Synthetic Biology Of Plant Specialised Metabolism Using NGS Information Of Non-Model Medicinal Plants
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Presented at Plant Genomics and Gene Editing Congress: Asia. For more information visit: www.global-engage.com Synthetic biology is powerful strategy to reconstruct biosynthetic pathways when molecular tools are available. Here, we report the potentials of this strategy as a case study in isoquinoline alkaloid biosynthesis. So far, we have characterized biosynthetic enzyme genes of specialized metabolism using a combination of transcriptome and metabolome. In this presentation, identification of several biosynthetic enzyme genes and production of some metabolites are discussed
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Synthetic Biology Of Plant Specialised Metabolism Using NGS Information Of Non-Model Medicinal Plants
- 1. Synthe'c biology of plant specialized metabolism using NGS informa'on of non-‐model medicinal plants Fumihiko Sato (Lab. Mol. Cell. Biol. To/potency, Grad. Sch. Biostudies, Kyoto University) Sanguinarine Rob Bertholf/ Flickr (CC BY 2.0) from California is burs/ng with flower hMp://www.sciencemag.org/news/siOer/california-‐burs/ng-‐flowers Plant Genomics & Gene Edi/ng, Asia Congress, Hong Kong, 11th April, 2017
- 2. Prehistory of synthetic biology of benzylisoquinoline alkaloid (BIA) biosynthesis tyrosine (S)-‐norcoclaurine (S)-‐re/culine salutaridine (S)-‐scoulerine dihydromacarpine dihydrosanguinarine Protopine (poppy; Histamine H1 antagonist) Morphine (opium poppy; narcotic) Magnoflorine (Coptis etc; anti- HIV, NSC150443) Berberine（Antimicrobial, antidiarrheal） Sanguinarine (poppy; antimicrobial)macarpine escholtzine TAT HPDC PO TDC NCS 6OMT CNMT MCH 4’OMT RE SAS SalR SalAT T6ODM COR CODM BBE SMT CAS THBO CHS STS TNMT MSH P6H P450 P450 OMT OMT CYP80G2 CNMT Coptis japonica Papaver somniferum California poppy Eschscholzia californica P450s DBOX DBOX
- 3. Major BIQ biosynthetic enzymes have been characterized in 1990s, and past 20-30 years major enzyme genes were isolated. ＊ ＊ ＊ ＊ ＊ High berberine producing C. japonica cells Protoberberine type alkaloids Benzophenanthridine type alkaloids Choi et al., J.B.C., 2002 Morishige et al., J.B.C., 2000 Morishige et al., J.B.C., 2000 Takeshita et al., P.C.P., 1995 Ikezawa et al., J.B.C., 2003 Gesell et al. Planta 2011 Matsushima et al., Plant Biotech. 2012 Ikezawa et al., J.B.C. 2008 Cis-‐NMT CYP719A14 CYP719B1 Pauli and Kutchan 1998 Ikezawa et al., J.B.C., 2003 Dittrich and Kutchan 1991 Minami et al. PNAS 2008 Gesell et al., J.B.C., 2009 Grothe et al., J.B.C., 2001 Hagel and Facchini Nat Chem Biol 2010 Hagel and Facchini Nat Chem Biol 2010 CODM Samanani et al. Plant J 2004 Minami et al., J.B.C., 2007 Lee and Facchini Plant Cell 2010 Unterlinn er et al. Plant J. 1999 CODM T6ODM T6ODM Ziegler et al., Plant J. 2006 Ikezawa et al. FEBS J 2007 PCR 2009 EcCYP719A5 Kraus & Kutchan 1995 Morishige et al., E. J. B. 2002 Liscombe and Facchini, JBC 2007 Takemura et al. Phytochem. 2013 P6H STORR Farrow et al. Nat. Chem Biol. 2015, Winzer et al. Sci. 2015 Enzyme purification/isolation of cDNA, Transcriptome analysis of specialized cell Recombinant expression
- 4. (S)-‐N-‐methylstylopine (S)-‐scoulerine (S)-‐tetrahydrocolumbamine berberine (S)-‐cheilanthiforine (S)-‐stylopine A: protopine (S)-‐tetrahydroberberine (S)-‐N-‐methylTHB B: allocryptopine 6-‐hydroxy-‐protopine 6-‐hydroxy-‐allocryptopine CYP719A3 CYP719A2 CYP719A5 CYP82N2v2 CjSMT CjSMT CYP719A5 CYP719A17 CYP719A2 Expression of genes except CjSMT were ploMed as reverse number. CYP719A3 products Ec cell lines CYP82N2v2 Integrated analysis of metabolome and transcriptome enabled the gene identification columbamine 4 Takemura et al. (2013) Phytochem. 91:100
- 5. Synthetic biology and reconstruction of BIA biosynthesis in microbes substrate product yield reference dopamine re/culine 55 mg/L Minami et al., 2008 glucose re/culine 46 mg/L Nakagawa et al., 2011 glycerol thebaine 2.1 mg/L Nakagawa et al., 2016 nor-‐ laudanosoline dihydro-‐ sanguinarine 43 μg/L Fossa/ et al., 2014 nor-‐ laudanosoline stylopine 676 μg/L Trenchard and Smolke, 2015 glucose thebaine 7.7 μg/L Galanie et al., 2015 E. coli Yeast Increased needs for enzyme genes !!!
- 6. Step1: Fermentive production of reticuline, key intermediate from low price substrates (collaboration with Dr. H. Minami group at Ishikawa Pref. Univ.) Nakagawa et al. (2011) Nature Commun. 2, Art. No. 326 Glucose Pentose phosphate pathway Glycolysis TKT PEPS E4P PEPPr-‐DAHPS DAHP Chorismate HPPPr-‐CM/PDH Dopamine 3,4-‐DHPAA L-‐DOPA L-‐Tyrosine TYRDODC MAO NCS 6OMT CNMT 4’OMT (S)-‐Norlaudano-‐ soline (S)-‐3’-‐Hydroxy-‐ coclaurine (S)-‐3’-‐Hydroxy-‐N-‐ methylcoclaurine(S)-‐Re'culine TAT 5 genes in 2 plasmids 11 genes in 3 plasmids from simple substrate E. coli 46 mg/L
- 7. Step2; Production of more complex and desired chemicals such as thebaine (Nakagawa et al. 2016 Nature Comm. 7:10390) key1; Improvement of reticuline production with the combination of three E. coli cells. (Nakagawa et al. 2014 Sci. Rep. 4: 6695) Key2: Successful expression of SalS (CYP719B1) in E. coli and stepwise conversion of chemicals using multiple E. coli cells (R,S)NLS (R,S)-‐NLS producer Key success Expression of SalS in E. coli
- 8. Step3; Easy reconstruction BIA biosynthesis using co-culture of recombinant Pichia yeast; Flexibility, Robustness, Efficiency （from hMp://bioinforma/cs.psb.ugent.be/genomes/） Pichia pastoris Ｃ Ｃ Ｃ Ｃ Ｃ BBE CYP719A5 CYP719A2/A3 (S)-‐re/culine (S)-‐scoulerine (S)-‐cheilanthifoline (S)-‐stylopine (S)-‐cop/sine case study in stylopine biosynthesis oxida/on Co-‐culture construct All-‐in-‐one (consolidated) construct Dr. Kentaro Hori Hori et al. (2016) Sci. Rep. 6: 22201
- 9. Stylopine production by all-in-one (B52) Pichia cells BBE CYP719A5 CYP719A2 B52 株 re/culine（substrate） BBE CYP719A5 CYP719A2 B52 cells stylopine cop/sine scoulerine cheilanthifoline medium cell extract Total (medium + cell extract) Rapid conversion of substrate by all-in-one cells (n=3, ± SE) Hori et al. (2016) Sci. Rep. 6: 22201
- 10. Stylopine production by co-culture of Pichia with each enzyme gene (HB+HA5+HA2) Longer incubation, but still high conversion was achieved as all-in-one cells. HB 株 BBE HA5 株 CYP719A5 HA2 株 CYP719A2 re/culine（substrate） HB cell BBE HA5 cell CYP719A5 HA2 cell CYP719A2 scoulerine cheilanthifoline stylopine cop/sine Medium Cell extract Total (medium+cell extract) (n=3, ± SE) Hori et al. (2016) Sci. Rep. 6: 22201
- 11. Potential advantage of co-culture system B52 （All-‐in-‐one） Hidden weakness of all-in-one strategy; interference by products might reduce the production at the feeding of substrate Co-culture system would be slow reaction but tolerant for continuous production at the feeding to products due to the isolation of reactions (n=3, ± SE) (n=3, ± SE) HB, HA5, HA2 （co-‐culture） Hori et al. (2016) Sci. Rep. 6: 22201
- 12. hMps://shop.takii.co.jp/CGI/shop/search/ image_loader.cgi? item_code=FHN110&type=1 ） *Scaffolds >1 kb * Step4; Mining of uncharacterized biosynthetic enzyme genes from the draft genome sequence DraO genome sequence of Eschscholzia californica cv Hitoezaki of 467 Mb (ca 90% of 502 Mb genome) was obtained by the support of MEXT KAKENHI (no. 221S0002). *data type: 454 GS FLX Titanium and, **data type: Illumina GAII are from PhytoMetaSyn (hMp://www.phytometasyn.ca/) Hori et al. unpublished data
- 13. Identification of all known biosynthetic enzyme genes in draft genome Query Accession number Func'on Loca'on re'culine biosynthesis Ec6OMT BAM37634.1 puta/ve norcoclaurine 6-‐O-‐methyltransferase scaffold194712 CjCNMT BAB71802.1 coclaurine N-‐methyltransferase scaffold11375 EcCYP80B1 AAC39453.1 (S)-‐N-‐methylcoclaurine 3'-‐hydroxylase scaffold252 Ec4'OMT BAM37633.1 3'-‐hydroxy-‐N-‐methylcoclaurine-‐ 4'-‐O-‐methyltransferase scaffold7259 sanguinarine biosynthesis EcBBE AAC39358.1 berberine bridge enzyme scaffold194677 scaffold194509 EcCYP719A5 BAG75113.1 cheilanthifoline synthase scaffold1365 EcCYP719A2 ACO90219.1 stylopine synthase scaffold194371 EcCYP719A3 BAD98249.1 stylopine synthase scaffold5603 EcTNMT ACO90222.1 tetrahydroprotoberberine N-‐ methyltransferase scaffold31885 EcCYP82N5 -‐ (S)-‐N-‐methylstylopine 14-‐ hydroxylase scaffold1139 EcCYP82N2v2 BAK20464.1 protopine 6-‐hydroxylase scaffold194758 EcSARED1 ADE41047.1 sanguinarine reductase scaffold3017 Hori et al. unpublished data
- 14. dihydromacarpine magnoflorine tylosine (S)-‐norcoclaurine salutaridine (S)-‐scoulerine dihydrosanguinarine morphine sanguinarine macarpine escholtzine TAT HPDC PO TDC NCS 6OMT CNMT MCH (CYP80B1) 4’OMT RE SAS (CYP719B1) SalR SalAT T6ODM COR CODM BBE SMT CAS (CYP719A1) THBO CHS(CYP719A5) STS(CYP719A2/A3) TNMT MSH(CYP82N5) P6H (CYP82N2v2) P450 P450 OMT OMT CYP80G2 CNMT Coptis japonica Eschscholzia californica P450s (S)-‐re/culine berberine protopine DBOX DBOX A. thaliana E. californica CYP80 0 2 CYP82 5 27 CYP719 0 6 Abundant presence of P450 genes of BIA biosynthesis in genome Hori et al. unpublished data
- 15. Expression analysis suggests some P450 candidates in macarpine biosynthesis MH cells ML cells MS intensity (×106) MS intensity (×106) TIC TIC m/z 348 m/z 354 m/z 364 m/z 370 m/z 362 m/z 364 m/z 392 reten/on /me (min) reten/on /me (min) Hori et al. unpublished data (n=3, ±SD, student’s t-‐test, * : p < 0.05, ** : p < 0.01) 1 2 3 4 3 5 6 macarpine 1: allocryptopine 2: protopine 3: 10-‐hydroxychelerythrine 4: chelerythrine 5: chelirubine 6: macarpine
- 16. Co-culture of yeast cells expressing CYP82-3 and G11-OMT confirmed 10-hydroxylase activity of CYP82-3 G11-‐OMT CYP82-‐3 Hori et al. unpublished data
- 17. Further characterization identified another P450 with similar hydroxylase activities but different specificity CYP82-‐3 G11-‐OMT ? CYP82-‐1L CYP82-‐1 like dihydrosanguinarine/ dihydrochelerythrine 10-‐hydroxylase CYP82-‐3 dihydrosanguinarine 10-‐hydroxylase dihydromacarpine 10-‐hydroxydihydrochelerythrine dihydrochelerythrine dihydrosanguinarine 10-‐hydroxydihydrosanguinarine dihydrochelerubine Hori et al. unpublished data
- 18. Step5: Beyond NGS Sequence itself did not show enzyme activities. We need more biochemical characterization to get gold enzyme. Purwanto et al. unpublished data G3/7OMT G3 G3/ 7OMT G3 SOMT S-‐scoulerine S-‐re/tuline
- 19. Take home message • Synthetic biology is powerful to reconstruct biosynthesis. • Genome sequence obtained by NGS would be useful platform to isolate novel candidates in biosynthesis. • But, sequence itself is not sufficient to predict the enzyme function. • More accumulation of biochemical data is needed. 19
- 20. Acknowledgments BIQ biosynthetic pathway and genes Dr. Yasuyuki Yamada (genome mining, TFs) Dr. Kentaro Hori (genome mining, P450) Mr. Purwanto (genome mining, OMTs) Synthetic biology Dr. K. Hori, Mr. Purwanto Dr. Hiromichi Minami (Ishikawa prefectural University) ＊Grant-in-Aid for Scientific Research from JSPS * Grant-in-Aid for Scientific Research on Innovative Areas from JSPS Collaborators Chemical analysis Dr. Kinuko Iwasa (Kobe Phamaceu. College) Genome sequencing Dr. Atsushi Toyota (NIG) Mitsui Petrochem. Inc. (alkaloids) Takeda Chem. (Coptis plants), Dr. Yabugasaki (pGYR), Dr. Mizutani (P450 spectra analysis) 20 Dr. Yamada Dr. Hori Mr. Purwanto Dr. Minami
- 21. Thank you for your attention 21