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

Abstract

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.

Keywords: Nicotiana benthamiana; alkaloid biosynthesis; gene editing; metabolic engineering; nicotine.

Fig. 1

Schematic diagram of pyridine alkaloid biosynthesis in Nicotiana spp. The three colored boxes highlight the different biosynthetic branches involved. Full arrows represent single characterized steps, and dotted arrows represent one or more putative steps. The intermediates shown in brackets are putative. The asterisk in nicotinic acid indicates position C6, whose hydrogen is lost during biosynthesis (Dawson et al., 1960). Enzymes are represented by dark gray ovals. Question marks indicate uncertainty about the precise reactions catalyzed by the mentioned enzymes. A622, PIP‐family reductase; AO, aspartate oxidase; BBL, berberine bridge enzyme‐like protein; l‐Lys, l‐lysine; l‐Orn, l‐ornithine; MPO, methylputrescine oxidase; NAD, nicotinamide adenine dinucleotide; ODC, ornithine decarboxylase; PMT, putrescine methyltransferase; QPT, quinolinate phosphorybosyltransferase; QS, quinolinate synthase; RibP, ribose phosphate residue.

Fig. 2

Nicotiana benthamiana berberine bridge enzyme‐like (NbBBL) enzymes. (a) Schematic of NbBBL enzymes indicating the locations and sequences of guide RNAs used for Cas9‐mediated targeted mutagenesis. Canonical NGG protospacer adjacent motifs are underlined; *, stop codon. Background shading of bases indicates a mismatch to the guide RNA sequence. (b) Expression of NbBBL genes relative to NbEF1a in roots of plants following treatment with 2.5 mM methyl jasmonate (MeJa). Expression was determined by reverse transcription‐quantitative PCR (RT‐qPCR). Error bars indicate the SE of the mean of three biological replicates (four technical replicates per sample). (c) Schematics of plasmid constructs used for Cas9‐mediated targeted mutagenesis by integration of T‐DNA (above) or transient expression of a mobile guide RNA (mbgRNA). 16k, 16k gene; 35s, cauliflower mosaic virus 35s promoter; CP, coat protein; FT, flowering locus T mobile signal; LB, left border; MP, movement protein; nos, nopaline synthase promoter or terminator; ocs, octopine synthase terminator; PEBV, pea early browning virus promoter; RB, right border; RDRP, RNA‐dependent RNA polymerase; RZ, self‐cleaving ribozyme. This figure contains graphics from Biorender (biorender.com).

Fig. 3

Workflow for the selection of gene‐edited lines, and genotypes of all Nicotiana benthamiana berberine bridge enzyme‐like (NbBBL) genes in each line. The size of insertions (+) and deletions (−) in base pairs (bp) are provided together with the last correct amino acid (*aa). +, 5 residues of NbBBLb AYINY (Alanine, Tyrosine, Isoleucine, Asparagine, Tyrosine) are replaced with D (Aspartic Acid). Green text, homozygous mutation; purple text, biallelic mutation; gray shading, due to the size, type or location, the mutation may not result in loss of activity. Details of all mutations can be found in Supporting Information Tables S6–S10. This figure contains graphics from Biorender (biorender.com).

Fig. 4

Pyridine alkaloid content in leaves of Nicotiana benthamiana berberine bridge enzyme‐like (BBL) mutant lines in comparison with control lines, as analyzed by LC–MS. Line names and their respective NbBBL genotypes are shown at the bottom, with green shading representing a homozygous knockout, purple shading indicating a biallelic knockout, and gray shading indicating uncertainty with respect to loss of function (due to size, type or location of the mutation; see details of all mutations in Fig. 3; Supporting Information Tables S6–S10). Three control lines were analyzed: WT, wildtype (control for all lines); TC WT, tissue culture control (additional control for lines 159–198; bold); and Cas9, transgenic line constitutively expressing Cas9 (additional control for lines 102 and 138; underlined). Graphs to the left represent the levels of nicotine, anabasine, and anatabine in leaves of glasshouse‐grown plants (nd, not determined). Graphs to the right represent the analogous measurements 5 d after induction with 0.1% (v/v) MeJa (equivalent to 4.6 mM MeJa). For each line, four to five biological replicates were measured (black diamonds). Compact letter display is used to visualize significant differences determined using ANOVA and post hoc Tukey tests on log‐transformed data. Adjusted P‐values for all pairs can be found in Table S11.

Fig. 5

Analysis of (S)‐ and (R)‐nicotine in leaves of the quintuple Nicotiana benthamiana berberine bridge enzyme‐like (NbBBL) mutant (line 102) in comparison with control lines (WT and Cas9). Traces correspond to extracted ion chromatograms (nicotine, [M + H]⁺) resulting from chiral LC–MS analyses (Lux® 3 μm AMP column). Traces on the left are from uninduced plants, while traces on the right are from the same plants 5 d after induction with MeJa. Higher injection volumes were used for all uninduced samples (10 μl compared with 2 μl) to obtain comparable peak sizes. A total of four to five biological replicates were analyzed. While only one replicate is shown in this figure, all data are provided in Supporting Information Fig. S3. The two bottom rows show the results of running a racemic nicotine standard and a (S)‐nicotine standard along with the uninduced and induced samples.

Fig. 6

Incorporation of deuterated (D4)‐nicotinamide into pyridine alkaloids in the quintuple Nicotiana benthamiana berberine bridge enzyme‐like (NbBBL) mutant (line 102) in comparison with control lines (WT and Cas9). Seedlings were grown under hydroponic conditions, induced with MeJa, and fed continuously for 5 d with either labeled (D4) or unlabeled (D0, control) nicotinamide via the roots. (a) Relative levels of differently labeled nicotine, anabasine, and anatabine in whole seedlings, as observed using LC–MS. For nicotine and anabasine, D0, D3, and D4 versions were observed and quantified. For anatabine, D0, D7, and D8 versions were observed and quantified. For each version, relative peak areas were obtained by dividing by the sum of peak areas of all labeled versions. Six biological replicates were analyzed (black diamonds). Bars represent mean values, and error bars represent the outlier range in plus direction with a coefficient of 1.5. Numbers above the arrows correspond to the fold difference between D3 and D4 versions (for nicotine and anabasine) or between the D7 and D8 versions (for anatabine), ± SD. Compact letter display is used to visualize significant differences determined using ANOVA and post hoc Tukey tests on log‐transformed data. Adjusted P‐values for all pairs can be found in Supporting Information Table S12. (b) Representative traces (extracted ion chromatograms, [M + H]⁺) of the differently labeled pyridine alkaloids in quintuple NbBBL mutants (line 102) compared with WT plants. The colors of the depicted traces correspond to the colors of the corresponding bars in panel a. Chemical structures of each of the analyzed compounds are shown inside the gray boxes under each pair of graphs. The position of the lost deuterium atom was inferred from the comprehensive feeding studies carried out previously (Dawson et al., ; Leete & Liu, ; Leete, 1978).

Fig. 7

Dihydrometanicotine (DMN) accumulation in roots of the quintuple NbBBL mutant (line 102) as analyzed by LC–MS. Seedlings were grown under hydroponic conditions, and roots were harvested 5 d after induction with MeJa. Traces are extracted ion chromatograms ([M + H]⁺) corresponding to nicotine (black) or DMN (blue). Results from mutant line 102 are compared with results from two control lines (WT and Cas9). A total of five to six biological replicates per line were analyzed. While only one replicate is shown in this figure, all data is provided in Supporting Information Fig. S4. Also shown are two traces corresponding to a (S)‐nicotine standard and a DMN standard. In addition, MS2 spectra for DMN ([M + H]⁺) from line 102 and from the DMN standard are shown to the right. The labels at the top of the MS spectra indicate the mass of the fragmented ion (parenthesis), the collision energy, and the retention time.

Fig. 8

Mechanistic proposals for the BBL‐catalyzed reaction(s). The proposals are based on our experimental results with the quintuple NbBBL knockout as well as previous results by Dawson and Leete (Dawson et al., ; Leete & Liu, ; Leete, 1978). A deuterium atom is shown at the C6 position in nicotinic acid (top molecule) to facilitate visualization of the fate of a hydrogen isotope at this position during biosynthesis. The stereochemistry of the initial reduction was established by Leete (1978). In the BBL‐catalyzed oxidations, the deuteride acceptor is likely to be FAD (not shown). [O], oxidation; DMN, dihydrometanicotine; spont., spontaneous. (a) Scenario in which BBL enzymes catalyze both a stereoselective coupling and a subsequent oxidation. (b) Alternative scenario in which the coupling is not stereoselective and is either spontaneous or catalyzed by an unknown enzyme. In this scenario, BBL enzymes are specific for the S intermediate, thus giving rise only to (S)‐nicotine. (c) Proposal for the pyridine alkaloid pathway in the absence of functional BBL enzymes.