Multiplex base editing of RBSs rewires Bacillus subtilis metabolism for lycopene overproduction.

Bacillus subtilis is a GRAS-certified chassis, yet scalable and precise multi-gene regulation remains a bottleneck in its metabolic engineering. Here, we present bsBETTER, a base editor-guided, template-free system enabling high-diversity expression tuning across multiple genomic loci. By editing ribosome binding sites (RBSs) of 12 lycopene biosynthetic genes, we generated thousands of combinatorial variants in situ, achieving up to 255 of 256 theoretical RBS combinations per gene. High-throughput screening identified variants exhibiting up to a 6.2-fold increase in lycopene production relative to strains carrying direct genomic overexpression of MEP pathway genes. RBS strength measurements revealed strong context dependence, highlighting the importance of genome-integrated expression optimization. Multi-omics analysis showed extensive transcriptional and metabolic rewiring, including enhanced MEP pathway flux and NADPH-generating capacity. bsBETTER offers a scalable, high-resolution approach for metabolic pathway engineering in B. subtilis, providing a generalizable framework for combinatorial gene expression modulation and metabolic pathway optimization.