Nicotine Biosynthesis Project

Bottom line: nicotine is made in tobacco roots from two branches, a
pyridine ring from nicotinic acid and a pyrrolidine ring from putrescine, and
2026 work in Nicotiana attenuata completed the pathway by adding a
glucosylation step (UGT1), a deglucosylation step (BGL1/2) and a vacuolar MATE
transporter. We seeded the project in N. attenuata (NICAT), mapped the
paper's gene names to current UniProt accessions by sequence, and created 22
gene reviews: the core enzymes plus the plausible paralogs where the mapping
was ambiguous. We did this because public annotation still lags the new
pathway, so no GO record yet connects most of these proteins to nicotine. All
22 reviews are at DRAFT status and cover 117 existing annotations; they propose
GO:0042179 nicotine biosynthetic process for six genes, remove electronic
spermidine synthase terms from the three PMT paralogs, and narrow generic
terms to specific activities such as GO:0050139 nicotinate-N-glucosyltransferase
activity for UGT1. NaNAMNH still has no stable public accession.

Editor Brief (2026-04-04):
Seed this project in Nicotiana attenuata (NICAT), not N. tabacum. The April 2, 2026
Cell paper from Chris directly completes nicotine biosynthesis in N. attenuata, identifies
the newly required glycosylation and deglycosylation steps, and provides a minimal
heterologous reconstruction path. This species also has strong prior pathway/evolution
literature and a well-used ecological/genomic model system in the repo's target domain.
The project starts from a conservative core pathway set, keeps regulators out of the seed
enzyme list, and explicitly separates canonical literature genes from accession-backed
review jobs where current public annotation still lags the paper nomenclature.

Goal

Review the core genes required to synthesize nicotine in one defensible Nicotiana species,
starting from a pathway-focused seed set that can be expanded only when the literature clearly
demands it.

This project owns three things:

1. The canonical single-species nicotine pathway gene list.
2. The review-job table used to fetch/create gene review stubs.
3. The accession-resolution backlog for Cell-era names that are not yet normalized in public
   annotation resources.

Why Nicotiana attenuata

N. attenuata is the best seed species because it now has:

• A direct "complete biosynthesis of nicotine" paper in the target species.
• Older pathway/evolution work already centered on N. attenuata rather than only cultivated
  tobacco.
• Root-expression and ecology literature that helps distinguish core biosynthetic genes from
  housekeeping paralogs and pathway regulators.

I am not seeding this in N. tabacum even though tobacco is the familiar nicotine species,
because the new Cell paper's mechanistic closure is in N. attenuata, and forcing a
cross-species translation at project start would add avoidable paralog/orthology noise.

Selection Rules

Include a gene in the seed set only if at least one of these is true:

• The Cell 2026 paper directly assigns a catalytic or transport role in nicotine biosynthesis.
• Earlier N. attenuata pathway literature already places it in the root-specific nicotine
  branch.
• A paralog-disambiguation review is required to decide which duplicated copy is the real
  pathway member.

Exclude from the initial seed set:

• Pure regulators unless they are needed later for pathway-control follow-up.
• General wound/jasmonate response genes that are upstream of many defenses.
• Transport or storage genes with only indirect nicotine associations unless the literature
  puts them into the biosynthetic/metabolon core.

Core Pathway Seed Set

Included now

Keep as follow-up, not seed-core

Cell Paper Impact

The incoming paper materially changes the project framing:

• It strengthens the species choice in favor of N. attenuata.
• It upgrades glycosylation and deglycosylation from side chemistry to pathway-core steps.
• It makes NaMATE1 a core transport gene rather than a peripheral transport annotation.
• It argues that the minimal heterologous production set is:
  NaODC, NaPMT1, NaMPO, NaUGT1, NaA622, NaBBL2, NaBGL1, and NaMATE1.
• It also supports keeping the upstream pyridine branch (NaAO2, NaNAMNH, NaQPT2) in
  scope for a complete species review rather than only reviewing the minimal yeast set.

Review Orchestration

Two sidecars drive execution:

• projects/NICOTINE_BIOSYNTHESIS/genes.csv
  Canonical literature-facing pathway list and inclusion rationale.
• projects/NICOTINE_BIOSYNTHESIS/review_jobs.csv
  Accession-backed job queue plus explicit pending rows where public annotation still trails
  the literature.

Launch jobs with:

uv run python projects/NICOTINE_BIOSYNTHESIS/launch_review_jobs.py --dry-run
uv run python projects/NICOTINE_BIOSYNTHESIS/launch_review_jobs.py --include-existing --dry-run
uv run python projects/NICOTINE_BIOSYNTHESIS/launch_review_jobs.py
uv run python projects/NICOTINE_BIOSYNTHESIS/launch_review_jobs.py --with-deep-research --deep-research-provider codex

Default behavior:

• Runs only rows marked launch_ready.
• Resolves paths from the repository root even when invoked outside the repo top level.
• Skips aliases that already have seeded review files unless --include-existing is passed.
• Fetches UniProt/GOA/stub review files with stable project aliases.
• Skips unresolved rows but reports them at the end.

Current Branch Snapshot

This branch is already past pure scaffold stage:

• All 22 current launch_ready aliases in review_jobs.csv have now been fetched into genes/NICAT/.
• All 22 current launched NICAT aliases have now been advanced to DRAFT review state.
• The 2026-04-05 mapping dive converted NaUGT1, NaMPO1, NaBGL2, and NaMATE1
  from pure accession backlog into fetched sequence-backed candidate rows.
• The same review pass promotes BGLU18_6 and BGLU18_1 as the leading beta-GD
  anchors and demotes BGLU42 to a lower-confidence historical comparator.
• NaNAMNH is now the main unresolved seed-core accession gap.
• NaNUP remains a tracked transport follow-up outside the minimal launch core.

Accession-Mapping Policy

For several Cell-era names, public resources still expose generic paralog names rather than the
paper's Na... symbols. This project therefore uses two layers:

• Canonical symbol layer for the biology and project framing.
• Launch job layer for the current fetchable accession(s).

If a public accession mapping is ambiguous, review all plausible paralogs early rather than
pretending the ambiguity does not exist.

Mapping Dive (2026-04-05)

A focused mapping pass used the Cell/bioRxiv tobacco locus IDs, the SGN tobacco 2017 proteome,
the SGN N. attenuata NIATv7 proteome, the UniProt NICAT proteome, and a spot remote BLAST
check for NaMPO1.

Current best candidates are:

• NaMPO1 -> AMO_3 / A0A314KPU1
  Tobacco Nitab4.5_0000983g0060 (NtMPO1) maps strongly to N. attenuata primary amine
  oxidase NIATv7_g14063, which in UniProt cross-release space best matches A0A314KPU1.
• NaMATE1 -> DTX40_3 / A0A314KVN4
  Tobacco Nitab4.5_0000884g0030 (MATE1) maps cleanly to NIATv7_g09978, then to UniProt
  DTX40_3.
• NaUGT1 -> UGT85A2_0 / A0A2H4GSI3
  Tobacco Nitab4.5_0006222g0020 (UGT1) maps strongly to NIATv7_g26396, then to UniProt
  UGT85A2_0.
• NaBGL1 -> BGLU18_6 / A0A1J6KFZ7
  Tobacco beta-GD1 (Nitab4.5_0000884g0020) maps best to NIATv7_g57285, then to UniProt
  BGLU18_6.
• NaBGL2 -> BGLU18_1 / A0A314KWB2
  Tobacco beta-GD2 (Nitab4.5_0000031g0400) maps best to NIATv7_g09974, then to UniProt
  BGLU18_1.

Important caveats:

• These are sequence-backed cross-release mappings, not official symbol normalizations from
  the Cell paper into current public NICAT nomenclature.
• The already-launched NaBGL1_candidate_BGLU42 row should now be treated as a weaker
  historical comparator, not the best orthology anchor for the paper's beta-GD genes.
• NaNAMNH remains unresolved: the Cell paper clearly assigns the function, but a stable public
  NICAT accession still does not surface cleanly from current annotation resources.

Immediate Worklist

• [x] Choose a single seed species.
• [x] Add the Cell 2026 paper to the project framing.
• [x] Create canonical pathway list and job queue.
• [x] Launch first-pass review jobs for all current launch_ready rows.
• [x] Curate the PMT paralog pair beyond seeded stubs.
• [x] Convert NaUGT1, NaMPO1, NaBGL2, and NaMATE1 from pure backlog rows into
  sequence-backed candidate jobs.
• [x] Decide that the new BGLU18 candidates replace BGLU42 as the primary
  deglycosylase review anchors, while retaining BGLU42 as a comparator.
• [x] Advance all currently launched NICAT aliases beyond INITIALIZED.
• [ ] Resolve a stable public accession for NaNAMNH.
• [ ] Revisit whether NaNUP belongs in the core transport batch after the minimal metabolon is reviewed.

Slides

• Slides (Marp source: NICOTINE_BIOSYNTHESIS-slides.md) — AI generated

Sources

1. Chang L, Xu Z, Deng P, Zhang N, He T, Liu X, He W, Zheng A, Hu W, Pan M, Li W, Halitschke R, Li R, Fan M, Baldwin IT, Zhang Y, Li D. 2026. Complete biosynthesis of nicotine. Cell. Chris-provided source: https://www.cell.com/cell/fulltext/S0092-8674(26)00335-1
   Accessible abstract mirror used during scaffolding: https://www.lifescience.net/entries/860228/complete-biosynthesis-of-nicotine/
2. Schwabe BTW, Angstman IM, Vollheyde K, Ingold Z, Li J, Stankevich KS, Spicer CD, Fascione MA, Grogan G, Geu-Flores F, Lichman BR. 2025. Nicotine biosynthesis completed by cryptic activating glucosylation. bioRxiv. DOI: 10.64898/2025.12.04.692101
   Full preprint consulted here: https://www.biorxiv.org/content/10.64898/2025.12.04.692101v1
   Lab pages confirming the preprint and its emphasis on cryptic glucosylation: https://lichmanlab.weebly.com/research-articles.html and https://lichmanlab.weebly.com/news.html
3. Xu S, Brockmoller T, Navarro-Quezada A, et al. 2017. Wild tobacco genomes reveal the evolution of nicotine biosynthesis. PNAS.
   https://pmc.ncbi.nlm.nih.gov/articles/PMC5468653/
4. Zhang et al. 2025. DNA methylation valley as a distinguishing feature occurs in root-specific expressed nicotine-related genes in Nicotiana attenuata. Frontiers in Plant Science.
   https://pmc.ncbi.nlm.nih.gov/articles/PMC12361153/
5. Sol Genomics Network FTP. Nicotiana tabacum Edwards et al. 2017 proteome.
   https://ftp.solgenomics.net/genomes/Nicotiana_tabacum/edwards_et_al_2017/annotation/Nitab-v4.5_proteins_Edwards2017.fasta
6. Sol Genomics Network FTP. Nicotiana attenuata genome release v2, annotation v5 proteome.
   https://ftp.solgenomics.net/genomes/Nicotiana_attenuata/NIATTr2.an5.aa.fa
7. UniProtKB stream for Nicotiana attenuata proteins.
   https://rest.uniprot.org/uniprotkb/stream?compressed=false&format=fasta&query=organism_name:%22Nicotiana%20attenuata%22