An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis
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An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis. / Vollheyde, Katharina; Dudley, Quentin M.; Yang, Ting; Oz, Mehmet T.; Mancinotti, Davide; Fedi, Mariano Olivera; Heavens, Darren; Linsmith, Gareth; Chhetry, Monika; Smedley, Mark A.; Harwood, Wendy A.; Swarbreck, David; Geu-Flores, Fernando; Patron, Nicola J.
I: New Phytologist, Bind 240, Nr. 1, 2023, s. 302-317.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis
AU - Vollheyde, Katharina
AU - Dudley, Quentin M.
AU - Yang, Ting
AU - Oz, Mehmet T.
AU - Mancinotti, Davide
AU - Fedi, Mariano Olivera
AU - Heavens, Darren
AU - Linsmith, Gareth
AU - Chhetry, Monika
AU - Smedley, Mark A.
AU - Harwood, Wendy A.
AU - Swarbreck, David
AU - Geu-Flores, Fernando
AU - Patron, Nicola J.
N1 - Publisher Copyright: © 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.
PY - 2023
Y1 - 2023
N2 - The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.
AB - The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.
KW - alkaloid biosynthesis
KW - gene editing
KW - metabolic engineering
KW - Nicotiana benthamiana
KW - nicotine
U2 - 10.1111/nph.19141
DO - 10.1111/nph.19141
M3 - Journal article
C2 - 37488711
AN - SCOPUS:85165581637
VL - 240
SP - 302
EP - 317
JO - New Phytologist
JF - New Phytologist
SN - 0028-646X
IS - 1
ER -
ID: 363060517