Let There Be Light: Genome Reduction Enables Bacillus subtilis to Produce Disulfide-Bonded Gaussia Luciferase

Abstract

Bacillus subtilis is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, B. subtilis presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, B. subtilis 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 B. subtilis to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Gaussia 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 Bacillus strains as promising expression platforms for proteins with multiple disulfide bonds.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production

“…Here, we present the engineered strain IIG-Bs-27-39, lacking 21.6% of the B. subtilis 168 genome, as a potential cell factory for heterologous secretory protein production. While previous studies have showcased particular advantages of the use of genome-reduced strains in the production of staphylococcal and other proteins, 6 , 31 , 39 , 40 this study was focused on exploring the behavior of the IIG-Bs-27-39 strain during fermentation in bioreactors. To pinpoint potential bottlenecks in the secretion of a difficult-to-produce protein, the IIG-Bs-27-39 strain was equipped with the genes for subtilin-inducible expression of the IsaA protein from S. aureus .…”