Researcher Profile and Settings

Master

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and Chemistry

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and Chemistry

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Profile and Settings

Profile and Settings

• Name (Japanese)

  Kitaoka

• Name (Kana)

  Naoki

• Name

  201601015832263622

Achievement

Research Interests

• Plant hormone   Secondary metabolite   biosynthesis   

Research Areas

• Life sciences / Bioorganic chemistry

Research Experience

• 2023/01 - Today Hokkaido University Research Faculty of Agriculture Associate Professor
• 2020/04 - 2022/12 Hokkaido University Research Faculty of Agriculture Assistant Professor
• 2017/04 - 2020/03 Toyama Prefectural University Assistant Professor
• 2014/07 - 2017/03 Tohoku University Postdoctoral Researcher
• 2012/10 - 2014/06 Iowa State University Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology Postdoctoral Researcher

Education

• 2008/04 - 2012/09  Hokkaido University  Graduate School of Agriculture
• 2004/04 - 2008/03  Hokkaido University  School of Agriculture

Awards

• 2024/03 Japan Society for Bioscience, Biotechnology, and Agrochemistry JSBBA Award for Young Scientists
  

Published Papers

• Isolation and Identification of the Antibacterial Compounds Produced by Maillard Reaction of Xylose with Phenylalanine or Proline.

  Kazuho Aonishi, Shungo Miyao, Lisa Yokoi, Naoki Kitaoka, Kento Koyama, Hideyuki Matsuura, Shigenobu Koseki

  Journal of Agricultural and Food Chemistry 2024/07 [Refereed][Not invited]
   

  Maillard reaction products (MRPs) of xylose with phenylalanine and xylose with proline exhibit high antibacterial activity. However, the active antibacterial compounds in MRPs have not yet been identified or isolated. This study aimed to isolate the active compounds in the two antibacterial MRPs. The organic layer of the MRP solution was separated and purified using silica gel chromatography and high-performance liquid chromatography. The chemical structures of the isolated compounds were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds inhibited the growth of Bacillus cereus and Salmonella Typhimurium at 25 °C for 7 days at a concentration of 0.25 mM. Furthermore, the isolated compounds inhibited the growth of naturally occurring microflora of lettuce and chicken thighs at 25 °C for 2 days at a concentration of 0.5-1.0 mM. The antibacterial compounds found in MRPs demonstrated a wide range of effectiveness and indicated their potential as alternative preservatives.
• Isolation and Structure Determination of cis-OPDA-α-Monoglyceride from Arabidopsis thaliana.

  Shotaro Hirota, Yusuke Ito, Shiro Inoue, Naoki Kitaoka, Tohru Taniguchi, Kenji Monde, Kosaku Takahashi, Hideyuki Matsuura

  Journal of Natural Products 87 1358 - 1367 2024/04 [Refereed][Not invited]
   

  cis-12-oxo-Phytodieneoic acid-α-monoglyceride (1) was isolated from Arabidopsis thaliana. The chemical structure of 1 was elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by FDMS and HRFDMS data. The absolute configuration of the cis-OPDA moiety in 1 was determined by comparison of 1H NMR spectra and ECD measurements. With respect to the absolute configuration of the β-position of the glycerol backbone, the 2:3 ratio of (S) to (R) was determined by making ester-bonded derivatives with (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride and comparing 1H NMR spectra. Wounding stress did not increase endogenous levels of 1, and it was revealed 1 had an inhibitory effect of A. thaliana post germination growth. Notably, the endogenous amount of 1 was higher than the amounts of (+)-7-iso-jasmonic acid and (+)-cis-OPDA in intact plants. 1 also showed antimicrobial activity against Gram-positive bacteria, but jasmonic acid did not. It was also found that α-linolenic acid-α-monoglyceride was converted into 1 in the A. thaliana plant, which implied α-linolenic acid-α-monoglyceride was a biosynthetic intermediate of 1.
• Isolation, structural elucidation, and biological activity of a novel isocoumarin from the dark septate endophytic fungus Phialocephala fortinii

  Kei Bando, Ryoga Kushibe, Naoki Kitaoka, Yutaka Tamai, Kazuhiko Narisawa, Hideyuki Matsuura

  Zeitschrift für Naturforschung C 79 89 - 92 2024/03 [Refereed]
  
• Investigation of the Biosynthesis Pathway that Generates cis-Jasmone.

  Shiro Inoue, Hiromu Tsuzuki, Kazuhiko Matsuda, Naoki Kitaoka, Hideyuki Matsuura

  ChemBioChem 25 e202300593  2023/11 [Refereed][Not invited]
   

  Researchers have established that (+)-7-iso-jasmonic acid ((+)-7-iso-JA) is an intermediate in the production of cis-jasmone (CJ); however, the biosynthetic pathway of CJ has not been fully described. Previous reports stated that CJ, a substructure of pyrethrin II produced by pyrethrum (Tanacetum cinerariifolium), is not biosynthesized through this biosynthetic pathway. To clarify the ambiguity, stable isotope-labelled jasmonates were synthesized, and compounds were applied to apple mint (Mentha suaveolens) via air propagation. The results showed that cis-jasmone is not generated from intermediate (+)-7-iso-JA, and (+)-7-iso-JA is not produced from 3,7-dideydro-JA (3,7-ddh-JA); however, 3,7-didehydro-JA and 4,5-didehydro-7-iso-JA were converted into CJ and JA, respectively.
• Airborne Methyl Jasmonate is Metabolized to Jasmonic Acid and 12-Hydroxyjasmonic Acid, and Induces Jasmonate Biosynthesis in Marchantia polymorpha

  Hiromu Tsuzuki, Naoki Kitaoka, Shiro Inoue, Kosaku Takahashi, Hideyuki Matsuura

  ChemistrySelect 8 (21) 2365-6549 2023/06 [Refereed]
   

  Methyl jasmonate (MeJA) is a volatile jasmonate compound commonly used to induce defense responses in spermatophytes. This study reports that airborne MeJA-d(3), deuterated MeJA, increases the levels of (dinor-)12-oxo-phytodienoic acids [(dn-)OPDAs] and jasmonic acid (JA) as well as JA-d(3) and 12-hydroxyjasmonic acid-d(2) (12-OH-JA-d(2)), MeJA-d(3) metabolites, in the model bryophyte Marchantia polymorpha. Enhancement of JA biosynthesis was substantiated by the expression of JA biosynthetic genes induced by airborne MeJA. Additionally, each of enantiomers, (+)-MeJA and (-)-MeJA, was observed to induce the accumulation of JA and (dn-)OPDAs in M. polymorpha. This study demonstrates that airborne MeJA is metabolized to JA and 12-OH-JA, and induces JA biosynthesis in M. polymorpha. Moreover, the transient increase in endogenous JA level after airborne MeJA treatment provides concrete evidence that M. polymorpha biosynthesizes JA.
• Biological Activity of Anti-bolting Compound, alpha-(7Z,10Z,13Z)-hexadeca-7,10,13-trienoic Acid Monoglyceride to Reduce the Endogenous Amount of Gibberellins

  Tsuyoshi Ogihara, Shunpei Shikama, Akihisa Ishii, Syotaro Hirota, Junichi Kashiwagi, Kaien Fujino, Yuki Mitsui, Takafumi Shimizu, Mitsunori Seo, Naoki Kitaoka, Yasunori Koda, Hideyuki Matsuura

  Journal of Plant Growth Regulation 0721-7595 2023/04 [Refereed]
   

  A monoacylglycerol, alpha-(7Z,10Z,13Z)-hexadeca-7,10,13-trienoic acid monoglyceride, extracted from leaves of Japanese radish (Raphanus sativus) has been reported as an anti-bolting compound (ABC); however, the mechanism how ABC inhibits the plant bolting has been remained to be elucidated. In this paper, it was found that exogenous applications of ABC led Arabidopsis thaliana seedlings decrease transcriptional levels of AtKO and increase those of AtGA2ox to reduce endogenous levels of gibberellins (GAs) to retard A. thaliana growth, whose physiological phenomena were counteracted by exogenous applications of ent-kaurenoic acid and GA(3), respectively. Furthermore, alpha-oleanolic acid monoglyceride having both activities of supressing KO and enhancing GA2ox inductions retarded A. thaliana growth post germination, although alpha-palmitic acid monoglyceride having only the effect of supressing KO induction was not enough to suppress the growth. These experimental data supported the idea that dual function was essential for ABC to show biological activity.
• Pennelliiside D, a New Acyl Glucose from Solanum pennellii and Chemical Synthesis of Pennelliisides.

  Rishni Masimbula, Hiroto Kobayashi, Tenki Nakashima, Yurika Nambu, Naoki Kitaoka, Hideyuki Matsuura

  Molecules (Basel, Switzerland) 27 (12) 3728  2022/06/09 [Refereed]
   

  Acyl glucoses are a group of specialized metabolites produced by Solanaceae. Solanum pennellii, a wild-type tomato plant, produces acyl glucoses in its hair-like epidermal structures known as trichomes. These compounds have been found to be herbicides, microbial growth inhibitors, or allelopathic compounds. However, there are a few reports regarding isolation and investigation of biological activities of acyl glucoses in its pure form due to the difficulty of isolation. Here, we report a new acyl glucose, pennelliiside D, isolated and identified from S. pennellii. Its structure was determined by 1D NMR and 2D NMR, together with FD-MS analysis. To clarify the absolute configuration of the acyl moiety of 2-methylbutyryl in the natural compound, two possible isomers were synthesized starting from β-D-glucose pentaacetate. By comparing the spectroscopic data of natural and synthesized compounds of isomers, the structure of pennelliiside D was confirmed to be 3,4-O-diisobutyryl-2-O-((S)-2-methylbutyryl)-D-glucose. Pennelliiside D and its constituent fatty acid moiety, (S)-2-methylbutanoic acid, did not show root growth-inhibitory activity. Additionally, in this study, chemical synthesis pathways toward pennelliisides A and B were adapted to give 1,6-O-dibenzylpennelliisides A and B.
• A carlactonoic acid methyltransferase that contributes to the inhibition of shoot branching in Arabidopsis

  Kiyoshi Mashiguchi, Yoshiya Seto, Yuta Onozuka, Sarina Suzuki, Kiyoko Takemoto, Yanting Wang, Lemeng Dong, Kei Asami, Ryota Noda, Takaya Kisugi, Naoki Kitaoka, Kohki Akiyama, Harro Bouwmeester, Shinjiro Yamaguchi

  Proceedings of the National Academy of Sciences 119 (14) e2111565119  0027-8424 2022/04/05 [Refereed]
   

  Significance Strigolactones (SLs) are a group of apocarotenoid hormones, which regulates shoot branching and other diverse developmental processes in plants. The major bioactive form(s) of SLs as endogenous hormones has not yet been clarified. Here, we identify an Arabidopsis methyltransferase, CLAMT, responsible for the conversion of an inactive precursor to a biologically active SL that can interact with the SL receptor in vitro. Reverse genetic analysis showed that this enzyme plays an essential role in inhibiting shoot branching. This mutant also contributed to specifying the SL-related metabolites that could move from root to shoot in grafting experiments. Our work has identified a key enzyme necessary for the production of the bioactive form(s) of SLs.
• AtGH3 .10 is another jasmonic acid‐amido synthetase in Arabidopsis thaliana

  Jay C. Delfin, Yuri Kanno, Mitsunori Seo, Naoki Kitaoka, Hideyuki Matsuura, Takayuki Tohge, Takafumi Shimizu

  The Plant Journal 110 1082 - 1096 0960-7412 2022/03/22 [Refereed]
  
• Potato tuber-inducing activities of jasmonic acid and related-compounds (II).

  Kanji Miyawaki, Shiro Inoue, Naoki Kitaoka, Hideyuki Matsuura

  Bioscience, Biotechnology, and Biochemistry 85 (12) 2378 - 2382 2021/11/24 

  New information is being accumulated for plant-derived oxylipins, such as jasmonic acid (JA) amino acid conjugates. However, these compounds have not being examined for their activity in promoting potato tuber formation. It was found that (-)-JA had the highest activity followed cis-(-)-OPDA, (+)-4, 5-didehydroJA, cis-(+)-OPDA-l-Ile, and (-)-JA-l-Ile, -Leu, -Phe, -Val, although iso-OPDA and 3,7-didehydroJA did not exhibit activity.
• Verification of the versatility of the in vitro enzymatic reaction giving (+)-cis-12-Oxo-phytodienoic acid.

  Yusuke Ito, Kento Sasaki, Tsuyoshi Ogihara, Naoki Kitaoka, Kosaku Takahahi, Hideyuki Matsuura

  Bioorganic & Medicinal Chemistry Letters 49 128284 - 128284 2021/10/01 [Refereed][Not invited]
   

  Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic pathway of arabidopsides could occur via MGDG.
• A promiscuous fatty acid ω‐hydroxylase CYP94A90 is likely to be involved in biosynthesis of a floral nitro compound in loquat ( Eriobotrya japonica )

  Takuya Yamaguchi, Yumi Matsui, Naoki Kitaoka, Yasumasa Kuwahara, Yasuhisa Asano, Hideyuki Matsuura, Yukari Sunohara, Hiroshi Matsumoto

  New Phytologist 231 (3) 1157 - 1170 0028-646X 2021/08/31 [Refereed][Not invited]
  
• Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol β-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase

  Naoki Kitaoka, Taiji Nomura, Shinjiro Ogita, Yasuo Kato

  Applied Biochemistry and Biotechnology 193 (7) 2061 - 2075 2021/02/05 [Refereed][Not invited]
   

  Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol β-primeveroside (4-VPP) and 4-vinylguaiacol β-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.
• Interdependent evolution of biosynthetic gene clusters for momilactone production in rice

  Naoki Kitaoka, Juan Zhang, Richard K Oyagbenro, Benjamin Brown, Yisheng Wu, Bing Yang, Zhaohu Li, Reuben J Peters

  The Plant Cell 2020/12/04 [Refereed][Not invited]
  
• Bioproduction of glucose conjugates of 4-hydroxybenzoic and vanillic acids using bamboo cells transformed to express bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase.

  Naoki Kitaoka, Taiji Nomura, Shinjiro Ogita, Yasuo Kato

  Journal of Bioscience and Bioengineering 130 (1) 89 - 97 1389-1723 2020/07 [Refereed][Not invited]
   

  Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.
• Cobalt-dependent inhibition of nitrite oxidation in Nitrobacter winogradskyi.

  Richard Metzner, Taiji Nomura, Naoki Kitaoka, Akinori Ando, Jun Ogawa, Yasuo Kato

  Journal of Bioscience and Bioengineering 128 (4) 463 - 467 2019/10 [Refereed][Not invited]
   

  Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH3) to nitrate (NO3-) via nitrite (NO2-), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO2- oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l-Histidine effectively restored cell growth and NO2- oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation.
• Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the third isozyme with a distinct expression profile.

  Taiji Nomura, Ryo Kuchida, Naoki Kitaoka, Yasuo Kato

  Bioscience, Biotechnology, and Biochemistry 82 (5) 1 - 11 2018/02/23 [Refereed][Not invited]
   

  6-Tuliposide B (PosB), a major secondary metabolite that accumulates in tulip (Tulipa gesneriana), is converted to the antibacterial lactone, tulipalin B (PaB), by PosB-converting enzyme (TCEB). TgTCEB1 and TgTCEB-R, which encode TCEB, are specifically expressed in tulip pollen and roots, respectively, but are hardly expressed in other tissues (e.g. leaves) despite the presence of substantial PosB-converting activity, suggesting the existence of another TCEB isozyme. Here, we describe the identification of TgTCEB-L ("L" for leaf), a paralog of TgTCEB1 and TgTCEB-R, from leaves via native enzyme purification. The enzymatic characters of TgTCEB-L, including catalytic activity and subcellular localization, were substantially the same as those of TgTCEB1 and TgTCEB-R. However, TgTCEB-L did not exhibit tissue-specific expression. Identification of TgTCEB-L explains the PosB-converting activity detected in tissues where TgTCEB1 and TgTCEB-R transcripts could not be detected, indicating that tulip subtilizes the three TgTCEB isozymes depending on the tissue.
• Investigating inducible short-chain alcohol dehydrogenases/reductases clarifies rice oryzalexin biosynthesis

  Naoki Kitaoka, Yisheng Wu, Jiachen Zi, Reuben J. Peters

  The Plant Journal 88 (2) 271 - 279 0960-7412 2016/10 [Refereed][Not invited]
   

  Rice (Oryza sativa) produces a variety of labdane-related diterpenoids as phytoalexins and allelochemicals. The production of these important natural products has been partially elucidated. However, the oxidases responsible for production of the keto groups found in many of these diterpenoids have largely remained unknown. Only one short-chain alcohol dehydrogenase/reductases (SDRs), which has been proposed to catalyze the last step in such a pathway, has been characterized to date. While rice contains >220 SDRs, only the transcription of five has been shown to be induced by the fungal cell wall elicitor chitin. This includes the momilactone A synthase (OsMAS/SDR110C-MS1), with the other four all falling in the same SDR110C family, further suggesting roles in diterpenoid biosynthesis. Here, biochemical characterization with simplified substrate analogs was first used to indicate potential functions, which were then supported by further analyses with key biosynthetic intermediates. Kinetic studies were then employed to further clarify these roles. Surprisingly, OsSDR110C-MS2 more efficiently catalyzes the final oxidation to produce momilactone A that was previously assigned to OsMAS/SDR110C-MS1, and we speculate that this latter SDR may have an alternative function instead. Conversely, two of these SDRs clearly appear to act in oryzalexin biosynthesis, with OsSDR110C-MI3 readily oxidizing the 3 alpha-hydroxyl of oryzalexin D, while OsSDR110C-MS3 can also oxidize the accompanying 7 beta-hydroxyl. Together, these SDRs then serve to produce oryzalexins A-C from oryzalexin D, essentially completing elucidation of the biosynthesis of this family of rice phytoalexins.
• Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana

  Tong Zhang, Arati N. Poudel, Jeremy B. Jewell, Naoki Kitaoka, Paul Staswick, Hideyuki Matsuura, Abraham J. Koo

  Journal of Experimental Botany 67 (7) 2107 - 2120 0022-0957 2016/03 [Refereed][Not invited]
   

  Wound-inducible and ER-located amidohydrolases with overlapping substrate specificities for IAA- and JA-amino acid conjugates regulate the production and destruction of active auxin and JA signals in wounded leaves.Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways.
• Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis

  Naoki Kitaoka, Yisheng Wu, Meimei Xu, Reuben J. Peters

  Applied Microbiology and Biotechnology 99 (18) 7549 - 7558 0175-7598 2015/09 [Refereed][Not invited]
   

  The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3 beta-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2 alpha,3 alpha-dihydroxy-ent-isokaurene via 2 alpha-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3 alpha-hydroxy-ent-cassadien-2-one via 2 alpha-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.
• The Application of Synthetic Biology to Elucidation of Plant Mono-, Sesqui-, and Diterpenoid Metabolism

  Naoki Kitaoka, Xuan Lu, Bing Yang, Reuben J. Peters

  Molecular Plant 8 (1) 6 - 16 1674-2052 2015/01 [Refereed][Not invited]
   

  Plants synthesize a huge variety of terpenoid natural products, including photosynthetic pigments, signaling molecules, and defensive substances. These are often produced as complex mixtures, presumably shaped by selective pressure over evolutionary timescales, some of which have been found to have pharmaceutical and other industrial uses. Elucidation of the relevant biosynthetic pathways can provide increased access (e.g., via molecular breeding or metabolic engineering) and enable reverse genetic approaches toward understanding the physiological role of these natural products in plants as well. While such information can be obtained via a variety of approaches, this review describes the emerging use of synthetic biology to recombinantly reconstitute plant terpenoid biosynthetic pathways in heterologous host organisms as a functional discovery tool, with a particular focus on incorporation of the historically problematic cytochrome P450 mono-oxygenases. Also falling under the synthetic biology rubric and discussed here is the nascent application of genome-editing tools to probe physiological function.
• Endoplasmic Reticulum-associated Inactivation of the Hormone Jasmonoyl-L-Isoleucine by Multiple Members of the Cytochrome P450 94 Family in Arabidopsis

  Abraham J. Koo, Caitlin Thireault, Starla Zemelis, Arati N. Poudel, Tong Zhang, Naoki Kitaoka, Federica Brandizzi, Hideyuki Matsuura, Gregg A. Howe

  Journal of Biological Chemistry 289 (43) 29728 - 29738 0021-9258 2014/10 [Refereed][Not invited]
   

  Background: Accumulation of the plant hormone jasmonoyl-l-isoleucine (JA-Ile) is tightly controlled to prevent overactivation of defense responses. Results: Cytochrome P450 94s (CYP94s) with distinct tissue expression patterns localize to ER and oxidize JA-Ile to a dicarboxy derivative that fails to assemble JA-Ile co-receptor complexes. Conclusion: Sequential CYP94-catalyzed oxidations block receptor activation and signaling. Significance: P450s inactivate fatty acid-derived signals in both plants and animals. The plant hormone jasmonate (JA) controls diverse aspects of plant immunity, growth, and development. The amplitude and duration of JA responses are controlled in large part by the intracellular level of jasmonoyl-l-isoleucine (JA-Ile). In contrast to detailed knowledge of the JA-Ile biosynthetic pathway, little is known about enzymes involved in JA-Ile metabolism and turnover. Cytochromes P450 (CYP) 94B3 and 94C1 were recently shown to sequentially oxidize JA-Ile to hydroxy (12OH-JA-Ile) and dicarboxy (12COOH-JA-Ile) derivatives. Here, we report that a third member (CYP94B1) of the CYP94 family also participates in oxidative turnover of JA-Ile in Arabidopsis. In vitro studies showed that recombinant CYP94B1 converts JA-Ile to 12OH-JA-Ile and lesser amounts of 12COOH-JA-Ile. Consistent with this finding, metabolic and physiological characterization of CYP94B1 loss-of-function and overexpressing plants demonstrated that CYP94B1 and CYP94B3 coordinately govern the majority (>95%) of 12-hydroxylation of JA-Ile in wounded leaves. Analysis of CYP94-promoter-GUS reporter lines indicated that CYP94B1 and CYP94B3 serve unique and overlapping spatio-temporal roles in JA-Ile homeostasis. Subcellular localization studies showed that CYP94s involved in conversion of JA-Ile to 12COOH-JA-Ile reside on endoplasmic reticulum (ER). In vitro studies further showed that 12COOH-JA-Ile, unlike JA-Ile, fails to promote assembly of COI1-JAZ co-receptor complexes. The double loss-of-function mutant of CYP94B3 and ILL6, a JA-Ile amidohydrolase, displayed a JA profile consistent with the collaborative action of the oxidative and the hydrolytic pathways in JA-Ile turnover. Collectively, our results provide an integrated view of how multiple ER-localized CYP94 and JA amidohydrolase enzymes attenuate JA signaling during stress responses.
• CYP94B3 activity against jasmonic acid amino acid conjugates and the elucidation of 12-O-beta-glucopyranosyl-jasmonoyl-L-isoleucine as an additional metabolite

  Naoki Kitaoka, Hiroshi Kawaide, Naruki Amano, Takuya Matsubara, Kensuke Nabeta, Kosaku Takahashi, Hideyuki Matsuura

  Phytochemistry 99 6 - 13 0031-9422 2014/03 [Refereed][Not invited]
   

  The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and inactivation. Oxidation of jasmonoyl-L-isoleucine at the C-12 position, which is catalyzed by cytochrome P450s CYP94B3 and CYP94C1, is thought to be one of the main inactivation pathways. In this study, an additional function of CYP94B3 was elucidated, as well additional jasmonoyl-L-isoleucine metabolites being investigated. It was found that CYP94B3 also catalyzes the hydroxylation of jasmonoyl-L-valine and jasmonoyl-L-phenylalanine, and that these hydroxyl compounds accumulated after wounding and possessed lower activity than non-hydroxylated compounds. Additionally, 12-O-beta-glucopyranosyl-jasmonoyl-t-isoleucine accumulated after wounding, suggesting that it is a metabolite of
• Transportation of de novo synthesized jasmonoyl isoleucine in tomato

  Hideyuki Matsuura, Syohei Takeishi, Naoki Kiatoka, Chizuru Sato, Kae Sueda, Chikara Masuta, Kensuke Nabeta

  Phytochemistry 83 25 - 33 0031-9422 2012/11 [Refereed][Not invited]
   

  In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[(C6P)-C-13]Ile was recovered in distal untreated leaves after wounded leaves were treated with [(C6P)-C-13]Ile. However, as [(C6P)-C-13]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [(C6P)-C-13]Ile and [H-2(6)]JA were applied separately into the wounds on two different leaves, JA-[(C6P)-C-13]Ile and [H-2(6)]JA-Ile were detected in the stem exudates but [H-2(6)]JA-[(C6P)-C-13]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [H-2(3)]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [H-2(3)]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal. (C) 2012 Elsevier Ltd. All rights reserved.
• Biological Activity and Tissue Specific Accumulation of Fluorescently Labeled Methyl Jasmonate

  Naoki Kitaoka, Yuzou Sano, Seizo Fujikawa, Kensuke Nabeta, Hideyuki Matsuura

  Natural Product Communications 7 (2) 215 - 218 1934-578X 2012/02 [Refereed][Not invited]
   

  The C-7 position of jasmonate is practical for synthesis of a probe to use for chemical biological studies. To confirm the utility, we synthesized fluorescent-labeled methyl jasmonate. The synthesized compound exhibited Arabidopsis thaliana root growth inhibitory and meandering activity, and potent fluorescence was observed inside the root and root hairs.
• Arabidopsis CYP94B3 Encodes Jasmonyl-L-Isoleucine 12-Hydroxylase, a Key Enzyme in the Oxidative Catabolism of Jasmonate

  Naoki Kitaoka, Takuya Matsubara, Michio Sato, Kosaku Takahashi, Shinji Wakuta, Hiroshi Kawaide, Hirokazu Matsui, Kensuke Nabeta, Hideyuki Matsuura

  Plant and Cell Physiology 52 (10) 1757 - 1765 0032-0781 2011/10 [Refereed][Not invited]
   

  The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-l-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants.
• Potato Tuber Cell Expansion-Inducing Activity of Stereochemically Restricted JA Analogs

  Naoki Kitaoka, Eri Fukushi, Yasunori Koda, Kensuke Nabeta, Hideyuki Matsuura

  Bioscience, Biotechnology, and Biochemistry 73 (8) 1872 - 1876 0916-8451 2009/08 [Refereed][Not invited]
   

  Stereochemically restricted analogues of C-7 substituted 7-epi-jasmonate, together with 12-hydroxy jasmonic acid, 12-hydroxy jasmonic acid glucoside, and jasmonic acid conjugated with L-isoleucine (JA-Ile), were synthesized and then tested for potato tuber cell expansion-inducing activity. JA-Ile showed almost the same activity as JA, while the C-7 substituted 7-epi-jasmonates exhibited weaker activity than JA and showed an antagonist effect against JA.
• Isolation and Structural Elucidation of a New Cyclohexenone Compound from Lasiodiplodia theobromae

  Naoki Kitaoka, Kensuke Nabeta, Hideyuki Matsuura

  Bioscience, Biotechnology, and Biochemistry 73 (8) 1890 - 1892 0916-8451 2009/08 [Refereed][Not invited]
   

  A new cyclohexenone compound was isolated as a mixture of enantiomers from a culture filtrate of Lasiodiplodia theobromae. The relative structure was determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-enone on the basis of MS, H-1-NMR, and C-13-NMR spectroscopic analyses, including 2D-NMR experiments. Resolution of the enantiomers was conducted by a coupling reaction with (S)-MTPA-Cl followed by HPLC separation.

MISC

• ゼニゴケにおけるジャスモン酸の生合成

  北岡 直樹, 都築 玄武, 高橋 公咲, 松浦 英幸  アグリバイオ  7-  (3)  60  -63  2023/03
• Evolution of biosynthesis and signaling pathway of jasmonates

  Naoki Kitaoka, Kosaku Takahashi  植物の生長調節  56-  (2)  77  -84  2021/12
• オリザレキシン生合成における酸化酵素の働き:化学構造の多様性はどのようにもたらされるか？

  北岡 直樹  化学と生物  55-  (9)  585  -586  2017
• 植物ホルモン・ジャスモン酸の主要な不活性化経路 : ジャスモン酸イソロイシン12位水酸化酵素CYP94B3の発見

  北岡 直樹, 松浦 英幸  化学と生物  50-  (3)  156  -157  2012/03/01  [Not refereed][Not invited]
  

Association Memberships

• The Japanese Society for Chemical Regulation of Plants   The Japanese Society of Plant Physiologists   Japan Society for Bioscience, Biotechnology, and Agrochemistry   

Research Projects

• Elucidation of the inhibitory mechanism of bolting in plants and development of bolting inhibitors

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 松浦 英幸, 北岡 直樹, 久保 友彦, 黒田 洋輔, 柏木 純一, 北崎 一義, 橋本 誠
• 苔類に特徴的な大環状芳香族化合物の生合成機構および生理作用の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2023/03 

  Author : 北岡 直樹

   

  苔類に属する様々な種からみつかっている一方で、苔類以外の植物種からはみつかっていない『苔類にユニークな代謝産物』が植物体内でどのように作られ（生合成され）、どのような役割を担っているか（生理作用を有するか）を明らかにすることを本研究課題では目的としている。具体的には候補となる酵素を大腸菌や酵母で発現させその機能を明らかにするとともに、同酵素遺伝子の破壊株を作製し代謝物プロファイルを調べる。さらに、作製した破壊株の表現型を調べることで、前述の目的の達成を目指す。
  【作製した遺伝子破壊株における代謝産物の分析】 ゲノム編集技術であるCRISPR/Cas9を用いて作製した標的遺伝子のゼニゴケ破壊株を作製した。現在までに、異なる二段階を触媒すると予想される２つの遺伝子群に加えて、標的化合物への関与が示唆される機能未知の遺伝子の破壊株の作製に成功している。遺伝子破壊株の代謝物分析を行なったところ、最初の段階を触媒すると予想される酵素遺伝子の破壊株で標的化合物の減少がみられた。その一方で、後半の段階を触媒すると予想される酵素遺伝子や未知の酵素遺伝子の遺伝子破壊株においては、予想していた標的化合物の減少がみられなかった。 【遺伝子のクローニング】コケ植物より、標的遺伝子をクローニングし大腸菌発現用のベクターに導入した。作製した発現用ベクターを用いて、タンパク質発現用の大腸菌の形質転換を行なった。同大腸菌を用いて発現、Niアフィニティーカラムによって発現したHisタグつき組換え酵素を用いて酵素活性を調べたところ、微弱ではあるが所望の酵素活性の検出に成功した。
• Creation of the plant cultured cells to produce the functional products

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2020/03 

  Author : Kitaoka Naoki

   

  We generated the transformed bamboo cells which efficiently produced the functional products. Firstly, we generated the bamboo cells which were transformed by hydroxycinnamic acid hydratase/aldolase (PpHCHL) from Pseudomonas putida. In the PpHCHL-transformed cells, the high accumulation of glucose conjugates of vanillic acid and 4-hydroxybenozic acid were observed. Secondary, we generated the bamboo cells which were transformed to express phenolic acid decarboxylase (BaPAD) from Bacillus amyloliquefacience. In the BaPAD transformed cells, primeverosides of 4-vinylpheol and 4-vinylguaiacol were produced.
• ジャスモン酸12位水酸化酵素の同定及び、傷害応答性移動シグナル分子の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2010 -2012 

  Author : 北岡 直樹

   

  昨年度はジャスモン酸不活性化に関する研究をさらに応用し、ジャスモン酸によって誘導されるイネのファイトアレキシンの一種であるPhytocassaneの生合成に関する研究を行った。Phytocassaneの生合成は、前駆体となるゲラニルゲラニル二リン酸(GGPP)が二種の環化酵素により環化を受け炭素骨格が形成されent-cassadieneとなり、その後シトクロムP450等により修飾を受け生合成される。先行研究により2種の環化酵素及びシトクロームP450であるCYP71Z7、CYP76M7、CYP701A8が同定されている。CYP71Z7、CYP701A8、CYP76M7はそれぞれent-cassadieneの2位、3位、11位を水酸化する活性を有する。私は、生合成に関与する環化酵素及びシトクロームP450をコードする遺伝子で形質転換した大腸菌を用い低分子化合物を得るmetabolic engineering systemを活用し、Phytocassane生合成に関与すると予想されるシトクロームP450のさらなる評価を行った。 前述のようにent-cassadieneの2位水酸化酵素として報告されているCYP71Z7の酵素活性をより詳細に解析し、CYP71Z7はent-cassadieneの2位に水酸基を導入するのみでなく、2位をさらに酸化しカルボニル基とし、その後3位に水酸基を導入する活性を有することが明らかとした。CYP71Z7には90%以上の相同性を示すCYP71Z6が存在する。またイネはent-cassadieneの他にも数種類のジテルペンを有することが知られている。そこで、metabolic engineering systemを用いてCYP71Z6及びCYP71Z7の基質特異性を調べた。その結果ent-コパニル二リン酸から生合成されるent-kaurene、ent-isokaurene、ent-sandaracopimaradiene、ent-cassadieneを基質とした際、CYP71Z6は2位のみを水酸化するが、CYP71Z7に関しては2位水酸化体に加えて3位水酸化体、2-keto-3-olを与えることが明らかとなった。水酸化体の位置選択性は、ent-cassadieneを基質とした際はほぼ2位水酸化体のみを与え、ent-sandaracopimaradieneを基質とした際は、2位水酸化体と3位水酸化体を同程度与えるといったように基質に依存することが示された。これら反応特異性は基質によって酵素の活性中心であるヘムとの位置関係が基質によって異なることによって生じると予想される。