Escherichia coli BL21 (DE3) is an excellent and widely used host for
recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific
proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed
glucose dehydrogenase (GDH), a significant industrial
enzyme, severe cell
autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other
recombinant proteins. This precludes a further increase of the produced
enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial
enzyme production costs. Analysis of membrane structure and
messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the
autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate
autolysis completely. Consequently, we attempted to develop a strong expression host resistant to
autolysis by controlling the speed of
recombinant protein expression. To find a more suitable
protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to
autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model
enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other
enzymes. The engineered E. coli strain constructed in this study conveniently optimizes
recombinant protein overexpression by suppressing cell
autolysis, and shows great potential for industrial applications.