Figure 3. Process characteristics of GFP-producing strains in chemostat culture. (A-C) Courses of the specific content of GFP (YP/X) and biomass. (D-F) Single-cell expression analysis. (A, D) B3<T7-GFP> wt, (B, E) B3<T7-GFP> #E7P6 S10, (C, F) BQ<A1-GFP> wt.
For B3<T7-GFP> wt, induction with IPTG resulted in very high specific content of 348 mg GFP/g CDM (Figure 3A), which was clearly higher than the product titer from a fed-batch culture with the same final biomass of 30 g/L (Figure 4A, Table 3). This productivity could be maintained for 72 hours after induction. An extreme decrease was observed in the biomass and product titer. This phenomenon was demonstrated in previous studies in which we also observed a decrease in the product titer 60 h after induction.[15] After approximately 120 hours, the biomass recovered and increased to the intended 30 g/L CDM, but without product formation. The specific GFP content remained at 0 mg/g until the end of the fermentation. As shown in Figure 3D, non-producers had completely asserted themselves, and the results of the microtiter experiments were reproduced. Similar behavior was observed with the derivative #E7P6 S10 (Figure 3B), with an extreme decrease in the biomass to almost 0 g/L after 72 hours in chemostat mode. After 120 hours, the cells recovered to the intended biomass of 30 g/L CDM. Unlike its wild-type ancestor, the specific GFP content did not decrease to zero, but decreased steadily until the end of the fermentation. The peak at a fluorescence intensity of 102 in the FL1-A channel (Figure 3E) suggests that a mixed population emerged in which non-producers have gradually overgrown the producing cells, causing the production to decrease over the course of the cultivation.
By determining the T7 RNAP gene sequence via PCR amplification of the corresponding chromosomal region and Sanger sequencing, we identified the insertion element insD-3 within the T7 RNAP gene of B3<T7-GFP> wt and a mutation within the lacUV5 promoter in derivative #E7P6 S10, which may have resulted in the complete loss of GFPmut3.1 productivity
In contrast, no extreme decrease in biomass was observed for the host RNAP-dependent expression system BQ<A1-GFP> wt, indicating an extraordinarily high stability (Figure 3C). Over the entire cultivation period of 312 hours and 45 generations, the specific product content remained constant at approximately 100 mg GFP/g CDM (Figure 3C). Flow cytometry confirmed the process stability. A homogenous population was confirmed at each time point (Figure 3F). The lower productivity of this expression system in combination with GFP apparently represents such a low metabolic load that there was no population collapse. Potential mutants did not have a growth advantage, which means that the population of producing cells could persist for a period of 45 generations.