Journal of Convergence for Information Technology e-ISSN 2586-4440 Vol. 10. No. 3, pp. 76-91, 2020 DOI : https://doi.org/10.22156/CS4SMB.2020.10.03.076

Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho

Jaeho Park1, Hong Xi2, Jiyun Han3, Jeongmin Lee3, Yongsung Kim4, Jun-mi Lee3, Janghyuk Son5, Joungjwa Ahn6, Taewon Jang7, Jisoo Choi8, Jongsun Park5* 1Professor, Department of Pharmaceutical Science, JungWon University 2Senior Researcher, InfoBoss Co., Ltd. & InfoBoss Research Center 3Researcher, InfoBoss Co., Ltd. & InfoBoss Research Center 4Team Leader, InfoBoss Co., Ltd. & InfoBoss Research Center 5CEO, InfoBoss Co., Ltd. & InfoBoss Research Center 6Professor, Department of Food Science & Technology, JungWon University 7Student, Department of Medicinal Resources, Andong National University 8Student, Department of Pharmaceutical Science, JungWon University 미선나무 품종 옥황 1호의 유전체를 활용한 Acteoside 생화학 합성과정 예측 및 확인

박재호1, 시 홍2, 한지윤3, 이정민3, 김용성4, 이준미3, 손장혁5, 안정좌6, 장태원7, 최지수8, 박종선5* 1중원대학교 제약공학과 교수, 2인포보스 주식회사 및 인포보스 기업부설연구소 선임연구원, 3인포보스 주식회사 및 인포보스 기업부설연구소 연구소 연구원, 4인포보스 주식회사 및 인포보스 기업부설연구소 팀장, 5인포보스 주식회사 및 인포보스 기업부설연구소 대표, 6중원대학교 식품공학과 교수, 7안동대학교 생약자원개발학과 학생, 8중원대학교 제약공학과 학생

Abstract Whole genome sequence of Abeliophyllum distichum Nakai () cultivar Ok Hwang 1 Ho, which is Korean endemic species, was recently sequenced to understand its characteristics. Acteoside is one of major useful compounds presenting various activities, and its several proposed biochemical pathways were reviewed and integrated to make precise biochemical pathway. Utilizing MetaPre-AITM which was developed for predicting secondary metabolites based on whole genome with the precise biochemical pathway of acteoside and the InfoBoss Pathway Database, we successfully rescued all enzymes involved in this pathway from the genome sequences, presenting that A. distichum cultivar Ok Hwang 1 Ho may produce acteoside. High-performance liquid chromatography result displayed that callus of A. distichum cultivar Ok Hwang 1 Ho contained acteoside as well as isoacteoside which may be derived from acteoside. Taken together, we successfully showed that MetaPre-AITM can predict secondary metabolite from plant whole genomes. In addition, this method will be efficient to predict secondary metabolites of many plant species because DNA can be analyzed more stability than chemical compounds.

Key Words : Acteoside, biochemical pathway, Whole genome, MetaPre-AITM, InfoBoss Pathway Database, Abeliophyllum distichum, cultivar Ok Hwang 1ho

요 약 최근에 한국 고유종인 미선나무 (Abeliophyllum distichum Nakai; Oleaceae) 품종 옥황1호의 유전체가 성공적으로 해독되었다. Acteoside는 다양한 활성을 가지는 물질이며, 여러개의 생화학합성과정이 제시되어왔고, 이들을 통합 검토하여 정 확한 생화학합성과정을 완성하였다. 유전체 데이터로부터 2차대사산물을 예측할 수 있는 MetaPre-AITM와 정확한 acteoside 생화학합성과정, InfoBoss Pathway Database를 활용하여, acteoside에 관여하는 모든 효소의 유전자를 옥황1호 유전체로부 터 성공적으로 확인하였다. 이는 옥황1호는 acteoside 물질을 생산할 수 있는 가능성이 있음을 의미한다. 이에 고성능액체크로 마토그래피를 사용하여 옥황1호의 캘러스 세포를 분석하여 acteoside과 이의 유도체인 isoacteoside를 확인하였다. 본 연구는 MetaPre-AITM은 유전체로부터 2차대사산물을 성공적으로 예측하였다. 이 방법은 화학물질보다 안정적인 DNA를 분석하여 2 차 대사산물을 예측하는 효율적인 방법이 될 것이다.

주제어 : Acteoside, 생화학 합성과정, 유전체, MetaPre-AITM, InfoBoss Pathway Database, Abeliophyllum distichum, 옥황1호

*This study was fully supported by the InfoBoss Research Grant(IBG-0032). *Corresponding Author : Jongsun Park([email protected]) Received January 6, 2020 Revised February 6, 2020 Accepted March 20, 2020 Published March 28, 2020 77 융합정보논문지 제10권 제3호

1. Background background is identical, which is good for controlling quality. Ok Hwang 1ho has already Abeliophyllum distichum Nakai belonging to been cultivated for long time in Oleaceae is Korean endemic species in GoesanBunjae-Nongwon (Goesan-gun, Chungbuk Abeliophyllum genus unique in Korea. This Province, Republic of Korea). Genome sequence genus is independent to neighbor genus, of A. distichum cultivar Ok Hwang 1ho can be , based on phylogenetic studies [1,2] as used for finding enzymes involved in biochemical well as complete chloroplast genome sequences pathways of secondary metabolites. One of [2-6]. It indicates that A. distichum is one of typical examples is Coffea canephora genome economically valuable our own biological for identifying caffeine biosynthesis: all resources under the Nagoya Protocol [7]. Till identified enzymes of this biochemical pathway now, ten natural habitats have been reported were identified from genome sequences and [8-11], which is a limited factor to prepare compared with the other species [24]. In enough amount of samples for extracting useful addition, tea (Camellia sinensis) genome was compounds. However, commercial cultivar, also sequenced with presenting enzymes of named as Ok Hwang 1ho, was successfully caffeine biosynthesis pathway [25]. registered and cultivated well, indicating that Acteoside, which is a caffeoyl phenylethanoid enough amount of samples can be prepared for glycoside isolated from various plant commercial use. species including Phlomis genus () Recent researches have reported that A. [26], Byblis liniflora (Byblidaceae) [27], distichum synthesize acteoside, eutigoside B, Verbascum phlomoides [28], Verbascum isoacteoside, rutin, cornoside, and hirsutrin [12, mallophorum () [29], Plantago 13]. Moreover, activities of extracts from A. lanceolata (Plantaginaceae) [30], Plantago distichum have been investigated showing asiatica (Plantaginaceae) [31], and Rehmannia anti-cancer activity [14,15], antioxidative glutinosa () [32], plays multiple activities [16-20], inhibition of DNA damage roles as a neuroprotective agent [33-35], an [16,21], anti-inflammatory effects [15,17,19,22], anti-inflammatory agent [36-38], an and whiting effect of skin [18,20,23]. These results antibacterial agent [36,39-41], and an antiviral strongly support that A. distichum is a good activity [31]. target to understand its usefulness as candidate In this study, we investigated three biochemical of medical materials and functional foods. In pathways of acteoside described in Fig. 1 and addition, these compounds and activities were proposed the integrated biochemical pathway identified in natural isolate of A. distichum. described in Fig. 2 Based on this pathway, we Recently, whole genome of A. distichum successfully identified the enzyme genes from cultivar Ok Hwang 1ho was successfully genome sequence of A. distichum cultivar Ok Hwang sequenced and assembled, presenting 1.01 Gbp 1ho collected from GoesanBunjae-Nongwon in length which is congruent to the result of (Goesan-gun, Chungbuk Province, Republic of k-mer analysis (1.02 Gbp; Park et al., in Korea) with utilizing MetaPre-AITM. Based on preparation). Official cultivar of A. distichum has prediction result, in total, 42 predicted genes two advantages: amount of plant samples can be were mapped to 24 enzymes in the three easily expanded based on already accumulated proposed pathways and 33 predicted genes cultivation experiences and its genetic Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 78 corresponding to 17 enzymes from the acteoside, the bioinformatic pipeline, named as integrated pathway of acteoside were MetaPre-AITM, was used, which was constructed successfully identified from the genome of A. together with the GenomeArchive® distichum cultivar Ok Hwang 1ho. After that we (http://www.genomearchive.info/; Park et al., in confirmed existence of acteoside in callus of A. preparation), the Genome Information System (GiS; distichum cultivar Ok Hwang 1ho via http://gis.infoboss.co.kr/), and the InfoBoss Pathway high-performance liquid chromatography Database (IPD; http://pathway.infoboss.co.kr/; Park experiment. Our results strongly suggested that et al., in preparation). IPD is a standardized plant secondary metabolites can be predicted database of curated biochemical pathways with from the genome sequences with the plant genomes supported by the Plant Genome MetaPre-AITM. Database (http://www.plantgenome.info; Park et al., in preparation). MetaPre-AITM contains three major 2. Materials & Methods components to identify enzymes from whole genome sequences as depicted in Fig. 3: i) 2.1 Preparation of callus of A. distichum homology-based enzyme identifier which selects cultivar Ok Hwang 1ho candidate genes based on already-known enzyme Fresh of A. distichum cultivar Ok Hwang amino acid sequences, ii) functional-domain based 1ho were collected from GoesanBunjae-Nongwon enzyme identifier which utilizes functional domains (Goesan-gun, Chungbuk Province, Republic of predicted by InterProScan [42], iii) Korea, Voucher in InfoBoss Cyber Herbarium (IN); phylogenetic-approach enzyme identifier which Y. Kim, IB-00589). Leaves were surface sterilized finds correct enzymes which display high similarity for 3 sec in 70% ethanol, soaked in sodium of amino acids within gene family, such as hypochlorite (5.25%), 10 min in sterile aquadest Cytochrome P450s, iv) amino acid pattern analyzer and then they were washed two times with sterile based on support vector machine (SVM) which aquadest in laminar air-flow hood. The callus classifies enzymes with forth-level of enzyme induction media was composed of MS (Murashige classification (EC) numbers displaying better than and Skoog) basal medium and supplemented with previous AI-based classifiers [43, 44], and v) 30 g/L sucrose, naphthalene acetic acid 1 mg/L, AI-based evaluator which makes final decision of and 2,4‑dichlorophenoxyacetic acid 1 mg/L. The enzyme functions together with analysis results from callus was induced 20 days later. Induced callus the first two engines. was subcultured in the same medium and sufficient amount was secured. Samples for HPLC 2.3 Protocol of high-performance liquid analysis were used callus 20 days after the chromatography (HPLC) for identifying subculture. Analytical samples were prepared by acteoside adding 10 ml of methanol to 3 g of callus, A Waters 2695 system equipped with Waters extracting by sonication for 20 minutes, and 2996 PDA was used for the analysis of filterated at 0.45 µM. acteoside. The separation was carried out on a Xbridge-C18 column (250 mm × 4.6 mm, 5 µm) 2.2 Identification of enzymes related to with a C18 guard column. The binary mobile biochemical pathways of acteoside phase consisted of acetonitrile (solvent A) and To identify enzymes of biochemical pathways of water containing 1% acetic acid (solvent B). All 79 융합정보논문지 제10권 제3호 the solvents were filtered through a 0.45 µm biochemical pathways [48-50]. Pathway A filter prior to use. The flowrate was kept mentioned primary-amine oxidase (CUAO; EC constant at 1.0 ml/min for a total run time of 1.4.3.21) and alcohol dehydrogenase (ADH; EC 40 min. The system was run with a gradient 1.1.1.1) involved in the reaction from tyramine to program: 0 min, 90% B; 0-5 min, 85% B; 5-15 tyrosol; while pathway C presented min, 85% B; 15-40 min, 85% to 60% B. The primary-amine dehydrogenase (AOC3; EC sample injection volume was 10 µl. The peaks 1.4.3.21) and aryl-alcohol dehydrogenase (AAD; of interest were monitored at 335 nm by a PDA EC 1.1.1.90). Since 4-hydroxyphenylacetaldehyde by comparing with an acteoside standard. is aromatic aldehyde and tyrosol is aromatic alcohol, ADH described with orange colored 3. Results & Discussions wave outlined box in Fig. 1A is not proper enzyme which catalyzes tyrosol from 3.1 Integrated biochemical pathways of 4-hydroxyphenylacetaldehyde. acteoside based on proposed multiple Moreover, tyrosinase (TYR) mentioned in all acteosides biochemical pathways three proposed pathways described in Fig. 1 Till now, three biochemical pathways of was changed to catechol oxidase (CO) because acteoside described in Fig. 1 have been proposed TYR gene was not found in plant genome. (here after three pathways are mentioned as To construct reasonable biochemical pathway pathway A, B, and C, corresponding for Fig. 1A, of acteoside, we identified all enzymes which 1B, and 1C [32, 45, 46]). Three pathways of were mentioned in the three proposed pathways, acteoside present critical differences among which were listed in Table 1. Some enzymes, such them: for example, process of as alcohol dehydrogenase (ADH), were used biochemical pathway in pathway C described multiple times in the three pathways. In total, we more intermediate products in comparison to identified 24 distinct enzymes from the three that of pathway B. Especially, pathway C proposed pathways, which are listed in Table 2. contains enzymes which do not exist in plant Two of them, aspartate aminotransferase, species highlighted as orange colored text in Fig. chloroplastic (ASP5) and aspartate 1C: dopamine beta monooxygenase (DBH; EC aminotransferase, mitochondrial (GOT2), have number is 1.14.17.1), which plays role to produce the same function but their subcellular locations L-noradrenaline from dopamine, does not have are different so that we considered those as any homologous gene in plant species in KEGG distinct enzyme; however, only one predicted database even though it is known that also protein of A. distichum (ADP131992.1.1) was produce dopamine from L-noradrenaline [47]. In found. During identification of 24 enzymes, we addition, pathway A contains typo of enzyme integrated two subpathways, name marked as orange color with wave outlined subpathway [51] and tyrosine-derived box in Fig. 1A: ALDH is typo of alcohol subpathway [52] referred to the pathway C: EC dehydrogenase (ADH) which produces number of trans-cinnamate 4-monooxygenase from dopamine. This kind of (C4H) mentioned in the pathway C was corrected phenomenon is natural because the proposed EC number from 1.14.13.11 to 1.14.14.91 because biochemical pathways of acteosides are not fully EC number 1.14.13.11 was deprecated in 2018. In probed by experiments like the other addition, both pathways B and C display one Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 80 ambiguous process in the last step of which pathway of acteoside became reasonable in substances are caffeic acid and hydroxytyrosol comparison to the three proposed pathways glucoside; however, we suggest that glucose of described in Fig. 1. hydroxytyrosol glucoside is originated from salidroside through catechol oxidase (CO; EC 3.2 Prediction of biochemical pathway of number is 1.10.3.1) which is replacement of acteoside from whole genome sequences polyphenol oxidase (PPO) described in pathway A of A. distichum cultivar Ok Hwang 1ho (EC number is 1.14.18.1). However, enzyme of We used the first version of gene model of A. which EC number is 1.14.18.1 is phenol oxidase distichum cultivar Ok Hwang 1ho whole and is distributed only in fungal and animal genome sequences (Park et al., in preparation). species. The result of MetaPre-AITM also presents Among 147,440 predicted genes, 33 predicted that plant genomes including A. distichum do not proteins which are corresponding to 17 contain PPO gene described in pathway A. In enzymes in the integrated pathway of acteoside addition, for construction of better quality of were identified via the MetaPre-AITM and listed acteoside biochemical pathway, we also refer to in Table 2. Usual way to identify enzymes in the KEGG pathway database [53], which is one of whole genome sequences is searching similar major databases of biochemical pathways. proteins using BLAST program which calculates Based on these results, we proposed the sequence similarity [54]. It is based on the integrated biochemical pathway of acteoside assumption that similar protein sequences may covering distinct 17 enzymes described in Fig. 2. have similar three-dimensional structure and its The integrated pathway of acteoside provides i) function will also be similar. However, there is the detailed process that phenylalanine and exceptional case, for example, Cytochrome tyrosine were generated from chorismite, ii) P450 [55-57] which involve in various incorporates the phenylpropanoid and biochemical pathways with presenting high tyrosine-derived pathways, and iii) probes the similarity of amino acid sequence to each final step that acteoside was generated from other. It causes trouble to identify correct caffeic acid and hydroxytyrosol glucoside with enzyme genes when only relying on sequence . In addition, this pathway also similarity. MetaPre-AITM contains the engine can provides correct EC numbers of each enzyme. identify correct enzyme which can cause One example of correction of EC numbers is problem, which is phylogenetic-approach prephenate dehydrogenase (PD; EC 1.3.1.12) enzyme identifier in Fig. 3. Taken together, the mentioned in the pathway C: the enzyme of 33 predicted proteins listed in Table 2 indicate which EC nunber is 1.3.1.2 was not found in A. that A. distichum cultivar Ok Hwang 1ho distichum genome sequences; instead, EC genome contains all enzymes involved in the 1.3.1.13 enzyme was found in the genome, integrated pathway of acteoside. It will be a indicating that EC 1.3.1.12 should be changed strong evidence that A. distichum cultivar Ok to EC 1.3.1.13. However, there is no clue to Hwang 1ho can synthesize acteoside as a determine the proper enzyme which produces secondary metabolite. acteoside by combining caffeic acid and hydroxy tyrosol glucoside in the last step. All steps except it in the integrated biochemical 81 융합정보논문지 제10권 제3호

3.3 Prediction of biochemical pathway of assembly and related bioinformatic analyses acteoside from whole genome have also been solved. Moreover, DNA is more stable and easier to be analyzed using computer sequences of A. distichum cultivar Ok than chemical compounds, e.g. HPLC or gas Hwang 1ho chromatography, suggesting that our approach To confirm whether acteoside exists in A. supported by MetaPre-AITM will become an distichum cultivar Ok Hwang 1ho or not, we efficient way to identify secondary metabolites chose callus for conducting high-performance from many plant species. In addition, we also liquid chromatography (HPLC; See Materials & know that suggested pathways of secondary Methods). The HPLC result depicted in Fig. 4 metabolites should be reviewed in various presents that acteoside and isoacteoside were aspects like what we did in this study. Taken clearly identified. Acteoside was already together, our result can be a first step to link TM predicted from the result of MetaPre-AI , plant genomes to its secondary metabolites. In probing that the prediction result was correct. near future, this method can be applied to In addition, isoacteoside identified together is many plant species for identifying useful isomer of acteoside, of which position of compounds efficiently and systematically. carbon of one residue is different from that of acteoside. It can be generated from the process REFERENCES of combing caffeic acids with hydroxytyrosol glucosides or be produced after accumulating [1] D. K. Kim & J. H. Kim. (2011). Molecular acteoside compound via specific enzyme. phylogeny of tribe Forsythieae (Oleaceae) based on nuclear ribosomal DNA internal transcribed However, this enzyme was not identified till spacers and plastid DNA trnL-F and matK gene now, which will be a next step for finding sequences. Journal of plant research, 124(3), proper enzyme for isoacteoside. 339-347. DOI : 10.1007/s10265-010-0383-9 [2] Y. H. Ha, C. Kim, K. Choi & J. H. Kim. (2018). 4. Conclusions Molecular phylogeny and dating of Forsythieae (Oleaceae) provide insight into the Miocene In this study, we predicted acteoside history of Eurasian temperate . Frontiers in compound from A. distichum genome analysis plant science, 9, 99. conducted by MetaPre-AITM and confirmed its DOI : 10.3389/fpls.2018.00099 existence using HPLC experiment. It suggests [3] J. Min et al. (2019). The complete chloroplast genome of a new candidate cultivar, Sang Jae, of that the new method conducted by Abeliophyllum distichum Nakai (Oleaceae): initial TM MetaPre-AI to identify useful secondary step of A. distichum intraspecies variations atlas. metabolites in plant species based on their Mitochondrial DNA Part B, 4(2), 3716-3718. DOI : 10.1080/23802359.2019.1679678 genome sequences works properly. This new [4] J. Park, Y. Kim, H. Xi, T. Jang & J. H. Park. (2019). method can be a practical candidate to improve The complete chloroplast genome of both accuracy and efficiency to identify Abeliophyllum distichum Nakai (Oleaceae), secondary metabolites from plant species. Due cultivar Ok Hwang 1ho: insights of cultivar specific variations of A. distichum. Mitochondrial to recent stabilized next generation and third DNA Part B, 4(1), 1640-1642. generation sequencing technologies [58-60], the DOI : 10.1080/23802359.2019.1605851 cost of genome sequencing project has been [5] J. Park et al. (2019). The complete chloroplast decreased as well as the difficulties of genome genome of a new candidate cultivar, Dae Ryun, of Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 82

Abeliophyllum distichum Nakai (Oleaceae). Abeliophyllum distichum Nakai in human Mitochondrial DNA Part B, 4(2), 3713-3715.DOI : colorectal cancer cells. BMC complementary and 10.1080/23802359.2019.1679676 alternative medicine, 14(1), 487. DOI : 10.1186/1472-6882-14-487 [6] H. W. Kim, H. L. Lee, D. K. Lee & K. J. Kim. (2016). Complete plastid genome sequences of [15] J. W. Lee & Y. J. Kang. (2018). Anti-inflammatory Abeliophyllum distichum Nakai (Oleaceae), a Effects of Abeliophyllum distichum Extract Korea endemic genus. Mitochondrial DNA Part B, and Associated MAPKs and NF-κB Pathway in 1(1), 596-598. Raw264. 7 Cells. Korean J Plant Res., 31(3), DOI : 10.1080/23802359.2016.1202741 202-210. DOI : 10.7732/kjpr.2018.31.3.202 [7] M. Buck & C. Hamilton. (2011). The Nagoya Protocol on access to genetic resources and the [16] J. Ahn & J. H. Park. (2013). Effects of fair and equitable sharing of benefits arising from Abeliophyllum distichum Nakai flower extracts their utilization to the Convention on Biological on antioxidative activities and inhibition of DNA Diversity. Review of European Community & damage. Korean Journal of Plant Resources, International Environmental Law, 20(1), 47-61. 26(3), 355-361. DOI : 10.1111/j.1467-9388.2011.00703.x DOI : 10.7732/kjpr.2013.26.3.355 [8] H. T. Shin, M. H. Yi, Y. S. Kim, B. C. Lee & J. W. [17] J. H. Choi et al. (2017). Polyphenolic compounds, Yoon. (2010). Recently augmented natural antioxidant and anti-inflammatory effects of habitats of Forsythia koreana (Rehder) Nakai and Abeliophyllum distichum Nakai extract. J Appl Bot Abeliophyllum distichum Nakai in Korea. Korean Food Qual, 90, 266-273. Journal of Plant , 40(4), 274-277. DOI : 10.5073/JABFQ.2017.090.033 DOI : 10.11110/kjpt.2010.40.4.274 [18] N. Y. Kim & H. Y. Lee. (2015). Effect of [9] J. H. You & C. H. Lee. (2005). Analysis on antioxidant and skin whitening of ethanol Herbaceous Communities and Flora around extracts from ultrasonic pretreated Abeliophyllum distichum Habitats. Korean Abeliophyllum distichum Nakai. Korean Journal Journal of Plant Resources, 18(2), 315-324. of Medicinal Crop Science, 23(2), 155-160. DOI : 10.7783/KJMCS.2015.23.2.155 [10] J. H. Shin, J. W. Son & J. J. Lee. (2016). A Literature review on the Aeliophyllum distichum [19] S. J. Chang, N. B. Jeon, J. W. Park, T. W. Jang, J. B. Nakai. Proc Korean Soc Environ Ecol Con, 26(1), Jeong & J. H. Park. (2018). Antioxidant activities 61. and anti-inflammatory effects of fresh and air-dried Abeliophyllum distichum Nakai leaves. [11] H. Y. Lee, T. G. Kim & C. H. Oh. (2014). Recently Korean J. Food Preserv., 25(1), 27-35. Augmented natural habitat of Abeliophyllum DOI : 10.11002/kjfp.2018.25.1.27 distichum Nakai in Yeoju-si, Gyunggi-do, Korea. Korean Journal of Environment and Ecology, [20] T. W. Jang & J. H. Park. (2017). Antioxidative 28(1), 62-70. DOI : activities and whitening effects of ethyl acetate 10.13047/KJEE.2014.28.1.62 fractions from the immature seeds of Abeliophyllum distichum. J. Life Sci, 27(5), [12] H. M. Li, J. K. Kim, J. M. Jang, C. B. Cui & S. S. 536-544. Lim. (2013). Analysis of the inhibitory activity of DOI : 10.5352/JLS.2017.27.5.536 Abeliophyllum distichum constituents against aldose reductase by using high-speed counter [21] T. W. Jang & J. H. Park. (2018). Antioxidant current chromatography. Archives of pharmacal activity and inhibitory effects on oxidative DNA research, 36(9), 1104-1112.DOI : damage of callus from Abeliophyllum distichum 10.1007/s12272-013-0127-1 Nakai. Korean Journal of Plant Resources, 31(3), 228-236. [13] H. Oh et al. (2003). Four glycosides from the DOI : 10.7732/kjpr.2018.31.3.228 leaves of Abeliophyllum distichum with inhibitory effects on angiotensin converting enzyme. [22] S. Y. Nam et al. (2015). Anti-inflammatory effects Phytotherapy Research, 17(7), 811-813. of isoacteoside from Abeliophyllum distichum. DOI : 10.1002/ptr.1199 Immunopharmacology and immunotoxicology, 37(3), 258-264. [14] G. H. Park et al. (2014). The induction of DOI : 10.3109/08923973.2015.1026604 activating transcription factor 3 (ATF3) contributes to anti-cancer activity of [23] T. W. Jang et al. (2018). Whitening activity of 83 융합정보논문지 제10권 제3호

Abeliophyllum distichum Nakai leaves according identification of genes involved in acteoside to the ratio of prethanol A in the extracts. biosynthesis. Frontiers in Plant Science, 8, 787. Korean Journal of Plant Resources, 31(6), DOI : 10.3389/fpls.2017.00787 667-674. [33] X. M. Peng, L. Gao, S. X. Huo, X. M. Liu & M. Yan. DOI : 10.7732/kjpr.2018.31.6.667 (2015). The mechanism of memory enhancement [24] F. Denoeud et al. (2014). The coffee genome of acteoside (verbascoside) in the senescent provides insight into the convergent evolution of mouse model induced by a combination of d‐gal caffeine biosynthesis. science, 345(6201), and AlCl3. Phytotherapy Research, 29(8), 1181-1184. 1137-1144. DOI : DOI : 10.1126/science.1255274 10.1002/ptr.5358 [25] E. H. Xia, H. B. Zhang, J. Sheng, K. Li, Q. J. Zhang, [34] H. Q. Wang, Y. X. Xu & C. Q. Zhu. (2012). C. Kim & H. Huang. (2017). The tea tree genome Upregulation of heme oxygenase-1 by acteoside provides insights into tea flavor and independent through ERK and PI3 K/Akt pathway confer evolution of caffeine biosynthesis. Molecular neuroprotection against beta-amyloid-induced plant, 10(6), 866-877. DOI : neurotoxicity. Neurotoxicity research, 21(4), 10.1016/j.molp.2017.04.002 368-378. DOI : 10.1007/s12640-011-9292-5 [26] P. Sarkhail et al. (2014). Quantification of verbascoside in medicinal species of Phlomis and [35] T. O. Elufioye, T. I. Berida & S. Habtemariam. their genetic relationships. DARU Journal of (2017). Plants-derived neuroprotective agents: Pharmaceutical Sciences, 22(1), 32. cutting the cycle of cell death through multiple DOI : 10.1186/2008-2231-22-32 mechanisms. Evidence-Based Complementary and Alternative Medicine, 2017. [27] J. Schlauer J. Budzianowski, K. Kukulczanka & L. DOI : 10.1155/2017/3574012 Ratajczak. (2004). Acteoside and related phenylethanoid glycosides in Byblis liniflora [36] J. H. Lee et al. (2006). The effect of acteoside on Salisb. plants propagated in vitro and its histamine release and arachidonic acid release in systematic significance. Acta Societatis RBL-2H3 mast cells. Archives of pharmacal Botanicorum Poloniae, 73(1). research, 29(6), 508. DOI : 10.5586/asbp.2004.002 DOI : 10.1007/BF02969425 [28] L. N. Gvazava, & V. S. Kikoladze. (2007). [37] K. H. Kim, S. Kim, M. Y. Jung, I. H. Ham & W. K. Verbascoside from Verbascum phlomoides. Whang. (2009). Anti-inflammatory phenylpropanoid Chemistry of Natural Compounds, 43(6), 710-711. glycosides from Clerodendron trichotomum leaves. DOI : Archives of pharmacal research, 32(1), 7-13. 10.1007/s10600-007-0240-9 DOI : 10.1007/s12272-009-1112-6 [29] L. Speranza et al. (2009). Anti-inflammatory [38] T. Ohno, M. Inoue, Y. Ogihara & I. Saracoglu. properties of the plant Verbascum mallophorum. (2002). Antimetastatic activity of acteoside, a Journal of biological regulators and homeostatic phenylethanoid glycoside. Biological and agents, 23(3), 189-195. Pharmaceutical Bulletin, 25(5), 666-668. DOI : 10.1248/bpb.25.666 [30] M. Murai, Y. Tamayama & S. Nishibe. (1995). Phenylethanoids in the Herb of Plantago [39] K. H. Kang, S. K. Jang, B. K. Kim & M. K. Park. lanceolata and Inhibitory Effect on Arachidonic (1994). Antibacterial phenylpropanoid glycosides Acid-Induced Mouse Ear Edema1. Planta Medica, from Paulownia tomentosa Steud. Archives of 61(5), 479-480. pharmacal research, 17(6), 470. DOI : 10.1055/s-2006-958143 DOI : 10.1007/BF02979128 [31] K. Chathuranga et al. (2019). Anti-respiratory [40] J. Molnar et al. (1989). Antimicrobial and syncytial virus activity of Plantago asiatica and immunomodulating effects of some phenolic Clerodendrum trichotomum extracts in vitro and glycosides. Acta Microbiologica Hungarica, 36(4), in vivo. Viruses, 11(7), 604. 425-432. DOI : 10.3390/v11070604 [41] J. Živković et al. (2014). Phenolic profile, [32] F. Wang et al. (2017). Transcriptome analysis of antibacterial, antimutagenic and antitumour salicylic acid treatment in Rehmannia glutinosa evaluation of Veronica urticifolia Jacq. journal of hairy roots using RNA-seq technique for functional foods, 9, 192-201. Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 84

DOI : 10.1016/j.jff.2014.04.024 medicinal herb Picrorhiza kurroa. Phytochemical Analysis, 24(6), 598-602. [42] P. Jones et al. (2014). InterProScan 5: DOI : 10.1002/pca.2437 genome-scale protein function classification. Bioinformatics, 30(9), 1236-1240. [51] C. M. Fraser & C. Chapple. (2011). The DOI : 10.1093/bioinformatics/btu031 phenylpropanoid pathway in Arabidopsis. The Arabidopsis Book/American Society of Plant [43] A. Dalkiran et al. (2018). ECPred: a tool for the Biologists, 9, e0152. prediction of the enzymatic functions of protein DOI : 10.1199/tab.0152 sequences based on the EC nomenclature. BMC Bioinformatics, 19(1), 1-13. [52] J. J. Xu, X. Fang, C. Y. Li, Q. Zhao, C. Martin, X. DOI : 10.1186/s12859-018-2368-y Y. Chen & L. Yang. (2018). Characterization of Arabidopsis thaliana hydroxyphenylpyruvate [44] Y. Li et al. (2018). DEEPre: sequence-based reductases in the tyrosine conversion pathway. enzyme EC number prediction by deep learning. Frontiers in plant science, 9, 1305. Bioinformatics, 34(5), 760-769. DOI : 10.3389/fpls.2018.01305 DOI : 10.1093/bioinformatics/btx680 [53] M. Kanehisa, M. Furumichi, M. Tanabe, Y. Sato & [45] A. Reyes-Martínez, J. R. Valle-Aguilera, M. K. Morishima. (2016). KEGG: new perspectives on Antunes-Ricardo, J. Gutiérrez-Uribe, C. Gonzalez genomes, pathways, diseases and drugs. Nucleic & M. del Socorro Santos-Díaz. (2019). Callus from acids research, 45(D1), D353-D361. DOI : Pyrostegia venusta (Ker Gawl.) Miers: a source of 10.1093/nar/gkw1092 phenylethanoid glycosides with vasorelaxant activities. Plant Cell, Tissue and Organ Culture [54] M. Johnson, L. Zaretskaya, I. Raytselis, Y. (PCTOC), 139(1), 119-129. Merezhuk, S. McGinnis & T. L. Madden. (2008). DOI : 10.1007/s11240-019-01669-5 NCBI BLAST: a better web interface. Nucleic acids research, 36(suppl_2), W5-W9. [46] Y. Zhou, X. Wang, W. Wang & H. Duan. (2016). DOI : 10.1093/nar/gkn201 De novo transcriptome sequencing-based discovery and expression analyses of [55] B. Siminszky. (2006). Plant cytochrome verbascoside biosynthesis-associated genes in P450-mediated herbicide metabolism. Rehmannia glutinosa tuberous roots. Molecular Phytochemistry Reviews, 5(2-3), 445-458. breeding, 36(10), 139. DOI : 10.1007/s11101-006-9011-7 DOI : 10.1007/s11032-016-0548-x [56] M. A. Schuler. (1996). Plant cytochrome P450 [47] A. Kulma & J. Szopa. (2007). Catecholamines are monooxygenases. Critical reviews in plant active compounds in plants. Plant Science, 172(3), sciences, 15(3), 235-284. 433-440. DOI : 10.1080/07352689609701942 DOI : 10.1016/j.plantsci.2006.10.013 [57] J. Park et al. (2008). Fungal cytochrome P450 [48] M. Cercós, G. Soler, D. J. Iglesias, J. Gadea, J. database. BMC genomics, 9(1), 402. Forment & M. Talón. (2006). Global analysis of DOI : 10.1186/1471-2164-9-402 gene expression during development and [58] M. L. Metzker. (2010). Sequencing technologies— ripening of citrus flesh. A proposed the next generation. Nature reviews genetics, mechanism for citric acid utilization. Plant 11(1), 31. molecular biology, 62(4-5), 513-527. DOI : 10.1038/nrg2626 DOI : 10.1007/s11103-006-9037-7 [59] S. Goodwin, J. D. McPherson & W. R. McCombie. [49] X. Li, Y. He, C. H. Ruiz, M. Koenig & M. D. (2016). Coming of age: ten years of Cameron. (2009). Characterization of dasatinib next-generation sequencing technologies. Nature and its structural analogs as CYP3A4 Reviews Genetics, 17(6), 333. mechanism-based inactivators and the proposed DOI : 10.1038/nrg.2016.49 bioactivation pathways. Drug Metabolism and Disposition, 37(6), 1242-1250. [60] C. Bleidorn. (2016). Third generation sequencing: DOI : 10.1124/dmd.108.025932 technology and its potential impact on evolutionary biodiversity research. Systematics [50] V. Kumar, H. Sood, M. Sharma & R. S. Chauhan. and biodiversity, 14(1), 1-8. (2013). A proposed biosynthetic pathway of DOI : 10.1080/14772000.2015.1099575 picrosides linked through the detection of biochemical intermediates in the endangered 85 융합정보논문지 제10권 제3호

Fig. 1. Possible three acteoside biochemical pathways Proposed pathways of acteoside were displayed as orange color with wave outline boxes which indicate incorrect information. (A) displays proposed biochemical pathway of acteoside from Figure 5 in Wang et al., 2017 [32]. (B) presents proposed biochemical pathway of acteoside from Figure 4 in Reyes-Martinez et al., 2019 [45]. (C) shows proposed biochemical pathway of acteoside from Figure 1 in Zhou et al., 2016 [46]. Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 86

Fig. 2. Biochemical pathway of Acteoside constructed from three pathways proposed previously Integrated pathway of acteoside was displayed with chemical compound structure drawed by ChemDraw. Numbers on black arrows indicate EC number of enzymes involved in the reaction. Compound names were displayed below chemical compound structure. Dotted arrows indicate that enzyme in that reaction is not identified fully. Black boxes including label starting with EC mean EC number of enzymes in each step. Labels starting with ADP indicate predicted gene names of A. distichum cultivar Ok Hwang 1ho genome corresponding to enzymes in each step. 87 융합정보논문지 제10권 제3호

Fig. 3. Structureof MetaPre-AITM Cylinder diagrams indicates databases used as input in MetaPre-AITM. Filled rectangle with dotted line means MetaPre-AITM solution used in this study. Blue rounded rectangles mean components of MetaPre-AITM which can generate intermediate results from input data. Orange-colored rounded rectangle indicates AI-based evaluator which merges all intermediate results and makes a final result. Green-colored rounded rectangle with pathway diagram displays final result of MetaPre-AITM.

Fig. 4. Identification of Acteoside and Isoacteoside by HPLC of extracts from callus of A. distichum Cultivar Ok Hwang 1ho Chromatogram indicate HPLC analysis of standard (A) and callus extracts (B). In the callus extract, the peaks of Acteoside and Isoacteoside wrere indified at the same retention time as standard, and the absorption spectrums (small graphs) of each peak were also the same. Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 88

Table 1. List of candidate enzyme genes related to possible two acteoside biochemical pathways identified from A. distichum whole genome sequences

No Enzyme name EC number Substrate Products Original pathway

Caffeic acid Caffeoyl CoA A 1 4-coumarate--CoA ligase (4CL) 6.2.1.12 Cinnamoyl-CoA C Tyramine Tyrosol A,B* 2 Alcohol dehydrogenase (ADH) 1.1.1.1 Dopamine Hydroxytyrosol A DOPAL Hydroxytyrosol B Arogenate dehydrogenase (NADP+), plant 3 1.3.1.78 L-arogenate Tyrosine C (TYRAAT) 4 Arogenate/prephenate dehydratase (ADT) 4.2.1.91 Prephenate Phenylpyrute C 4-hydroxyphenylacetalde 5 aryl-alcohol dehydrogenase (AAD) 1.1.1.90 4-hydroxyphenylethanol C hyde Aspartate aminotransferase, chloroplastic 6 2.6.1.1 Phenylpyrute Phenylalnine C (ASP5) Aspartate aminotransferase, mitochondrial 7 2.6.1.1 4-hydroxyphenylpyruvate Tyrosine C (GOT2) Aspartate-prephenateaminotransferase 8 2.6.1.79 Prephenate L-arogenate C (PAT) 5-O-Caffeoyl shikimic 9 Caffeoylshikimate esterase (CSE) 3.1.1.- Caffeic acid C acid 10 Catechol-O-methyltransferase (COMT) 2.1.1.6 Caffeic acid B 11 Chorismate mutase (CM) 5.4.99.5 Chorismate Prephenate C 4-coumaric acid Caffeic acid A, B Coumaroylquinate (coumaroylshikimate) 12 1.14.14.96 5-O-Caffeoyl shikimic 3′-monooxygenase(C3H) 4-Coumaroyl shikimate C acid 13 Cyclohexadienyl dehydratase (Phe) 4.2.1.51 L-arogenate Phenylalanine C 14 Dopamine betamonooxygenase (DBH) 1.14.17.1 Dopamine L-noradrenaline C 15 Monoamine oxidase (MAO) 1.4.3.4 Dopamine DOPAL B 16 Phenylalanine ammonia-lyase (PAL) 4.3.1.24 L-phenylalanine 4-cinnamic acid A, B, C 17 Prephenate dehydrogenase (PD) 1.3.1.13 Prephenate 4-hydroxyphenylpyruvate C Tyramine, 4-hydroxyphenylacetalde 18 Primary-amine oxidase (AOC3) 1.4.3.21 C 4-hydroxyphenylpyruvate hyde

19 Dopamine Hydroxytyrosol A Primary-amine oxidase (CuAO) 1.4.3.21 Tyramine Tyrosol A. B Caffeoyl CoA, Shikimate O-hydroxycinnamoyltransferase Acteoside A 20 2.3.1.133 Hydroxytyrosol glucoside (HCT) p-Coumaroyl-CoA 4-coumaroyl shikimate C

Trans-cinnamate 4-monooxygenase 4-cinnamic acid 4-coumaric acid A, B 21 1.14.14.91 (C4H) Cinnamoyl-CoA p-Coumaroyl-CoA C Hydropytyrosol Salidroside A glucoside

22 Tyrosinase (TYR) 1.14.18.1 Tyrosine DOPA A, B, C Tyramine Dopamine A Tyrosol Hydroxytyrosol A Tyrosine Tyramine A, B, C 23 Tyrosine decarboxylase (TyDC) 4.1.1.25 DOPA Dopamine A, B Tyrosol Salidroside A, B Hydroxytyrosol UDP-glucose:(glucosyl) LPS Hydroxytyrosol A 24 2.4.1.- glucoside alpha-1,2-glucosyltransferase (UGT) Caffeoyl CoA, Acteoside A Hydroxytyrosol glucoside 89 융합정보논문지 제10권 제3호

Table 2. List of enzymes identified from the integrated pathway of acteoside

No Enzyme name EC number Predicted gene

1 Chorismate mutase (CM) 5.4.99.5 ADP088311.1.1 ADP067318.1.1 2 Arogenate/prephenate dehydratase (ADT) 4.2.1.91 ADP090928.1.1 3 Aspartate-prephenateaminotransferase (PAT) 2.6.1.79 ADP087260.1.1 4 Aspartate aminotransferase, chloroplastic (ASP5) 2.6.1.1 ADP131992.1.1 5 Aspartate aminotransferase, mitochondrial (GOT2) 2.6.1.1 ADP131992.1.1 6 Prephenate dehydrogenase (PD) 1.3.1.13 ADP033243.1.1 ADP048895.1.1 7 Cyclohexadienyl dehydratase (Phe) 4.2.1.51 ADP000570.1.1 8 Arogenate dehydrogenase (NADP+), plant (TYRAAT) 1.3.1.78 ADP059693.1.1 9 Phenylalanine ammonia-lyase (PAL) 4.3.1.24 ADP115622.1.1 ADP047556.1.1 10 Catechol oxidase (CO) 1.10.3.1 ADP097461.1.1 11 Tyrosine decarboxylase (TyDC) 4.1.1.25 ADP042753.1.1 ADP143827.1.1 ADP143816.1.1 12 Trans-cinnamate 4-monooxygenase (C4H) 1.14.14.91 ADP098858.1.1 ADP074900.1.1 ADP094567.1.1 13 Primary-amine oxidase (CuAO) 1.4.3.21 ADP135933.1.1 ADP035792.1.1 14 Coumaroylquinate (coumaroylshikimate) 3′-monooxygenase(C3H) 1.14.14.96 ADP104543.1.1 ADP007159.1.1 15 Alcohol dehydrogenase (ADH) 1.1.1.1 ADP068307.1.1 ADP041117.1.1 ADP073020.1.1 ADP075198.1.1 16 Aryl-alcohol dehydrogenase (AAD) 1.1.1.90 ADP034058.1.1 ADP062724.1.1 ADP135289.1.1

17 2.4.1.- ADP077969.1.1 UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase (UGT) ADP000154.1.1 ADP032128.1.1 18 4-coumarate-CoA ligase (4CL) 6.2.1.12 ADP056394.1.1 19 Caffeoylshikimate esterase (CSE) 3.1.1.- ADP144392.1.1 ADP013856.1.1 ADP001950.1.1 20 Catechol-O-methyltransferase (COMT) 2.1.1.6 ADP058351.1.1 ADP086664.1.1 ADP099608.1.1 21 Dopamine betamonooxygenase (DBH) 1.14.17.1 N/A 22 Monoamine oxidase (MAO) 1.4.3.4 ADP086112.1.1 ADP110810.1.1 23 Shikimate O-hydroxycinnamoyltransferase (HCT) 2.3.1.133 ADP090962.1.1 24 Tyrosinase (TYR) 1.14.18.1 N/A Prediction and Identification of Biochemical Pathway of Acteoside from Whole Genome Sequences of Abeliophyllum Distichum Nakai, Cultivar Ok Hwang 1ho 90

박 재 호(Jaeho Park) [정회원] 김 용 성(Yongsung Kim) [정회원] ․ 1998년 2월 : 안동대학교 자원식물 ․ 2012년 2월 : 성균관대학교 생명 학과(농학사) 과학과(이학사) ․ 2000년 2월 : 안동대학교 생물학과 ․ 2014년 8월 : 성균관대학교 생명 (이학석사) 과학과(이학석사) ․ 2004년 2월 : 안동대학교 생물학과 ․ 2017년 2월 ∼ 현재 : 성균관대학교 (이학박사) 생명과학과 박사과정

․ 2010년 3월 ∼ 현재 : 중원대학교 제약공학과 교수 ․ 2015년 5월 ∼ 현재 : 인포보스(주) 생물다양성팀 팀장 ․ 관심분야 : 약용식물학, 천연물소재학 ․ 관심분야 : 식물분류학 ․ E-Mail : [email protected] ․ E-Mail : [email protected]

시 홍(Hong Xi) [정회원] 이 준 미(Jun-mi Lee) [정회원] ․ 1995년 2월 : 중국교통대학교 ․ International Economy and 2020년 2월 : 서울대학교 응용생 Trade, Biotechnology (이학사) 물화학부(농학사, 졸업예정) ․ ․ 1997년 2월 : 중국교통대학교 생물 2019년 10월 ~ 현재: Infoboss 정보학 (석사) 인턴 ․ ․ 1997년 2월 : 중국교통대학교 생물 관심분야 : 생화학 ․ 정보학 (박사) E-Mail : [email protected]

․ 1997년 2월 : 인포보스(주) 선임연구원 ․ 관심분야 : 생물정보학 손 장 혁(Janghyuk Son) [정회원] ․ E-Mail : [email protected] ․ 2015년 8월 : 창업진흥원 기업멘토 한 지 윤(Jiyun Han) [정회원] ․ 2016년 6월 : 창업진흥원 아이디어 마루 멘토 ․ 2020년 2월 : 서울대학교 응용생물 ․ 2016년 4월 : 서울산업진흥원 인재 화학부(농학사, 졸업예정) 추천단 ․ 2020년 1월 ∼ 현재 : 인포보스(주) ․ 2017년 2월 : 고려대학교 경제학 석사 인턴 ․ 관심분야 : 생화학, 면역학, 미생물학 ․ 2019년 12월 ∼ 현재 : 인포보스(주) 공동대표 ․ E-Mail : [email protected] ․ 관심분야 : 정보처리 및 IT, 사업계획 타당성분석 지적재산권 ․ E-Mail : [email protected]

이 정 민(Jeongmin Lee) [정회원] 안 정 좌(Joungjwa Ahn) [정회원] ․ 2020년 2월 : 서울대학교 응용생 ․ 1987년 2월 : 서울대학교 지구과학 물화학부(농학사, 졸업예정) 교육과(이학사) ․ 2020년 1월 ∼ 현재 : 인포보스(주) ․ 1991년 5월 : 캔사스주립대 식품영 인턴 양학과 (이학석사) ․ 관심분야 : 유기화학, 미생물학, 세 ․ 1994년 12월 : 캔사스주립대 식품 포생물학 영양학과 (이학박사) ․ E-Mail : [email protected] ․ 2009년 3월 ~ 현재 : 중원대학교 식품공학과 교수 ․ 관심분야 : 기능성식품, 식품가공 ․ E-mail : [email protected] 91 융합정보논문지 제10권 제3호

장 태 원(Taewon Jang) [정회원]

․ 2015년 2월 : 중원대학교 생약자원 개발학과 (이학사) ․ 2017년 2월 : 중원대학교 생약자원 개발학과 (이학석사) ․ 2017년 9월 ∼ 현재 : 안동대학교 생약자원학 박사과정

․ 관심분야 : 세포생물학, 분자생물학, 분석화학 ․ E-Mail : [email protected]

최 지 수(Jisoo Choi) [정회원] ․ 2018년 2월 : 중원대학교 생약자원 개발학과 (이학사) ․ 2020년 2월 : 중원대학교 생약자원 개발학과 (이학석사) ․ 관심분야 : 세포생물학, 분자생물학, 분석화학 ․ E-Mail : [email protected]

박 종 선(Jongsun Park) [정회원]

․ 2006년 8월 : 서울대학교 농업생명 과학대학 응용생물화학부, 공과대 학 컴퓨터공학부 학사 ․ 2010년 8월 : 서울대학교 농업생명 과학대학 협동과정 농업생물공학 박사

․ 2015년 5월 ∼ 현재 : 인포보스(주) 공동대표 ․ 관심분야 : 생물정보학 ․ E-Mail : [email protected]