Onaka Lab | Gakushuin University

Onaka Lab.

Onaka lab was established in April 2023 in the Department of Life Science, Faculty of Science, Gakushuin University. Its predecessors were the Laboratory of Microbial Metabolic Potential, Institute for Fermentation Osaka (2012-2017) and the Laboratory of Microbial Enzyme Potential, Amano Enzyme (2017-2023), which were endowed courses that existed in the Faculty of Agriculture at the University of Tokyo.

“Microorganism” is a generic term for tiny invisible organisms. These ubiquitous organisms are soil decomposers, and are involved in maintaining the global environment, such as the carbon and nitrogen cycles. In addition, they are normal inhabitants in the human body, pathogens for mammals and plants, and producers of various organic materials. However, we usually do not mind their presence in our daily lives.

The recent advances in microbial genome analysis have changed the perception of microorganisms. Microbial genomic information revealed that microorganisms have biological diversity far beyond that between mammals and plants, and we are expecting to discover novel microbial functions from these genomic data.

In Onaka lab, we are exploring microorganisms' hidden gifts and talents and applying them in industrial processes. In particular, we would focus on actinomycetes, soil bacteria known for their ability to produce antibiotics, and identifying novel potent biosynthetic machinery in their secondary metabolism.

The current research interests of our laboratory are (i) the development of technology to promote antibiotic production capacity of actinomycetes, (ii) biosynthetic studies of RiPPs for drug discovery based on medium-sized molecules, (iii) Natural organic arsenic compounds, (iv) studies regarding fermented foods and beverages: conventional fermented foods and their relationship to new fermentation methods.

Our Research

Development of technology to promote antibiotic production capacity of actinomycetes

Biosynthetic studies of RiPPs for drug discovery based on medium-sized molecules

Medium-sized molecules with molecular weights between 500 and 2,000 benefit drug discovery as they exhibit substrate specificities comparable with those of biopharmaceuticals, such as antibodies, and can be produced at a low cost, similar to small molecules. Among the medium-sized molecules, peptide-based natural products have received attention because they can be produced via fermentation. Fermentation is more cost effective than the production of synthetic peptides.

We focused on RiPPs, which is a group of peptide-based compounds in which the amino acids are modified by post-translationally modified enzymes after ribosome synthesis. RiPPs undergo post-translational modifications, therefore, they are more likely to form a stable three-dimensional structure and exhibit a higher affinity for the target as compared with non-modified peptides. We have investigated the biosynthesis of GS, lactazoles, and goadvionins, and have synthesized hundreds of RiPP analogues exchanged the amino acid sequences (Fig. 4-2). RiPPs are stable in living cells because they are less likely to be degraded by peptidases and other enzymes.

(1) Lactazole: thiopeptide produced via minimum biosynthetic gene set

Thiopeptides are produced primarily by actinomycetes and typically contain highly modified sulfur-containing peptides; the peptides feature a characteristic macrocycle knotted with pyridine or piperidine; pyridine and piperidine are a six-membered nitrogen-containing ring structures. Thiopeptides are a group of antibiotics that, similar to GS, are biosynthesized via the ribosomal translation system. More than 100 thiopeptides have been identified to date. We performed genome mining to identify a cryptic thiopeptide biosynthetic gene cluster that participates in the biosynthesis of lactazoles from Streptomyces lactacystinaeus OM-6519 (Fig. 3-1).

Fig. 3-1 : To date, the ring size of macrocyclic thiopeptides is limited to 26, 29, or 35 atoms, whereas lactazoles are structurally novel thiopeptides comprising a 32-membered macrocycle. The 2-oxazolyl-6-thiazolylpyridine core has three positions that connect to tryptophan via an amino linkage, and provides a unique structure in thiopeptides.

Lactazoles originate from the simplest cluster, which contains only six unidirectional genes (lazA to lazF). This cluster is the smallest cluster among the known thiopeptide biosynthetic gene clusters (Fig. 3-2). The structure gene, lazA, contains the precursor peptide sequence and is classified into a phylogenetically distinct clade. lazC participates in the macrocyclization process and aids the formation of a central pyridine moiety via gene disruption. This compact biosynthetic machinery is expected to exhibit high potency, which will result in diverse thiopeptide core structures. Our approach facilitates the production of a more diverse set of thiopeptide structures, thereby increasing the range of semi synthetic RiPPs for use in drug development.

Fig. 3-2 : Thiopeptide biosynthesis gene clusters reported previously. Lactazole (topmost) comprises the minimum gene set. All the genes are in the same direction and have the same transcriptional unit, rendering gene modification easier and more suitable for the production of useful chemicals compared with other thiopeptide clusters.

Key reference paper pertaining to lactazole biosynthesis

This paper by S. Hayashi, T. Ozaki, S. Asamizu, H. Ikeda, S. Ōmura, N. Oku, Y. Igarashi, H. Tomoda, & H. Onaka (Genome mining reveals a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides lactazoles. Chem Biol 21(5):679-88 (2014). https://doi.org/10.1016/j.chembiol.2014.03.008) mentions the identification of lactazoles via genome mining.

(2) Development of technology to extend structural diversity of peptide antibiotics

We are the first group in the world to successfully reconstitute an entire RiPP biosynthetic pathway using an in vitro one-pot reaction. The system comprises a cell-free translation system and an in vitro post-translational modification system, in which the precursor peptide gene is first introduced into the reaction tube, and GS, lactazole, and their analogues are synthesized overnight (Fig. 4-1). This epoch-generating system can rapidly and easily synthesize various RiPP analogues containing different amino acid sequences in one day.

Fig. 4-1 : Overview of one-pot system for synthesizing goadsporin with only DNA input. Chemical structure of goadsporin (GS) (bottom); GS comprises six oxazole and thiazole rings (inside region shown in blue) and two dehydroalanine rings (inside region shown in green), that are produced by the dehydration and condensation of serine, threonine, and cysteine. Structural gene *godA* is a gene that encodes the precursor peptide of GS. By feeding structural gene *godA* into the reaction tube, cell-free translation and post-translational modification reactions are sequentially performed to synthesize GS. Using this system, various analogues can be synthesized rapidly and easily in 24 h using mutated *godA* as templates.

As for the synthesis of lactazole analogues, since the substrate recognition of the macrocyclophilic enzyme LazC was found to be permissive, we successfully created analogues comprising various ring sizes, and analogues comprising amino acid sequences within the ring; the analogues differed completely from the original. Additionally, we successfully introduced non-proteinaceous amino acids by using the cell-free translation system (Fig. 4-2).

Fig. 4-2 : Syntheses of various thiopeptide derivatives using lactazole as an original structure. This study extended the chemical diversity of thiopeptides beyond the chemical structural diversity existing in natural environments. (1) In artificial amino acid insertion, we successfully introduced amino acids other than the 20 amino acids that constitute proteins. (2) A variety of macrocycle sizes can be created, from a minimum of 5 residues to a maximum of 21 residues. (3) In the tail extension, the amino acid residues in the tail are extended, and the amino acid sequence differs completely from that of the original lactazole; the structure is absolutely different from the original lactazole structure.

Key reference papers pertaining to syntheses of various RiPPs analogues

In this paper, (Minimal lactazole scaffold for in vitro thiopeptide bioengineering. Nature Comm 11, 2272 (2020). https://doi.org/10.1038/s41467-020-16145-4), the authors, A. A. Vinogradov, M. Shimomura, Y. Goto, T. Ozaki, S. Asamizu, Y. Sugai, H. Suga, & H. Onaka report the in vitro one-pot reaction of lactazole biosynthesis and the synthesis of diverse lactazole analogues.
The paper written by T. Ozaki, K. Yamashita, Y. Goto, M. Shimomura, S. Hayashi, S. Asamizu, Y. Sugai, H. Ikeda, H. Suga, & H. Onaka, (Dissection of goadsporin biosynthesis by in vitro reconstitution leads to designer analogues expressed in vivo. Nature Comm e14207 (2017). https://doi.org/10.1038/ncomms14207) reports the in vitro one-pot reaction of GS biosynthesis and the syntheses of 52 GS analogues.

(3) Novel lipopeptide goadvionins

Goadvionins are hybrid polyketide (PK)/RiPP hybrid compounds synthesized via a biosynthetic pathway, which differs from the typical lipopeptide biosynthetic pathway (Fig. 5-1). Whereas most lipopeptides are composed of non-ribosomal ribosomal synthase (NRPS), goadvionins are biosynthesized via the condensation of RiPPs and PK by a novel acyltransferase, GdvG (Fig. 5-2).

Fig. 5-1 : Chemical structure of goadvionin.

Fig. 5-2 : Schematic illustration of goadvionin biosynthesis, in which PK and RiPP moieties are biosynthesized separately and condensed by the novel acyltransferase GdvG to form goadvionin.

Fig. 5-3 : Although only three PK/RiPPs hybrid natural products have been discovered to date, including goadvionin, many biosynthetic gene clusters exist in microbial genomes that are used to synthesize PK/RiPP hybrid natural products. Among them, many clusters are expected to exhibit chemical structures that differ significantly from those of known PK/RiPP hybrid natural products, and further relevant studies should be performed.

Key reference paper pertaining to goadvionin

The paper (Acyltransferase that catalyses the condensation of polyketide and peptide moieties of goadvionin hybrid lipopeptides. Nature Chem 12, 869-877 (2020). doi: 10.1038/s41557-020-0508-2 ) by R. Kozakai, T. Ono, S. Hoshino, H. Takahashi, Y. Katsuyama, Y. Sugai, T. Ozaki, K. Teramoto, K.Teramoto, K. Tanaka, I. Abe, S. Asamizu, & H. Onaka, reports the isolation, structural analysis, biosynthesis, and bioactivity of goadvionins.

Natural organic arsenic compounds.

Studies regarding fermented foods and beverages: conventional fermented foods and their relationship to new fermentation methods

(1) Development of Alcoholic Beverages Using Wild Yeasts

Professor Onaka isolated wild sake brewing yeast from barley malt in Toyama and assisted in the development of Toyama-brewed craft beer as part of the university’s regional collaboration activities. “Yeast born in Toyama” (Fig. 6-1) exhibits a high alcohol fermentation ability and has been used for brewing Toyama-brewed craft beer, and for brewing Japanese sake, wine, and whiskey.

In addition, ccPTM18, a wild yeast isolated from wine fermenting in barrels at COCOFARM & WINERY (Ashikaga, Japan), is characterized by the acidity it yields. When it is used in Japanese sake brewing, it produces sake that is similar to white wine and has a pleasant floral note and a sour taste.

Each year, sake is produced using these wild yeasts and is enjoyed by many people (Fig. 6-2).

Fig. 6-1 : Barley malt used for isolation of “Yeast born in Toyama” (top) and SEM image of isolated yeast “Yeast born in Toyama” (bottom).

Fig. 6-2 : “Onaka” sake produced in 2021, which was released through crowdfunding (https://www.makuake.com/project/onaka07/). Wild wine yeast ccPTM18 was used to produce the Onaka sake. Oak barrel-aged sake (right) and sparkling sake (left).

(2) Fermented Foods Worldwide

Various fermentation cultures exist worldwide. Traditional fermentation is used primarily to preserve fresh foods for long durations; however, recently, the benefits of consuming of fermented foods, have been prioritized. By independently creating various fermented foods from countries worldwide, ourselves, we can obtain a deeper understanding of fermented foods, which will promote the development of new fermented foods.

We welcome joint research and consultation with companies, stores, etc. Contact email

Click to read an article that describes each fermented food. It is available in Japanese only, however, many pictures are included and will aid your understanding.

Address

Onaka Lab.

Faculty of Science, Gakushuin University, 1-5-1, Mejiro, Toshima, Tokyo, 171-8588 Japan.
Contact email

Current members

Hiroyasu Onaka

Specially-appointed Professor (Ph.D.)
Contact email

Education

The University of Tokyo, Tokyo, Japan A.B. 1993
The University of Tokyo, Tokyo, Japan Ph.D. 1998
Postdoctoral Fellow at Cold Spring Harbor lab., NY, U.S.A. & Penn State University, PA, U.S.A. 1998-1999

Professional Experience

1997-1999 JSPS (Japan Society for the Promotion of Science) Research Fellowship for Young Scientist
1999-2006 Assistant professor at the Department of Biotechnology, Toyama Prefectural University, Toyama, Japan.
2006-2010 Lecturer at the Department of Biotechnology, Toyama Prefectural University, Toyama, Japan.
2010-2012, Associate Professor at the Department of Biotechnology, Toyama Prefectural University, Toyama, Japan.
2012-2023 Specially-appointed professor of Graduate school of Agricultural and Life Sciences, The University of Tokyo
2023- Professor of Department of Life Science, Faculty of Science, Gakushuin University

Society Memberships and Awards

Hamada Award, the Society for Actinomycetes Japan, 2005
The Japan Bioscience, Biotechnology and Agrochemistry Society Award for the Encouragement of Young Scientists, Japan society for Bioscience, Biotechnology and Agrochemistry, 2008
Toyama Award, Toyama Prefecture Citizens' Personal Development Foundation, 2010
Specially appointed Fellow of Toyama Prefectural University, 2012-
The Sumiki-Umezawa Memorial Award, The Japan Antibiotics Research Association, 2016
Omura Award, the Society for Actinomycetes Japan, 2022

Shotaro Hoshino

Specially-appointed Assistant Professor (Ph.D.)
Contact email

Education

2010.4-2014.3 The University of Tokyo (Bachelor of Pharmaceutical Science)
2014.4-2016.3 The University of Tokyo (Master of Pharmaceutical Science)
2016.4-2019.3 The University of Tokyo (Doctor of Pharmaceutical Science)

Professional Experience

2016.4-2019.3 JSPS Research Fellow (DC1)
2019.6-2021.11 JSPS Overseas Research Fellowships
2019.6-2022.2 Princeton University, Department of Chemistry, Postdoctoral Researcher (Prof. Mohammad R. Seyedsayamdost Lab.)
2022.4-2023.3 Specially-appointed Assistant Professor of Graduate school of Agricultural and Life Sciences, The University of Tokyo
2023.4- Assistant Professor of Department of Life Science, Faculty of Science, Gakushuin University

Ph.D. Thesis

Searching for rare-actinomycetes-derived novel drug seeds by combined-culture method (The University of Tokyo, 2019.3)

Interests, Keywords

Natural product chemistry, Isolation & structural elucidation, microbial interaction, Natural product biosynthesis, Regulation of secondary metabolism, Enzymology, Actinobacteria

Shinta Ijichi

Ph.D. student (D2)

Ayari Kuboki

Master's student (M1)

Saho Fukuhara

Master's student (M1)

Chihiro Hoshino

Master's student (M1)

Suzuna Inaba

Undergraduate student (B1)

Makoto Kato

Undergraduate student (B1)

Hanaha Kawaguchi

Undergraduate student (B1)

Gaku Suzuki

Undergraduate student (B1)

Nozomi Takaku

Undergraduate student (B1)

Miki Yoshimoto

Undergraduate student (B1)

Publications

2024

Y. Lei, H. Onaka, S. Asamizu. Transcriptionally induced nucleoid-associated protein-like ccr1 in combined-culture serves as a global effector of Streptomyces secondary metabolism. Frontiers in Microbiology. 15 (2024) https://doi.org/10.3389/fmicb.2024.14229772024
K. M. Do, S. Hoshino, T. Kodama, H. M. Nguyen, N. Ikumi, H. Onaka, H. Morita. Five pimarane diterpenoids from Kaempferia champasakensis and their cytotoxic activities. Journal of Natural Medicine, online ahead of print. doi 10.1007/s11418-024-01829-8.
N. N. Hoang^#, S. Hoshino^#, T. Kodama, T. X. Hoang, H. Onaka, H. Morita. Two new neo-clerodane diterpenoids from Tinospora cordifolia stems. Phytochemistry Letters, 63: 54-59 (2024). ^#Equal contributions doi: 10.1016/j.phytol.2024.08.004.
N. N. Hoang, S. Hoshino, T. Kodama, Y. Nakashima, K. M. Do, H. X. Thao, N. Ikumi, H. Onaka, H. Morita. Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities. Chemical and Pharmaceutical Bulletin, 72(6): 540-546. doi 10.1248/cpb.c24-00240.
C. Pan, H. Ikeda, M. Minote, T. Tokuda, T. Kuranaga, T. Taniguchi, N. Shinzato, H. Onaka, H. Kakeya. Amoxetamide A, a new anoikis inducer, produced by combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis. Journal of Antibiotics. 77(1):66-70 (2024). doi: 10.1038/s41429-023-00668-1
K. M. Do, S. Hoshino, T. Kodama, H. M. Nguyen, S. Van Le, N. Ikumi, H. Onaka, H. Morita. A new 3,4-seco-isopimarane and three new isopimarane diterpenoids from Kaempferia champasakensis collected from Vietnam and their cytotoxic activities. Journal of Natural Medicine, 78: 537-546 (2024). doi 10.1007/s11418-024-01789-z.
S. Hoshino, H. Onaka, I. Abe. Recent advances in the biosynthetic studies of bacterial organoarsenic natural products, Natural Product Reports, in press DOI: 10.1039/d4np00036f.

2023

2022

A. A. Vinogradov, Y. Zhang, K. Hamada, J.S. Chang, C. Okada, H. Nishimura, N. Terasaka, Y. Goto, K. Ogata, T. Sengoku, H. Onaka, H. Suga. De novo discovery of thiopeptide pseudo-natural products acting as potent and selective TNIK kinase inhibitors. Journal of the American Chemical Society, 144(44):20332-20341 (2022) doi: 10.1021/jacs.2c07937
S. Asamizu, S. Ijichi, S. Hoshino, H. Jo, H. Takahashi, Y. Itoh, S. Matsumoto, H. Onaka. Stable isotope-guided metabolomics reveals polar-functionalized fatty-acylated RiPPs from Streptomyces. ACS Chemical Biology 17(10): 2936-2944 (2022) doi: 10.1021/acschembio.2c00601
S. Asamizu, A.A.C. Pramana, S. Kawai, Y Arakawa, H. Onaka. Comparative metabolomics reveals a bifunctional antibacterial conjugate from combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. ACS Chemical Biology 17(9): 2664-2672 (2022) doi: 10.1021/acschembio.2c00585
M. Yanagisawa, S. Asamizu, K. Satoh, Y. Oono, H. Onaka. Effects of carbon ion beam-induced mutagenesis for the screening of RED production-deficient mutants of Streptomyces coelicolor JCM4020. PLoS One 17(7): e0270379 (2022) https://doi.org/10.1371/journal.pone.0270379
A.A. Vinogradov, Chang, H. Onaka, Y. Goto, H. Suga. Accurate models of substrate preferences of post-translational modification enzymes from a combination of mRNA display and deep learning. ACS Central Science 8(6): 814-824 (2022) https://doi.org/10.1021/acscentsci.2c00223
M. Kato, S. Asamizu, H. Onaka. Intimate relationships among soil bacteria: actinomycetes and mycolic acid-containing bacteria. Scientific Reports 12: 7222 (2022) https://doi.org/10.1038/s41598-022-11406-2
Y. Asai, T. Hiratsuka, M. Ueda, Y. Kawamura, S. Asamizu, H. Onaka, M. Arioka, S. Nishimura, M. Yoshida. Differential biosynthesis and roles of two ferrichrome-type siderophores, ASP2397/AS2488053 and ferricrocin, in Acremonium persicinum. ACS Chemical Biology, 17(1), 207-216 (2022) https://doi.org/10.1021/acschembio.1c00867
A. Hikima, S. Asamizu, H. Onaka, H. Zhang, H. Tomoda, N. Koyama. Kimidinomycin, a new antibiotic against Mycobacterium avium complex, produced by Streptomyces sp. KKTA-0263. The Journal of Antibiotics, 75: 72-76 (2022) https://doi.org/10.1038/s41429-021-00494-3
E. J. Han, S. R. Lee, S. Hoshino, M. R. Seyedsayamdost. Targeted discovery of cryptic metabolites with antiproliferative activity. ACS Chemical Biology, 17: 3121-3130 (2022) https://doi.org/10.1021/acschembio.2c00588
S. M. Horwitz, T. C. Blue, J. A. Ambarian, S. Hoshino, M. R. Seyedsayamdost, K. M. Davis. Structural insights into inhibition of the drug target dihydroorotate dehydrogenase by bacterial hydroxyalkylquinolines. RSC Chemical Biology 3: 420-425 (2022) https://doi.org/10.1039/d1cb00255d
T. Mori, Y. Nakashima, H. Chen, S. Hoshino, T. Mitsuhashi, I. Abe. Structure-based redesign of Fe(II)/2-oxoglutarate-dependent oxygenase AndA to catalyze spiro-ring formation. Chemical Communications 58(36): 5510-5513 (2022) https://doi.org/10.1039/d2cc00736c
N. Oku, S. Takemura, H. Onaka*, Y. Igarashi*. NMR characterization of streptogramin B and L-156,587, a non-synergistic pair of the streptogramin family antibiotic complexes produced inductively by a combined culture of Streptomyces albogriseolus and Tsukamurella pulmonis. MRC 60(2): 261-270 (2022)

Onaka Lab.

Our Research

Development of technology to promote antibiotic production capacity of actinomycetes

(1) Combined-culture, a new co-culture method for screening of natural products

Key reference papers pertaining to combined culture

(2) Goadsporin (GS): a natural product that induces secondary metabolism in various actinomycetes

Key reference papers pertaining to GS

Biosynthetic studies of RiPPs for drug discovery based on medium-sized molecules

(1) Lactazole: thiopeptide produced via minimum biosynthetic gene set

Key reference paper pertaining to lactazole biosynthesis

(2) Development of technology to extend structural diversity of peptide antibiotics

Key reference papers pertaining to syntheses of various RiPPs analogues

(3) Novel lipopeptide goadvionins

Key reference paper pertaining to goadvionin

Natural organic arsenic compounds.

Studies regarding fermented foods and beverages: conventional fermented foods and their relationship to new fermentation methods

(1) Development of Alcoholic Beverages Using Wild Yeasts

(2) Fermented Foods Worldwide

Address

Onaka Lab.

Current members

Hiroyasu Onaka

Education

Professional Experience

Society Memberships and Awards

Shotaro Hoshino

Education

Professional Experience

Ph.D. Thesis

Interests, Keywords

Publications

2024

2023

2022

2021

2020

2019

2018

2017

2016

2015

2014

2013

2012