An improvedNicotiana benthamianabioproduction chassis provides novel insights into nicotine biosynthesis

Abstract

AbstractThe model plantNicotiana benthamianais an increasingly attractive organism for the production of high-value, biologically active molecules. However,N. benthamianaaccumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improvedN. benthamianagenome 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 3-4 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 improvedN. benthamianachassis for bioproduction and opens the possibility that BBLs are the sought-after coupling enzymes in nicotine biosynthesis.