glu
variousLys

60,0004,000
70,00015,000(withdrawn)“
20,00010,00010,0003,000
400
90,00040,00050,000100,000
40,00050,000

Thr
Lys
Thr
Lys
Thr
Glu
“
“

40,000(withdrawn)“
160,000
10,00060,000200,000
20,000

http://www.ajinomoto.co.jp/kyushu/info.html
http://www.ajinomoto.co.jp/kawasaki/profile.html
http://www.kyowa.co.jp/csr/sustain/report/houfu.html
http://www.ajinomoto.com/ar/i_r/pdf/fact/Feed-useAA-Oct2007.pdf#search=’
Based on the above websites as well as articles in the Health Food Journal, Nikkan
Kogyo Shimbun, The Chemical Daily and other sources.

Survey Report on the Systematization of Amino Acid Fermentation Technology29

Although Tanabe Pharmaceuticals actively publishedresearch on amino acid fermentation up until the 1980s,
it is no longer doing this. It has had a change inmanagement policy due to its enterprise integration
with Mitsubishi and it is thought to be likely to focus
its research and development goals towards
pharmaceutical products.

30<References>(1) Nikkei-Bio Yearbook 2005: p. 538.
(2) S. Matsunaga: “The Chinese MSG Industry”,
http://www.cin.or.jp/beijing/members/quarterly/in
dustry99_1.htm (01/31/2008)

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

10 Amino Acids and Environmental Issues
10.1 Fermentation Process

10.2 Livestock Ammonia Emission Reduction
Effect from Amino Acid Supplements in

The fermentation process itself is a natural process, likebrewing sake or soy sauce, and has no problematic
impact on the environment. Systems are being
developed that are even more actively in harmony withthe environment. For example, in the process of
producing glutamic acid by fermentation fromsugarcane, Ajinomoto factories in Indonesia and Brazil
produce a waste liquid byproduct containing microbes,
ammonia, phosphoric acid, calcium, and magnesium.
They have established a system of returning thisbyproduct to the land in the form of a near-complete
fertilizer. This resource-circulation type of production
process is called a bio-cycle. Fertilizer made from thisbyproduct has been shown to effectively increase theyield and quality of the agricultural produce and is not
only used on sugarcane, but also on corn, coffee, fruit
trees, and flowers (1).

farmorganic
fertilizer

sugarcaneorganic nitrogen(microbe)
inorganic nitrogenmineralssugar factory

factory

molasses
raw sugar

Animal Fodder
Emissions of nitrogen (ammonia) from animal
excrement are thought to be a leading cause ofenvironmental pollution, affecting the air quality or thewater quality of rivers and lakes. Nitrogen emissions
are caused by an imbalance in the amino acids in the
animal fodder. The amino acid composition in the cornand other grains used for animal feed lacks particularamino acids when compared to the amino acidcomposition of eggs or milk, meaning a poor aminoacid balance. Accordingly, even if there are a lot ofother amino acids present, they will be broken down
into ammonia and excreted rather than being used. Thisis the cause of nitrogen emissions. Consequently,
supplementing the animal fodder with the lysine andthreonine lacking in the corn and other grains not only
improves nutrition, but also increases the utilization of
the amino acids and significantly reduces the amountof nitrogen emissions.(2) Thus, effective use of aminoacids helps to conserve the environment.
<References>(1) Ajinomoto Co., Inc. Public Relations Department:
Amino-san Hakurankai [Amino Acid Exposition],p. 17, September 2004.
(2) Ajinomoto Co., Inc. Public Relations Department:
Amino-san Hakurankai [Amino Acid Exposition],p. 15, September 2004.

sugar

Fig. 10.1 Raw Materials and Byproduct Cycle System at an
Amino Acid Fermentation Factory (revised from (1))

Survey Report on the Systematization of Amino Acid Fermentation Technology

31

11 Safety of Amino Acids
11.1 Debate over the Safety of MSG
Amino acids are nutrients routinely ingested as food or
fodder in the form of protein or in isolated form. They
are believed to be extremely safe compared with
non-routinely ingested products such as medicines.
However, concern and anxiety over the safety ofchemical compounds used in pharmaceutical products
and food additives spread throughout the world fromthe late 1960s onwards, extending to amino acids aswell. Amidst these circumstances, the following tworeports emerged on MSG.
(1) A report on the so-called Chinese restaurant
syndrome claiming that excessive ingestion of MSG in
Chinese cuisine caused hot flashes, headaches, heart
palpitation and other symptoms (1)(2) A report claiming that newborn juvenile mice
forcibly administered with large amounts of MSG(0.5-4.0 g/kg bodyweight) incurred neuronal cell deathin the hypothalamus and inner layers of the retina (2)These reports were widely picked up on by newspapers
and other media, with Asahi Shimbun in particular
reporting in a one-sided and sensational manner, “Et Tu,
Monosodium Glutamate?” (likening it to sodium
cyclamate, which was banned from being sold due tocarcinogenic issues). Based on results by J. W. Olney,author of report (2) above, the United States Senate
Select Committee on Nutrition and Human Needs
recommended that MSG not be used in baby food.
In June 1970, the WHO/FAO Joint Expert Committee
on Food Additives (JECFA) recommended an
acceptable daily intake (ADI) of 120 mg/kg
bodyweight for MSG. With the consumer movement
led by R. Nadar intensifying in the United States,
re-evaluation began on food additives andpharmaceutical products that had been designated as
GRAS (Generally Recognized as Safe).
Meanwhile, MSG manufacturers’ committees aroundthe world formed into the International Glutamate
Technical Committee to demand a verifiable judgement,
promoting the collection of scientific and impartial dataand holding open symposia. Consequent reports statedthat there were no signs of neurotoxicity resulting fromoral ingestion of MSG and that a double-blind trial
using a placebo had proven that there was nocorrelation between MSG ingestion and the adverse
symptoms reported above (the so-called Chinese
restaurant syndrome).(3), (4) In May 1980, the FDA
announced its final conclusion that MSG was “safe as afood additive at its current usage level,” even for baby
food.
Even with this scientific “safety declaration” for MSG,

32

there is still a persistent theory that MSG is harmful,with products and stores using catchphrases assuringthat “no chemical seasonings are used in our products.”
A survey carried out by the author on university
students revealed that over 90 % of students have theimpression that MSG is “not good for health.” It is theauthor’s belief that proper understanding is necessary
and that publicity activities would play a major role inthis. There is a problem in the current situation with awidespread, deep-rooted sense of antagonism based on
misunderstanding and completely removed frompeople’s likes or dislikes.

11.2 Amino Acid Safety Data
Table 11.1 shows the median lethal dose LD50 (thedosage required to kill 50 % of the members of a tested
population = acute toxicity) of various amino acids.
The values for the salts and acetic acids used ineveryday food are also given here for the sake ofcomparison; the values for most of the amino acids arehigher than these, in other words, they are far safer.
Subacute toxicity testing (testing involving 1-3 monthsof continuous administration to rats and then checking
for weight change and histopathology of visceralorgans), two-year chronic toxicity testing on mice, rats,and dogs, and carcinogenicity testing have proven thatno changes occurred at all compared to the control
groups.(6)Table 11.1 LD50 Values of Amino Acids and Other
Food Ingredients (5)Substance
Salt
Cys
Acetic acid
Vitamin B12
Sodium carbonate
NiacinLys(Cys)2Citric acid
Vitamin C
GlycerolSer
Arg, Phe, Gly
Ala, Asn, Ile, Gln, Glu,
Thr, Tyr, Trp, Val, His,
Pro, Met, Leu
SucroseLD50(g/kg bodyweight, orally administered
to rats)
3.0
3.1
3.3
4.0
4.1
7.0
10.6
11.2
11.7
11.9
12.6
14.0
16.0
>16.0
29.7

All of the amino acids examined were L-compounds.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

11.3 Issues with Chemical Compounds Derived
from Tryptophan
There is a case of an incident presumed to be caused bysecondary substances produced from amino acids. In
the early 1980s, there were instances of
eosinophilia-myalgia syndrome (EMS) believed to becaused by a particular batch of tryptophan, resulting inover 30 fatalities. The substances thought to be thecause were identified within a relatively short space oftime to be ethylene-bis (tryptophan) and (phenylamino)
alanine. These compounds were detected in productsthat used a particular batch of tryptophan that had low
purity and are believed to have formed as thetryptophan transformed in the presence of impurities.
The supplier, Showa Denko, had to pay a high amountof indemnity because of this incident. While it does
depend on the type of amino acid, this incident
particularly highlights the importance of product purity
and content quality control.

<References>(1) R. H. M. Kwok: “Chinese-Restaurant Syndrome”,
New Engl. J. Med., 278, 796, 1968.
(2) J. W. Olney: “Brain Lesions, Obesity, and Other
Disturbances in Mice Treated with Monosodium
Glutamate”, Science 164, 719, 1969.
(3) Y. Takasaki: “Studies on Brain Lesions after
Administration of Monosodium Glutamate toMice. II. Absence of Brain Damage Following
Administration of Monosodium Glutamate in theDiet”, Toxicology, 9, 307, 1978.
(4) R. S. Gela, et al.: “Multicenter, Double-Blind,
Placebo-Controlled, Multiple-Challenge
Evaluation of Reported Reactions to Monosodium
Glutamate”, J. Allergy Clin. Immunol.106, 973,
2000.
(5) Ajinomoto Co., Inc. Amino Acid Division (ed.):Ajinomoto’s Amino Acids Handbook, pp. 74-75,
January 2003.
(6) S. Ota: Umami Chōmiryō no Chishiki [Knowledgeof Flavor Seasoning], pp. 100-103, Saiwai Shobo,
June 1992.

Survey Report on the Systematization of Amino Acid Fermentation Technology

33

12 Systematization of Amino Acid Fermentation
Technology

It has been a century since the beginning of industrial
production of amino acid, and 50 years since thebeginning of industrial production by fermentation.
Both of these technologies are original to Japan. Whileamino acids are now mainly produced by fermentation,
this is one of three production methods, along withprotein hydrolysis and chemical synthesis. Eachmethod was developed with a lot of support fromscience and technology. Table 12.1 summarizes the
progression of this technology, focusing on thefermentation method, the main topic of this report.
The first amino acid commercialized was glutamic acidin 1908, produced by protein hydrolysis. Production
using this method continued until around 1960;in otherwords, although increasingly fewer amino acids were
produced using the protein hydrolysis method due tothe establishment of the revolutionary fermentation
method around that time, the method is still used today
for some amino acids, including tyrosine, cysteine, andasparagine. The amino acid separation and purification
technology established for this method was also used in
the fermentation method.
The method of producing glutamic acid by chemical
synthesis was developed to overcome a few issues withthe proteolysis method. This technology originatedfrom synthetic chemistry and although it wasindustrialized for a short time, it was not well received
by consumers and was largely discontinued. Currently
some amino acids are still produced using the chemical
synthesis method, including glycine, DL-methionine,
and DL-alanine.
The fermentation trend began with the major discoveryof glutamic-acid-producing microbes and glutamic acidfermentation, which still occupies a central position inamino acid fermentation. This technology was
undergirded by interest from many researchers due tothe fact that there was already a successful business forthe attractive target product, namely sodium glutamate(“Ajinomoto”). The technology was also supported byexisting science and technology that could be used as34

an approach to glutamic acid production, namely
microbe screening and cultivation technology in thefields of microbiology, biochemistry, and molecularbiology. The technological development is thought tohave been significantly influenced by prior antibiotics
production methods using fermentation.
It appears that it was not considered possible at thetime to produce glutamic acid directly from glucose
and the successful isolation of a new microbe called aglutamic acid microbe was deemed a successful“defiance of common sense.” The key to the successlay in the screening method. Interestingly, thistechnology was produced by Kyowa Hakko, acompany that specialized in fermentation technology
rather than glutamic acid. Technology developed forthe protein hydrolysis method was used for separating
and purifying the glutamic acid from the culture
solution.
The method of inducing auxotrophic mutants andanalog-resistant strains, particularly the latter, fromglutamic acid microbes resulted in manyamino-acid-producing microbes and amino acidfermentation suddenly skyrocketed. At the same time,
another production method was established involving
the use of a bioreactor, screening the microbes used infermentation and developing the enzyme method.
Genetic engineering technology that emerged in the
1980s has already made major progress on existingproducing strains as a “booster” to the existing
technologies and further significant technologicaldevelopments can be expected in future. Around 75 %of amino acid products and 85 % of amino acid productvolume is produced using the fermentation and enzyme
methods.
Amino acid fermentation will continue to increase
quantitatively as the population increases. Further
significant developments are also likely with the recent
establishment of peptide synthesis technology,
produced from amino acids.

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

Survey Report on the Systematization of Amino Acid Fermentation Technology

35

13 Future Developments
Amino acid fermentation is original Japanesetechnology that has now spread around the world,
contributing to medical care and food supplies. Japan isat the center of these technologies and leads the worldin this industry. Amino acids are essentially naturalcompounds and there is no alternative protein
compound for them, especially nutritionally. The
demand for them will certainly increase, given the
expected future worldwide population growth and foodshortage.
Although the belief is that large growth is expected in
the market and that superior technology will produce
large profits, the fact is that this is not always the case.
The main reasons for this are increases in the price ofraw materials and decreases in product prices. This isparticularly true of glutamic acid and lysine, which areproduced in large quantities and the expense of raw
materials takes up a large proportion of the cost. The
basic patents for the technology have already expired,
meaning that Japan no longer has the advantageous
position it held to begin with and is now in increasingly
fierce competition with overseas manufacturers, whohave access to cheap raw materials. The cost increase
can no longer simply be incorporated into the productprice, necessitating further cost reductions andoptimized production efficiency through increased
yields, efficient use of main auxiliary materials,
reductions in energy, reductions in byproducts, anddevelopment of environmentally friendly processes.
Technological developments have a very high relative
importance in cost reduction. Let us consider what kindof possibilities there are for improvement anddevelopment.

13.1 Strain Breeding / Improving Existing Strains
A field that also considers possibilities fortechnological improvements. In other words, it is basedon new technology that utilizes DNA information and
DNA technology and involves the use of DNA andmRNA to comprehensively analyze the movements ofproteins and intermediates as well as the metabolic
flow within microbial cells, with the ultimate aim oftrying to optimize the entire metabolism of the
producing strain. Some of these attempts have alreadyproduced some powerful results. For example, there isa general trend for production performance to reach apeak in the late stage of the culture and the rmf genecharacteristically increases its level of expression
during this stage. Knocking out this gene made it
possible to suppress the peaking and boost amino acidproduction performance. Research has shown thatknocking out the arc gene, which regulates theexpression of enzyme genes related to and including
the TCA cycle, boosts enzymatic activity related to the
TCA cycle and boosts the production performance of36

glutamic acid, arginine, and lysine, which arebiosynthesized through the TCA cycle.
A gene has even been identified as related being to thesecretion of substance from the cell, which often
presented issues in using microbes to produce
substances. Amplifying this gene has been shown to
improve the secretion efficiency, or in other words, toimprove the amino acid production performance.(1) Abold attempt is also being undertaken to create
microbes as minimum genome factories with theminimum genomes (sets of genetic instructions)necessary for producing useful substances, with theaim of efficient production.(2) These research findings
have been put into practical use and can be expected toincrease production even further.

13.2 Strain Breeding / Developing and Using
New Raw Materials
While bioethanol has suddenly become a talking point
in recent years, as long as the price of crude oil
continues to rise, starch and sugar, the main raw
materials used in amino acid fermentation, will always
compete with bioethanol and food, and price increases
will be an issue. Therefore, there is a need to consider
using raw materials other than starch and sugar. For
example, it is worth investigating breeding producing
strains from microbes that can assimilate methanol, orfrom photosynthetic bacteria that can utilize CO2.
Consideration is also being given to using rice straw,
chaff, sugarcane stalks, wood scraps, and other types ofbiomass. Although there is the problem of sugar
conversion and collection from these materials, further
development of raw materials for amino acidfermentation can be expected.

13.3 Cultivation Technology
Since continuous cultures have unresolved issues withmicrobe contamination and the like, no suchproduction processes have been implemented otherthan activated sludge processes. This is the mostanticipated technology in terms of energy and resource
conservation.

13.4 Separation / Purification Technology
This technology is quite complete and there isconsidered to be little prospect for any radical
improvements. Acidic cultivation is being considered
as a means of reducing the byproduct at the purification
stage. For example, cultivation for glutamic acid

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

fermentation is now carried out under neutral
conditions by neutralizing with ammonia; sulfuric acidis added for crystallization, then it is finally neutralizedwith NaOH to produce MSG. This producesammonium sulfate as a byproduct. The idea is that if
cultivation could be carried out in acidic conditions,
there would be no ammonium sulfate byproduct.(1)13.5 Environmental Issues
This report mentioned that environmentally friendly
systems have been created to recycle raw materials andbyproducts at fermentation plants. These systems are
now expected to be more complete than ever.
Ajinomoto Co., Inc. has already launched its“Awareness Project for High Utilization Technology of
Cassava” in collaboration with agricultural experimentstations in Indonesia, aimed at intensively utilizing thewaste liquid from its fermentation plants as fertilizer.(1)Future issues for consideration include converting
sugar-extracted sugarcanes (biomass) into sugar or fueland reducing CO2 emissions from factories.
Faced with these technological challenges, hopefully
Japan will maintain and even advance its predominantposition in amino acid technology.

13.6 Development of Peptide Technology
Peptides are substances that are closely linked to aminoacids. Peptides link amino acids and form when the
carboxyl group of one α-amino acid joins to the amidegroup of another α-amino acid through dehydrated
condensation (see Fig. 2.2). While these have a numberof physiological functions, such as hormones, and havepotential use as pharmaceutical products or similar, todate only small-scale production has been carried out
through chemical synthesis. There are complex issues
involved, such as introducing/eliminating protecting
groups to/from functional groups, and no practicalproduction methods are being carried out this way. By
contrast, biochemical production methods haverecently been developed and are gaining attention. One

such method has been developed by Ajinomoto Co.,
Inc. and has been used to successfully screen microbial
enzymes that catalyze the following peptide synthesis
reaction from isolated amino acid (aa1) and amino acidmethyl ester (aa2-COOCH3).(3)aa1+aa2-COOCH3+H2O→aa1-aa2+CH3OH
aa1-aa2+aa3-COOCH3+H2O→aa1-aa2-aa3+CH3OH
This method thus successively lengthens the peptide
chain.
A method developed by Kyowa Hakko performs an
enzymatic reaction (4) using ATP, with isolated aminoacids as the energy source. This produces both forward
and reverse peptides using the following bonding
method and is thought to lower the yield of intended
particular peptides.
aa1+aa2+ATP → aa1-aa2+aa2-aa1
The fact that these technological developments havemade peptide production possible indicates a greater
likelihood of discovering promising peptides fromamong unlimited combinations of amino acids and it isvery likely that peptide fermentation will emerge as a
new technological field in the near future.
<References>(1) O. Kurahashi: “Amino-san Hakkō no Shinka to
Mirai [Evolution and Future of Amino Acid
Fermentation]”, Abstracts from the TechnicalCommittee on Fermentation and Metabolism
Symposium, p. 25, July 13, 2007.
(2) S. Anazawa: “Hakkō Kōgyō ni okeru Shin-Seikino Gijutsu Tenkai [New-Century Technological
Expansion in the Fermentation Industry]”,
Abstracts from the Technical Committee on
Fermentation and Metabolism Symposium, p. 31,
July 13, 2007.
(3) K. Yokozeki and S. Hara: J. Biotechnology, 115,
211, 2005.
(4) K. Tabata, et al: J. Bacteriol., 187, 5195, 2005.

Survey Report on the Systematization of Amino Acid Fermentation Technology

37

14 Acknowledgements
The author wishes to express sincere thanks to the following people who assisted with the provision of resources andthe like.
Ajinomoto Co., Inc.

Kyowa Hakko Kogyo Co., Ltd.
Mitsubishi Tanabe Pharma
Corporation
Japan Bioindustry Association

Ryuichiro Tsugawa
Osamu Kurahashi
Takashi Utagawa
Sadao Teshiba
Keiko Ochiai
Tetsuya Tosa
Hiroaki Matsumae
Eikou Shimizu

List of Registration Candidates

38

No.

Name

1

Japan’s oldest
sample ofglutamic acid(prepared by
Kikunae Ikeda)

2

Domyōji-gamepots

3

immobilized
enzyme cutting
machineFormatLocation

Manufacturer

exhibitAjinomotoCorporate
Museum, Minato,Tokyo

original
item

AjinomotoCorporate
Museum, Minato,Tokyo

original
item

A memorable productproduced by Tanabe
Tanabe Seiyaku
Tanabe1974-1976 Pharmaceuticals, a
Yamaguchi Co.,
Pharmaceuticals
pioneer in enzyme
Ltd., Sanyo-Onoda
immobilization

Kikunae Ikeda

unknownYear

Comments

1908

The first glutamic acidextracted from kombu,
which became the major
product “Ajinomoto”

Memorable ceramic pots
developed as
1912-1925 acid-resistant reaction
vessels to withstandhydrochloric acid

National Museum of Nature and Science Survey Report on the Systematization of Technology, Vol. 11 March 2008

This report comprises findings for “Research on Japanese Technological Innovations Based on
Historical Industrial Technology Materials” (17074009), as part of Japanese TechnologicalInnovations: Compiling Experience and Forming a Knowledge Base, a field-specific research
project funded by a Technology Grant-in-Aid for Scientific Research in 2007.

National Museum of Nature and Science
Survey Report on the Systematization ofTechnology, Vol. 11
19 March 2008
EditorNational Museum of Nature and Science(Independent Administrative Institution)
Center of the History of Japanese Industrial
Technology(Under charge of: coordinating editor: Takayuki Nagata; editors:
Toshihiko Okura, Toshio Kubota)

Publisher:

National Museum of Nature and Science(Independent Administrative Institution)〒7-20 Ueno Park, Taito-ku, Tokyo 110-8718
Tel: 03-3822-0111

Design and Printing: J-Spark Co., Ltd.