Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations

Abstract

Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.

Deletion of Rap-phosphatases for quorum sensing control in Bacillus and its effect on surfactin production

“…The strains used in this study were derived from B. subtilis JABs24 (Geissler et al 2019 ) and are listed in Table 1 . For ComX pheromone bioactivity assay, strain B. subtilis CT2 (Treinen et al 2021 ) was used. Cells were conserved in lysogeny broth (LB), containing 15–25% ( v / v ) glycerol and stored at -80 °C.…”

“…All cultivation experiments were conducted in baffled shake flasks at 120 rpm and 37 °C using incubator shakers (Newbrunswick TM /Innova® 44, Eppendorf AG, Hamburg, Germany). Precultures were prepared as described in Treinen et al ( 2021 ) with relative filling volumes of 0.2 mL/mL (20%). In detail, preculture I was carried out in LB medium and incubated for 15–16 h. Preculture I was then used to inoculate preculture II, which was carried out in MSM and incubated for 12 h. Exponentially growing cells were used for inoculation of the main culture, which was performed in 1000 mL shake flasks using a relative filling volume of 0.1 mL/mL (10%) and an initial OD 600 of 0.1.…”

“…Quantifications that were not possible with an application volume of ≤ 5 µL of undiluted sample were assessed as not detectable. ComX bioassay was performed as described in Treinen et al ( 2021 ) with minor adjustments. In brief, reporter strain B. subtilis CT2 was used to determine ComX-dependent expression of lacZ .…”

“…For example, the native P srfA promoter can be exchanged to uncouple surfactin production and the quorum sensing system (Coutte et al 2010 ; Hoffmann et al 2021 ; Willenbacher et al 2016 ). Another option is to incorporate the quorum sensing system for lipopeptide production into the context of molecular process control, for example by modeling the kinetics of quorum sensing molecules (Treinen et al 2021 ). However, in order to exploit the Bacillus quorum sensing system for bioprocess design, a deeper understanding of its components and their effects on the cells on both the qualitative and quantitative level is required.…”

Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food-related sources

“…To enable a controlled biosynthesis of surfactin, the expression of the srfA operon is targeted by various regulatory networks. One of the predominant control mechanisms is based on the ComX-mediated quorum sensing, which leads to a ComA-dependent activation of the srfA operon expression (Nakano et al 1991;Treinen et al 2021).…”