Figure captions
Figure 1 . GDH overproduction triggers autolysis of BL21 (DE3). (A) GDH activity and OD were monitored during the whole fermentation period. (B) SDS-PAGE results of intracellular and extracellular protein in BL21 (DE3) at 46h. The white rectangle was used to indicate the GDH expression band. (C) SDS-PAGE results of intracellular and extracellular protein in BL21 (DE3)-GDH and BL21 (DE3)-GDH-mutant strain at 31h. (D) The cell membrane changes in BL21 (DE3) strain between 24h and 43h were observed by SEM. The red arrow indicates the protrusions or shrinking of cell membrane. Values and error bars represent the means and the deviations from triplicate experiments.
Figure 2. PCD was found to be induced in the process of GDH overproduction, and GDH activity was analysis after blocking the reported PCD pathway. (A) Quantitative real-time PCR results at 24h and 43h for the three key genes (recA , mazEF and yohJk ) involved in the PCD pathway. (B) The changes of intracellular and extracellular GDH activity was measured at 24h and 43h in BL21 (DE3) as well as △recA, △yohJK, △mazEF, and △recA+ yohJK+ mazEF derivatives. Values and error bars represent the means and the deviations from triplicate experiments.
Figure 3 . BL21 (DE3-lac1G) greatly reduced autolysis and enhanced GDH overproduction. (A) Sequence variation between the four types of promoters. Substitutions are marked in red. (B) The extracellular GDH activity under four different promoters was measured during the entire fermentation period. (C) Comparison between the BL21 (DE3) and BL21 (DE3-lac1G) for GDH activity and SDS-PAGE at 43h. (D) The cell membrane changes of BL21 (DE3-lac1G) strain between 24h and 43h was investigated by SEM. Values and error bars represent the means and the deviations from triplicate experiments.
Figure 4. Comparison between BL21 (DE3) and BL21 (DE3-lac1G) for GDH activity and productivity in scale-up fermentation. Values and error bars represent the means and the deviations from triplicate experiments.
Figure 5. Performance assessment of the BL21 (DE3-lac1G) strain. (A) The relative mRNA expression of T7 RNAP under four different promoters. (B) The GDH activity changes under different combinations with weak/strong T7 RNAP expression levels and high/low plasmid copy numbers. (C) Model explaining the differences in behavior between BL21 (DE3-lac1G) and BL21 (DE3). Values and error bars represent the means and the deviations from triplicate experiments.
Figure 6. Plasmid stability test of BL21 (DE3-lac1G) strain. (A) The percentage of plasmid-carrying cells of BL21 (DE3)-PET24a (empty), BL21 (DE3) and BL21 (DE3-lac1G) was tested during the entire fermentation period. (B) Overview of stability determination during the fermentation (above), and the GDH activity of 45 clonds isolated at the end of fermentation (below).
Figure 7. The BL21 (DE3-lac1G) strain exhibited a superior overexpression capacity with 10 additional different heterologous proteins compared with the parental strain BL21 (DE3). SDS-PAGE with intracellular and extracellular proteins expressed in BL21 (DE3) and BL21 (DE3-lac1G) was showed and compared. The white rectangle indicates the target protein expression band.
Figure S1. OD600 analysis of BL21 (DE3) with expression vector without RBS. Values and error bars represent the means and the standard deviations of triplicate experiments.
Figure S2. Comparison between BL21 (DE3) and C41 (DE3) for GDH activity. Values and error bars represent the means and the standard deviations of triplicate experiments.