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\begin{document}

\title{Downregulation of T7 RNA polymerase transcription enhances pET-based
recombinant protein production via suppression of Escherichia coli BL21
(DE3) programmed cell death}



\author[1]{Xiao-Man Sun}%
\author[1]{Zi-Xu Zhang}%
\author[1]{Ling-Ru Wang}%
\author[2]{Jing-Gang Wang}%
\author[2]{Yan Liang}%
\author[3]{Hai-Feng Yang}%
\author[4]{Rongsheng Tao}%
\author[5]{Yu Jiang}%
\author[6]{Jun-Jie Yang}%
\author[7]{Sheng Yang}%
\affil[1]{Nanjing Normal University}%
\affil[2]{HuaRui Biotechnology Company}%
\affil[3]{Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences}%
\affil[4]{Shanghai Institutes for Biological Sciences}%
\affil[5]{Shanghai TaoYuSheng Biotechnology Co., Ltd}%
\affil[6]{Shanghai Institutes for Biological Sciences Chinese Academy of Sciences}%
\affil[7]{CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
E. coli BL21 (DE3) is an excellent and widely used host for recombinant
protein production. Many variant hosts were developed from 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, serious 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. The membrane structure and mRNA
expression analysis showed that cells suffered programmed cell death
(PCD) during autolysis period. However, blocking three known PCD pathway
in BL21 (DE3) cannot alleviate autolysis completely. Furthermore, 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 a host of BL21 (DE3-lac1G), which
successfully withstand the PCD of the host. The GDH activity at 43h was
greatly increased from 37.5 U/mL 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 43h, compared to the 3 U/mL/h of BL21
(DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the
production of other 10 enzymes. The engineered E. coli strain in the
study conveniently optimizes recombinant protein overexpression by
suppressing cell autolysis, and shows potential industrial applications.%
\end{abstract}%



\sloppy


\textbf{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 \emph{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 \emph{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.

\textbf{2. Materials and methods}

\textbf{2.1 Construction of strains and plasmids}

All plasmids used in this study are listed in Supporting information
Table S1, and the primers are listed in Table S2. \emph{E. coli} strain
DH5\selectlanguage{greek}α \selectlanguage{english}was used as the cloning host. The GDH plasmids variants with
site-directed mutations and deletions of the RBS site (AAGGAG) were
constructed using megaprimer PCR. The modified pET plasmids with RK2
replicon were constructed by standard restriction cloning based on
Gibson assembly. All mutant strains were generated from BL21 (DE3) using
CRISPR-Cas9 technology (Jiang et al., 2015). The template DNA with
1,000bp homologous arms was prepared by PCR. Strains harboring the pCas
plasmid were transformed with the pTarget plasmid and the template DNA
by electroporation (1.85 kV, 200 Ohm, 25 uF) and regenerated for 1 h in
1 mL LB medium at 37 . The mutants were confirmed by colony PCR.
Correct colonies were then screened and the plasmids cured.

\textbf{2.2 Culture conditions}

The \emph{E. coli} strains was grown in Luria Bertani (LB) or Terrific
Broth (TB) medium at 37 , supplemented where necessary with 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL
kanamycin (Kan). Flask fermentation was performed as follows: \emph{E.
coli} BL21 (DE3) harboring pET24a-GDH was grown in 3 mL of LB liquid
medium overnight at 37. Then, 300 uL of the resulting culture were used
to inoculate 30 mL of Terrific Broth (TB) medium in a 250ml shake flask.
Cells were cultured at 37  to an OD\textsubscript{600} of 2-4, at which
point IPTG was added to a final concentration of 0.3 mM, and the
fermentation was allowed to continue at 28 for an additional 40 h.

The GDH expression strains were first pre-cultured at 37 , 220 rpm for
16 h seeded into a 500 mL shake flask containing 100 mL of TB medium,
and cultured at 37 \textsuperscript{o}C, 220 rpm for 5 h. In the
scale-up fermentation experiments, the resulting 100 mL seed culture was
used to inoculate a 7.5-L fermenter containing 4 L of TY medium (Tao et
al., 2014) at 37  at an aeration rate of 2.5 vvm. Agitation was
steadily increased to a maximum of 800 rpm to maintain 20\% dissolved
oxygen (DO). When the DO further increased to more than 60\%, 400 g/L
glycerol, 50 g/L yeast extract, and 25 g/L tryptone were added into the
medium, and the fermentation was continued at 800 rpm at an aeration
rate of 2.5 vvm. When the OD\textsubscript{600} reached 20, 0.2 mM IPTG
was added to the medium and the cells were allowed to express the target
protein at 28  for 43 h.

\textbf{2.3 Scanning electron microscopy}

BL21 (DE3) and BL21 (DE3-lac1G) cells harboring the pET24a-GDH
expression vector were cultured as described above, and samples were
collected at 24h and 43h, respectively. Samples were fixed overnight at
4\selectlanguage{ngerman}°C in 2.5\% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and then
stained with 2\% OsO4 in the same buffer. Then, samples were dried at
critical point and coated with platinum. Observations were made using a
Tecnai FEG ESEM Quanta 200 microscope (FEI Corp, USA), and images were
recorded using SIS iTEM software (Olympus Corp, Japan).

\textbf{2.4 GDH activity assay}

To measure GDH activity, cells from 1 mL of fermentation broth were
collected by centrifuging at 1,500 ×g for 10 minutes at 4\selectlanguage{english}. The culture
supernatant was discarded, and the cells were resuspended in 1 mL of 100
mM Tris-HCl (pH 7.5). The cell suspensions was lysed by sonication on
ice: 200 W, working 3 s, interval 5 s for 40 times, to yield the crude
enzyme solution. The reaction mixture contained 100 mM glucose, 100 mM
Tris-HCl (pH 7.5), 2 mM NAD\textsuperscript{+}, and an 8000-fold
dilution of crude enzyme solution. The enzyme activity was assayed at 25
\selectlanguage{english}, by recording the increase of the absorbance at OD\textsubscript{340}.
The enzyme activity was calculated using the formula U/mL = {[}\selectlanguage{greek}Δ\selectlanguage{english}A/min{]}
\selectlanguage{ngerman}× {[}1/\selectlanguage{english}\selectlanguage{greek}ε{]} \selectlanguage{ngerman}× 8000 (\selectlanguage{greek}ε \selectlanguage{english}= A/cL = 6.402 mL / (mol * cm)) (Bucher et al.,
1974).

\textbf{2.5 SDS-PAGE analysis}

The cells from 1 mL of fermentation broth were collected by
centrifugation at 12000rpm for 10 min, and the supernatant was
discarded. The cells were resuspended in 1 mL of 20 mM Tris-HCl buffer
(pH 7.5) and disrupted using an Ultrasonic Cell Disruptor (operating for
5s, pausing for 5s, 40 times). The cell lysate was centrifuged at 4 \selectlanguage{ngerman}°C,
12000 rpm for 10 min, and the soluble protein remained in the
supernatant. An 80 \selectlanguage{greek}μ\selectlanguage{english}L aliquot of the supernatant was mixed with 20 \selectlanguage{greek}μ\selectlanguage{english}L of
5\selectlanguage{ngerman}×loading buffer, and heated in a 100 °C metal bath for 10 min. The
final protein samples were used for SDS-PAGE. Samples corresponding to
0.15 OD (10-well gel) or 0.075 OD (15-well gel) were added to the
corresponding protein gel wells, and powered on until the
electrophoresis was complete.

\textbf{2.6 Quantitative real-time PCR}

The relative transcription of genes was evaluated by qRT-PCR using the
StepOnePlus\selectlanguage{english} Real-Time PCR System (Applied Biosystems, USA). Specific
primers for qRT-PCR were designed to bind inside the T7RNAP gene, and
amplify a fragment of approximately 220 bp. Total RNA was extracted
using the Total RNA Isolation Kit (Blood/Cultured Cell/Fungus)
(GeneDireX, Germany). The cDNA was produced using the SuperScript\selectlanguage{english} III
First-Strand Synthesis System (Invitrogen USA). An aliquot containing
200 ng of cDNA was subjected to qRT-PCR using the EvaGreen qPCR
System-ROX I (GeneDireX, Germany). The reaction (20 \selectlanguage{greek}μ\selectlanguage{english}L) contained 10 \selectlanguage{greek}μ\selectlanguage{english}L
of SsoAdvanced Universal SYBR Green Supermix (BioRad, USA), 20 \selectlanguage{greek}μ\selectlanguage{english}M
primers , 10 ng DNA, and adequate H\textsubscript{2}O. Reactions were
performed in white 96-well PCR plates (BioRad) and measured on a CFX384
real-time PCR detection system (BioRad) using the following thermal
cycling parameters: 1 cycle, 95 \selectlanguage{ngerman}° C, 2 min; 40 cycles , 95 ° C, 10 s, 59
°C , 20s, 72°C , 20s; terminal melt-curve analysis (59-95 °C in 0.5 °C /
5 s increments). G6PDH was used as the endogenous reference gene to
calculate the relative mRNA expression via the
2\selectlanguage{english}\textsuperscript{-^^CT}method.

\textbf{3 Results}

\textbf{3.1 GDH overproduction triggers autolysis}

GDH is an important industrial enzyme, but high level production of GDH
in BL21 (DE3) still presents a great technical challenge. In this study,
the GDH activity suddenly decreased from 217 U / mL at 31h to 37.5 U /
mL at 43h (Fig. 1A). Therefore, the fermentation time must be shortened,
making it impossible to continue fermentation to obtain higher yields.
Moreover, the OD in the later fermentation stage also showed a decline,
indicating that autolysis may have caused cell death and a release of
GDH into the extracellular medium. As shown in Fig. 1B, the SDS-PAGE
showed a reduction of the intracellular heterologous protein, while
there was a lot of the target protein in the extracellular medium. In
order to investigate if autolysis was caused by the enzyme activity of
GDH, the loss-of--function mutant GDH\textsubscript{Tyr253Cys}was
constructed (Makino et al., 1989), and the enzyme activity was decreased
from 214.3 U / mL to 23.1 U / mL. However, the SDS-PAGE results showed
that the mutant GDH\textsubscript{Tyr253Cys} still caused autolysis
(Fig. 1C), which suggested that the autolysis was not related to enzyme
activity. In addition, a mutant plasmid without RBS site was constructed
to investigate if autolysis was caused by transcription rather than
translation. The results showed that the cell growth was inhibited even
after removing RBS site (Fig. S1), but no autolysis observed, which
suggested that the intense burst of mRNA synthesis by T7 RNAP also had
some toxic effects on \emph{E. coli} . Furthermore, the membrane
structure of \emph{E. coli} cells during lysis due to GDH overexpression
was visually inspected by means of scanning electron microscopy (SEM).
The images showed extensive membrane elongation and shrinking (Fig. 1D),
which confirmed the phenomenon of autolysis. Importantly, when amide
hydrolase, Cephalosporin C acylase, formate dehydrogenase, R-carbonyl
reductase, L-lactate dehydrogenase, L-2-hydroxyisocaproate
dehydrogenase, L-phenylalanine dehydrogenase, secondary alcohol
dehydrogenase and other 10 enzymes were individually overexpressed each
also induced autolysis of BL21 (DE3). Therefore, solving the problem of
autolysis in this workhorse strain is of great importance for industrial
production.

\textbf{3.2GDH expression resulted in programmed cell death}

The combination of deceased OD and concomitant phenotypic changes
provided convincing evidence that autolysis induced by GDH
overexpression may be caused by triggering of a programmed cell death
(PCD) pathway. The three major PCD pathways in bacteria are
apoptotic-like death, mazEF-mediated death, and the cidA/lrgA
holin-antiholin system. The RecA meditated apoptosis-like death pathway
is thought to be activated by the SOS response (Lee et al., 2019b). The
cidA/lrgA loci are regulators of the holin-antiholin system in
\emph{Staphylococcus aureus} , and the homologous locus of yohJK locus
of \emph{E.coli} was considered to have the same effect in \emph{E.
coli} (Dewachter et al., 2015). As shown in Fig. 2A, mRNA expression
analysis showed that the expression of recA, mazEF, and yohJK at 43h was
increased by 2.5-3.6 folds over their respective expression at 24h,
which implied that autolysis could be triggered by PCD. To inhibit PCD
mediated by known pathways, corresponding single and multiple deletion
mutants were constructed. As shown in Fig. 2B, the \selectlanguage{english}^recA strain can
exhibited reduction of the extracellular fraction of GDH enzyme activity
from 90 to 65\%, but it did not markedly inhibit the autolysis.
Similarly, deletion mazEF and yohJK did not alter the cell viability
under GDH overexpression. Even the combined deletion of \selectlanguage{english}^recA- mazEF-and
yohJK did not alleviate the PCD, suggesting that PCD induced by GDH
overproduction was not triggered by the reported three pathways.

\textbf{3.3 Development of the BL21 (DE3-lac1G) strain that is resistant
toPCD}

The autolysis of DE3 strains may be controlled by various fermentation
strategies, but obtaining a strong expression host that is intrinsically
resistant to PCD is more meaningful and convenient for
industrialization. As a common solution, C41 (DE3) was chosen as
expression host, and the results showed that the GDH activity was
increased from 173.7 U / mL at 24h to 207.3 U / mL at 43h (Fig. S2). It
is important to note that there was no significant autolysis in the C41
(DE3) strain, although there was no great increase in GDH activity.
Compared to BL21 (DE3), an important difference of the C41 (DE3) strain
is the exchange of lacUV5 into a weaker lac promoter. In fact, the
lacUV5 and lac (also named lac-1A) promoters differ only in two places
and three points, so other two different promoter variants lacUV5-1A and
lac-1G was obtained by shuffling and recombining of lacUV5 and lac-1A
(Fig. 3A). Interestingly, the GDH activity increased significantly in
the new strain BL21 (DE3-lac1G), resulting in 452.0 U / mL enzyme
activity at 43h, which was about 12-times higher than that of BL21 (DE3)
(Fig. 3B). However, the enzyme activity of the BL21 (DE3-1A) strain was
lower than that of the starting strain BL21 (DE3). It is worth noting
that although the enzyme activity of BL21 (DE3-lac1A) was low, there was
no decrease of enzyme activity (Fig. 3B). More importantly, a very low
extracellular GDH activity was obtained in BL21 (DE3-lac1G) strain (Fig.
3C), which implied that an appropriate T7 RNAP expression level was
realized using the lac-1G promoter. Further SEM images indicated that
the membranes of BL21 (DE3-lac1G) cells were smooth and robust at 43h
(Fig. 3D), indicating that no PCD occurred in this new host. In the
scale-up fed-batch fermentation, the GDH activity of BL21 (DE3) peaked
at 31h and remained nearly unchanged thereafter, while that of BL21
(DE3-lac1G) was enhanced during the entire fermentation period, reaching
yields of up to 3142.4 U / mL (Fig. 4). Notably, with increasing culture
time, the productivity of GDH in BL21 (DE3) and BL21 (DE3-lac1G)
followed opposite trajectories. In the BL21 (DE3) strain, the highest
productivity was obtained at 7-19h, and then declined to only 3 U/mL/h
at 31-43h. By contrast, the productivity of BL21 (DE3-lac1G) was
increased constantly, and reaching 77.68 U/mL/h at 43-50h.

\textbf{3.4 Analysis of key factors for controlling autolysis}

To investigate whether the changes in the transcription level of T7 RNAP
caused the difference in protein expression, the transcription levels of
4 promoters were measured by qRT-PCR. As shown in Figure 5A, the
strength of the 4 promoters were lac-1A
\textless{}lac-1G\textless{}lacUV5\textless{}lacUV5-1A. The
transcription level of T7 RNAP from P\textsubscript{lac-1G} was only
16.4\% that of P\textsubscript{lacUV5}. Consequently, the former
maintained GDH expression at a slightly lower level, which might be the
key reason for suppression of PCD. In a previous study, the expression
capacity of T7 RNAP within a wide range was realized by designing RBS,
which precisely regulate the expression of recombinant proteins (Liang
et al., 2018). However, compared to P\textsubscript{lac-1A}, the
relative transcription level of P\textsubscript{lac-1G} was increased by
2.05 times, which resulted in faster GDH accumulation than in BL21
(DE3-lac1A), which finally led to the highest performance.
Interestingly, the transcription level of the lacUV5-1A promoter showed
a 2.68 folds increase, indicating that a strong promoter is not
conducive to the expression of GDH and may be the key reason for PCD.

In addition to controlling the expression level of T7 RNAP, we
investigated if it could also be prevented by modifying the copy number
of the plasmid carrying the target gene. High copy number plasmid for
protein production puts extra metabolic load on host cells, resulting
inhibition of cell growth and plasmid instability (Bentley et al.,
2009). In a previous study, sequence homology between CoIE1 RNA I/ RNA
II and tRNAs was abolished to keep the plasmid copy number constant,
then metabolic activity can be prolonged (Grabherr et al., 2002). Based
on this, the replicon of the pET plasmid (CoIE1) with 15 copies was
replaced by the RK2 replicon with 5 copies. Then, the BL21
(DE3-lac1G)-R2K and BL21 (DE3)-R2K strains with combinations of ``strong
+ low copies'' and ``weak + low copies'' were obtained, respectively. As
shown in Fig. 5B, although the GDH activity of BL21 (DE3)-R2K was higher
than that of BL21 (DE3) -colE1, it was only 51.62\% that of the BL21
(DE3-lac1G)-colE1. The ``weak + low copies'' group exhibited much lower
enzyme activity than the other groups. Overall, controlling the
expression level of T7 RNAP is more effective than controlling the copy
number of the plasmid, implying that T7 RNAP plays the role of the lysis
``switch'' in the pET system for recombinant protein overexpression
(Fig. 5C).

\textbf{3.5 Stability and versatility of BL21 (DE3-lac1G) strain}

Stability and versatility are key indicators for investigating the
quality of protein expression hosts. As shown in Fig. 6A, overall, the
ration of plasmid-carrying cells in BL21 (DE3) continued to decline with
increasing fermentation time, reaching a low of 40\% at 36h and
decreasing further to 0 at 43h. By contrast, the ratio of
plasmid-carrying cells in BL21 (DE3-lac1G) remained about 96.5\% at 43h,
and about 49\% of the cells maintained enzyme activity at normal levels
(\textasciitilde{} 350 U / mL) (Fig. 6B). Therefore, the stability of
the new protein expression host is much higher than that of the parent
strain. Moreover, the SDS-PAGE images showed that BL21 (DE3-lac1G) is
also resistant to autolysis that is triggered by a further 10 tested
recombinant proteins, including amide hydrolase, cephalosporin C
acylase, formate dehydrogenase, R-carbonyl reductase, L-lactate
dehydrogenase, L-2-hydroxyisocaproate dehydrogenase, L-phenylalanine
dehydrogenase, secondary alcohol dehydrogenase, D-2-hydroxyisocaproate
dehydrogenase, S-carbonyl reductase (Fig. 7).

\textbf{Discussion}

In the last decades of the 20\textsuperscript{th} century,
\emph{E.coli}BL21 (DE3) has become the preferred host for recombinant
protein production. However\selectlanguage{english},some recombinant proteins may impose a high
metabolic burden or lead to toxicity in the host cell, which may result
in reduced growth rate, low final cell density, and even cell death
(Bhattacharya \& Dubey, 1995). After 24h, GDH expression inhibited cell
growth and induced severe autolysis (Fig. 1A and B). For the expression
of toxic membrane protein, a common strategy is to decrease the
expression of the toxin protein by governing the expression of T7 RNAP,
such as C41 (DE3) and Lemo21 (DE3). Notably, the C41 (DE3) strain weaken
the lacUV5 promoter by recA-dependent recombination with the lac
promoter (Susan et al., 2015), indicating that the strength of the lac
promoter may not be optimal. However, the C41 (DE3) strain could not
effectively improve the expression of GDH compared to BL21 (DE3), which
implied that the toxic effect of GDH is different from that of toxic
membrane proteins. Most importantly, the autolysis phenotype appeared
during the expression of a variety of proteins, which necessitates
shortening the fermentation time, and eventually reduces the protein
yield. The host mutation of BL21 (DE3-lac1G) changes one base in the
lac-1A promoter, which had an enormous positive impact on autolysis,
ie., a high rate of 89.55 U/mL/h was obtained at 43h in comparison of
that of BL21 (DE3) with 3 U/mL/h. Taken together, our results proved
that the new strain BL21 (DE3-lac1G) strain can effectively suppressing
PCD and maintain plasmid stability, which will make it a popular host
for protein production, especially for proteins requiring a longer
fermentation time or maturation with after processing.

The production of foreign proteins often imposes a metabolic burden on
the host by triggering local and global cellular stress responses
(Bhattacharya \& Dubey, 1995). In particular, this metabolic burden
often manifests as an increase of the energy demand or maintenance
energy requirement such as amino acids, ribosomes or other precursors
(Mairhofer et al., 2013). The metabolic burden on host cells can be
improved by making right choices for promoter (Pasini et al., 2016),
plasmid copy number (Flores et al., 2004), and removal of codon bias
(Rahmen et al., 2015). Moreover, optimizing environment conditions also
can reduced metabolic burden, such as addition of amino acids or using
complex medium (Fong \& Wood, 2010). As a first measure, if the growth
rate of recombinant strain is inhibited, then two causes may explain the
phenotype: gene toxicity and basal expression of the toxic mRNA/protein.
Protein toxicity refers to the toxicity of the target protein itself,
such as its enzyme activity or cell damage due to misfolded aggregates
(Binepal et al., 2012). On the other hand, studies have reported that
excessive T7 RNAP itself can be lethal to cells (Davanloo et al., 1984).
Moreover, it was found that the toxicity caused by mRNA may be neither
related to plasmid abundance nor to the abundance of the encoded mRNA
(Mittal et al., 2018). Previous studies also proved that the toxicity of
certain gene was only dependent on transcription but independent of
protein translation (Li \& Rinas, 2020). In this study, a GDH mutant
with dramatically reduced activity and a construct without RBS proved
that autolysis was not caused by the activity or amount of protein, so
it is likely that the autolysis is induced by certain mRNA elements. In
this study, the T7 RNAP expression from lac-1G promoter was found to be
higher than from lac-1A promoter, but lower than from lacUV5.
Consequently, the rate of protein overexpression in BL21 (DE3-lac1G) was
manipulated at a will level, not only transcription with a higher level
but also no more than the host tolerance during the whole fermentation
stage. A recent study of a plasmid-driven T7 (PDT7) system also
suggested that high expression of T7 RNAP affected cell metabolism and
led to toxicity and instability (Tan \& Ng, 2020). In fact, the T7 RNAP
itself could be not toxic, but when it combined with a strong promoter,
it can induce severe growth defects. A excessively strong T7 RNAP system
robbed energy from the basal metabolism, which is the major reason of T7
RNAP toxicity (Tan \& Ng, 2020). By inhibiting endogenous RNAP or
reducing parts of non-essential proteome production, it is possible to
balance cell growth and recombinant protein production on resources
allocation (Kim et al., 2019). Recently, the evolved T7 phage RNAP
inhibitor Gp2 was used in BL21 (DE3) to decouple recombinant protein
production from cell growth, which enhanced protein yields up to
3.4-fold (Stargardt et al., 2020). Moreover, the resources can be
selectively allocated for transcription or translation of target genes
by orthogonal molecular elements (Darlington et al., 2018;
Segall-Shapiro et al., 2014), which were beneficial to reduce the
metabolic burden of the host cells. This study proved that the key to
regulating autolysis and protein overexpression is controlling the
expression of T7 RNAP, which plays the role of the main on-off
``switch'' in the pET system. Moreover, controlling the rate of
transcription of T7 RNAP can mitigate the metabolic burden effectively
and easily. The new host strain of BL21 (DE3-lac1G) can effectively
produce recombinant protein without affecting growth.

Under excessive stress, the repair mechanisms will be overwhelmed and
cells will undergo programmed cell death (PCD). This program offers no
direct advantage to individual cells, but could benefit its siblings by
releasing nutrients for other cells in the colony or preventing the
spread of viruses (Tanouchi et al., 2013). The death involved PCD
pathway is meditated by an intracellular program (Nagamalleswari et al.,
2017), mainly including the toxin-antitoxin system, holin-antiholin
system and ALD pathway. The membrane integrity of BL21 (DE3) was
impaired when overexpressing GDH. More importantly, the expression level
of PCD markers including recA, mazEF, and yohJK were showed to be
up-regulated at 43h compared to 24h (Fig. 2A), which suggested that
BL21(DE3) suffered PCD at 43h. In general, deletion of the key genes
involved in the PCD pathway can restore cell viability. For example,
periplanetasin-2 was proven to induce apoptosis-like death
in\emph{E.coli} according to physiological changes, which was proven
when the antibacterial activity of periplanetasin-2 was decreased by
deletion of recA (Lee et al., 2019a). However, deletion of three key
genes did not restore viability in the GDH production process,
indicating that cell death of BL21 (DE3) does not proceed only through
these three general bacterial PCD pathway. In the new strain BL21
(DE3-lac1G), the lower protein expression rate did not exceed the
cellular metabolic capacity, and therefore could not induce PCD.
Nevertheless, the common characteristics of proteins that cause PCD and
how it is promoted needs further study.

In the future, this novel autolysis system can be used in secretory
protein production. The production of extracellular proteins has
distinct advantages, such as simplifying the disruption of the cell wall
and purification processes (Su et al., 2013). Previous studies applied
holins and endolysins to promote cell lysis, combined with various
inducible promoters to prevent cell lysis before sufficient cell growth
(Choi \& Lee, 2004). However, expression of lytic proteins still uses
valuable cellular energy resources. Inspired by this study, the promoter
of T7 RNAP can be designed to let cells overexpress too much of the
target protein at a desired time-point induce autolysis, so that the
host produces the target protein from beginning to end, without relying
on exogenous lysing proteins. In addition, four promoters with different
strength can form a series of protein expression hosts with different T7
RNAP expression levels, providing a variety of host choices. Moreover,
the promoter strength of lacUV5-1A is 2.68 times higher than that of the
strong promoter lacUV5, which indicated that lacUV5-1A promoter can
satisfy the requirements of high expression of target genes in metabolic
engineering in the future.

\textbf{Acknowledgements}

These studies were supported by the National Science Foundation for
Distinguished Young Scholars of China (Number: 21825804) and National
Science \& Technology Major Project ``Key New Drug Creation and
Manufacturing Program'', China (Number: 2018ZX09711002).

\textbf{Competing interests}

The authors declare that they have no competing interests.



\section*{Supporting Information}

{\label{supporting-information}}

Table S1: Strains and plasmids used in this work.

Table S2. Primers used in this work.

Figure S1. OD600 analysis of BL21 (DE3) with expression vector without
RBS.

Figure S2. Comparison between BL21 (DE3) and C41 (DE3) for GDH activity.

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\textbf{Figure captions}

\textbf{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.

\textbf{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 (\emph{recA} , \emph{mazEF} and \emph{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 \selectlanguage{english}^recA, \selectlanguage{english}^yohJK, \selectlanguage{english}^mazEF, and \selectlanguage{english}^recA+ yohJK+ mazEF derivatives.
Values and error bars represent the means and the deviations from
triplicate experiments.

\textbf{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.

\textbf{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.

\textbf{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.

\textbf{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).

\textbf{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.

\textbf{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.

\textbf{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.

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