R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2022

Molecular Microbiology and Biotechnology Group
Members (As of Dec. 2021)
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
Shinichi Oide, Senior Researcher
Kiyoshi Oi, Senior Researcher
Tetsu Shimizu, Researcher
Akiyoshi Higo, Researcher
Natalia Maria Theresia, Researcher
Norimasa Kashiwagi, Researcher

Jyumpei Kobayashi, Researcher
Ryoma Hashimoto, Researcher
Naoki Saruya, Researcher
Yuki Nozaki, Researcher
Dyah Candra Hapsari Subagyo, 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 a Biorefinery Technology that Contributes to Carbon
Neutrality
1. Introduction
In October 2020, the Japanese government declared“2050 carbon neutral.” Therefore, the government willalternative aircraft fuels using microalgae and carbonrecycled chemicals. Biomass resources, CO2, waste plastics, and waste rubber are used as raw materials.

reduce greenhouse gas emissions by 46% by 2030 and

Biotechnological fields, such as synthetic biology

virtually zero greenhouse gas emissions by 2050. The

and genome editing technology, have rapidly and re-aim was to attain a carbon-free society in 2050.

cently advanced. In addition, the technological innova-

As a policy to realize this declaration, the “Greention of “bio-digital technology,” a fusion of biotechnol-

Growth Strategy Through Achieving Carbon Neutrality

ogy and information technology (digital), such as IoT

in 2050” was developed in December 2020 and revisedand AI, is rapidly developing. Therefore, “bio-manufac-

in June 2021. Fourteen fields have also been set toturing,” using this bio-digital technology and bio-re-achieve this goal. These fields are expected to grow assources, is expected to significantly contribute to car-an industry and are considered indispensable for reduc-bon neutrality and carbon negative.

ing greenhouse gas emissions.

Our group has been developing biorefinery technol-

Among these, ⑪ “The Carbon Recycling Industry”

ogies, which are technologies to produce biofuels andcan be mentioned as a field in which biotechnology cangreen chemicals using renewable resources (biomass)

influence. Specifically, it is the production field of car-as raw materials using microorganisms, with the aim of

bon-recycled fuels, such as alcohol-to-jet (ATJ) and bio-balancing the global environment and the economy.

31

R&D Activities ● Molecular Microbiology and Biotechnology Group

These are technologies that can contribute to ⑪ “TheRITE Today 2022

Chapter 4).

Carbon Recycling Industry” above. In this development,This overview explains our core technology, thewe have observed that although coryneform bacteria“RITE Bioprocess®,” and its features. Next, we will intro-(typical industrial microorganisms) suppress theirduce these developments, highlighting their target uses.

growth under reducing conditions, they maintain met-

Then, as crucial technological development, national

abolic functions. They can metabolize saccharides toprojects based on the “bio-digital technology” that has

produce organic acids and related compounds effi-

made remarkable progress will be described. Finally, we

ciently. Therefore, we developed a growth-independent

want to commercialize our efforts.

bioprocess “RITE Bioprocess®” and established “thecomplete and simultaneous use of mixed sugars de-

2. The core technology of our group “RITE Bioprocess®”

rived from non-food biomass” and “high resistance to

Our group has established the innovative biopro-

fermentation inhibitors” that are essential and ele-

cess “RITE Bioprocess®” based on a new concept, “coremental technologies for industrialization (see Chapter

technology of RITE.” This concept has achieved out-

2).

standing results in developing technologies for theWith these technologies, we are reporting thehighly efficient production of biofuels and green chem-

world’s highest level of high-efficiency biofuel products,

icals, such as amino acids and aromatic compounds. It

including ethanol, butanol, green jet fuels, biohydro-

has also been significantly evaluated in Japan and over-gens, and green chemicals, including lactic acid, succinic

seas (Fig. 1).

acid, alanine, valine, shikimic acid, protocatechuic acid,
4-aminobenzoic acid, and 4-hydroxybenzoic acid. Furthermore, we are currently focusing on developing production technologies for aromatic compounds, raw materials for higher value-added fragrances, cosmetics,
pharmaceuticals, and so on (see Chapter 3).
Alternatively, we participated in the New Energy andIndustrial

Technology

Development

RITE Bioprocess®

Non-food
biomass(Growth-arrested bioprocess)Pretreatment
Saccharification

Coryneformbacteria

Biofuels
Green chemicals

Recovery
Purification

Metabolicengineeringetc.

O

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

HO

OH O

OH

OH

OH

NH2

Fig. 1 Biorefinery concept using the “RITE Bioprocess®”

Organization(NEDO) “Smart Cell” project, SIP Strategic Innovation

2.1. Features of the “RITE Bioprocess®”

Creation Program, and NEDO “Bio-Manufacturing” pro-

2.1.1. Feature 1: Growth-independent bioprocess

ject to develop the latest technology, integrating bio-In the “RITE Bioprocess®,” first, many coryneform

technology and digital technology. Through these na-bacteria that are highly and metabolically designed totional projects, we are progressing with R&D to improve

help efficiently produce the target substance are culti-

the efficiency of biosynthesizing high-performancevated. Then, after densely filling the reaction vessel withchemicals, which were difficult to produce through con-cells, the reaction is conducted in a state where cell di-ventional synthetic methods. Likewise, since 2020, we

vision is stopped by removing anaerobic conditions andhave participated in the NEDO “Moonshot” project andfactors essential for proliferation (Fig. 2).

are working on researching and developing multi-lock
type biopolymers that can be decomposed into theocean using non-food biomass as a raw material (see32

R&D Activities ● Molecular Microbiology and Biotechnology Group

Alternatively, by further introducing a C5 sugar trans-

RITE Bioprocess®

porter gene, we succeeded in increasing the usage rate

High-density packing of microbial cells
Patent: JP3869788
JP4451393
US7368268
EP1647594

C5, C6 sugars
Complete simultaneous
utilization of mix sugars

JP4712716
US8604180
DE602004026192.0
FR1647594

JP4927297
US7598063
JP4294373

High resistance tofermentation inhibitors

Biofuels
Biochemicals

RITE Today 2022

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

Conventional bioprocessof the C5 sugar to the same level as the C6 sugar (seethe graph on the right in Fig. 3). Therefore, C5 and C6
saccharides can be used simultaneously, and cellulosic
raw materials can be used efficiently.

Production is accompanied by microbial growth

growthGrowth-nonarrested bioprocess■Cell-growth-dependent productivity■Carbon loss by cell growthFig. 2 Feature 1 of the “RITE Bioprocess®”

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

The key to its high efficiency lies in the growth-independent bioprocess of producing compounds while
suppressing the growth of microorganisms, which eliminates the need for nutrients and energy required forConcentration of sugars (mM)

(Growth-independent bioprocess)the development. Specifically, although raw sugar ma-

200

200

glucosexylosearabinose

150

100

100

50

50

0

0

2

4

6

Time(h)810glucosexylosearabinose

150

12

0

0

2

4

6

8

10

12

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

terials are not used for growth, they produce target
substances, making it possible to use microbial cells re-Fig. 3 Feature 2 of the “RITE Bioprocess®”markably and efficiently like a chemical catalyst. This

(Simultaneous usage of mixed sugars)

method also achieved a bioprocess with high productivity equal to or higher than that of a typical chemical

2.1.3. Feature 3: High tolerance to fermentation inhibi-

process.

tors
Fermentation-inhibiting substances, such as phe-

2.1.2. Feature 2: Complete simultaneous use of C5 andnols and furans produced during the pretreatment of

C6 mixed sugars

lignocellulosic biomass, sturdily inhibit creating the tar-

Cellulose-based biomass comprises C5 sugars, suchget substance. Therefore, increasing the microorgan-

as xylose and arabinose, and C6 sugars, such as glucose.

isms (bacteria) resistance to the fermentation inhibitor

Therefore, simultaneously using C5 and C6 sugars is in-

is indispensable to produce the target substance effi-dispensable for efficient production processes using

ciently. The “RITE Bioprocess®” demonstrates that it iscellulosic biomass.

highly resistant to fermentation inhibitors because it

Coryneform bacteria into which a C5 glucose me-does not grow as described above (Fig. 4).

tabolism gene has been introduced have slower usage
rates with xylose (C5 sugar) and arabinose (C5 sugar)
than glucose (C6 sugar) (see the graph on the left in Fig.
3). Therefore, when the raw materials are continuously
added, the C5 sugar accumulates, eventually decreasing
the production efficiency of the compound.

33

R&D Activities ● Molecular Microbiology and Biotechnology Group3. Development of target productsComparison of resistance tofermentation inhibitors

Relative ethanol
productivity（%）

4-HB

3.1.1. Biobutanol

100

80

Butanol is more suitable as a gasoline additive than

80

O

60

O

40

20

ethanol owing to its better physicochemical properties,

60

40

0

3.1. Biofuel

Furfural100

CHO

including higher energy content, lower vapor pressure,

20

OH

0

RITE Today 202260

and lower water solubility. It can also be used as a base

●RITE Bioprocess ■Zymomonas mobilis ▲Saccharomyces cerevisiae

material for producing bio-jet fuels using conventional

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

chemical reactions. In turn, the bio-jet fuel synthesized

0

5

10

15

Concentration (mM)

20

0

20

40

Concentration (mM)

Fig. 4 Feature 3 of the “RITE Bioprocess®”from biobutanol can be used in airplanes. Airlines and(High tolerance to fermentation inhibitors)

aircraft manufacturers have paid considerable attention
to the importance of bio-jet fuel as critical for reducing

2.2. Main substances produced by the “RITE Biopro-

CO2 emissions because it uses plant-based materials as

cess®”

feedstock instead of petroleum. The bio-jet fuel synthe-

Figure 5 shows some substances currently achieving

sized from butanol is called "alcohol-to-jet" (ATJ) fuel.

high production by the group. As mentioned above,

In 2016, it cleared the standards of the America Society

many materials have reached the world’s highest level

for Testing and Materials (ASTM) and became availableof productivity. Nevertheless, we are expanding fromfor commercial flights.

ethanol and biohydrogen to butanol and high-perfor-

Prior to these movements, we have been developing

mance bio-jet fuel materials in biofuels. We also ex-

a highly efficient biobutanol production process using

panded from L-lactic acid, D-lactic acid, and amino acids"RITE Bioprocess®". As part of this development, we

to high-performance chemicals, such as aromatic com-conducted the “International Joint Research and Devel-

pounds in green chemicals.

opment Project for Innovative Energy Technology” by
the Ministry of Economy, Trade, and Industry.

Biofuels

Green chemicals

Gasoline additives• Ethanol*

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)
Alcohols• Isopropanol (Propylene raw materials)• Xylitol (Sweetener)*：Polymer raw materials

Bio-jet fuels• Isobutanol*• n-butanol*• C9~C15 Saturatedhydrocarbon+ Aromatics
Biohydrogen

Red character：World’s highest productivity achieved

In thisproject, through joint research with the US National Renewable Energy Laboratory (NREL), we developed biobutanol production technology using mixed sugar derived from non-food biomass as a raw material. In addition, through joint research with the US Pacific Northwest National Laboratories (PNNL), we proceeded withthe development of technology to convert butanol into

Fig. 5 Examples of substances produced bydrop-in fuel such as jet fuel. Furthermore, our group im-

the “RITE Bioprocess®”

proved the butanol resistance of the production strains,
optimized the metabolic pathways of the production
strains, and developed energy-saving butanol recovery
technology. As a result, we have achieved the world's
highest level of high productivity in the bioproductionof butanol (Fig. 6).

34

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE

Bio-butanol-producing bacteria with high yield

RITE‐NREL

Utilization ofunused acetate

Concentration and purificationof bio-butanol by combinationof PV membrane and distillation

RITE Bioprocess®

RITE‐PNNL
Drop-in fuels (motor fuel, jet fuel)Total energy for concentrationand purification can be
reduced below 10%bons comprising n-paraffins, isoparaffins, cycloparaffins,and aromatic compounds with 9–15 carbon atoms. So
far, ASTM International has approved seven production

Bio-butanol

Oligomerization

NREL：National renewable energy laboratory (U.S.)

Petroleum-based jet fuels are mixtures of hydrocar-

Butanol-resistant genes are obtained

Mixed sugars
NREL
Pretreatedbiomass

RITE Today 2022

PNNL：Pacific Northwest National Laboratory (U.S.)

pathways for bio-jet fuels, such as producing HEFA fuelby hydroprocessing fatty-acid esters and ATJ fuel by ol-

Fig. 6 Production of biobutanol and bio-jet fueligomerizing ethanol or isobutanol. In 2020, ATJ fuelusing the “RITE Bioprocess®”

coupled with our biobutanol production technology
was also approved. However, these certified bio-jet

Meanwhile, as an initiative for commercialization,

fuels primarily consist of isoparaffins and lack other es-

RITE provided technical cooperation in the “Let's Fly bysential components: cycloparaffins and aromatics.

Recycling 100,000 Clothes!” project (2018–2020) spon-

Therefore, they do not meet ASTM standards on theirsored by JAL. This project manufactured bio-jet fuels

own and should be blended with petroleum-based jetfrom used clothes collected in cooperation with JAL andfuel to 50% or less when used.

JEPLAN, Inc., and our technology was selected for it.

To overcome this blending ratio limitation of certi-

The Green Earth Institute Co., Ltd. (GEI), a venture

fied bio-jet fuels, we are developing a high-perfor-

company originating from RITE, participated in this pro-mance green jet fuel containing cycloparaffins, aromat-

ject together with RITE. Consequently, isobutanol was

ics, and isoparaffins. The novel jet fuel is expected to be

produced by the “RITE Bioprocess®” using coryneform

used alone without being blended with petroleum-bacteria developed by RITE. Hence, in 2020, the bio-jet

based jet fuel. In the R&D of high-performance bio-jet

fuel produced from this isobutanol passed the interna-fuel, we achieved promising results, such as a novel bi-

tional standard ASTM D7566 Annex5 Neat for the firstocatalyst that enabled cross-coupling reactions be-time as a purely domestic bio-jet fuel. The first flighttween C2 and C8 compounds to synthesize C9–C15

equipped with domestic bio-jet fuel was realized onbranched and cyclic compounds, which can then be

February 4, 2021 on JAL's Haneda-Fukuoka route.

chemically converted to jet fuel components (Fig. 7).

Therefore, in the future, by combining these elemental technologies and various procedural knowledge,

Fermentation

ATJ

we propose to produce jet fuels from biobutanol for use
and commercialization.

SugarsOH

Microbecatalyst

Chemicalconversion

High performancegreen jet fuelFermentation・Bioconversion

3.1.2. Green jet fuel
There is an urgent need to reduce the aviation in-

C１２
Isoparaffin

Isobutanol

SugarsMicrobecatalyst

C9-C15
Branched-,
cyclicprecursors

Chemicalconversion

C9-C15Isoparaffins
Cycloparaffins
Aromatics

dustry’s environmental load. As part of the reduction
measures, electric or hydrogen aircraft are being devel-

Fig. 7 R&D of a novel high-performance bio-jet fueloped, but considerable technical issues have been observed. Thus, disseminating drop-in biofuels derived
from renewable feedstocks is unavoidable.

3.1.3. Biohydrogen
Hydrogen is an energy carrier key to realizing carbon neutrality because (i) its combustion generates only35

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2022

water; (ii) it can be produced from diverse energy

mechanism of acetate metabolism in photosynthetic

sources, including renewable ones; (iii) it can be stored

bacteria for use in photofermentation, which has beenin large quantities for long periods; and (iv) it can be

successfully applied to improve the acetate-hydrogen

distributed and used for power generation, transporta-

conversion efficiency (Fig. 8).

tion, and various industrial processes. However, CO2
emissions during hydrogen production processes cur-

Dark fermentation

rently used are a problematic issue because fossil resources are used as feedstock. Therefore, a Basic Hydrogen Strategy was drawn up at the Ministerial Council[NiFe]H2[Mo]

C6H12O6(Glucose)

2 H+

2 NADH

CO2

meeting on Renewable Energy, Hydrogen, and Related

Formate

2 CO2

2 C3H4O3(Pyruvate)

4 H2Issues in 2017. The conference stated the importance of

2 Fd2-

developing innovative technologies for hydrogen pro-

2 H2duction, storage, and distribution to realize a hydrogen

8 H2

society over the medium to long term up to 2050.

2 CH2O2(Formate)
2 C2 H 4 O 2(Acetate)

2 CO2
2 C2H3O-CoA(Acetyl-CoA)

Photofermentation■ High-yield H2
production pathway■ Efficient conversionof acetate to H2

Therefore, hydrogen strategies around the world have
moved. As noted, the US Department of Energy

Fig. 8 Metabolic engineering of dark fermentative andlaunched the “Energy Earthshots Initiative,” in which thephotofermentative hydrogen-producing microorganisms

first “Hydrogen Shot” was set as a challenging goal, i.e.,
reducing the cost of CO2-free hydrogen by 80% in one
decade.

3.2. Amino acids (alanine and valine)
Usually, amino acid fermentation is conducted un-

Furthermore, although bioprocesses have signifi-

der aerobic conditions, where high productivity requires

cant potential for CO2-free hydrogen production, inno-

the aeration and agitation of the system to be ade-vative improvements in technology should establish aquately controlled. However, this process is difficult tocost-effective process for producing biohydrogen.

achieve in large-scale fermenters because their internal

Therefore, along with Sharp Corporation, our group has

oxygen concentration is not homogeneous. To over-developed a biohydrogen production process with ancome this problem, we have developed a new and ge-

overwhelming production rate (max 300 L H2/h/L) using

netically modified Corynebacterium strain using thethe hydrogen production pathway via formate during“RITE Bioprocess®” to produce amino acids under an-

dark fermentation. On the basis of this achievement, ouraerobic conditions. Furthermore, under anaerobic con-group is now working on improving hydrogen yieldditions, the technological hurdle for amino acid produc-from biomass by integration with photofermentation.

tion is to balance the redox reaction without oxygen as

The formate-dependent hydrogen production path-an electron acceptor. To this end, we successfully intro-

way uses only a part of the reducing power generated

duced an artificial pathway for amino acid biosynthesis

by degrading sugars, such as glucose, derived from bi-

into microbial cells, solving the technological hurdle.

omass. Subsequently, a high-yield hydrogen produc-

Our group published this accomplishment in an inter-tion pathway using both NADH and ferredoxin (Fd) as

national journal in 2010 (Appl. Microbiol. Biotechnol. 87:

the reducing power was engineered and demonstrated

159–165).

to work. Our group also elucidated a unique regulatory

GEI was established in 2011 for industrializing the36

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2022

“RITE Bioprocess®.” In 2011, RITE and GEI began col-tion processes. Bacterial cells synthesize various aro-

laborative research on amino acid production using thematic compounds, including amino acids (phenylala-“RITE Bioprocess®” and developed technologies fornine, tyrosine, and tryptophan), folate (vitamin B9), andscaling up production, growing efficient productioncoenzyme Q, all of which are derived from the shikimate

strains, and reducing production costs. Subsequently, in

pathway (Fig. 9). Therefore, by employing the metabol-

2019, RITE succeeded in producing a strain that yielded

ically engineered C. glutamicum, we successfully estab-

the world’s highest production concentration of L-valine

lished a highly efficient bioprocess for producing thewith the best production efficiency. Furthermore, RITE

following aromatic compounds from non-food feed-

has completed commercialization projects with GEI andstocks: shikimate, an essential building block of theoverseas partners to achieve the commercial produc-anti-influenza drug, Tamiflu; 4-aminobenzoate, thetion of these amino acids. We aimed to produce thisbuilding block of a potentially useful functional poly-amino acid from renewable resources to reduce the life

mer; and aromatic hydroxy acids, having potential ap-

cycle carbon footprint.

plications in polymer, pharmaceutical, cosmetic, adhe-

In 2016, we succeeded in demonstrating the feasi-

sive material, and flavor (vanillin) industries. Currently,

bility of L-alanine’s production technique using com-

we are seeking to develop new strains to produce usefulmercial-scale facilities of our partner company, whicharomatic compounds that the wild-type C. glutamicum

was an important milestone for its industrialization. One

is unable to produce. These strains will be achieved byof our group members also participated in the first op-

introducing genes derived from versatile biological re-eration and worked with local employees to lead thesources into the bacterium. The techniques developed

project to a successful conclusion. After an evaluationin the Smart Cell Project, as described earlier, will also

by the Food Safety Committee in August 2017, thehelp to accelerate the development of strains and im-safety of the L-alanine produced by our strain as a foodprove their productivity.

additive was confirmed, allowing it to be made commercially available for this purpose besides its use forindustrial applications. We are now working on a joint
research project to produce other amino acids.

3.3. Green-aromatic compounds
Aromatic compounds are essential industrial chemicals used for synthesizing polymers and various valueadded chemicals for use in pharmaceutical, nutraceutical, flavor, cosmetic, and food industries. Although they
are currently derived from petroleum or natural plantresources, their environmentally friendly biotechnological production from renewable feedstocks is desirable

Fig. 9 The biosynthetic pathway for various

to create a sustainable society that is no longer depend-aromatic compounds

ent on petroleum resources and has efficient produc-37

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2022

4. Core technologies

Creation Technology” to solve problems associated with

4.1. The NEDO Carbon Recycle Project

the practical application of fermentative production

On a national strategy, realizing the world’s mosttechnologies using production strains developed in theadvanced bioeconomy society in 2030, the develop-

Smart Cell Project and accumulated omics data (metab-ment of material production technologies as the basis

olome, transcriptome, proteome). We will also validateof the bio-manufacturing industry is an urgent issue. By

the technologies (Fig. 10).

utilizing biological functions, it is possible to produce
materials from biomass without depending on fossil resources as raw materials. These technologies will contribute to the realization of a carbon recycling society.
In response to this social situation, NEDO launched
a new project in 2020, “Development of Bio-based Production Technology to Accelerate Carbon Recycling.”
Participating institutions will develop and validate nextgeneration production technologies based on the fer-

Fig. 10 Construction of an advanced bioproduction

mentation technologies cultivated in Japan so far orsystem platforms

that did not involve conventional methods.
Our Group participated in the predecessor of this4.2. The Cross-ministerial strategic innovation promo-

project, “Development of Production Techniques fortion program (SIP)

Highly Functional Biomaterials Using Plants and Other(Development of biomonomers for high-performance

Organisms” (NEDO Smart Cell Project). Along with pro-plastics)

ject participants (universities, research institutes, and

RITE is participating in the theme “Technologies forcompanies), we developed technologies to design a

Smart Bio-industry and Agriculture” in the SIP, which

Smart Cell (defined as a finely designed and expression-seeks to promote R&D, from the basic research stage

controlled cell). We also validated the technologies by

to the final outcome, in a seamless manner by endeav-

breeding production strains in a short period of time.

oring to strengthen cooperation among industries, ac-

In the Carbon Recycling Project, in addition to fur-ademia, and governments beyond the framework ofther improving the technologies developed during thegovernment ministries and traditional disciplines. The

Smart Cell Project, production process technologies, in-theme aims to realize a sustainable growing society thatcluding scale-up and refining, will be developed. Fur-uses manufacturing technologies developed throughthermore, advanced bioproduction system platforms

biotechnology and digital resources.

and peripheral technologies will be developed by cre-In the program, RITE participates in the consortium

ating information analysis technologies that can control“Development of Technologies for Functional Design

production processes. These results will accelerate theand Production of Innovative Biomaterials,” which aimssocial implementation of bio-based materials.

for synthesizing polymers with new functions desired by

Our group participated in this project from the firstmarkets using monomers biosynthesized from cheap

year. Our target is catechol, a highly toxic aromatic

raw materials, such as biomass. Thus far, polymers withcompound. The aim is to develop “Industrial Smart Cell

38

R&D Activities ● Molecular Microbiology and Biotechnology Groupultra-high heat resistance and those for battery materi-

RITE Today 2022

4.3. The NEDO moonshot-type R&D project

als have been developed (Fig. 11). To design the biosyn-

Plastics are lightweight, inexpensive, and durable

thetic route for monomers comprising these polymers,

polymer materials indispensable for daily life. However,

the group led by RITE has been developing and validat-

because they are chemically stable, they are not easily

ing technologies for functional modification of enzymes

decomposed in the natural environment.

using enzymes involved in monomer precursor biosyn-

It has been reported that there is a typical trade-off

thesis as targets. The technology enables the efficient

between biodegradability and durability/toughness of

extraction of combined functional mutations by provid-

polymers, such as plastics. Hence, biodegradable poly-

ing a machine learning algorithm with data of manymers are not durable because they degrade quickly inmutant enzymes to train it. The technology for seeking

the natural environment and their mechanical proper-

novel enzymes with desired functions using multiple

ties are insufficient. Thus, only limited applications areamino acid sequence data from enzyme databases ispossible. Although conventional plastics have excellent

also being developed. These technologies allowed us todurability and toughness, their biodegradability is poor.

modify the substrate specificity of target enzymes andSuppose that it is possible to create new plastics

improve their activities. In the fiscal year of 2021,

with durability and toughness with biodegradability,

through collaboration between the consortia, RITE has

these plastics can be used in several applications as en-

been working on the task to improve the productivity

vironmentally friendly plastic. They can also be recycled.

of the target monomer biosynthesized by a bacterial

Notably, plastic litters scattered in the marine environ-strain by optimizing its culture conditions. Thus far, thement are challenging to recover and have a negative

productivity of the monomer has successfully been im-

effect on the ecosystem.

proved twofold by seeking optimal culture conditions.

Therefore, developing plastics that can control the

Subsequently, RITE will evaluate the monomer’stiming and speed of biodegradation (multi-lock biopol-

productivity using biomass-derived raw materials pro-ymers) is critical for resource recycling.

vided by a collaborative consortium.

In the NEDO moonshot-type R&D project “Development of Multi-Lock Biopolymers Degradable in Ocean
from Non-Food Biomasses”, we will introduce a “multilock mechanism” for plastic degradation to break the
trade-off relationship between plastics. Hence, throughmultiple and simultaneous stimuli, such as light, heat,
oxygen, water, enzymes, microorganisms, and catalysts
during decomposition, the multi-lock mechanism will
suppress the decomposition to maintain durability andtoughness and prevent degradation. Products to becommercialized in this project are plastics, tires, textiles,

Fig. 11 Synthesis of developing monomers and polymers

fishing nets, and fishing gears made from non-food bi-

and polymer applications

omass. Material design guidelines for multi-lock biodegradable plastics will also be established through col-39

R&D Activities ● Molecular Microbiology and Biotechnology GroupRITE Today 2022

laboration between industry, academia, and the gov-

industrialization of our biomass-derived phenol-pro-ernment. In this context, our group is promoting R&D

ducing technology, named the “Two-Stage Bioprocess.”on the bioproduction of various monomers that can be

This was the first example in demutualization of a tech-used as raw materials for these products from non-foodnology research association. Then, in April 2018, GPD

biomass materials. This project includes the functional-changed its name to Green Chemicals Co., Ltd. (GCC).

ization of plastic-degrading enzymes that can be used

Since GCC’s phenol-producing technology andin the multi-lock mechanism (Fig. 12)

knowledge apply to the production of various other ar-(Here is the HP of the project: http://www.moon-

omatic compounds, establishing a bioprocess for eachshot.k.u-tokyo.ac.jp/en/index.html).

higher value-added chemical and commercializing
products that meet customer needs are in progress (seeUncollectable ocean
plastic litters

Fishing
tools litter

Fiber litter

Multipleenvironmental
stimuli to unlock

EnzymeOO

Bioprocess

RITE② High-functionalization of polymerdegrading enzyme

Environmental,
chemical, and enzymatical

HO

Multi-lockbiopolymer

The present three significant GCC products are

On-demand
decomposition

Plastic litter

Spread by accidentHigh
durability
in useSection 3.3).

Light
Ion
etc….

shown in Fig. 13. In 2021, several companies started
quality evaluation of 4-hydroxybenzoic acid (4-HBA).

H2O

OH

Monomer

RITE

Inedible
biomass① High production of biomonomers

CO2

Photosynthesis

Fig. 12 R&D of marine-degradable multi-lockbiopolymers made from non-food biomass
for resource recycling

5. Toward the industrialization of our technologies
5.1. Green Chemicals Co., Ltd.
(Head Office ・ Laboratory: in Kyoto headquarters,
RITE; Shizuoka Laboratory: in Shizuoka plant, Sumitomo
Bakelite Co., Ltd.)(Click here for GCC)
Currently, commercial phenol can only be derived

Fig. 13 Three major products of Green Chemicals Co., Ltd.

from petroleum. We have taken on the challenge of developing the world’s first bio-manufacturing process forbiomass-derived phenol. The aim is to aid global environmental conservation and greenhouse gas reduction.
In February 2010, Sumitomo Bakelite Co., Ltd. and5.2. Green Earth Institute Co., Ltd.
(Headquarters: Bunkyo-ku, Tokyo, Japan; ResearchInstitute: Kazusa, Kisarazu City, Chiba, Japan)(Click here for GEI homepage)

RITE established Green Phenol/High Performance Phe-GEI is a RITE-launched venture company establishednolic Resin Manufacturing Technology Research Associ-on September 1, 2011, to facilitate the quick commer-

ation (GP Union).

cialization of research results based on those above-

In May 2014, GP Union was reorganized into Green

mentioned innovative “RITE Bioprocess®.” GEI is cur-Phenol Development Co., Ltd. (GPD), to accelerate therently conducting joint research and activities aimed at40

R&D Activities ● Molecular Microbiology and Biotechnology Group

commercialization with RITE to realize the practical usesof green chemicals and biofuel production technologies

RITE Today 2022

term R&D project.
B)

At an early stage, we will take measures tomanufactured using microorganisms. The company waschange the major raw materials purchased

listed on TSE Mothers in December 2021.

from other companies from fossil resource-de-With amino acids, as mentioned earlier, GEI has suc-rived materials to bio-derived materials toward

ceeded in producing L-alanine and L-valine on a com-

“2050 carbon neutral.” This project is also being

mercial scale using the production strain developed by

implemented as a medium- to long-term R&D

RITE (see Section 3.2). In addition, the Ministry of Health,goal.

Labor and Welfare confirmed the safety of L-alanine asC)

Changing the substance, a product of the com-a food additive, paving the way for its use in the foodpany, or the substance and raw material fromindustry.

fossil resources to bio-derived ones is another

However, based on bio-jet fuels made from non-goal. With this project, substances that can be

food biomass, which are highly expected to reduce CO2

commercialized quickly at high unit prices are

emissions from aircraft, GEI is continuously conducting

being implemented as short-term R&D goals.

joint research with RITE in this area and is working toward commercialization. Due to this initiative, we suc-

6. Closing remarks

ceeded in producing “Japan’s first purely domestic bio-Due to the abovementioned technological innova-

jet fuel flight” on February 4, 2021, for the JL319 flighttion of “bio-digital technology,” the understanding of(from Haneda Airport to Fukuoka Airport) (see Chapter

life phenomena by the “data-driven” approach of dis-

3.1).

covering the law from huge life information has recently

Furthermore, GEI is developing green chemicals inprogressed. Under such a background, research on thecooperation with RITE for marketing, commercialization,

biology of synthesis that accumulates data and under-and scaling up the mass production.

stands biological functions by repeating the Design-

GEI will continue to influence developing biorefinery

Build-Test-Learn (DBTL) cycle is also rapidly developing.

businesses toward realizing a society that does not rely

All the national projects introduced in Chapter 4 also

on fossil resources.

use these technological innovations, new knowledge,
and methods to achieve dramatic improvements in de-

5.3. Joint research with companies

velopment efficiency. In these projects, the biorefinery

In response to requests from companies, we aretechnology using the abovementioned smart cell also

conducting joint research on the production of many

plays a significant role as a core technology and is ex-

substances other than the biofuels and green chemicals

pected to have a tremendous ripple effect in the indus-

introduced in this overview. Although these contents

trial (manufacturing) and energy fields (Fig. 14).

cannot be presented here, they include the following:A)

At an early stage, we will take measures tochange the substance, the company’s main
product, from fossil resource-derived to bioderived, toward “2050 carbon neutral.” Here, it
is being implemented as a medium- to long-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 2022

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

In 2022, the group will continue to research and develop green chemicals, such as aromatic compounds,
and biofuel production using the “RITE Bioprocess®”and “industrial smart cell design system.” In addition, we
also want to focus on developing practical production
technologies and contribute to the “realization of carbon neutral by green bioprocesses.”
Since the declaration of the “2050 carbon neutral” in
October 2020, inquiries from companies have increased,
and the number of joint research projects with companies has also increased. However, the group is looking
for research partner companies, including cases otherthan those introduced in Chapter 5, Section 3. There isalso a possibility that compounds that are difficult to
produce microorganisms can be highly manufactured
using the latest elemental technologies’ developmental
results, such as the abovementioned aromatic compounds. Therefore, if there is a compound that you wantto make bio-derived, please contact us.
※ “RITE Bioprocess®” is a registered trademark of RITE.

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