TY - JOUR
T1 - Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids
AU - Vavricka, Christopher J.
AU - Yoshida, Takanobu
AU - Kuriya, Yuki
AU - Takahashi, Shunsuke
AU - Ogawa, Teppei
AU - Ono, Fumie
AU - Agari, Kazuko
AU - Kiyota, Hiromasa
AU - Li, Jianyong
AU - Ishii, Jun
AU - Tsuge, Kenji
AU - Minami, Hiromichi
AU - Araki, Michihiro
AU - Hasunuma, Tomohisa
AU - Kondo, Akihiko
N1 - Funding Information:
This 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.
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/5/1
Y1 - 2019/5/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-019-09610-2
DO - 10.1038/s41467-019-09610-2
M3 - Article
C2 - 31043610
AN - SCOPUS:85065169335
VL - 10
SP - 2015
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 2015
ER -