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Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids

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dc.contributor.authorVavricka, Christopher J.en
dc.contributor.authorYoshida, Takanobuen
dc.contributor.authorKuriya, Yukien
dc.contributor.authorTakahashi, Shunsukeen
dc.contributor.authorOgawa, Teppeien
dc.contributor.authorOno, Fumieen
dc.contributor.authorAgari, Kazukoen
dc.contributor.authorKiyota, Hiromasaen
dc.contributor.authorLi, Jianyongen
dc.contributor.authorIshii, Junen
dc.contributor.authorTsuge, Kenjien
dc.contributor.authorMinami, Hiromichien
dc.contributor.authorAraki, Michihiroen
dc.contributor.authorHasunuma, Tomohisaen
dc.contributor.authorKondo, Akihikoen
dc.contributor.departmentBiochemistryen
dc.date.accessioned2019-07-30T16:43:01Zen
dc.date.available2019-07-30T16:43:01Zen
dc.date.issued2019-05-01en
dc.description.abstractPrevious studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.en
dc.description.notesThis work was supported by project P16009, Development of Production Techniques for Highly Functional Biomaterials Using Smart Cells of Plants and Other Organisms (Smart Cell Project), from the New Energy and Industrial Technology Development Organization (NEDO). This research is also partly supported by KAKENHI (V18K065770) and the Kato Memorial Bioscience Foundation (2017M-014). Special thanks to Ryo Inoue, Associate Professor Kentaro Okano and Professor Atsunori Mori at the Graduate School of Engineering, Department of Chemical Science and Engineering, Kobe University, for analysis of NMR samples. Molecular biology support from Dr. Hitoshi Mitsunobu, Dr. Jun Teramoto and other members in the research group of Professor Keiji Nishida is appreciated. We acknowledge Ms. Yoshimi Hori and Dr. Musashi Takenaka for the assistance with metabolomics analysis. Discussions with Dr. Hideo Kawaguchi and Dr. Takeshi Yamauchi were critical to the development of this study. Computational analyses by Masahiro Murata and Saeko Fujihana were also helpful. The authors are very thankful to Dr. Akira Nakagawa for support with downstream BIA production.en
dc.description.sponsorshipDevelopment of Production Techniques for Highly Functional Biomaterials Using Smart Cells of Plants and Other Organisms (Smart Cell Project), from the New Energy and Industrial Technology Development Organization (NEDO) [P16009]en
dc.description.sponsorshipKAKENHI [V18K065770]en
dc.description.sponsorshipKato Memorial Bioscience Foundation [2017M-014]en
dc.identifier.doihttps://doi.org/10.1038/s41467-019-09610-2en
dc.identifier.issn2041-1723en
dc.identifier.other2015en
dc.identifier.pmid31043610en
dc.identifier.urihttp://hdl.handle.net/10919/92047en
dc.identifier.volume10en
dc.language.isoenen
dc.publisherSpringer Natureen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleMechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloidsen
dc.title.serialNature Communicationsen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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