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.