Development of a fluorescent biosensor for monitoring protein secretion at a single-cell level in bacillus subtilis.

Abstract

Microorganisms are widely exploited as cellular factories for recombinant protein production. However, this process imposes a high metabolic burden on the cells and can lead to the emergence of non-producing subpopulations. The Segregostat, which combines continuous culture with automated control through flow cytometry measurements, has already demonstrated its efficiency in dynamically controlling populations. For this approach to work, recombinant protein production must generate a sufficient burden to trigger population heterogeneity, as shown in Escherichia coli. In contrast, in hosts such as Bacillus subtilis, the main source of stress is protein secretion, but whether secretion-related stress can induce population heterogeneity remains unknown. Consequently, in order to extend the use of the Segregostat to B. subtilis, the presence of secretion-related heterogeneity must first be demonstrated. To address this, B. subtilis strains were genetically engineered with a secretion-stress-sensitive promoter (htrA-CssRS) fused to a fluorescent protein. In parallel, the secretion of an alginate lyase was monitored using another fluorescent reporter. Analyses revealed little to no effect of secretion on growth. In continuous culture, enrichment of the medium did not enhance protein production but instead led to increased cell size. The combination of nutrient withdrawal and heat shock favored the emergence of a subpopulation, although their respective contributions could not be distinguished. Overall, alginate lyase production alone did not induce detectable secretion stress, while the low performance of the fluorescent stress reporter raises questions about its reliability.