Discussion
In the last decades of the 20th century, E.coliBL21 (DE3) has become the preferred host for recombinant protein
production. However,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 inE.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.