Computational Design of Ten Superhot Capsicum Cultivars: Vanillylamine Precursor Supply, Not Pun1/AT3, Controls the Scoville Ceiling

An integrated computational pipeline combining developmental RNA-seq (Salmon 2.8.0), PyDESeq2 differential expression (4,365 significant genes, 28,074 tested), constraint-based metabolic flux modeling (COBRApy 0.32.1, 37 metabolites, 44 reactions), ESM2 protein language model mutagenesis (650M parameters), and CRISPR-SpCas9 construct design produces ten engineered Capsicum cultivar specifications spanning 866,611–2,956,310 SHU. Analysis of 4,365 differentially expressed genes across a C. chinense developmental time series identifies vanillylamine precursor supply — not Pun1/AT3 acyltransferase activity — as the principal bottleneck controlling the capsaicinoid ceiling in existing high-SHU germplasm.