Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Abstract

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.

Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

“…Here, most approaches are related to reducing recombinant gene expression, for example by decreasing gene copy numbers, promoter strength or inducer concentration e.g. [ 24 – 26 ]. The exact nature of the “disturbing impact” of enhanced recombinant gene transcription on cell growth is not yet known.…”

Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation

“…3D). Besides, E. coli BL21(DE3) strain usually suffered from cell autolysis (Wagner et al 2008;Narayanan et al 2008), which is obscure to control the right chance for cell harvest at the fermentation anaphase. Recombinant P. pastoris therefore exhibited great operation stability for fermentation process.…”

Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

“…Studies had shown that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), an autolysable protein, severe cell autolysis was induced, resulting in low protein production [12]. Therefore, we investigated whether the three engineered strains can improve the expression of GDH through uorescence intensity (Fig.…”

“…Recently, a gene growth switch based on CRISPRi was developed to achieve e cient production of target proteins by targeting genes related to cell growth and DNA replication in E. coli [11]. Sun et al constructed a non-autolytic strain capable of e ciently producing recombinant proteins by hybridizing the weak Lac promoter with the strong P LacUV5 [12]. On another hand, the P LacUV5 has a higher basic leakage expression, which cannot provide precise control of gene expression [13,14].…”