Binding pocket optimization of Pueraria mirifica type II chalcone isomerase synergizing with a necrosis-attenuating...

Pathway reconstitution of leguminous isoflavonoids in non-legume plant chassis offers a tractably controlled environment to probe specialized metabolism, yet it remains critically bottlenecked by suboptimal heterologous enzyme efficiency and severe host tissue necrosis. Here, we overcome these long-standing architectural limitations by establishing a highly synergistic, necrosis-attenuated Nicotiana benthamiana synthetic biology platform. We characterized chalcone isomerases from Pueraria mirifica (PmCHIs), revealing that PmCHI Type II operates as a biochemical junction exhibiting a specialized substrate-dependent alkaline optimum (pH 7.5-8.0) exclusively toward isoliquiritigenin. Combining 500-ns molecular dynamics simulations with structure-guided mutagenesis, active-site engineering identified an I195V variant with enhanced isoliquiritigenin preference. The conservative I195V substitution was predicted to reduce local steric crowding and produced an isoliquiritigenin selectivity ratio of 1.63 in vitro. For in planta pathway integration, we delivered a polycistronic cascade using P2A ribosome-skipping peptides to streamline multiplex multi-gene stacking. Crucially, substituting the conventional Arabidopsis thaliana MYB12 regulator with a host-derived N. benthamiana MYB60 transcription factor successfully attenuated tissue necrosis, accompanied by reduced electrolyte leakage. Quantitative proteomics showed no statistically significant difference in PmCHI abundance between the I195V and WT-II treatments in planta, while independent plant batches consistently showed higher daidzein accumulation with I195V, although the magnitude of enhancement varied among experiments. The NbMYB60-based platform extended the operational viability window up to 9 days, delivering a robust daidzein titer of 1.5 mg/g DW at 6 days post-infiltration, with individual high-performing replicates reaching up to 2.5 mg/g DW. Together, these results establish an integrated enzyme-engineering and necrosis-attenuating regulatory framework for persistent daidzein biosynthesis and specialized pathway reconstitution in planta.