In the Laboratory of Microbial Metabolic Potential, we are exploring the hidden gifts and talents of the microorganisms and applying them in industrial processes.

About the Laboratory of Microbial Metabolic Potential (Onaka Lab.)

The Laboratory of Microbial Metabolic Potential is an IFO-endowed course at the Graduate School of Agricultural and Life Sciences, the University of Tokyo, since 2012. The IFO, the Institution for Fermentation, Osaka, is a public interest incorporated foundation in Japan, and it provides grants for microbial researches that contribute to the further development of microbiology.

“Microorganism” is a generic term for tiny invisible organisms. These ubiquitous organisms are soil decomposers, and are involved in maintaining 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 a wide variety of organic materials. However, usually, we do not mind their presence in our daily life.

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 the Laboratory of Microbial Metabolic Potential, we are exploring the hidden gifts and talents of the microorganisms and applying them in industrial processes. In particular, we would be focusing on actinomycetes, soil bacteria known for their ability to produce antibiotics, and on identifying novel potent biosynthetic machinery in their secondary metabolism.

The current research interests of our laboratory are (i) development of drug screening from natural compounds with combined bacterial culture, (ii) biosynthesis of Goadsporin, which is a liner peptide including thiazole and oxazole rings, (iii) indolocarbazole biosynthesis.

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Our Research

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

The development of new drugs relies heavily on the discovery of novel natural products produced by microorganisms. The recent genomic analysis of some Streptomyces strains has revealed the presence of biosynthetic gene clusters for about 30 secondary metabolites; these data imply that a single Streptomyces strain can produce over 30 secondary metabolites. However, some of these secondary-metabolite genes are cryptic in fermentation culture. Because many biosynthetic genes for secondary metabolites remain silent under normal laboratory culture conditions, the valuable secondary metabolites encoded by them are not studied.

We found that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the physical contact between the mycolic-acid-containing bacteria and Streptomyces. The production of red pigment by Streptomyces lividans TK23 was induced by co-culture with Tsukamurella pulmonis TP-B0596, however there is no pigment when these two bacterium were partitioned. (Fig. 1-1)

Observation of combined-culture by scanning electron microscopy (SEM) indicated that adhesion of live MACB to S. lividans mycelia were a significant interaction that resulted in formation of co-aggregation. (Fig. 1-2)

We used these results to develop a new co-culture method, called 'combined culture' method, which facilitates the screening of natural products. To date, we have already discovered 5 types, 17 novel compounds with combined-culture. (Fig. 1-3) (Fig. 1-4)

Fig. 1-1 : The stimulation of mycolic acid containing bacteria were directly transmitted by physical contact. When Streptomyces and mycolic acid-containing bacteria were grown in a dialysis flask, which contains two compartments to grow both bacteria separately through a dialysis membrane, S. lividans did not produce red pigments. (ref. 1-1)
Red pigment production was only detected in the border of both bacteria, suggesting that physical contact triggers to produce the red pigments. (ref. 1-1)

Fig. 1-2 : Co-aggregation of S. lividans and Rhodococcus erythropolis (left) and Rhodococcus opacus (right) observed using SEM.

Fig. 1-3 : Novel compounds isolated from the combined-culture method. (ref. 1-3, 1-4, 1-5)

Fig. 1-4 : Combined-culture affects the production increment for heterologous expressions in Streptomyces lividans (ref. 1-2)

Reference

ref. 1-1 : Onaka H, Mori Y, Igarashi Y, & Furumai T. Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species. Appl Environ Microbiol 77(2): 400-406 (2011)

ref. 1-2 : Onaka H, Ozaki T, Mori Y, Izawa M, Hayashi S, & Asamizu S. Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans. J Antibiot (Tokyo) 2015 Apr 1. doi: 10.1038/ja.2015.31.

ref. 1-3 : Sugiyama R, Nishimura S, Ozaki T, Asamizu S, Onaka H, & Kakeya H. 5-Alkyl-1,2,3,4-tetrahydroquinolines, New Membrane-Interacting Lipophilic Metabolites Produced by Combined Culture of Streptomyces nigrescens and Tsukamurella pulmonis. Org Lett 2015 Apr 17;17(8):1918-21. doi: 10.1021/acs.orglett.5b00607.

ref. 1-4 : Hoshino S, Wakimoto T, Onaka H, & Abe I. Chojalactones A-C, Cytotoxic Butanolides Isolated from Streptomyces sp. Cultivated with Mycolic Acid Containing Bacterium. Org Lett 2015 Mar 20;17(6):1501-4. doi: 10.1021/acs.orglett.5b00385.

ref. 1-5 : Hoshino S, Zhang L, Awakawa T, Wakimoto T, Onaka H, & Abe I. Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823. J Antibiot (Tokyo) 2014 Oct 22. doi: 10.1038/ja.2014.147.

(2) Biosynthesis and genetic engineering of goadsporin, a liner azole-containing peptide produced by Streptomyces sp. TP-A0584

With the progress of genome-mining techniques for secondary metabolite screening, many kinds of a liner azole-containing peptide (LAPs) synthesized by ribosome were discovered from a wide variety of microorganisms. The common feature of LAPs is the peptide backbone containing thiazole, metylthiazole, and oxazole rings, which are derived from serine, threonine, and cycteine, respectively, and these heterocyles contribute for the bioactivity and the structure stability.

LAPs biosynthetic gene clusters consist of the structure gene, the post-translational-modificaion enzyme genes, transcriptional regulator genes, and the immunity genes. Interestingly, although the all of them contains enzymatic genes for thiazole or oxazole formation, these genes have very low similarities among these producing strains.

Goadsporin (GS) is a linear polypeptide antibiotic produced by Streptomyces sp. TP-A0584 (Fig. 2-1). Goadsporin consists of 19 amino acids, which included 8 unusual amino acids, oxazole, thiazole and dehydroalanine. GS promotes the secondary metabolite and morphogenesis at low concentrations, and induces growth inhibition at high concentrations in actinomycetes (Fig. 2-2).

The GS biosynthetic gene cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity, and transcriptional regulation spanning 20 kb. GS biosynthesis is initiated by the translation of 49 amino acid godA polypeptide. The subsequent cyclization, dehydration to form oxazole and thiazole rings are probably catalyzed by godD, godE, godF, and godG. Finally, it would be digested the N-terminal 30 amino acids leader sequence and acetylated by godH acetyltransferase homolog to afford GS.

Over 50 GS analogs were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is applied for heterocyclization of peptide. This approach will open the door to biosynthesize the new biological active peptides.

Fig. 2-1 : The chemical structure of goadsporin (ref. 2-1)

Fig. 2-2 : Bioactivity of GS on other Streptomyces species (ref. 2-1)

Fig. 2-3 : Proposed goadsporin biosynthetic pathway (ref. 2-2)

Reference

ref. 2-1 : Onaka H, Tabata H, Igarashi Y, Sato Y, & Furumai T, Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and Characterization, The Journal of Antibiotics 54, 1036-1044, (2001)

ref. 2-2 : Onaka H, Nakaho M, Hayashi K, Igarashi Y, & Furumai T. Cloning and characterization of goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584. Microbiology 151: 3923-3933 (2005)

ref. 2-3 : Haginaka K, Asamizu S, Ozaki T, Igarashi Y, Furumai T, & Onaka H. Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis. Biosci Biotechnol Biochem 78(3): 394-399 (2014)

ref. 2-4 : Ozaki T, Kurokawa Y, Hayashi S, Oku N, Asamizu S, Igarashi Y, Onaka H. Insights into the biosynthesis of dehydroalanines in goadsporin. ChemBioChem 2015 Dec 2. doi: 10.1002/cbic.201500541.

(3) Genome mining reveals a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides, lactazoles.

Thiopeptides are produced mainly by actinomycetes and typically contain highly modified sulfur-containing peptides, which have a characteristic macrocycle knotted with pyridine or piperidine, a six-membered nitrogen-containing ring. Although more than 100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We used genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles from Streptomyces lactacystinaeus OM-6519. To date, the ring size of macrocyclic thiopeptide is limited to 26, 29, or 35 atoms, while lactazoles are structurally novel thiopeptides with a 32-membered macrocycle (Fig. 3-1). The 2-oxazolyl-6-thiazolylpyridine core with the 3-position connected to tryptophan through an amino linkage also provides a unique structure in thiopeptides. Lactazoles did not show any antimicrobial activities, however, we found that inhibitory activities for the bone morphogenetic protein (BMP) signal cascade in vivo, which could add to a new aspect of therapeutic treatment against to thiopeptide family antibiotics.

Lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). It is the smallest cluster among the known thiopeptide biosynthetic gene clusters (Fig. 3-2). The structure gene, lazA contains the precursor peptide sequence, and it is classified into a phylogenetically distinct clade. lazC is involved in the macrocyclization process, leading to central pyridine moiety formation by gene disruption.

Substitution of the endogenous promoter with that of godA, a gene involved in goadsporin biosynthesis results in an approximately 30-fold increase in lactazole A production. A using the godA promoter to regulate the lactazole biosynthetic machinery, production of two analogs, S11C and W2S, was achieved.

We expect that this compact biosynthetic machinery has high potency to lend large diversity to the thiopeptide core structures. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

Fig. 3-1 : Chemical structures of thiopeptides and goadsporin

Fig. 3-2 : Gene organization of the laz cluster and comparison with other thiopeptide biosynthetic gene clusters

Reference

ref. 3-1 : Hayashi S, Ozaki T, Asamizu S, Ikeda H, Ōmura S, Oku N, Igarashi Y, Tomoda H, & Onaka H. (2014) Genome mining reveals a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides lactazoles. Chem Biol 21(5):679-688.

(4) Characterization of indolocarbazole biosynthetic enzymes in actinomycetes

Indolocarbazoles are compounds with a 6-ring-fused plane structure, which mimics ATP. Therefore, compounds containing an indolocarbazole core could be used to inhibit protein kinases such as Protein Kinase A (PKA) and Protein Kinase C (PKC), and are potential candidates of antitumor drugs. Staurosporine, an indolocarbazole compound, is biosynthesized by Streptomyces sp. TP-A0274. A whole set of biosynthetic genes have been already cloned, and these studies revealed that 4 enzymes, StaO, StaD, StaP, and StaC, are responsible for indolocarbazole core biosynthesis. The starter unit for the indolocarbazole core is 2 molecules of tryptophane. In the biosynthesis, StaO that encodes a monooxygenase, catalyzes the conversion from tryptophane to the indolepyruvic acid imine form (IPA imine). StaD then catalyzes the intermolecular-coupling reaction of 2 IPA imines to give a chromopyrrolic acid (CPA), which is a key intermediate of indolocarbazoles. Finally, intramolecular C-C bonds are formed between indole rings, followed by oxidation of the pyrrole moiety by StaP and StaC to yield K252c, an indolocarbazole core.

StaP, the C-C coupling enzyme, is a cytochrome P450. The X-ray crystal structures of CPA-bound and free forms of StaP demonstrate the molecular mechanism of substrate recognition by StaP. StaP is a typical P450 fold; however, it catalyzes the reaction by means of an indole cation radical intermediate, which is similar to the peroxidase reaction.

Fig. 4-1 : Indolocarbazolo biosynthetic pathway

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Address

Laboratory of Microbial Metabolic Potential

Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo.
1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.

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Current members

Hiroyasu Onaka

Specially-appointed Professor (Ph.D.)

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

Society Memberships and Awards

Shumpei Asamizu

Specially-appointed Assistant Professor (Ph.D.)

Education

  • 2001.4-2005.3 Toyama Prefectural University (Bachelor of Agriculture from NIAD-UE)
  • 2005.4-2007.3 Toyama Prefectural University (Master of Engineering)
  • 2007.4-2010.3 Toyama Prefectural University (Doctor of Engineering)

Professional Experience

  • 2008.4-2010.3 JSPS Research Fellow (DC2)
  • 2010.4-2013.3 Oregon State University, Postdoctoral Research Associate (Taifo Mahmud Lab.)
  • 2013- Specially-appointed Assistant Professor of Graduate school of Agricultural and Life Sciences, The University of Tokyo

Ph.D. Thesis

Studies on Bis-indole Natural Products Biosynthesis from Bacteria (Toyama Prefectural University, 2010.3)

Interests, Keywords

Secondary metabolism, Biosynthesis, Enzyme chemistry, Secondary metabolism gene regulation, Natural products chemistry, Synthetic biology, Metabolic engineering, Protein Engineering

Yoshinori Sugai

Specially-appointed Assistant Professor (Ph.D.)

Education

  • Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan, B.Agr. 2006
  • Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan, Ph.D. 2011

Professional Experience

  • 2010-2012 JSPS Research Fellowship for Young Scientist (DC2)
  • 2012-2016 The University of Tokyo, Postdoctoral Research Associate (Hakko Lab.)
  • 2016- Specially-appointed Assistant Professor of Graduate school of Agricultural and life Science, The University of Tokyo

Ph.D. Thesis

Enzymatic total synthesis of fully 13C-labeled compound (Tokyo University of Agriculture and Technology, 2011.3)

Interests, Keywords

Natural product chemistry, Secondary metabolism, Biosynthesis

Morito Shimomura

Student in the first year of the master's program

Masaomi Yanagisawa

Student in the first year of the master's program

Abrory Agus Cahya Pramana

Student in the first year of the master's program

Akira Kanada

Undergraduate Student

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Publications

2016

  1. T. Ozaki, K. Yamashita, Y. Goto, M. Shimomura, S. Hayashi, S. Asamizu, Y. Sugai, H. Ikeda, H. Suga*, and H. Onaka*. Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogs expressed in vivo. Nature communications, in press
  2. Sugiyama R, Nishimura S, Ozaki T, Asamizu S, Onaka H, Kakeya H. Discovery and Total Synthesis of Streptoaminals: Antimicrobial [5,5]-Spirohemiaminals from  the Combined-Culture of Streptomyces nigrescens and Tsukamurella pulmonis. Angew Chem Int Ed Engl. 2016 Jul 27. 55(35): 10278–10282 doi: 10.1002/anie.201604126.
  3. Du D, Katsuyama Y, Onaka H, Fujie M, Satoh N, Shin-Ya K, Ohnishi Y. Production of a Novel Amide-Containing Polyene by Activating a Cryptic Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08. Chembiochem. 2016 Jun 17. doi: 10.1002/cbic.201600167.

2015

  1. Ozaki T, Kurokawa Y, Hayashi S, Oku N, Asamizu S, Igarashi Y, Onaka H. Insights into the biosynthesis of dehydroalanines in goadsporin. ChemBioChem 2015 Dec 2. doi: 10.1002/cbic.201500541.
  2. Hoshino S, Okada M, Wakimoto T, Zhang H, Hayashi F, Onaka H, Abe I. Niizalactams A-C, Multicyclic Macrolactams Isolated from Combined Culture of Streptomyces with Mycolic Acid-Containing Bacterium. J Nat Prod 2015 Dec 1. in press
  3. Asamizu S, Ozaki T, Teramoto K, Satoh K, Onaka H. Killing of Mycolic Acid-Containing Bacteria Aborted Induction of Antibiotic Production by Streptomyces in Combined-Culture. PLoS One 2015 Nov 6;10(11):e0142372. doi: 10.1371/journal.pone.0142372.
  4. Hoshino S, Wakimoto T, Onaka H, Abe I., Chojalactones A-C, Cytotoxic Butanolides Isolated from Streptomyces sp. Cultivated with Mycolic Acid Containing Bacterium. Org Lett 2015 Mar 20;17(6):1501-4. doi: 10.1021/acs.orglett.5b00385.
  5. Sugiyama R, Nishimura S, Ozaki T, Asamizu S, Onaka H, Kakeya H. 5-Alkyl-1,2,3,4-tetrahydroquinolines, New Membrane-Interacting Lipophilic Metabolites Produced by Combined Culture of Streptomyces nigrescens and Tsukamurella pulmonis. Org Lett 2015 Apr 17;17(8):1918-21. doi: 10.1021/acs.orglett.5b00607.
  6. H. Onaka, T. Ozaki, Y. Mori, M. Izawa, S. Hayashi, and S. Asamizu. Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans. J Antibiot (Tokyo). 2015 Apr 1. doi: 10.1038/ja.2015.31.

2014

  1. Hoshino S, Zhang L, Awakawa T, Wakimoto T, Onaka H, Abe I. Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823. J Antibiot (Tokyo) 2014 Oct 22. doi: 10.1038/ja.2014.147.
  2. K.Haginaka, S. Asamizu, T. Ozaki, Y. Igarashi, T. Furumai, and H. Onaka*. Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis. Biosci Biotechnol Biochem 78(3): 394-399 (2014)
  3. R. Uchida, M. Nakai, S. Ohte, H. Onaka, T. Katagiri, and H. Tomoda*. 5-Prenyltryptophol, a new inhibitor of bone morphogenetic protein-induced alkaline phosphatase expression in myoblasts, produced by Streptomyces colinus subsp. albescens HEK608. J Antibiot (Tokyo) 2014 May 14. doi: 10.1038/ja.2014.44.
  4. S. Hayashi, T. Ozaki, S. Asamizu, H. Ikeda, S. Ōmura, N. Oku, Y. Igarashi, H. Tomoda, and 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)

2013

  1. Y. Kim, Y. In, T. Ishida, H. Onaka, and Y. Igarashi*. Biosynthetic origin of alchivemycin A, a new polyketide from Streptomyces and absolute configuration of alchivemycin B. Org Lett 15(14): 3514-3517 (2013)
  2. M. Kameya, H. Onaka, and Y. Asano*. Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis., Anal Biochem 438(2): 124-132 (2013)
  3. N. Koyama, M. Yotsumoto, H. Onaka. and H. Tomoda*. New structural scaffold 14-membered macrocyclic lactone ring for selective inhibitors of cell wall peptidoglycan biosynthesis in Staphylococcus aureus., J Antibiot (Tokyo). 66(5): 303-304 (2013)

2012

  1. Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Göransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Müller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJ, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Süssmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, and van der Donk WA*. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30(1): 108-160 (2012)
  2. S. Asamizu, S. Hirano, H. Onaka*, H. Koshino, Y. Shiro, and S. Nagano*. Coupling Reaction of Indolepyruvic Acid in the Presence of StaD and Its Product: Implications for Biosynthesis of Indolocarbazole and Violacein. ChemBioChem 13(17): 2495-2500 (2012)
  3. K. Sakai, N. Koyama, T. Fukuda, Y. Mori, H. Onaka, and H. Tomoda*. Search method for inhibitors of Staphyloxanthin production by methicillin-resistant Staphylococcus aureus. Biol Pharm Bull 35(1): 48-53 (2012)

Past publications