Lawaetz, A. C. | bioRxiv Science

bioRxiv2026

SpoVG and the Kre-ComK Regulatory Module Orchestrate Production of the EPE Toxin in Bacillus subtilis

Survival of bacteria in their natural habitat requires dynamic responses and adaptation to environmental cues. In Bacillus subtilis, one adaptive strategy is cannibalism, a form of programmed cell death during post-exponential development. Cannibalism enhances multicellular differentiation by prolonging or preventing commitment to endospore formation under starvation conditions. B. subtilis produces three cannibalism toxins: the sporulation delay protein, the sporulation killing factor, and the epipeptide EPE. Production of the latter is encoded in the epeXEPAB operon. Expression of this operon is transcriptionally controlled by the stationary phase regulators Spo0A and AbrB. Here, we demonstrate that EPE production is also post-transcriptionally regulated by two RNA binding proteins, Kre and SpoVG. Deletion of comK, the master regulator of competence development, abolished EPE production. This defect was reversed by additionally deleting kre. The RNA-binding protein, Kre, binds the epeX transcript and acts as a bidirectional ComK repressor, indicating that ComK indirectly regulates EPE biosynthesis via Kre. A second RNA-binding protein, SpoVG, also binds to the epeX mRNA. While Kre acts as a negative regulator, SpoVG was essential for EPE production. These findings reveal a novel regulatory connection between competence and cannibalism, expanding our understanding of how programmed cell death is coordinated in B. subtilis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/716078v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1c71c02org.highwire.dtl.DTLVardef@ffdcbborg.highwire.dtl.DTLVardef@1f4a181org.highwire.dtl.DTLVardef@1975dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Genome mining of amylases and amylase inhibitors from Streptomyces

Most antibiotics are derived from Streptomyces bacteria and produced industrially via fermentations relying on food-grade feedstocks, which has a substantial environmental impact. Efficient utilisation of starch-rich organic wastes such as bread and potato waste as alternative feedstocks remains limited by incomplete knowledge of starch metabolism in Streptomyces. Here, a genus-wide analysis of 295 Streptomyces strains was performed, identifying 645 amylases grouped into pullulanases, -1,4-amylases, and cyclomaltodextrin-like amylases, alongside biosynthetic gene clusters encoding amylase inhibitors such as acarbose and tendamistat. Structural and sequence analyses revealed that inhibitor resistance arises from subtle modifications in the -amylase catalytic pocket. This comprehensive analysis of amylases and inhibitors provides a foundation for engineering inhibitor-resistant enzymes tailored to diverse starch substrates, facilitating the development of sustainable, starch-based Streptomyces fermentations. Impact statementPharmaceutical manufacturing contributes substantially to global carbon emissions, with feedstocks used in natural product fermentations being a major factor. Replacing conventional feedstocks with organic waste streams, such as starch-rich bread or potato waste, could reduce the environmental impact, but two key challenges remain: production strains may lack the enzymes required to efficiently degrade alternative substrates, and nutrient changes may trigger carbon catabolite repression, reducing product yields. While previous studies have linked carbohydrate-active enzymes with biosynthetic gene clusters, no comprehensive analysis has focused specifically on starch degradation in Streptomyces. To fill this gap, a genus-wide analysis of amylases and amylase inhibitors across 295 Streptomyces genomes was carried out. This created an atlas of 645 amylases which may serve as the foundation for the targeted selection of enzymes optimised for catabolising organic waste from various sources, thereby improving the sustainably of industrial natural product production such as antibiotics. Data summaryScripts used along with supplementary files and figures can be accessed through GitHub at https://github.com/ALawaetz/Amylases_and_amylase_inhibitors_in_Streptomycetes.

microbiology↗