R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

Molecular Microbiology and Biotechnology Group
Members (As of Apr. 2024)
Masayuki Inui, Group Leader, Chief Researcher
Haruhiko Teramoto, Associate Chief Researcher
Kazumi Hiraga, Associate Chief Researcher
Hajime Terasaki, Associate Chief Researcher
Masato Miyamoto, Associate Chief Researcher
Nobutake Fugono, Associate Chief Researcher
Yuya Tanaka, Senior Researcher
Masako Suda, Senior Researcher
Yukihiro Kitade, Senior Researcher
Satoshi Hasegawa, Senior Researcher
Akira Watanabe, Senior Researcher
Takahisa Kogure, Senior Researcher
Takeshi Kubota, Senior Researcher
Kiyoshi Oi, Senior Researcher
Akiyoshi Higo, Researcher
Natalia Maria Theresia, Researcher
Norimasa Kashiwagi, Researcher
Ryoma Hashimoto, Researcher
Naoki Saruya, Researcher
Yuki Nozaki, Researcher
Dyah Candra Hapsari Subagyo, Researcher

Takafumi Shimizu, Researcher
Dita Grinanda, Researcher
Miyuki Shintaku, Researcher
Takayuki Kuroishikawa, Researcher
Naoyuki Tajima, Researcher
Junko Watanabe, Assistant Researcher
Yuko Ikenaga, Assistant Researcher
Shoko Minakuchi, Assistant Researcher
Miyuki Nagamori, Assistant Researcher
Kae Naito, Assistant Researcher
Noriko Ikeda, Assistant Researcher
Kazuyo Yoneda, Assistant Researcher
Mayu Koizumi, Assistant Researcher
Junko Nishi, Assistant Researcher
Motomi Iwashima, Assistant Researcher
Kayo Yoshida, Assistant Researcher
Aya Okada, Assistant Researcher
Maki Faulkner, Assistant Researcher
Satomi Kayamura, Assistant Researcher
Ryoko Hatsugai, Assistant Researcher

Development of Bio-manufacturing Technologies that Contribute to
Carbon Neutrality
1. Introduction

synthetic biology and genome editing technology) withSince bioprocesses can be manufactured under

digital technology (e.g., the internet of things artificial

ambient temperature and pressure, they are expected

intelligence) and the growing awareness of global

to reduce CO2 emissions, unlike chemical processes,

environmental issues, such as the need to disengage

which are manufactured under high temperature andfrom fossil resources for resource autonomy, thepressure. This also has an advantage over chemical

practical application of this technology is expected toprocesses in producing complex compounds with highaccelerate and the market size to expand rapidly in the

carbon counts because they are generally synthesized

future. Major investments are being made in this fieldbyin the U.S. and China, and international competition ismultistep

biomanufacturing

reactionsisinattracting

the

cell.

attention

Thus,
as

an

intensifying. In Japan, the Green Innovation (GI) Fundinnovation that enables both economic growth andProject

andthe

Biomanufacturingsolutions to global-scale social issues such as resource

Promotion Project were launched this fiscal year as

autonomy and a breakaway from dependence on fossil

large-scale projects to develop and socially implement

resources. With the development of “bio x digitaltechnologies for the design and development of newtechnology” through the fusion of biotechnology (e.g.

microorganisms

and

the

Revolution

advancementof36

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

manufacturing processes using microorganisms. These

Manufacturing Demonstration" project as a joint

projects aim to be carbon neutral and carbon negative

development with private companies using the sameby switching from petroleum as a raw material totechnology

andatmospheric CO2 and unused resources.

development

to

are

conducting

commercialize

researchandbioproductionofAgainst this background, the Research Institute ofcarotenoids and flavors (see Chapter 3, Section 4 andInnovative Technology for the Earth (RITE) has been

Chapter 3, Section 5). In this fiscal year, the company

developing biorefinery technology to produce biofuels

began participating in the GI Fund Project and the Bio-and green chemicals from non-food biomass at highmanufacturing

efficiency

developthroughmicrobial

bioprocesses.

RITE

Revolution

bioproduction

Promotion

Projecttechnology

for

to

high-

discovered that coryneform bacteria, a typical industrial

performance adhesive raw materials from CO2 and bio-

microorganism, maintain metabolic functions underupcycling technology to produce useful chemicals fromreducing conditions despite growth inhibition and that

unused resources (see Chapter 3, Section 1 and Chapter

they efficiently metabolize sugars and produce organic

3, Section 2). In addition, RITE is participating in theacids. Consequently, RITE developed the growth-

NEDO "Moonshot" project to research and develop

independent bioprocess "RITE Bioprocess" . In addition,

ocean degradable multilock biopolymers made fromwe established elemental technologies essential for

non-food biomass (see Chapter 3, Section 6).

*1

industrialization, such as "complete simultaneous use of

In

this

overview,

we

first

explainourcore

mixed sugars derived from non-food biomass" andtechnologies such as "RITE Bioprocess"*1 and "Smart"high tolerance to fermentation inhibitors" (see Chapter

Cell Creation Technology." Next, we discuss a national

2, Section 1). Using these technologies, we reported the

project based on bio x digital technology innovation,world's highest efficiency production of ethanol,

which has been making remarkable progress in recent

butanol, green jet fuel, and biohydrogen as biofuels,

years as a basic technology development, and finally,and lactic acid, succinic acid, alanine, valine, tryptophan,

we introduce our efforts for commercialization.

shikimic acid, protocatechuic acid, 4-aminobenzoic acid,and 4-hydroxybenzoic acid as green chemicals.

2. The Core Technologies of RITE

Currently, RITE is focusing on developing production

2.1. “RITE Bioprocess”*1

technologies for aromatic compounds that can be used

“RITE Bioprocess”*1, developed by RITE, is a

as raw materials for high-value–added fragrances,proprietary technology that enables highly efficient

cosmetics, pharmaceuticals, fibers, and polymers, as

production of biofuels and green chemicals, such aswell as bio-manufacturing technologies that directly

amino acids and aromatic compounds (Fig. 1). The threeuse CO2 as a raw material.

features of “RITE Bioprocess”*1 are described below (forSo far, we have participated in the New Energy andIndustrial

Technology

Development

details, see RITE Today 2022).

Organization(NEDO) "Smartcell" project and the "Data-driven
Integrated Bioproduction Management System" projectand have been developing "Smart Cell Creation
Technology," a bio x digital technology (see Chapter 2,
Section 2). We are also participating in the NEDO "Bio-Fig. 1 Biorefinery concept using the “RITE Bioprocess”*1

37

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

Feature 1: Growth-arrested bioprocess
Anaerobic conditions and removing factors essential
for proliferation allow the desired series of reactions tooccur while cell division is arrested (Fig. 2). In otherwords, nutrients and energy previously used for
multiplication are now used to produce the target
substance. This has enabled microbial cells to be usedextremely efficiently similar to a chemical catalyst,
realizing a bioprocess with high productivity equal to or
greater than that of ordinary chemical processes.
Fig. 3 Feature 2 of the “RITE Bioprocess”*1(Simultaneous usage of mixed sugars)

Feature 3: High tolerance to fermentation inhibitors“RITE

Bioprocess”*1

hasdemonstrated

highresistance to fermentation inhibitors because themicroorganisms do not grow as described above (Fig.
4).

Therefore,

this

can

be

appliedtousingsaccharification liquids containing various fermentation
inhibitors
Fig. 2 Feature 1 of the “RITE Bioprocess”*1

and

even

to

producingfermentation

inhibitors.

(Growth-arrested bioprocess)Feature 2: Complete simultaneous use of C5 and C6
mixed sugars
Most inedible biomass (cellulosic biomass) comprisea mixture of C5 sugars such as xylose and arabinose andC6 sugars such as glucose.
RITE has succeeded in increasing the utilization rateof C5 sugars to that of C6 sugars by introducing a C5
sugar transporter gene in addition to the C5 sugar

Fig. 4 Feature 3 of the “RITE Bioprocess”*1

metabolism gene (Fig. 3). This enables the full(High tolerance to fermentation inhibitors)

simultaneous use of C5 and C6 sugars and efficient use
of cellulosic (non-food biomass) feedstock.

2.2. Smart Cell Creation Technologies
With the fusion of cutting-edge biotechnology anddigital technology, the potential of biological cells canbe maximized to create “smart cells” that are optimized
for biomanufacturing. This type of technology is called38

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

Smart Cell Creation Technology and has made thecompound

from

the

reaction

systemdevelopment of smart cells dramatically more efficient.

constructing

a continuous

RITE is involved in developing these technologies bycombines resin adsorption and membrane separation,

participating in the NEDO Smart Cell Project (2016–

Fig. 6). By applying this system to catechol production,2020). During the project, we demonstrated thea dramatically high production of catechol was

effectiveness of the Smart Cell Creation Technology byachieved.

reaction(e.g.,

bysystemthatefficiently creating high-producing strains for the target
chemical. By incorporating these technologies, RITE has
succeeded

in upgradingtheproduction

strain–breeding technology and fermentation-production
technology (Fig. 5). Additionally, the Smart Cell Creation
Technologies have been overtaken by the NEDO Biomanufacturing project, and improvements are being
made for the practical application of microbial

Fig. 6 A continuous reaction system using

production.

membrane separation and resin adsorption

2.4. Substances Produced by the “RITE Bioprocess”*1
Several materials currently in high production by
RITE are shown in Figure 7. As mentioned above, thecompany has achieved world-class productivity in many
substances. In biofuels, the company is expanding into
production of butanol and high-performance biojet fuelFig. 5 Smart Cell Creation Technologies

materials

inaddition

to

that

of

ethanol

andbiohydrogen. Furthermore, in green chemicals, RITE is
2.3. Continuous Reaction System
RITE has developed biotechnological processes for
producing various compounds. In this development

developing

a

wide

range

of

high-performancechemicals such as aromatic compounds in addition to
L-lactic acid, D-lactic acid, and amino acids.

activity, we have faced the problem that several target
compounds have extremely strong cytotoxic effects,
and production is halted because of the toxicity of thecompounds that have accumulated during production.
For example, catechol production, which is being
developed in the NEDO Smart Cell Project and the
NEDO Bio-manufacturing Project, plateaued after
reaching a specific concentration in the conventional
batch method. To avoid cytotoxicity and achieve highproduction, we constructed a continuous reaction

Fig. 7 Substances produced using the “RITE Bioprocess”*1

system that selectively removes and recovers the target

39

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

3. Fundamental Technology Development (National

purification, and recovery, and accelerate the creation

Projects)of bioderived products by demonstrating industrial

3.1. NEDO GI Fund Projectproduction systems. Specifically, teams in this project*2

The GI fund project entitled “Promotion of carbon

are conducting research and development in threerecycling using CO2 as a direct raw material throughareas: “Development of basic technology to promotebiomanufacturing technology” aims to contribute to

the utilization of bioresources,” “Development of basic

realizingnew

technology for biofoundry production processes,” andbiomanufacturing products using CO2 as a raw material“Demonstration of industrial material production

and implementing them in society to transform thesystems.”

carbonneutrality

bydeveloping

industrial structure by using CO2 as a resource and toachieve “carbon neutrality by 2050.”

RITE has been involved in this project since the firstyear and is currently developing a new group of

In this context, RITE, in collaboration with Sekisui

technologies(Industrial

Smart

Cell

Creation

Chemical Co., Ltd, has started a project entitledTechnology) to solve the problems associated with the“Commercialization of high-value–added chemicals

practical application of biomanufacturing technology

using CO2 as raw material through bioproduction(Fig. 8). In FY2023, RITE mainly worked with partner

technology” from FY2023 and is currently ongoing

research institutions to develop technology that(project period: eight years from FY2023 to FY2030).

eliminates productivity declines caused by uneven(For details, see Special Feature)

culture3.2. Research and Development of Technologies toPromote Biomanufacturing*2
This project aims to solve both social issues such asenvironmental problems and economic growth byusing unused resources such as discarded domestic
biomass as raw materials and converting them into
useful substances using biotechnology. (For details, see
Special Feature).

environments

contributed

to

in

large

constructing

fermentation-production

fermenters.
andsimulation

RITE

validating

a

model

by

acquiring and providing detailed gene expression data
and metabolite data in an environment that reproduces
culture stress. By creating technologies to solve
problems that may occur during large-scale production,
we aim to eliminate rework in developing production
strains and accelerate the social implementation of biobased products.

3.3. NEDO Bio-manufacturing Project*2
The NEDO project “Development of bio-based
product production technology to accelerate therealization of carbon recycling,” also known as the Biomanufacturing

project,

conducts

researchanddevelopment that combines biotechnology and digitaltechnology to produce materials from biomass that
does not depend on fossil resources (from 2020). The
Smartcell Project is the predecessor of this project. This
project aims to expand new bioresources, develop

Fig. 8 Bio-manufacturing project: Development

bioproductionof Industrial Smart Cell Creation Technology

processes

includingseparation,40

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

3.4. NEDO Bio-manufacturing Demonstration Project3.5. NEDO Bio-manufacturing Demonstration Project(Carotenoid)*2(Rose Aroma Ingredient）*2

Carotenoids are natural pigments produced bySince 2022, RITE has participated in the NEDO “Bio-plants and some bacteria. They are popular as

manufacturing” project with Takasago International

functional ingredients for lifestyle disease prevention

Corporation. We are driving the development of anand antiaging because of their high antioxidant activity,"industrial smart cell" that can produce a rose aroma

and their market size is growing. However, theingredient and a bioproduction system that can avoid

carotenoid content in natural sources is low, and mostmicrobial “product inhibition” derived from theof marketed carotenoids are produced by chemicalfragrance materials (Fig. 10).

synthesis from petroleum. Moreover, most carotenoids

In FY2023, RITE succeeded in increasing theare characterized by low-absorption because of theirproductivity by metabolic engineering and optimizing

chemical structure, and their bioavailability is also low.

production conditions, by using the production system

Several chemical processes that converting carotenoid

described

structures from low- to high-adsorption types have

improvement of the smart cells, testing of scale-up, and

been

are

modification of the equipment. We aim to achieve

unsatisfactory. Recently, as new sources of carotenoids,

Japan’s first social implementation of production of asmart cells producing high amounts of carotenoids

fragrance ingredient by precision fermentation.

devised,

but

their

conversion

rates

above.

We

will

continue

with

the

have been reported, but their carotenoids are also lowabsorption types.
Since 2022, RITE has participated in the NEDO “Biomanufacturing” project with Harima Chemicals, Inc. forsocial implementation of a bio-based mass production
system for highly bioavailable carotenoids (Fig. 9). We

Fig. 10 Production of a rose aroma ingredient

have developed a smart cell that specifically produces
high-adsorption types of carotenoids. We are currently

3.6. NEDO Moonshot-type Research and Development

improving the smart cell and developing other

Project*2

processes such as fermentation, carotenoid-extraction,and purification.

The project entitled ”Development of Multilock
Biopolymers Degradable in Ocean from Nonfood
Biomasses” is performing research and development tointroducea"multilock

mechanism"forplastic

degradation (Fig. 11). Multiple stimuli such as light, heat,
oxygen, water, enzymes, microorganisms, and catalysts
can be used as triggers to start degradation of plastics,
but avoid their degradation and maintain their
durability and toughness while the plastics are in use.
Fig. 9 Outline of our project for bio-based production of

However, when accidentally dispersed into the marine

highly bioavailable carotenoids

environment, the multilock mechanism is unlocked toenable fast on-demand degradation.

41

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

Products targeted for practical application in thisproject include tires and textiles, which generatesecondary fine debris when used, as well as plastic
bottles, fishing nets and fishing tackle that contribute toghost fishing, all of which negatively impact theenvironment due to runoff into the ocean.
In FY2023, we focused on developing a technologythat enables the artificial control of the initiation timingofthedegradationofmultilocked

plastics

Fig. 11 Marine-degradable multilock biopolymers(Developments of new technologies using degradativefrom nonfood biomass and their circulation

enzymes). First, the thermostability of the enzyme wasdramatically improved by electrostatic binding of thethermostable-plastic–degrading

enzymeto

3.7. Japan Science and Technology Agency COI-NEXTabiodegradable carrier.

RITE has participated in the program on open
innovation platform (COI-NEXT) commissioned by the

Next, we produced a film where the enzyme wasJapan Science and Technology Agency. In the platform

mixed into plastic by thermal melting, exposed this to“Carbon Cultivation Hub Challenging the Limits ofseawater, and succeeded in proving that rapid

Carbon Negativity” started in 2023, we are working on

enzymatic degradation at the laboratory level (on-developing biohydrogen production and liquid biofuel

demand degradation) occurred.

production technologies for establishing carbon-

We

will

aim

to

achieve

fasteron-demand

cultivation–based fuel-production technology. In thisdegradation by improving the functionality of the

project,

weplastic–degrading enzyme and optimizing the mixing

technologies to efficiently produce fuels (hydrogen/conditions with the plastic. In addition to degradation

liquid fuel) from various biomass feedstocks intests in marine fields (e.g. Ainan Town, Ehime

collaboration

Prefecture), an international joint research with the U.S.

working

Department of Energy's ARPA-E will be launched.

technologies, enabling an increase in CO2 fixation by(The HP of the project can be found at:

photosynthesis (Fig. 12). Hydrogen is expected to be

http://www.moonshot.k.u-tokyo.ac.jp/en/index.html).

the ultimate clean energy and is key in realizing carbon

on

will

with

developthe

biologicalparticipant

developing

conversion

organizations

biomass

cultivation

neutrality/negativity. In this context, a medium- tolong-term theme is to develop CO2-free hydrogenproduction processes. A short- to medium-term theme
is to develop liquid fuel-production processes that use
the same fundamental technologies as the other longterm theme. One of the key challenges for the social
implementation of biomass fuel-production technology
isreducing

production

costs.

In

addition,

thecomponents of biomass feedstock are diverse, and theircomposition considerably varies depending on the type42

R&D Activities ● Molecular Microbiology and Biotechnology Groupof feedstock, making it difficult to meet a wide range ofdemands with uniform technology. To solve these

RITE Today 2024

Bakelite Co., Ltd.) (Click here for GCC)In February 2010, RITE established the "Green

issues, this project will promote the development of

Phenoltechnologies in different fields, including various

Production Technology Research Association" (GPthermochemical

conversion

Association) with and Sumitomo Bakelite Co., Ltd. totechnologies in an integrated manner for enabling thedevelop fundamental technologies related to phenol

construction and expansion of a flexible biomass fuelproduction and phenolic resin production through the

supply system tailored to regional and feedstock needs.

application of bioprocesses that use cellulosic raw

andbiological

RITE has developed a biohydrogen production

andHigh-Performance

PhenolicResinmaterials (non-food biomass).

process with high production rate. Building on thisIn May 2014, the GP Association was reorganized asachievement, we are developing a microbial catalyst for"Green Phenol Development Co., Ltd." (GPD). This was

improving the hydrogen yield from biomass-derived

the first example of demutualization of a technology

sugars to a large extent. We constructed a geneticallyresearch association.

engineered microorganism with a novel hydrogenproduction

pathwayenhancedbymetabolic

In April 2018, given that GPD technology can
produce useful compounds in parallel with phenol

engineering. In addition, RITE has established a

production,

bioprocess that efficiently converts mixed C6 and C5Development Corporation was changed to Greensugars to ethanol. Using this technology, we will

Chemicals Co., Ltd., (GCC).

develop an alcohol to jet process to produce asustainableaviationvariousnamephenol-producing

of

Green

Phenoltechnology

andknowledge of Green Chemicals Co., Ltd can be applied

feedstocks, such as energy crops, rice with high CO 2

to producing other aromatic compounds (Fig. 13), we

fixationare developing a bioprocess for other high-value–

and

using

the

trade

biomass

capability,

fuel

Since

the

microalgae

with

high

carbohydrate productivity.

added chemicals and commercializing products thatmeet customer needs.
In FY2023, approval for industrial use of the two
production strains of GCC’s products (4-HBA, PCA) was
obtained by the Ministry of Economy, Trade andIndustry.

Fig. 12 Development of biohydrogen/bioethanol
production technologies

4. Future Industrialization of Our Technologies
4.1. Green Chemicals Co., Ltd. (GCC)(Head Office・Laboratory: in Kyoto headquarters, RITE;
Shizuoka Laboratory: in Shizuoka plant, Sumitomo

43

R&D Activities ● Molecular Microbiology and Biotechnology GroupInternational

RITE Today 2024

Corporation,

in

the

NEDO

Bio-

manufacturing Demonstration Project, we are also
conducting joint research with many other companies.
These include research and development to convert acompany’s product (fossil resource-derived material) tobioderived materials at an early stage, as well as
research and development to convert company's main
products or main raw materials (fossil resource-derived
materials) to bioderived materials in the medium tolong term.

5. Closing remarks

Fig. 13 Major product lineup of

In recent years, technological innovations and new

Green Chemicals Co., Ltd.

knowledge and methods in the aforementioned "bio x
4.2. Green Earth Institute Co., Ltd.

digital technology" have dramatically improved the(Head office: 6F Q Plaza Shinjuku 3-chome, 3-5-6

efficiency of smart cell development, especially in the

Shinjuku, Shinjuku-ku, Tokyo, Laboratory: 2-5-9 Kazusa-

national

kamatari, Kisarazu-shi Chiba)

production(Click here for the Green Earth Institute Inc. web site)

underway for social implementation of the smart cells

projects

described

demonstrationin

Chapterefforts

are

3.

And

vigorously

In September 2011, RITE established the Green Earthcreated by these results. These are expected to create aInstitute Inc. to commercialize "RITE Bioprocess" . In

new industry (smart cell industry) and have a significant

December 2021, due to successful business results, theripple effect on the industrial sector (manufacturing) incompany was listed on the Tokyo Stock Exchange

addition to the energy sector (Fig. 14).

*1

(Mothers), and in April 2022, moved to the Tokyo
Growth Market due to market reorganization.

RITE will continue to promote the development ofbio-manufacturing technologies that contribute toCurrently, the company is promoting research andcarbon neutrality by utilizing the "Smart Cell Creation

development with domestic and overseas partner

Technology" and "RITE Bioprocess"*1 to focus oncompanies, including "Biofoundry Base" (based on the

research and development using unused resources andgovernment's "Biostrategy 2020), NEDO’s "Green

CO2 in the atmosphere as raw materials, as well as theInnovation

development of practical production technologies for

Fund

Project,"

and

NEDO's"Bio-manufacturing Revolution Promotion Project."green chemicals.
Since the declaration of "carbon neutrality by 2050"

4.3. Joint Research with Companies

in October 2020, inquiries from companies have been

In addition to the main substances produced (seeincreasing, and the number of joint research projects

Chapter 2, Section 4) introduced in this overview, we arewith companies has also been increasing. RITE is still

conducting joint research on many other substances inlooking for companies to collaborate with. Compounds

response to requests from companies.

that have generally been considered difficult to produce

In addition to Harima Chemicals, Inc. and Takasago

by microorganisms may be produced at high levels by

44

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2024

using the latest elemental technology development
results. If you have a compound you would like tobiologize, we would like to hear from you.

Fig. 14 Fusion of industrial / energy fields impacted
by new bio and digital technologies*1“RITE Bioprocess” is a registered trademark of RITE.

*2

This article is based on results obtained from a projectcommissioned or subsidized by the New Energy andIndustrial

Technology

Development

Organization(NEDO).

45