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An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis

2023; Wiley; Volume: 240; Issue: 1 Linguagem: Inglês

10.1111/nph.19141

1469-8137

Katharina Vollheyde, Quentin M. Dudley, Ting Yang, Mehmet Tufan Öz, Davide Mancinotti, Mariano Olivera Fedi, Darren Heavens, Gareth Linsmith, Monika Chhetry, Mark A. Smedley, Wendy Harwood, David Swarbreck, Fernando Geu‐Flores, Nicola J. Patron,

RNA and protein synthesis mechanisms

Summary 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.