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

The Rap-phosphatases RapC, RapF and RapH, which are known to affect response regulator ComA, were deleted in the laboratory production strain JABs24 derived from B. subtilis 168. This allowed to assess the deletion of quorum sensing elements as a technique for molecular process control. The key outcomes of this study are listed in the following:

Quantitative surfactin analysis to determine the impact of individual rapC, rapF and rapH deletions.

Promoter activity PsrfA increased particularly for rapH deletion, whereas surfactin titers were not as strongly affected.

Increased product yield per biomass YP/X and specific surfactin productivity q for deletion mutants.

Increase in surfactin titer for rapC and rapF deletion only visible after prolonged incubation period.

Deletion of Rap-phosphatases did not have any appreciable effect on ComX activity.

Additionally, a negative control was constructed by deleting oppA, which is important for a correct function of oligopeptide ABC transporter Opp. Considering that this strain was used as a reference for a low surfactin production, an additional PsrfA-lacZ was not further investigated. But it is not only the Rap-phosphatases that influence the activity of ComA. Conversely, the expression of rapC as well as rapF is also regulated via ComA ~ P and thus induced by ComX (Comella and Grossman 2005). This led to the hypothesis in a previous study by Treinen et al. (2021) that high ComX levels during cultivation of Bacillus strains might negatively influence the surfactin production in form of a negative feedback loop. In turn, a deletion of the mentioned Rap-phosphatases could provide a checkpoint for strain improvement and the absence of the counteracting proteins could increase both surfactin titer and specific surfactin productivity qsurfactin (Fig. 1). At the same time, it is hypothesised that ComX concentration should remain constant or unaffected.

Influence of rap deletion on PsrfA activity

Not considering the initial high values in the lag phase due to the transfer of the preculture, the maximal measured promoter activity of reference strain KM1016 was comparable to literature data. Here, a PsrfA-lacZ of 197.2 ± 24.1 MU was obtained which is in agreement with Hoffmann et al. (2021), where about 200 MU were achieved with the same strain and a filling volume of 10% in shake flasks. In contrast, the deletion strains CT10 and CT11 deviated only moderately from the reference values. This was not unexpected as previous findings showed that a deletion of these Rap-phosphatases did not always lead to a large increase in promoter activity (Auchtung et al. 2006; Bongiorni et al. 2005; Core and Perego 2003). However, the deletion strain CT12 showed considerably higher promoter activities than the reference strain KM1016 in both conducted cultivations. In detail, a 2.7-fold increase was achieved at t = 14 h for cultivation 1 (Fig. 2A) and a 1.9-fold increase at t = 18 h for cultivation 2 (Fig. 3A). This was in contrast to a study of Hayashi et al. (2006) who stated that although overexpression of RapH resulted in a lower expression of srfA, only a marginal effect could be observed after the deletion of the aforementioned Rap-phosphatase in comparison to the control strain B. subtilis OSM100. The question now arises as to why the deletion of RapH stood out in comparison to RapC and RapF. In this context, it should be mentioned that RapH takes on a special role, since not only the response regulator ComA, but also sporulation is affected by dephosphorylation of Spo0F ~ P, which is required for sporulation initiation (Smits et al. 2007). Due to its influence on the DNA binding activity of ComA (Smits et al. 2007), a deletion of rapH would most likely also have an indirect effect on the expression of rapC and rapF, which in turn again influence ComA activity (Comella and Grossman 2005).Lazazzera et al. (1999) claimed that RapC negatively controls its own production and Core and Perego (2003) previously found that deletion of rapC positively affected transcription of rapC-lacZ. This suggests that a deletion of rapH might also positively influence transcription of rapC and rapF. A therefore increased RapC and RapF production may have masked the effect of the rapH deletion, keeping the surfactin titer in a similar range as the reference. The assumption that active Rap-phosphatases could mask the effect of rapH deletion was previously postulated in a study by Smits et al. (2007).

Deletion strains showed higher Y P/X and q overall after reaching P max of reference

Reference strain KM1016 achieved the overall highest surfactin titer after 16 h during the first cultivation with a concentration of Pmax of 920.3 ± 65.1 mg/L (Table 2). This was in range of surfactin titers typically measured for JABs24 in shake flask cultivations, as shown by Geissler et al. (2019), with 1147.03 mg/L surfactin at CDWmax after 21 h. However, the obtained process parameters such as YP/X and specific surfactin productivity qoverall were higher for the deletion strains CT10–CT12, while biomass titers were overall lower compared to the reference KM1016. Accordingly, the productivity per cell of the mutant strains was higher and thus supported the hypothesis that a deletion of Rap-phosphatases might positively influence surfactin production. The differences in achieved CDWmax were even more drastic for negative control ΔoppA. This indicated a trend that as surfactin production increased, the biomass production was lowered. This was also observed in a study by Vahidinasab et al. (2020) in which strain BMV12, with a deleted srfA operon showed higher OD600 values compared to the strains in which the operon was active. This suggests the possible explanation, that if surfactin production is low or absent, more metabolic energy would be available for biomass growth. Conversely, this could explain why strains CT10–CT12 achieved a lower CDWmax after 16 h than the reference strain KM1016 considering the higher qsurfactin for the mutant strains.

Deletion of Rap-phosphatases only slightly affected ComX level

In Treinen et al. (2021), it was found that specific surfactin productivity qsurfactin did not increase linearly with the ComX activity. Since ComA ~ P also affects the expression of rapC and rapF (Comella and Grossman 2005), it was suggested that high ComX activity leads to high RapC and RapF concentration, which in turn lowers surfactin titer and surfactin productivity (Treinen et al. 2021). Conversely, this would imply that deletion of these Rap-phosphatases would abolish or attenuate this negative feedback and that, while ComX levels would remain constant, productivity as well as surfactin titer would increase. The ComX activities of the deletion strains were in a comparable range with the reference KM1016 or even lower. Furthermore, the obtained values were consistent with a previous study (Treinen et al. 2021), in which maximum ComX activities of 357.3 ± 41.9 MU were measured for strain B. subtilis DSM10T in shake flask cultivations with 40 g/L glucose. Nonetheless, all deletion mutants showed higher overall specific surfactin productivities qoverall. This supported the hypothesis already formulated in Treinen et al. (2021) and addressed again here. It also showed that the differences in measured promoter activity PsrfA-lacZ may have been in fact due to the deletion of the Rap-phosphatases and not due to the differences in ComX levels.

Negative control ΔoppA showed low surfactin concentrations

A crucial difference to the mutants with deletion of rap is that in the ΔoppA variant no Phr, the antagonist of Rap, should be taken up into the cell (Lazazzera et al. 1997). Therefore, all Rap-phosphatases should be fully active in this strain (Fig. 1), as previously suspected by Auchtung et al. (2006), who found low PsrfA-lacZ activity in a Δopp mutant. Consistent with this, low surfactin titers and consequently low specific surfactin productivities were obtained with the negative control CT5. Since the ABC transporter Opp is affected by the deletion of oppA, this strain has a special role as further effects on the cell might occur that are not considered in the context of this study (Lazazzera et al. 1997).

Prolonged cultivation time increased surfactin titer in ΔrapC and ΔrapF mutant

After prolonging the cultivation time, it was observed, that strains CT10 and CT11 exhibited elevated surfactin titers and product yield per biomass YP/X. However, the specific surfactin productivities qsurfactin were at the same level or even lower. This can be explained by the fact that although the mutants showed increased product titers, these were only reached at a later stage of cultivation. Future process design should therefore consider whether the focus is on productivity or yield. One explanation for the fact that the effects on surfactin titer are now noticeable could be that the stationary phase has not yet been reached in the previous experiment, although it is known that surfactin production takes place in the late exponential to early stationary phase (Ongena and Jacques 2008). Especially in cultivations with low glucose concentrations this growth phase hardly occurs because the cell growth stops immediately upon glucose depletion, as seen in an aerobic cultivation of B. subtilis JABs24 with 1% glucose (w/v) (Geissler et al. 2019). In a similar manner, the deletion mutants were cultured in mineral salt medium containing 8 g/L glucose during preliminary experiments (Figure S3), and a decrease in CDW was observed after 16–20 h of cultivation without entering the stationary phase. At the same time, surfactin remained in a comparable range of approximately 900–1000 mg/L without a noticeable effect of the rap deletions. In the previously discussed studies, the medium of choice was either Schaeffer’s sporulation medium (Bongiorni et al. 2005; Core and Perego 2003; Smits et al. 2007) or a mineral salt medium with 10 g/L glucose (Auchtung et al. 2006). The amount of carbohydrate source and the associated growth behaviour of B. subtilis could therefore explain the not always knowledgeable effect of rap deletions observed so far. This assumption is strengthened by a study of Sun et al. (2018) who investigated the influence of rapC deletion on bacillomycin D production in B. amyloliquefaciens using 20 g/L glucose. There, a 1.5-fold increase in bacillomycin D from 240.7 ± 18.9 mg/L to 360.8 ± 30.7 mg/L was observed with prolonged cultivation, after the titer initially remained comparable. A possible influence of the chosen medium on the results was also mentioned in the study by Auchtung et al. (2006), who pointed also to the various promoters tested as a further explanation for inconsistent effects.

To exploit quorum sensing for process control, it is essential to understand the individual processes within the cell. In this study, it was hypothesised that deletion of Rap-phosphatases RapC, RapF and RapH would increase surfactin titer and specific surfactin productivity qsurfactin. To the best of our knowledge, this is the first time that quantitative data on surfactin concentration have been collected in this context. Up to the Pmax of the reference KM1016, all three deletion mutants showed an increased product yield per biomass YP/X and qoverall with consistent ComX levels. With prolonged cultivation time, the surfactin titer could even be enhanced by 2.7-fold for CT10 (ΔrapC) and 2.5-fold for CT11 (ΔrapF), which further supports the previously established hypothesis. The insights into surfactin production at the quantitative level obtained in this study contribute to the integration of the quorum sensing mechanism into process control instead of perceiving it as a constraint. For instance, incorporating a model and thus combining the influence of ComX and Rap-phosphatases on surfactin production could be used in the scope of molecular process control. At the genetic level, this study lays the groundwork to investigate other Rap-phosphatases following the same protocol or to create combinatorial mutants to examine a possible masking effect. Since metabolic regulation in Bacillus is very extensive, it would also be interesting to study the influence of rap deletion in relation to other parts of the system. For example, with respect to other lipopeptides produced by Bacillus, this study can be used as an example to gain deeper insights into the broader spectrum of the regulatory mechanism and further exploit its potential.

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