Lee, J. A.; Nair, N. U.

Bacillus subtilis is an important chassis for biotechnology, but its use in multiplex genome engineering is limited by low natural transformation efficiency. Here, we compared inducible promoter systems for synthetic activation of the competence regulator ComK and evaluated their effects on the comG operon competence reporter and transformation efficiency. Xylose- and mannitol-inducible systems outperformed IPTG-based constructs and shifted 96-99% of cells into a reporter-positive competent state. However, reporter activation alone did not predict transformation potential. Optimization of culture density and induction timing increased transformant yield 45-fold relative to the initial protocol and 2800-fold relative to the conventional Spizizen method. Disruption of native competence regulatory genes did not improve performance and often reduced transformation output, highlighting the importance of endogenous regulatory circuitry. Using the optimized strain and protocol, we achieved co-transformation frequencies of 11-18% and constructed multiplex spore-display libraries containing fluorescent protein fusions integrated at multiple loci. Screening identified strong dual-display combinations and showed that cargo loading depends on anchor protein, integration locus, and genetic background. SscA fusions supported the highest display capacity and promoted synergistic co-display. Together, these results show improvements in natural transformation-based genome engineering in B. subtilis and provide insight into the construction of multifunctional engineered spores.