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.