Browsing by Author "Ogawa, Teppei"

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    Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids
    Vavricka, Christopher J.; Yoshida, Takanobu; Kuriya, Yuki; Takahashi, Shunsuke; Ogawa, Teppei; Ono, Fumie; Agari, Kazuko; Kiyota, Hiromasa; Li, Jianyong; Ishii, Jun; Tsuge, Kenji; Minami, Hiromichi; Araki, Michihiro; Hasunuma, Tomohisa; Kondo, Akihiko (Springer Nature, 2019-05-01)
    Previous 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.