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

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

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.

TidsskriftNew Phytologist
Udgave nummer1
Sider (fra-til)302-317
Antal sider16
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
Plasmids pNJB069 (pTRV1), pEE393, and pEE515 along with seeds of expressing SpCas9 were a generous gift from Dan Voytas. We thank Oleg Raitskin for advice on the design and construction of gene‐editing vectors; Wilfried Haerty for his advice and management of the genome assembly; and the John Innes Centre Horticultural Services team for help with plant husbandry. This work was funded by the United Kingdom Research and Innovation's Biotechnology and Biological Sciences Research Council (UKRI BBSRC) Strategic Programme Grant (BB/CSP1720/1) and its constituent work packages BBS/E/T/000PR9818 (Signatures of Domestication and Adaptation) and BBS/E/T/000PR9819 (Regulatory Interactions and Complex Phenotypes) and an industrial partnership award with Leaf Expression Systems (BB/P010490/1). Part of this work was delivered via the BBSRC National Capability in Genomics and Single Cell Analysis (BBS/E/T/000PR9816) by members of the Genomics Pipelines and Core Bioinformatics Group. The work was further funded by the Novo Nordisk Foundation (grants NNF16OC0019608 and NNF17OC0027744) and the Danish National Research Foundation (grant 0136‐00410B). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. N. benthamiana

Publisher Copyright:
© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.

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