R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2021

Molecular Microbiology and Biotechnology Group
Members (As of Dec. 2020)
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
Yuya Tanaka, Senior Researcher
Masako Suda, Senior Researcher
Yukihiro Kitade, Senior Researcher
Koichi Toyoda, Senior Researcher
Naoto Kato, Senior Researcher

Satoshi Hasegawa, Senior Researcher
Akira Watanabe, Senior Researcher
Takahisa Kogure, Senior Researcher
Takeshi Kubota, Senior Researcher
Toshihiro Oshima, Senior Researcher
Shinichi Oide, Senior Researcher
Tetsu Shimizu, Researcher
Tomoaki Hara, Researcher
Akiyoshi Higo, Researcher
Natalia Maria Theresia, Researcher
Norimasa Kashiwagi, Researcher
Jyumpei Kobayashi, Researcher
Ryoma Hashimoto, Researcher
Naoki Saruya, Researcher
Yuki Nozaki, 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
Kayoko Mori, Assistant Researcher
Motomi Iwashima,
Assistant Researcher
Kayo Yoshida, Assistant Researcher
Aya Okada, Assistant Researcher
Kimiyo Kishi, Assistant Researcher

Development of Biorefinery Technology to Realize a Sustainable
Society
1. Introduction
The coronavirus disease 2019 (COVID-19) pandemic
has had a great impact on economic and social activities
worldwide. Meanwhile, environmental problems suchas global warming and marine plastic pollution remain

Agricultural
technology
Agriculture,
Forestry

are becoming more serious. Under these circumstances,the promotion of the bioeconomy is becoming increasingly important for economic recovery after the conver-

CO2 absorption

Renewable
resources,
Materials,
Biomass

RecycleCO2,
Waste

Biorefinery
Chemicals,
Medicines,
Energy, Foods

Consumption

gence of infectious diseases and for solving environmental problems.
The concept of “bio-economy,” which aims to ex-

Created by RITE based on BioVale materials

Fig. 1 Economic contribution of biotechnology

pand a sustainable, renewable and recycling-oriented
economic society while solving global issues by utilizing

According to the Organisation for Economic Co-op-

biotechnology and renewable biological resources

eration and Development (OECD), “the biotechnology（Fig.1）.

market in 2030 will expand to about 190 trillion yen in
all member countries and the manufacturing sector willreach about 40%” (Fig. 2). It is said that all of these industries are bio-based.

30

R&D Activities ● Molecular Microbiology and Biotechnology Group

2030, $1.7 trillion

RITE Today 2021

bioethanol was produced in the U.S. in 2019. Similarly
the Agricultural Outlook 2020–2029 report, publishedjointly by the OECD and the Food and Agriculture Or-

Agriculture
36%

Industry
39%

Health25%

ganization, states that 129 million kL of bioethanol was
produced worldwide in 2019, with the U.S. accounting
for approximately half of this production.
Cellulose ethanol, a second-generation biofuel, is
produced from raw materials that do not compete withfood resources, such as the agricultural waste corn

Source：The Bioeconomy to 2030. OECD(2009), NEDO

Fig. 2 Economic contribution of biotechnology

stover. On the basis of Renewable Fuel Standard (RFS)
rules, the Environmental Protection Agency's final volume for the production of cellulosic biofuel in 2020 was

Under such circumstances, the government formu-

590 million gallons (2.23 million kL), which was slightly

lated “Bio-Strategy 2019” in June 2019, with the overall

more than 5.6% of the RFS target set in 2007. The ac-

goal of “achieving the world's most advanced bioecon-celeration of biofuel commercialization is required inomy society in 2030.” This was to be promoted while

the future. The Research Institute of Innovative Technol-

being updated every year, and in June 2020, "Bio-Strat-

ogy for the Earth (RITE) is developing a bioprocess thategy 2020 (Basic Measures)" was announced, followed

can efficiently utilize cellulosic biomass (see Chapter 2).

by "Bio-Strategy 2020 (Final version of Market Area

With regard to aviation fuel, the International Civil

Measures)" in January 2021.

Aviation Organization decided at their 2010 General As-

Our group is promoting the development of biore-

sembly not to increase total greenhouse gas emissions

finery technology, the core of bioeconomy, by using mi-after 2020. At the 2016 General Assembly, a decision

croorganisms. Biorefinery technology is used for pro-

was made to introduce a greenhouse gas reduction sys-ducing biofuels and green chemicals, using renewable

tem using market mechanisms (GMBM: global market-

resources (biomass) as raw materials.

based measures) after 2020. On the basis of this deci-

This first section provides an overview of the current

sion, the International Air Transport Association has for-status of biofuel and bioplastic (a substitute for general-mulated a concrete action plan, including the uses of

purpose plastics) research and development (R&D).

bio-jet fuel and emissions trading (Fig. 3). The GMBM
are expected to be launched in 2021. Along with these,

1.1. Biofuels

bio-jet fuel has been widely spread mainly in Europe

Bioethanol, a major biofuel, is produced from raw

and the U.S. every year, and commercial flight has beenmaterials such as corn (in the U.S.) and sugarcane (incontinued as a sustainable aviation fuel using cooking

Brazil). It is mixed with 10%–25% gasoline for use in au-

waste oil and the like.

tomobile engines. The highest production and con-

Our group is also conducting R&D on biofuels. In a

sumption of bioethanol occurs in the United States

project sponsored by Japan Airlines Co., Ltd. (JAL), we(U.S.) where corn is a major crop. According to esti-

have succeeded in producing domestic bio-jet fuel formates provided by the U.S. Energy Information Admin-the first time in Japan, and this bio-jet fuel was refueled

istration, 15.8 billion gallons (59.81 million kL) of

on a regular domestic flight (see Section 4.1).

31

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2021

We are also conducting R&D on a green jet fuel that

Ocean from Non-food Biomasses) of the New Energy

can be used as is, that is, without limitation of mixing

and Industrial Technology Development Organization

ratio to petroleum fuels.

(NEDO). Participation in the project “Production of Biomonomers from Non-food Biomass and Development

CO2 emissions
from aviation
(Mt)
3500

Predicting CO2 emissions
from aviation
Improvementof tech, ops,
infra

3000
2500

No action

1800 Mt

2000
1500

Biofuels &add. tech

1000

Carbonoffsetting

500
002000 2005 2010

2020

2030

Ref.） ICAO Environmental Report 2016

800 Mt
300 Mt

2040

2050＜ICAO’s prediction＞
Minimum carbon
emissions in 2050

＜ICAO’s target＞
Carbon neutral
Growth from 2020
＜IATA’s target＞
Net reduction incarbon emissions of
50% by 2050
compared to 2005

ICAO: International Civil Aviation Organization
IATA : International Air Transport Association

Fig. 3 Emissions reduction roadmapof Polymer-degrading Enzymes” will be undertaken.

2. Core technology of our group
Our group has established an innovative bioprocess
on the basis of a new technological concept. This trademark, registered RITE Bioprocess®, has achieved great
results in the development of technologies for producing green chemicals (e.g., biofuels, amino acids, and aromatic compounds), exhibiting high efficiency. Further-

With regard to marine fuels, Japan's International
Shipping Greenhouse Gas Zero Emission Project, estab-more, it has received high praise both in Japan andabroad (Fig. 4).

lished in collaboration with the Japan Ship Technology
Research Association, aims for a zero emissions target
by 2050 through the promotion of technological inno-

Non-food
biomass

Metabolicengineering etc.

generation like biohydrogen. The Project has developed

emissions.

Biofuels
Green chemicals

Pretreatment
Saccharification

vations, such as alternative low-carbon fuels of the next

a roadmap toward accelerating efforts to achieving zero

RITE Bioprocess®
(Growth-arrested bioprocess)

Recovery&
Purification

O

OH

OH
OH

Smart cells(Coryneform bacteria)

O

OH

HO

NH2

Production proceeds without cell growth・Simultaneous utilization of mixed sugar
・Tolerance to growth inhibitors
・High productivity

Fig. 4 Biorefinery concept using the RITE Bioprocess®

1.2. Bioplastic
Efforts to reduce greenhouse gases by spreading
and generalizing the use of plastics that do not depend

2.1. Features of the RITE Bioprocess®
2.1.1. Feature ①: Growth-independent bioprocess

on fossil resources have stagnated, and the environ-

The RITE Bioprocess® involves cultivating a large

mental pollution caused by marine plastic wastes has

amount of coryneform bacteria (smart cells) that arebecome a serious global issue. Against this background,metabolically designed to produce the target substancethere are great expectations from bioplastics and bio-

efficiently; filling a reaction tank with a high density of

degradable plastics made from biomass, which is a re-these cells; and finally, creating anaerobic conditions.

newable resource.

Alternatively, the reaction is performed in a state where“Bio-Strategy 2020” places great importance on theimmediate and continuous promotion of bioplastics (acell division is stopped by removing the factors essential for cell proliferation (Fig. 5).

general-purpose plastic alternative), which is one of thenine market areas shown in “Bio-Strategy 2019.”
Our group is involved in the Moonshot R&D project(Development of Multi-lock Biopolymers Degradable in32

R&D Activities ● Molecular Microbiology and Biotechnology Grouplevel as that of C6 sugar (see the graph on the right side

RITE Bioprocess®
High-density packing of microbial cells
C5, C6 sugars
Complete simultaneous
utilization of mix sugars

High resistance tofermentation inhibitors

RITE Today 2021

Patent:JP4712716
JP3869788 US8604180
JP4451393 DE602004026192.0
US7368268 FR1647594
EP1647594 JP4927297
US7598063
JP4294373

Growth-arrested bioprocess
Biofuels
Biochemicals ■ High yield and high productivity■ Simple system

of Fig. 6).
As a result, C5 and C6 saccharides can be used at thesame time (simultaneous utilization of mixed sugars),and cellulosic raw materials can be used efficiently.

Conventional bioprocess
Production is accompanied by microbial growth

growthImprovement of simultaneous
utilization of C6 & C5 sugars by introducing
pentose transporter gene（araE）

Growth-nonarrested bioprocess■Cell-growth-dependent productivity■Carbon loss by cell growth(Growth-independent bioprocess)The key to high efficiency is to produce compounds
in a state in which the growth of the microorganisms is
suppressed (i.e., a growth-independent bioprocess)Concentration of sugars (mM)

Fig. 5 Features of the RITE Bioprocess® ①

thereby eliminating the need for nutrients or energy to

200

200

glucosexylosearabinose

150

100

100

50

50

0

0

2

4

6

8

10

Time(h)glucosexylosearabinose

150

12

0

0

2

4

6

8

10

12

Time(h)RITE’s report: Appl. Microbiol. Biotechnol. 85:105-115. 2009.

achieve microbial growth. As a result, it is possible touse microbial cells extremely efficiently. The RITE Bio-

Fig. 6 Features of the RITE Bioprocess® ②

process® has succeeded in realizing a bioprocess with

(Simultaneous utilization of mixed sugars)

high productivity equal to or higher than that of a normal chemical process.

2.1.3. Feature ③: High tolerance to fermentation inhibitors

2.1.2. Feature ②: Simultaneous utilization of mixed
sugarsFermentation inhibitors (phenols, furans, etc.) thatare formed during the pretreatment of lignocellulosic

A cellulosic biomass consists of mixtures of hemicel-

biomass are known to exhibit strong inhibitory activity

lulose-derived pentoses (C5) and hexoses (C6). The sim-in the bioethanol production process. Therefore, in or-

ultaneous utilization of both pentoses and hexoses is

der to be able to produce the target substance effi-

essential for microbial biofuel production.

ciently, an increase in the tolerance of microorganisms

However, wild coryneform bacteria use xylose (C5sugar) and arabinose (C5 sugar) at a slower rate than(bacteria) to the fermentation inhibitors is indispensable.

they do glucose (C6 sugar) (see the graph on the left

The coryneform bacterium developed by our groupside of Fig. 6). When raw materials are continuously

has been demonstrated to have high tolerance to fer-added, the C5 sugars accumulate, and their production

mentation inhibitors rendered through the introduction

efficiency eventually decreases.

of several genes (Fig. 7).

Our group has succeeded in improving the metabolic system of coryneform bacteria by introducing into
them several genes involved in the utilization of C5
sugar thereby increasing its utilization rate to the same33

R&D Activities ● Molecular Microbiology and Biotechnology Group3. Next core technology

Comparison of resistance tofermentation inhibitors

Relative ethanol
productivity（%）

4-HB

NEDO launched the project “Development of Pro-

100

80

duction Techniques for Highly Functional Biomaterials

80

O

60

O

40

20

Using Smart Cells of Plants and Other Organisms”

60

40

0

3.1. NEDO Smart Cell ProjectFurfural100

CHO(Smart Cell Project) in 2016. Our group has participated

20

OH

0

RITE Today 2021

60

in this project since its inception. Members of the pro-

●RITE Bioprocess ■Zymomonas mobilis ▲Saccharomyces cerevisiae

ject develop technologies for designing the Smart Cell

RITE’s report: Appl. Environ. Microbiol. 73:2349-2353. 2007.

(defined as a finely designed and expression-controlled

0

5

10

15

Concentration (mM)

20

0

20

40

Concentration (mM)

Fig. 7 Features of the RITE Bioprocess® ③(High tolerance to fermentation inhibitors)cell) and validate these Smart Cell design systems.
We have selected catechol as our target compoundin the project. Catechol has not been produced in high2.2. Examples of substances produced by the RITE Bio-concentration by fermentation until now, even thoughprocess®

it is in high demand in various industrial fields. It is pre-Currently, in addition to the highly efficient produc-

sumed that the reasons are its high toxicity to microor-tion of various substances, such as ethanol, L-lactic acid,ganisms and the high complexity of the metabolic path-D-lactic acid, and amino acids, we are expanding ourways from glucose to catechol. Furthermore, wild-typebusiness to the production of high-performance chem-

Corynebacterium glutamicum does not have its own

icals, such as butanol, jet fuel materials, and aromatic

genes for catechol production. To solve these problems,compounds (Fig. 8).

we applied the Smart Cell design systems to create a C.

Biofuels
Gasoline additives★ Ethanol *Bio-jet fuels★ Isobutanol *☆ n-butanol *★ C9~C15Saturatedhydrocarbon+ Aromatics★ Biohydrogen

Green chemicals
Aromatics★ Shikimic acid (Anti-influenza drug; Tamiflu raw materials)★ Phenol * (Phenolic resins, Polycarbonates)★ 4-hydroxybenzoic acid * (Polymer raw materials)★ Aniline * (Natural resource tire (Age resistor))★ 4-aminobenzoic acid * (Pharmaceutical raw materials)★ Protocatechuic acid * (Cosmetic raw materials)
Organic acids★ D-lactate *, L-lactate * (Stereo-complex PLA)
☆ Succinate *Amino acids★ Alanine (Chelators)★ Valine (Next-generation feed-use amino acids)☆ Tryptophan (Next-generation feed-use amino acids)☆ Methionine (Feed-use amino acids, Seasoning)
Alcohols☆ Isopropanol (Propylene raw materials)★ Xylitol (Sweetener)*：Polymer raw materials★ Red character：World’s highest productivity achieved

glutamicum catechol overproducer in a short period oftime.
In collaboration with universities, research institutes,
and one company, which all have their own original
Smart Cell design systems, several genetic modification
points were proposed for improving catechol productivity. The proposals were integrated into a C. glutamicum strain to realize its high catechol-producing potential. As a result, the concentrations of catechol were

Fig. 8 Examples of substances produced by

gradually increased in stages, and the final strain

the RITE Bioprocess®

achieved a very high production concentration, far exceeding the highest reported worldwide (Fig. 9).

In the following sections, we describe the efforts inThis fiscal year (FY 2020) is the final year of the pro-the national projects in which our group is participating,

ject, and we have already achieved productivity that ex-

the development of production technology for green

ceeds its target value of the project. Continued efforts

chemicals containing biofuels and aromatic compounds,

towards development for practical use is being under-

which are the main targets, and introduce the efforts fortaken, which will contribute to the realization of a new

their practical application.

bio-based industry, the Smart Cell industry.

34

R&D Activities ● Molecular Microbiology and Biotechnology Group

Smart Cell design system andMetabolic pathways of C. glutamicum
Fundamental
technology

Fundamental
technology

Smart Cell
Design System

x500
x380 x400x1600

Condition
optimization

Fundamental
technology

x320

Production
・
Productization

Catechol

Fundamental
technology
Fundamental
technology

Catecholproduction

Industrial Smart Cell
creation technology
Solving problems associated withcommercialization

Fundamental
technology

GlucoseGlucoseRITE Today 2021

Industrial
Smart Cell

Smart Cell

Carbonrecyclingsociety
Sustainableeconomic
growthRange of the projectx1

Application of
fundamental
technologies

Fig. 10 Development of bio-derived material

Fig. 9 Development of catechol production by applying

production technologies

Smart Cell design systems

4. Development of target products
3.2. NEDO Carbon Recycle Project
NEDO has launched a new project, “Development of

4.1. Biofuel production research and development
4.1.1 Biobutanol

Bio-derived Material Production Technologies that Ac-

Butanol is more suitable as a gasoline additive thancelerate the Realization of Carbon Recycling.” In the on-

ethanol owing to its better physicochemical properties,

going Smart Cell Project (from 2016 to 2020), Smart Cell

including higher energy content, lower vapor pressure,design systems have been developed to create smart

and lower water solubility. It can also be used as thecells. On the basis of this achievement, the developmentbase material for the production of bio-jet fuel usingof industrial material production technology using bio-

conventional chemical reactions. The bio-jet fuel syn-logical functions is being carried out in the new project.

thesized from biobutanol can be used in airplanes. Air-The aim is to accelerate the social implementation oflines and aircraft manufacturers have paid great atten-

bio-derived products by developing technologies re-tion to the importance of bio-jet fuel, which has been

lated to production processes for industrialization, in-recognized as being critical for reducing CO2 emissions

cluding bioreactor scale-up and refining processes (Fig.

because it uses plant-based materials as feedstock in-

10).

stead of petroleum. The bio-jet fuel synthesized from

Our group participated in this project with the goals

butanol is often referred to as “alcohol-to-jet” (ATJ) fuelof identifying problems associated with the practicaland has been approved by the American Society for

application of microbial fermentation and contributing

Testing and Materials (ASTM) and is ready for use into the development of "Industrial Smart Cell creationcommercial aircraft.

technology" to solve the problems. Furthermore, we se-

We have developed a genetically engineered C. glu-

lected terpenoids as our target chemical compounds

tamicum strain that is highly efficient in producing bio-and started developing industrially applicable overpro-butanol. Furthermore, we had conducted a research

ducers.

project from 2015 to 2019 to investigate cellulosic butanol production (see Topics in RITE Today, 2016). The
project was funded by the Ministry of Economy, Tradeand Industry. The advantages of our production process
are as follows: (i) cellulosic biomass-derived mixed35

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2021

sugars can be used as feedstock, and (ii) production istolerance and optimizing the metabolic pathway of thefast and generates a high product yield (Fig. 11).

producing bacterial strain, and developing an energy-

Because butanol is highly toxic, we have developedsaving butanol recovery technology.

a genetically engineered Corynebacterium strain that

Meanwhile, as an initiative for commercialization,

has high tolerance to its toxicity. The strain has further

RITE provided technical cooperation in the “Let's Fly byenhanced the high productivity of the RITE Biopro-

Recycling 100,000 Clothes!” project (2018–2020) spon-cess®. Through collaboration with the U.S. National Re-

sored by JAL. This project aimed to manufacture bio-jet

newable Energy Laboratory, we have accelerated thefuel from used clothes collected in cooperation with JAL

R&D of biobutanol production from non-food biomass.

and JEPLAN, Inc.
Green Earth Institute Co., Ltd. (GEI), a venture com-

RITE
RITE‐NREL

Bio-butanol-producing bacteriawith high yield

Mixed sugar
NREL
Pretreatedbiomass

Utilization ofunused acetate

RITE Bioprocess®
RITE‐PNNL

Drop-in fuels (motor fuel, jet fuel)Chemicaloligomerization

Concentration andpurification of biobutanol by
combination of PV
membrane anddistillation

Bio-butanolpany originating from RITE, also participated in this project, and isobutanol was produced by the RITE Bioprocess® using coryneform bacteria developed by RITE. In
2020, the bio-jet fuel produced from this isobutanol
passed the international standard ASTM D7566 Annex5
Neat for the first time as a purely domestic bio-jet fuel.

Fig. 11 Production of biobutanol and bio-jet fuel using

On February 4, 2021, it was used as the first domesticallythe RITE Bioprocess®

produced bio-jet fuel on a JAL commercial flight, JL319,
from Tokyo Haneda to Fukuoka Airport.

Since 2017, we have also collaborated with the U.S.

In the future, by combining these elemental tech-

Pacific Northwest National Laboratories in researching

nologies and a variety of procedural knowledge, we aim

and developing the production of jet fuels by the chem-

to produce jet fuel from biobutanol and put it into ap-

ical oligomerization of biobutanol. This was based on aplication and commercialization.

new idea that if acetic acid is included in mixed sugars
from a pretreated biomass, it can be converted to eth-

4.1.2. Green jet fuelanol by an engineered C. glutamicum strain into which

Petroleum-based jet fuels are mixtures of hydrocar-

new genes have been introduced. Therefore, the mix-bons that consist of n-paraffins, isoparaffins, cyclopar-tures are utilized for the bioproduction of butanol andaffins, and aromatic compounds with 9–15 carbon at-

ethanol, which can then be subjected to chemical oli-oms. Any jet fuel must meet strict standards with regard

gomerization for use as jet fuels.

to its physical properties, such as a specified freezing

The distillation of a biobutanol solution requires apoint and density.

large amount of energy. To reduce the amount of en-

So far, ASTM International has approved six produc-

ergy needed, we developed an energy-saving biobuta-tion pathways for bio-jet fuels, such as the production

nol recovery process using a combination of distillationof HEFA fuel by the hydroprocessing of fatty acid esters,

and pervaporation. This resulted in energy savings of up

FT-SPK fuel by the Fischer–Tropsch synthesis of hydro-

to 90% and the achievement of the highest biobutanol

carbons from syngas, and ATJ fuel by the oligomeriza-

productivity in the world. The project successfullytion of alcohol. In 2020, ATJ fuel coupled with our bio-

achieved the original goals of improving the butanol

butanol production technology was approved. However,36

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2021

these certified bio-jet fuels consist mostly of isoparaf-

Ministerial Council on Renewable Energy, Hydrogen

fins and are lacking in other essential components: cy-

and Related Issues in 2017 states the importance of the

cloparaffins and aromatics. They do not meet ASTM

development of innovative CO2-free hydrogen produc-standards on their own and are required to be blended

tion technologies to realize a hydrogen society over thewith petroleum-based jet fuel so as to be 50% or less inmedium to long term. The aim is to achieve this by 2050,

total when used. Therefore, even when the production

based on the goals set to develop commercial-scale hy-

capacity of bio-jet fuels catches up with demands, moredrogen supply chains by circa 2030.

than 50% of fuel demands will still be occupied by petroleum-based jet fuel.

Although bioprocesses have significant potential for
CO2-free hydrogen production, innovative improve-

To overcome this blending ratio limitation of thements in technology are necessary to establish a cost-certified bio-jet fuels, we are additionally developing aeffective process for producing biohydrogen. In collab-

high-performance green jet fuel that contains cyclopar-

oration with the Sharp Corporation, our group has de-affins and aromatics in addition to isoparaffins. The

veloped a biohydrogen production process. The hydro-

novel jet fuel meets ASTM standards and is expected togen production rate achieved by our process is two or-

be used alone. In the R&D of the high-performance bio-ders of magnitude higher than that of conventional fer-

jet fuel, we have achieved some promising results, suchmentation processes. On the basis of this achievement,

as a novel biocatalyst that enables the cross-coupling

our group is now working on the metabolic engineering

reactions between C2 and C8 compounds for the syn-of hydrogen-producing microorganisms to improve hy-

thesis of C9–C15 branched and cyclic compounds,

drogen yields from cellulosic biomass.

which can then be chemically converted to jet fuel components.

Photosynthetic bacteria produce hydrogen gas (H2)using nitrogenase (a nitrogen-fixing enzyme) and the
reducing power generated from the degradation of or-

Bio-jet fuelscertified by ASTM international
HCJ
HEFA
SIP
FT-SPK(A) ATJ

Isoparaffins
Need to be blended
with petroleum-based jet fuelA novel high-performancegreen jet fuelIsoparaffins＋
Cycloparaffins
Aromatics
Expected to be used alone

ganic compounds (Fig. 13). Although H2 is a byproductof the nitrogenase reaction that generates ammonia(NH3) from nitrogen gas (N2), only H2 is produced by the
reduction of H+ in the absence of N2. This photofermentative process can produce hydrogen from acetate(a thermodynamically unfavorable reaction) using light

Fig. 12 Research and development of a novel high-energy. Thus, a major improvement in hydrogen yield isperformance bio-jet fuelexpected by integrating this process with the dark fermentative process of hydrogen production, which pro-

4.1.3. Biohydrogenduces acetate as a byproduct. However, in photosyn-

Hydrogen, the combustion of which generates only

thetic bacteria, reducing power is consumed by CO2 fix-

water, is considered the ultimate clean energy source.

ation and polymer synthesis for carbon storage, thereby

However, CO2 emissions during the hydrogen produc-

limiting its use for hydrogen production. Moreover, hy-tion processes currently in use are a problematic issue,

drogen is reused by uptake hydrogenase for providing

because fossil resources are used as the feedstock. The

cells with reducing power. On the basis of these findings,

Basic Hydrogen Strategy drawn up at a meeting of thethemetabolicengineering

of

H/C/N

metabolic37

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2021

pathways has resulted in a marked increase in the hy-of the RITE Bioprocess®. In 2011, RITE and GEI began

drogen yield from acetate.

collaborative research on amino acid production using
the RITE Bioprocess® and developed technologies for

Acetatescaling up production, growing efficient production

Light energy
Photosynthesis

Organic
compounddegradation

ATPe-Hydrogenase
Nitrogenase

H2 uptakeN2 fixation(H2 production)

Photosynthetic
bacteria
CO2 fixation
Microbial
growthCarbon storage
substance(polymer)synthesis

H2
N2 + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi
※ in the absence of N2：
2H+ + 2e- + 4ATP → H2 + 4ADP + 4Pi

strains, and reducing production costs. In 2019, RITE
succeeded in producing a strain that yielded the world's
highest production concentration of L-valine with thebest production efficiency. Furthermore, RITE has completed commercialization projects with GEI and overseas partners to achieve the commercial production ofthese amino acids. Our goal was to produce this aminoacid from renewable resources thereby reducing the life
cycle carbon footprint.
In 2016, we succeeded in demonstrating the feasibility of L-alanine production technique by using the

Fig. 13 Competition with H2 production (N2 fixation) for

commercial-scale facilities of our partner company,

reducing power (e−) in photosynthetic bacteria

which was an important milestone for its industrialization. One of our group members also participated in the

4.2. Amino acids (alanine and valine)

first operation and worked with local employees to lead

Normally, amino acid fermentation is carried out un-

the project to a successful conclusion. As the result ofder aerobic conditions, where high productivity requires

an evaluation by the Food Safety Committee in August

the aeration and agitation of the system to be properly

2017, the safety of the L-alanine produced by our strain

controlled. However, this is often difficult to achieve infor use as a food additive was confirmed, allowing it tolarge-scale fermenters because their internal oxygen

be made commercially available for this purpose be-concentration is not homogeneous. To overcome thissides its use for industrial applications. We are now

problem, we have developed a new, genetically modi-

working on a joint research project for the production

fied Corynebacterium strain with the RITE Bioprocess®

of other amino acids.

that allows the production of amino acids to be carriedout under anaerobic conditions. The technological hur-4.3. Green-aromatic compounds

dle for amino acid production under anaerobic condi-

Aromatic compounds are important industrial

tions is to balance the redox reaction without oxygen as

chemicals used for the synthesis of polymers as well as

an electron acceptor. To this end, we successfully intro-

a diverse group of value-added chemicals that are ap-

duced an artificial pathway for amino acid biosynthesis

plied in the pharmaceutical, nutraceutical, flavor, cos-

into the microbial cells thereby solving the technologi-

metic, and food industries. Although they are currently

cal hurdle. Our group published this accomplishment inderived from petroleum or natural plant resources, theiran international journal in 2010 (Appl. Microbiol. Bio-environmentally friendly biotechnological production

technol. 87: 159–165).

from renewable feedstocks is desirable from the view-

GEI was established in 2011 for the industrialization

point of creating a sustainable society that is no longer

38

R&D Activities ● Molecular Microbiology and Biotechnology Groupdependent on petroleum resources and has efficient

RITE Today 2021

4.4. Strategic Innovation Promotion Program (SIP)

production processes. Bacterial cells synthesize various

The cross-ministerial Strategic Innovation Promo-

aromatic compounds, including amino acids (phenylal-tion Program seeks to promote R&D, from the basic re-anine, tyrosine, and tryptophan), folate (vitamin B9), andsearch stage to the final outcome, in a seamless mannercoenzyme Q, all of which are derived from the shikimate

by endeavoring to strengthen cooperation among in-pathway (Fig. 14). By employing the metabolically engi-

dustries, academia, and governments beyond theneered C. glutamicum, we have successfully establishedframework of government ministries and traditionala highly efficient bioprocess for producing the following

disciplines. The theme “Technologies for Smart Bio-in-

aromatic compounds from non-food feedstocks: shiki-

dustry and Agriculture”, one of the 12 themes in the SIP

mate, a key building block of the anti-influenza drug

at Second Phase, aims to realize a sustainable growing

Tamiflu; 4-aminobenzoate, which is used as a building

society that uses manufacturing technologies devel-

block of a potentially useful functional polymer; and ar-oped through the integration of biotechnology andomatic hydroxy acids, which have potential applications

digital resources.

in the polymer, pharmaceutical, cosmetic, and adhesive

RITE is participating in “Development of Technolo-

material industries. Currently, we are seeking to develop

gies for Functional Design and Production of Innovative

new strains for the production of useful aromatic com-

Biomaterials (Fig. 15),” a consortium comprising thepounds that the wild-type C. glutamicum is unable totheme. This consortium consists of two groups: the pol-produce. This will be achieved by introducing genes de-ymer group for designing polymers with marketable

rived from versatile biological resources into the bacte-

properties and predicting the function of polymers con-rium. The techniques developed in the Smart Cell Pro-

sisting of particular monomers; and the monomer

ject, as described earlier, will help to accelerate the de-group for selecting biosynthesizable monomers andvelopment of strains and improve their productivity.

designing biosynthetic pathways and enzymes for themonomers required for polymer synthesis. As a leader

CH2OH

HOH2C

O

Glucose

OH
OH
OH

HO

PTS

PEP

OH

O
OH
OH

XyloseArabinose

Pentosetransporter

PYR
XyloseNADP+ NADPH

EM pathway

Pentose phosphatepathway

G-6-P
F-6-P

6-PGlucono-lactone

GAP

DHAP

NAD+

6-PGluconate

Rib5P

PGP

PYR

：Target products

PEP

Xylulose

Ribulose

xylB

araB

Ribu5P

Quinate

araD
Xlu5P

GAP

Sed-7-P

F-6-P
F-6-P
GAP

Ery-4-P

Shikimate DHS
pathway

ShikimateTyr-derivatives

Trp-derivatives

Trp

ANT

Chorismate

Tyr

PPY

Phe

Phe-derivatives

4-HBA

Phenol4-ABA

didates and enzyme modifications that are predicted tobe required for the synthesis of target monomers by bioinformatics teams in the group. The enzymes involvedin the synthesis of an aromatic diol and a precursor ofProtocatechuateand derivatives

DHQof the monomer group, RITE is evaluating enzyme can-Ribulose-5P

NADPH

DAHP

Pyruvate：Intermediate metabolite

araA

CO2

GAPDH

NADH

OH

OH

Arabinose

xylA

NADP+

F-1,6-P2

O
OH

HOH2C

Phenolan aromatic diamine are current targets to be modified.
Thus far, the predicted modification of amino acid sequences has successfully improved the activity of the
enzymes and altered their substrate specificity. We are
improving the accuracy of the technologies by evaluat-

Chorismate-derivatives

ing more enzymes.

Fig. 14 Biosynthetic pathway for various
aromatic compounds

39

R&D Activities ● Molecular Microbiology and Biotechnology GroupHigh heatresistant／high stiffnesspolymers
Rubbers andelastomers
Biodegradable
polymers

RITE Today 2021

In this project, RITE will promote the production of

Input

biomonomers and the development of polymer-deOutput

Highly functional
polymers

OutputBiosynthesizable
polymers

OutputMetabolic pathway・
improved enzymes

Input

grading enzymes from non-food biomasses. Specifically,(1) in order to establish a bioprocess that enables thehigh production of biomonomers, which are the raw

Targetpolymerproperties

materials for multi-lock biopolymers, RITE will promote
Designing of highly
functional polymers・
biomonomers

Efficient production of
biomonomers

the construction of a high biomonomer-producing
strain and the development of scale-up production

Fig. 15 Integrated design and production technology

technology. Additionally, (2) for the practical use of

system for biomaterials

multi-lock biopolymers, we will promote the development of highly functionalized polymer-degrading en-

4.5. NEDO Moonshot-type R&D Project: Development

zymes for efficient enzymatic degradation in the multi-of Multi-lock Biopolymers Degradable in Ocean fromlock mechanism and of high production technologies

Non-food Biomasses

for these enzymes.

This project aims to develop a “multi-lock-type biopolymer” that is as tough as conventional petroleum-

5. Toward the industrialization of our technologies

derived polymers when in use but can be rapidly de-

5.1. Green Chemicals Co., Ltd.

graded by external stimuli in the marine environment(Head Office ・ Laboratory: in Kyoto headquarters,

after its use (Fig. 16). We aim to simultaneously solve

RITE; Shizuoka Laboratory: in Shizuoka plant, Sumitomo

the problems of global warming and environmental

Bakelite Co., Ltd.)

pollution in the field of polymers. Generally, there is a

Currently, commercial phenol can only be derived

trade-off between plastic toughness and marine degra-

from petroleum. We have taken on the challenge of de-dability (biodegradability), but this project aims toveloping the world's first biomanufacturing process forachieve both.

biomass-derived phenol, with the ultimate goal being
to aid global environmental conservation and greenhouse gas reduction.
In May 2014, Sumitomo Bakelite Co., Ltd., and RITE
established Green Phenol Development Co., Ltd. (GPD),
to accelerate the industrialization of our biomass-derived phenol-producing technology, named the “TwoStage Bioprocess.” In April 2018, GPD changed its nameto Green Chemicals Co., Ltd. (GCC).
Because GCC’s phenol-producing technology andknowledge are applicable to the production of various

Fig. 16 Research and development of marine-degradable

other aromatic compounds, the establishment of a bio-

multi-lock biopolymers made from non-food biomass

process for each higher value-added chemical and thecommercialization of products that meet customer

https://www.nedo.go.jp/english/news/ZZCA_100007.html

needs are in progress (see Section 4.3).

40

R&D Activities ● Molecular Microbiology and Biotechnology GroupThe present product lineup of GCC is shown in Fig.
17. In 2020, using the pilot-scale facilities of GCC, we

RITE Today 2021

and Welfare, paving the way for its use in the food industry.

succeeded in demonstrating the high-concentration

With regard to bio-jet fuels made from non-food bi-

production of two target compounds, protocatechuic

omass, which are highly expected to reduce CO 2 emis-acid and shikimic acid, which was an important mile-sions from aircraft, GEI is continuously conducting jointstone for industrialization.

research with RITE in this area and is working towardcommercialization (see Section 4.1).

Product lineup
PhenolProtocatechuic Shikimic
acid
acid
O

O

OH

OH

4-HBA
O

OH

OH

cosmetic materials) in cooperation with RITE, and themarketing for commercialization and the scaling up for

OH
OH

Additionally, GEI is developing green chemicals (e.g.,

HO

OH

OH

OH

mass production are under way.
As a venture company that realizes the commercialization of RITE-originated technology and by contributing to the development of the biorefinery industry,
GEI will continue toward the realization of a society thatFig.17 Product lineup of Green Chemicals Co., Ltd.

5.2. Green Earth Institute Co., Ltd.
(Headquarters: Bunkyo-ku, Tokyo, Japan; ResearchInstitute: Kazusa, Kisarazu City, Chiba, Japan)
GEI is a RITE-launched venture company that wasestablished on September 01, 2011, to facilitate thedoes not rely on fossil resources.

6. Closing remarks
As mentioned earlier, in anticipation of a rapid economic recovery after the COVID-19 pandemic has subsided, the promotion of the bioeconomy is expected to
become increasingly important in the future.

quick commercialization of the research results of theUnder such circumstances, in recent years, the un-

aforementioned innovative RITE Bioprocess®. GEI isderstanding of life phenomena by the "data-driven" ap-

conducting both joint research and activities aimed at

proach of discovering the law from a large amount ofcommercialization with RITE in order to realize the prac-life information has been progressing.

tical uses of the green chemicals and biofuel production

Against this background, research on the biology oftechnologies produced using the microorganisms

synthesis (fusion of bio and digital processes) that ac-(coryneform bacteria) generated in the RITE Biopro-

cumulates data and understands biological functions bycess®.

repeating the cycle of design (Design), build (Build),

With regard to amino acids, as mentioned earlier,evaluation (Test), and learning (Learn) (i.e., the DBTL cy-

GEI has succeeded in producing L-alanine and L-valinecle) is developing rapidly. Our group also participateson a commercial scale using the production strain de-in multiple national projects (see Section 3).

veloped by RITE. Currently, commercial production is

In these projects, the biorefinery technology based

being realized through license agreements with domes-on the Smart Cell described earlier is expected to play a

tic and overseas partner companies (see Section 4.1).

major role as a core technology and have a large ripple

Additionally, the safety of L-alanine as a food additiveeffect on the industrial (manufacturing) and energy

has been confirmed by the Ministry of Health, Labour

fields (Fig. 18).

41

R&D Activities ● Molecular Microbiology and Biotechnology Group

Biologicalinformation
DNA
RNA
Protein
Metabolites
etc.

Advanced AI・
IT technology
Deep learning
Machine learning
Evolutionary
engineering
Simulation etc.

Smart Cell
Finely-designed andexpression-controlled cell

Design of biological function
Design

Build

Learn

Test

RITE Today 2021

Advanced biotechnology
Genome editing
High speed DNA sequencing
Metabolic control
Culture, breeding technology etc.

Control of functional expression

New industry creation”Smart Cell” Industry
Industry & energy production based on “smart-cell”

Industry

EnergyRenewable resource/energy saving

Non-fossil resources

Green chemicals
bioplastics
BiofiberEngineering plastics
Medicine, intermediateof medicine etc.

Biofuelsbioethanol
biojet fuels
biodieselbiogas etc.

Source: International trends for realization of Bioeconomy & efforts being by Japan (METI）25 Sept. 2018

Fig. 18 Fusion of industrial/energy fields changed by bio
and digital processes

In 2021, we will continue to develop innovative biorefinery production technologies centered on the cutting-edge biotechnology of the Smart Cell and contribute to the construction of a sustainable low-carbon society.
※ RITE Bioprocess® is a registered trademark of RITE.

42