Let There Be Light: Genome Reduction Enables to Produce Disulfide-Bonded Luciferase.

is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds. In the present study, we deployed genome minimization, signal peptide screening, and coexpression of recombinant thiol oxidases as strategies to improve the ability of to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Luciferase (GLuc), which contains five disulfide bonds. These chassis lack extracellular proteases, prophages, and key sporulation genes. Importantly, compared to the reference strain with a full-size genome, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc while growing to lower cell densities. Our results show that high-level GLuc secretion relates, at least in part, to the absence of major extracellular proteases. In addition, we show that the thiol-disulfide oxidoreductase requirements for disulfide bonding have changed upon genome reduction. Altogether, our results highlight genome-engineered strains as promising expression platforms for proteins with multiple disulfide bonds.