Genetic Insights into Growth, Sporulation, and Performance of Bacillus subtilis during Industrial Fermentation Conditions

Bacillus subtilis is widely used in industrial bioprocesses due to its GRAS (generally regarded as safe) status, rapid growth on inexpensive substrates, secretory capabilities, and extensive genetic manipulation toolkit. Its range of industrial relevant products include among other amylases, cellulases, proteases, xylanases, lipases, β-galactosidase, and alkaline serine proteases, which are crucial for the food, feed, detergent, textile, leather, paper, and pharmaceutical industries.

The feasibility of many industrial bioprocesses depends on the activity of vegetative cells. A significant challenge in such processes with Bacillus subtilis is its ability to procedure differentiated cell types which it used as survival strategies in nature but may not be productive towards desired products. Specifically, sporulation represents a dormant phase that reduces metabolic activity, productivity, and increases energy consumption. Different strategies for the mitigation of sporulation are already implemented for production purposes. The sigF gene deletion has been identified as a strategy to prevent sporulation, yet its comprehensive effects on the transcriptome and metabolism during growth conditions relevant to industrial producPon have not been fully explored. This study investigates the use of a ΔsigF mutant strain to mitigate sporulation and enhance industrial bioprocess efficiency.

The research involves the investigaPon of growth profiles and a comparative transcriptomic analysis between the wildtype Bacillus subtilis strain 168 and the ΔsigF mutant during high cell density fed-batch cultivation in a nutrient-rich defined medium. The study focuses on sporulation regulatory network genes and enzymes involved in general metabolism to assess the overall fitness of the ΔsigF strain as a production organism. In addition, the presence of small RNAs potentially orchestratong and fine-tuning expression and regulation during these conditions are explored.

In growth experiments, the ΔsigF mutant shows preliminary indications of reduced biomass yield in glucose minimal medium compared to the wildtype but grow very similar during nutrient-rich fed-batch fermentation. Key findings from a comprehensive transcriptomic analysis reveals the induction of sporulation process even under vegetative growth conditions. Otherwise limited differential gene expression suggests the effective abortion of sporulation without altering important metabolic functions or production properties.

However, upregulation of genes which may be undesirable in industrial production strains (genes involved in motility and chemotaxis, lysis, and conjugative machinery) are observed. Additional computational analysis of small RNAs revealed the surprisingly large abundance of RNAs with predicted targets of regulation spanning ribosomes, and proteins involved in trans-translation(ssrA), biosynthesis of surfacPn(srfAA, srfAB, and srfAC), cannibalism(spd) and regulating differentiation and stress responses(rapA and spoVG, hag). In conclusion, the ΔsigF  strain showed to be a good candidate as production strain for industrial bioprocesses when sporulation is undesirable. However, these findings also highlight that there is still room for improvement, and it is crucial to construct the deletion mutant correctly to maximize its efficacy and ensure optimal performance in industrial applications.

This thesis underscores the importance of genetic and environmental interactions in optimizing Bacillus subtilis strains for industrial bioprocesses and highlights the potential of the ΔsigF mutant for high-density fermentation applications. Further recommendations include exploring additional gene deletions to enhance strain performance, conducting single-cell analysis to understand population heterogeneity, and optimizing bioprocess parameters for improved industrial scalability.