Section 1: Abstract Engineering Brighter Autonomous Bioluminescence Autonomous bioluminescence offers a major advantage over conventional bioluminescent systems because it eliminates the need for repeated addition of external luciferin. In plants, the fungal bioluminescence pathway is especially attractive because it uses caffeic acid, a metabolite that already exists in plant metabolism. However, current glowing plants are still limited by pathway flux, enzyme efficiency, and overall light output. The goal of this project is to engineer a brighter autonomous bioluminescence system by combining improved fungal bioluminescence enzymes with upstream metabolic enhancements that increase precursor availability. My hypothesis is that replacing the original pathway enzymes with brighter variants, including truncated nnLuz v4, nnH3H v2, and mcitHispS, and then increasing caffeic acid supply through added metabolic modules such as BnC3’H1 and TAL/HpaB/HpaC, will produce stronger self-sustained light emission than earlier fungal pathway designs. The specific aims are to first validate improved core pathway components, then compare enhancer strategies in modular construct designs, and finally identify the best architecture for future transient expression in Nicotiana benthamiana and stable transformation in Nicotiana tabacum. Methods include modular DNA design in Benchling, synthesis of gene cassettes, hierarchical DNA assembly, sequence verification, and comparative testing of optimized enzyme combinations. For the current class stage, an initial simplified test will evaluate whether nnLuz v4 truncated and nnH3H v2 can generate light in an inducible expression system, providing an early validation step before full plant pathway assembly.