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
GDHTyr253Cyswas 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 GDHTyr253Cys 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 E. coli .
Furthermore,
the membrane structure of 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.
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 Staphylococcus
aureus , and the homologous locus of yohJK locus of E.coli was
considered to have the same effect in 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 △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 △recA- mazEF-and yohJK did
not alleviate the PCD, suggesting that PCD induced by GDH overproduction
was not triggered by the reported three pathways.
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.