{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T04:18:52Z","timestamp":1790482732807,"version":"4.1.0"},"reference-count":39,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2016,2,5]],"date-time":"2016-02-05T00:00:00Z","timestamp":1454630400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2016,2,5]],"date-time":"2016-02-05T00:00:00Z","timestamp":1454630400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Nat Commun"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1\u2009mg\u2009l\n                    <jats:sup>\u22121<\/jats:sup>\n                    from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (\n                    <jats:italic>R<\/jats:italic>\n                    )-reticuline in\n                    <jats:italic>E. coli<\/jats:italic>\n                    . Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this\n                    <jats:italic>E. coli<\/jats:italic>\n                    system represent a major step towards the development of alternative opiate production systems.\n                  <\/jats:p>","DOI":"10.1038\/ncomms10390","type":"journal-article","created":{"date-parts":[[2016,2,5]],"date-time":"2016-02-05T05:05:14Z","timestamp":1454648714000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":195,"title":["Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli"],"prefix":"10.1038","volume":"7","author":[{"given":"Akira","family":"Nakagawa","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eitaro","family":"Matsumura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Koyanagi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takane","family":"Katayama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noriaki","family":"Kawano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kayo","family":"Yoshimatsu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenji","family":"Yamamoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hidehiko","family":"Kumagai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fumihiko","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiromichi","family":"Minami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,2,5]]},"reference":[{"key":"BFncomms10390_CR1","doi-asserted-by":"publisher","first-page":"1559","DOI":"10.1038\/nbt1033","volume":"22","author":"RS Allen","year":"2004","unstructured":"Allen, R. S. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nature Biotechnol. 22, 1559\u20131566 (2004).","journal-title":"Nature Biotechnol."},{"key":"BFncomms10390_CR2","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1016\/j.ymben.2006.10.004","volume":"9","author":"S Frick","year":"2007","unstructured":"Frick, S., Kramell, R. & Kutchan, T. M. Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding. Metab. Eng. 9, 169\u2013176 (2007).","journal-title":"Metab. Eng."},{"key":"BFncomms10390_CR3","doi-asserted-by":"publisher","first-page":"252","DOI":"10.1093\/pcp\/pcl062","volume":"48","author":"T Inui","year":"2007","unstructured":"Inui, T., Tamura, K., Fujii, N., Morishige, T. & Sato, F. Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in Benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol. 48, 252\u2013262 (2007).","journal-title":"Plant Cell Physiol."},{"key":"BFncomms10390_CR4","doi-asserted-by":"publisher","first-page":"1380","DOI":"10.1021\/ja01588a033","volume":"78","author":"M Gates","year":"1956","unstructured":"Gates, M. & Tschudi, G. The synthesis of morphine. J. Am. Chem. Soc. 78, 1380\u20131393 (1956).","journal-title":"J. Am. Chem. Soc."},{"key":"BFncomms10390_CR5","doi-asserted-by":"publisher","first-page":"326","DOI":"10.1038\/ncomms1327","volume":"2","author":"A Nakagawa","year":"2011","unstructured":"Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. 2, 326 (2011).","journal-title":"Nat. Commun."},{"key":"BFncomms10390_CR6","doi-asserted-by":"publisher","first-page":"465","DOI":"10.1038\/nchembio.1816","volume":"11","author":"WC DeLoache","year":"2015","unstructured":"DeLoache, W. C. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 11, 465\u2013471 (2015).","journal-title":"Nat. Chem. Biol."},{"key":"BFncomms10390_CR7","doi-asserted-by":"publisher","first-page":"e0124459","DOI":"10.1371\/journal.pone.0124459","volume":"10","author":"E Fossati","year":"2015","unstructured":"Fossati, E., Narcross, L., Ekins, A., Falgueyret, J. P. & Martin, V. J. Synthesis of morphinan alkaloids in Saccharomyces cerevisiae. PLoS ONE 10, e0124459 (2015).","journal-title":"PLoS ONE"},{"key":"BFncomms10390_CR8","doi-asserted-by":"publisher","first-page":"1095","DOI":"10.1126\/science.aac9373","volume":"349","author":"S Galanie","year":"2015","unstructured":"Galanie, S., Thodey, K., Trenchard, I. J., Filsinger Interrante, M. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095\u20131100 (2015).","journal-title":"Science"},{"key":"BFncomms10390_CR9","first-page":"1152","volume":"33","author":"I Shiio","year":"1969","unstructured":"Shiio, I. & Nakamori, S. Microbial production of L-threonine part I.Production by Escherichia coli mutant resistant to \u03b1-amino-\u03b2-hydroxyvaleric acid. Agric. Biol. Chem. 33, 1152\u20131160 (1969).","journal-title":"Agric. Biol. Chem."},{"key":"BFncomms10390_CR10","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1007\/s00253-006-0792-9","volume":"75","author":"T L\u00fctke-Eversloh","year":"2007","unstructured":"L\u00fctke-Eversloh, T. & Stephanopoulos, G. L-tyrosine production by deregulated strains of Escherichia coli. Appl. Microbiol. Biotechnol. 75, 103\u2013110 (2007).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"BFncomms10390_CR11","doi-asserted-by":"publisher","first-page":"603","DOI":"10.1016\/j.ymben.2012.08.002","volume":"14","author":"Y Satoh","year":"2012","unstructured":"Satoh, Y., Tajima, K., Munekata, M., Keasling, J. D. & Lee, T. S. Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol. Metab. Eng. 14, 603\u2013610 (2012).","journal-title":"Metab. Eng."},{"key":"BFncomms10390_CR12","doi-asserted-by":"publisher","first-page":"6695","DOI":"10.1038\/srep06695","volume":"4","author":"A Nakagawa","year":"2014","unstructured":"Nakagawa, A. et al. (R,S)-Tetrahydropapaveroline production by stepwise fermentation using engineered Escherichia coli. Sci. Rep. 4, 6695 (2014).","journal-title":"Sci. Rep."},{"key":"BFncomms10390_CR13","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1016\/j.ymben.2015.06.010","volume":"31","author":"IJ Trenchard","year":"2015","unstructured":"Trenchard, I. J., Siddiqui, M. S., Thodey, K. & Smolke, C. D. De novo production of the key branch point benzylisoquinoline alkaloid reticuline in yeast. Metab. Eng. 31, 74\u201383 (2015).","journal-title":"Metab. Eng."},{"key":"BFncomms10390_CR14","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1186\/s12934-015-0252-2","volume":"14","author":"ND Gold","year":"2015","unstructured":"Gold, N. D. et al. Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics. Microb. Cell Fact. 14, 73 (2015).","journal-title":"Microb. Cell Fact."},{"key":"BFncomms10390_CR15","first-page":"49","volume":"3","author":"A Nakagawa","year":"2012","unstructured":"Nakagawa, A. et al. Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform. Bioeng. Bugs 3, 49\u201353 (2012).","journal-title":"Bioeng. Bugs"},{"key":"BFncomms10390_CR16","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1111\/j.1432-1033.1994.00125.x","volume":"225","author":"F Sato","year":"1994","unstructured":"Sato, F., Tsujita, T., Katagiri, Y., Yoshida, S. & Yamada, Y. Purification and characterization of S-adenosyl-L-methionine: norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 225, 125\u2013131 (1994).","journal-title":"Eur. J. Biochem."},{"key":"BFncomms10390_CR17","doi-asserted-by":"publisher","first-page":"830","DOI":"10.1074\/jbc.M106405200","volume":"277","author":"KB Choi","year":"2002","unstructured":"Choi, K. B., Morishige, T., Shitan, N., Yazaki, K. & Sato, F. Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem. 277, 830\u2013835 (2002).","journal-title":"J. Biol. Chem."},{"key":"BFncomms10390_CR18","doi-asserted-by":"publisher","first-page":"23398","DOI":"10.1074\/jbc.M002439200","volume":"275","author":"T Morishige","year":"2000","unstructured":"Morishige, T., Tsujita, T., Yamada, Y. & Sato, F. Molecular characterization of the S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275, 23398\u201323405 (2000).","journal-title":"J. Biol. Chem."},{"key":"BFncomms10390_CR19","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1126\/science.aab1852","volume":"349","author":"T Winzer","year":"2015","unstructured":"Winzer, T. et al. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein. Science 349, 309\u2013312 (2015).","journal-title":"Science"},{"key":"BFncomms10390_CR20","doi-asserted-by":"publisher","first-page":"728","DOI":"10.1038\/nchembio.1879","volume":"11","author":"SC Farrow","year":"2015","unstructured":"Farrow, S. C., Hagel, J. M., Beaudoin, G. A., Burns, D. C. & Facchini, P. J. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Biol. 11, 728\u2013732 (2015).","journal-title":"Nat. Chem. Biol."},{"key":"BFncomms10390_CR21","doi-asserted-by":"publisher","first-page":"310","DOI":"10.1006\/prep.1999.1195","volume":"18","author":"AK Hull","year":"2000","unstructured":"Hull, A. K. & Celenza, J. L. Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2. Protein Expr. Purif. 18, 310\u2013315 (2000).","journal-title":"Protein Expr. Purif."},{"key":"BFncomms10390_CR22","doi-asserted-by":"publisher","first-page":"325","DOI":"10.1007\/s00449-012-0787-0","volume":"36","author":"S Park","year":"2013","unstructured":"Park, S., Kim, Y. S., Rupasinghe, S. G., Schuler, M. A. & Back, K. Rice P450 reductases differentially affect P450-mediated metabolism in bacterial expression systems. Bioprocess Biosyst. Eng. 36, 325\u2013331 (2013).","journal-title":"Bioprocess Biosyst. Eng."},{"key":"BFncomms10390_CR23","doi-asserted-by":"publisher","first-page":"1387","DOI":"10.1007\/s00253-010-2994-4","volume":"89","author":"S Park","year":"2011","unstructured":"Park, S. et al. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli. Appl. Microbiol. Biotechnol. 89, 1387\u20131394 (2011).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"BFncomms10390_CR24","doi-asserted-by":"publisher","first-page":"6828","DOI":"10.1128\/AEM.01623-14","volume":"80","author":"Y Miki","year":"2014","unstructured":"Miki, Y. & Asano, Y. Biosynthetic pathway for the cyanide-free production of phenylacetonitrile in Escherichia coli by utilizing plant cytochrome P450 79A2 and bacterial aldoxime dehydratase. Appl. Environ. Microbiol. 80, 6828\u20136836 (2014).","journal-title":"Appl. Environ. Microbiol."},{"key":"BFncomms10390_CR25","doi-asserted-by":"publisher","first-page":"117","DOI":"10.1016\/S0960-0760(97)80002-7","volume":"61","author":"Y Osawa","year":"1997","unstructured":"Osawa, Y., Higashiyama, T., Toma, Y. & Yarborough, C. Diverse function of aromatase and the N-terminal sequence deleted form. J. Steroid Biochem. Mol. Biol. 61, 117\u2013126 (1997).","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"BFncomms10390_CR26","doi-asserted-by":"publisher","first-page":"33","DOI":"10.5487\/TR.2014.30.1.033","volume":"30","author":"HG Park","year":"2014","unstructured":"Park, H. G., Lim, Y. R., Han, S. & Kim, D. Expression and characterization of truncated recombinant human cytochrome P450 2J2. Toxicol. Res. 30, 33\u201338 (2014).","journal-title":"Toxicol. Res."},{"key":"BFncomms10390_CR27","doi-asserted-by":"publisher","first-page":"540","DOI":"10.1006\/abbi.1995.1329","volume":"319","author":"EM Gillam","year":"1995","unstructured":"Gillam, E. M., Guo, Z., Martin, M. V., Jenkins, C. M. & Guengerich, F. P. Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 319, 540\u2013550 (1995).","journal-title":"Arch. Biochem. Biophys."},{"key":"BFncomms10390_CR28","doi-asserted-by":"publisher","first-page":"24432","DOI":"10.1074\/jbc.M109.033373","volume":"284","author":"A Gesell","year":"2009","unstructured":"Gesell, A. et al. CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J. Biol. Chem. 284, 24432\u201324442 (2009).","journal-title":"J. Biol. Chem."},{"key":"BFncomms10390_CR29","doi-asserted-by":"publisher","first-page":"24425","DOI":"10.1074\/jbc.M109.011320","volume":"284","author":"N Grobe","year":"2009","unstructured":"Grobe, N. et al. Mammalian cytochrome P450 enzymes catalyze the phenol-coupling step in endogenous morphine biosynthesis. J. Biol. Chem. 284, 24425\u201324431 (2009).","journal-title":"J. Biol. Chem."},{"key":"BFncomms10390_CR30","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1006\/abbi.1993.1401","volume":"305","author":"EM Gillam","year":"1993","unstructured":"Gillam, E. M., Baba, T., Kim, B. R., Ohmori, S. & Guengerich, F. P. Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305, 123\u2013131 (1993).","journal-title":"Arch. Biochem. Biophys."},{"key":"BFncomms10390_CR31","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1111\/j.1365-313X.2006.02860.x","volume":"48","author":"J Ziegler","year":"2006","unstructured":"Ziegler, J. et al. Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase\/reductase involved in morphine biosynthesis. Plant J. 48, 177\u2013192 (2006).","journal-title":"Plant J."},{"key":"BFncomms10390_CR32","doi-asserted-by":"publisher","first-page":"31091","DOI":"10.1074\/jbc.270.52.31091","volume":"270","author":"R Lenz","year":"1995","unstructured":"Lenz, R. & Zenk, M. H. Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from papaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebaine in morphine biosynthesis. J. Biol. Chem. 270, 31091\u201331096 (1995).","journal-title":"J. Biol. Chem."},{"key":"BFncomms10390_CR33","doi-asserted-by":"publisher","first-page":"837","DOI":"10.1038\/nchembio.1613","volume":"10","author":"K Thodey","year":"2014","unstructured":"Thodey, K., Galanie, S. & Smolke, C. D. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat. Chem. Biol. 10, 837\u2013844 (2014).","journal-title":"Nat. Chem. Biol."},{"key":"BFncomms10390_CR34","doi-asserted-by":"publisher","first-page":"564","DOI":"10.1038\/nchembio.105","volume":"4","author":"KM Hawkins","year":"2008","unstructured":"Hawkins, K. M. & Smolke, C. D. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564\u2013573 (2008).","journal-title":"Nat. Chem. Biol."},{"key":"BFncomms10390_CR35","doi-asserted-by":"publisher","first-page":"7393","DOI":"10.1073\/pnas.0802981105","volume":"105","author":"H Minami","year":"2008","unstructured":"Minami, H. et al. Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl Acad. Sci. USA 105, 7393\u20137398 (2008).","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"BFncomms10390_CR36","doi-asserted-by":"publisher","first-page":"528","DOI":"10.1038\/nature12051","volume":"496","author":"CJ Paddon","year":"2013","unstructured":"Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528\u2013532 (2013).","journal-title":"Nature"},{"key":"BFncomms10390_CR37","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1038\/nbt.3095","volume":"33","author":"K Zhou","year":"2015","unstructured":"Zhou, K., Qiao, K., Edgar, S. & Stephanopoulos, G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nature Biotechnol. 33, 377\u2013383 (2015).","journal-title":"Nature Biotechnol."},{"key":"BFncomms10390_CR38","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1186\/s12934-015-0332-3","volume":"14","author":"S Galanie","year":"2015","unstructured":"Galanie, S. & Smolke, C. D.. et al. Optimization of yeast-based production of medicinal protoberberine alkaloids. Microb. Cell Fact. 14, 144 (2015).","journal-title":"Microb. Cell Fact."},{"key":"BFncomms10390_CR39","doi-asserted-by":"publisher","first-page":"3283","DOI":"10.1038\/ncomms4283","volume":"5","author":"E Fossati","year":"2014","unstructured":"Fossati, E. et al. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 5, 3283 (2014).","journal-title":"Nat. Commun."}],"container-title":["Nature Communications"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/ncomms10390.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/ncomms10390","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/ncomms10390.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T06:53:10Z","timestamp":1672815190000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/ncomms10390"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,2,5]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2016,4,22]]}},"alternative-id":["BFncomms10390"],"URL":"https:\/\/doi.org\/10.1038\/ncomms10390","relation":{},"ISSN":["2041-1723"],"issn-type":[{"value":"2041-1723","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,2,5]]},"assertion":[{"value":"30 October 2015","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"7 December 2015","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 February 2016","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"10390"}}