Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, Chemical Induced Chromosomal Expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers, while maintaining stability in the absence of antibiotic selection pressure. Here, we report Parallel Integration and Chromosomal Expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multi-gene components, and introduce a heat-curable plasmid to obviate <
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deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in <
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DH1, and to then generate libraries of pathway component copy-number variants to screen for improved titers. As a result, polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.