Introduction
The pET system is a powerful tool for recombinant protein overexpression. It is based on host strains lysogenic for the DE3 prophage, with an integrated T7 RNA polymerase (RNAP) gene into the host genome, and cognate plasmids containing the T7 promoter. The transcription rate of T7 RNAP is about 8 times faster than that of the native E. coli RNAP (Jeong et al., 2009; Studier & Moffatt, 1986). BL21 (DE3) is arguably the most widely used protein production host. The promoter of the T7 RNAP gene in BL21 (DE3) is the lacUV5 mutant variant of the lac promoter, which is stronger than the original (Jeong et al., 2009). The reasoning behind the choice of these components for the production of proteins was straight-forward, based on the premise that more mRNA is beneficial for protein overexpression. To date, protein expression systems have been optimized widely by multiple approaches, including host reconstruction, expression vector redesign, and optimization of fermentation conditions (Costello et al., 2019; Li et al., 2016; Rosano et al., 2019).
However, BL21 (DE3) still cannot effectively produce certain proteins, especially toxic membrane proteins. Subsequently, the C41 (DE3) and C43 (DE3) strains were developed for membrane protein production. Studies have shown that C41 (DE3), in which the lacUV5 promoter was mutated into the weaker lac promoter, can effectively express toxic proteins (Schlegel et al., 2015). This mutant had a lower transcription rate of T7 RNAP, by which the toxic effect caused by overexpression of membrane proteins could be effectivity relieved (Kwon et al., 2015). Before, a derivative strain of BL21 (DE3), named Lemo21 (DE3), was engineered in which the activity of the T7 RNAP can be controlled by using the inhibitor T7 lysozyme (Wagner et al., 2008). Mutant56 (DE3) was isolated from a library of BL21 (DE3) variants, ant it was found that one amino acid was changed in T7 RNAP, which weakens the binding of the T7 RNAP to T7 promoter, thereby increasing membrane protein yields (Baumgarten et al., 2017).
In addition to lower expression of toxic proteins, BL21 (DE3) is also unable to effectively overexpress certain proteins that induced a physiological burden such as growth inhibition, cell lysis, or even death (Bhattacharya & Dubey, 1995). The production of this type of protein is normal during the early fermentation stage, but cells suffer autolysis at the later fermentation stage. For example, the presence of penicillin acylase inclusion bodies inhibited cell growth and caused serious cell lysis, so that approximately 76% of penicillin acylase was found in the extracellular medium (Narayanan et al., 2008). This phenomenon caused the fermentation time to be shortened, which ultimately reduced the yield of recombinant protein. Previous studies indicated that autolysis is not a direct result of the amount of the heterologous protein (Spada et al., 2002), but rather the global stress response induced by recombinant gene transcription or translation (Hoffmann & Rinas, 2004). Recently, many studies suggested that recombinant gene transcription, the mRNA level or even the speed of translation are the major causes of the growth inhibition or metabolic collapse (Li & Rinas, 2020). For example, under certain culture conditions, the accumulation of GFP can affect the growth of E. coli , but the effect could not be alleviated by removing the RBS of GFP (Mittal et al., 2018). In some cases, if the codons encoding the same amino acid in the same recombinant protein are different, the effect on inhibiting cell growth will also be different (Mittal et al., 2018; Natalie et al., 2015). However, the molecular mechanisms of this apparent RNA toxicity are presently unclear.
The cell lysis may be remedied by lowering expression, decreasing the growth temperature, or reducing target gene promoter activity. However, it is more meaningful and convenient for industrialization to construct a strong expression host that is intrinsically resistant to autolysis. In this study, an important industrial enzyme, GDH (EC:1.1.1.47), was applied as reporter protein. Blocking the known PCD pathway and controlling the speed of protein expression was used to suppress PCD, which promises to solve the urgent problems of industrial protein production.