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Academic Editor: Giuseppe Comi
Received: 24 December 2024
Revised: 14 January 2025
Accepted: 16 January 2025
Published: 18 January 2025
Citation: Sui, X.; Guo, L.; Bao, Z.;
Xian, M.; Zhao, G. Efflux Pumps and
Porins Enhance Bacterial Tolerance to
Phenolic Compounds by Inhibiting
Hydroxyl Radical Generation.
Microorganisms 2025,13, 202.
https://doi.org/10.3390/
microorganisms13010202
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
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(https://creativecommons.org/
licenses/by/4.0/).
Article
Efflux Pumps and Porins Enhance Bacterial Tolerance to Phenolic
Compounds by Inhibiting Hydroxyl Radical Generation
Xinyue Sui 1, Likun Guo 1, Zixian Bao 1, Mo Xian 2and Guang Zhao 1, *
1State Key Laboratory of Microbial Technology and Institute of Microbial Technology, Shandong University,
Qingdao 266237, China
2CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese
Academy of Sciences, Qingdao 266101, China
*Correspondence: zhaoguang@sdu.edu.cn
Abstract: Phenolic compounds are industrially versatile chemicals that have been success-
fully produced in microbial cell factories. Unfortunately, most phenolic compounds are
highly toxic to cells in specific cellular environments or above a particular concentration
because they form a complex with iron and promote hydroxyl radical production in Fenton
reactions, resulting in the ferroptosis of cells. Here, we demonstrated that overexpression
of efflux pumps and porins, including porins LamB and OmpN, and efflux pumps EmrAB,
MdtABC, and SrpB, can enhance Escherichia coli phloroglucinol (PG) tolerance by inhibit-
ing the generation of hydroxyl radicals. In addition, LamB and OmpN overexpression
improved the bioproduction of PG. Furthermore, efflux pumps and porins can enhance
bacterial tolerance to various phenolic compounds, including phenol, catechol, resorcinol,
pyrogallol, and 2-naphthol. LamB and MdtABC confer a generalized tolerance to phenols.
However, EmrAB, OmpN, and SrpB showed inconsistent effects of bacterial tolerance
to different phenolic compounds. Our results will theoretically support the construction
of phenolic compound-tolerant bacteria strains, which should be more efficient in the
biosynthesis of phenols.
Keywords: phenolic compounds; efflux pumps; porins; toxicity; hydroxyl radical; phloroglucinol
1. Introduction
As a broad class of fine or bulk chemicals, phenolic compounds clearly have wide
applications in the industrial and consumer fields. For example, phenol is an important
commodity chemical, conventionally used as a precursor for the synthesis of various
plastics, synthetic fibers, phenolic resins, and synthetic rubber [
1
]. Phloroglucinol (PG)
is an important aromatic platform compound, which itself is an excellent smooth muscle
antispasmodic agent with good specificity [2]. It also has important applications in textile
dyeing, artificial rainfall, and synthesizing an array of plastics [3].
Currently, phenol production is largely dependent on petrochemicals, but it often
raises unsustainable, environmentally unfriendly, and economic and security concerns [
1
,
4
].
As a result, there is a strong interest in the use of microbial cell factories for the production
of various phenolics from renewable biomass feedstocks [
5
–
7
]. Unfortunately, in specific
cellular environments, most phenolic compounds have strong bactericidal properties even
at a low concentration [
6
–
9
]; for example, the survival rate of E. coli BL21(DE3) was only
5% with the presence of 0.5 g/L PG in a minimal salt medium (MSM) [
10
], and when 5 mM
phenol accumulated in the fermentation medium, the growth of the solvent-resistant strain
Pseudomonas putida S12 (P. putida S12) was inhibited thereby limiting phenol production [
11
].
Microorganisms 2025,13, 202 https://doi.org/10.3390/microorganisms13010202
Microorganisms 2025,13, 202 2 of 12
As reported, due to their hydrophobicity, phenolics can interact with the lipid bilayer of
the cell membrane, disrupting membrane integrity and fluidity, affecting the function of the
cell membrane, and releasing intracellular substances out of the cytoplasm, which can lead
to impaired protein function and nutrient translocation [
12
,
13
]. Phenolic compounds with
lower molecular masses exhibit greater toxicity compared to those with higher molecular
masses, as compounds with a lower molecular weight could more rapidly enter the cell,
enhancing the hindrance of glucose assimilation [
14
]. Additionally, phenolic compounds
can increase intracellular reactive oxygen species (ROS) generation which causes oxidative
stress, leading to denaturation and inactivation of proteins or enzymes as well as damage
to DNA [
13
,
15
]. According to our previous reports, the mechanism of phenol-induced ROS
production is elucidated, showing that complexes composed of phenols and iron were
formed, promoting hydroxyl radical production in Fenton reactions, and, in turn, inducing
ferroptosis-like cell death of E. coli [10].
In recent years, it has been reported that bacterial multidrug resistance is partially
dependent on the active exocytosis of drugs; that is, exocytosis proteins or regulatory
proteins are induced by antimicrobial drugs and expel the drugs, resulting in low intra-
cellular drug concentration [
16
,
17
]. Multidrug efflux pumps are membrane proteins and
can be categorized into six different families [
18
–
20
], including ATP-binding cassette (ABC)
transporter, resistance nodulation division (RND), major facilitator superfamily (MFS),
multidrug and toxic compound extrusion (MATE), small multidrug resistance (SMR), and
proteobacterial antimicrobial compound efflux (PACE) [
21
]. The RND family has clinical
significance because it confers endogenous resistance to Gram-negative bacteria and the
associated infections are more difficult to treat [
22
,
23
]. The RND family consists of the inner
membrane protein, the outer membrane channel protein, and the membrane-connected
protein [
24
]. AcrAB-TolC, the most important efflux pump protein in E. coli, belongs to the
RND family and non-selectively expels antibiotics from bacterial cells [
23
,
25
]. Upregulation
of this efflux pump results in an elevated resistance against carbapenem, aminoglycosides,
cephalosporins, and carbapenems [
25
]. In addition, MdtABC-TolC of E. coli and SrpABC of
P. putida are also part of the RND family and are associated with resistance to antibiotics
or organic solvents [
26
,
27
]. The MdtABC efflux system plays a dual function in bacteria,
mediating multidrug resistance and maintaining bacterial iron homeostasis [
26
]. Heterol-
ogous expression of the efflux pump SrpABC in Escherichia coli improves tolerance to
n-butanol [
27
]. The EmrAB-TolC belongs to MFS which is the largest and most diverse
superfamily of secondary transporters, which not only play a crucial role in the transport
of many substances but are also closely related to immunological issues such as viral inva-
sion and bacterial resistance [
20
,
28
,
29
]. Porins constitute a significant proportion of outer
membrane proteins within Enterobacteriaceae. It extends through the outer membrane,
facilitating passive diffusion of small hydrophilic molecules into the periplasm, effectively
reducing the intracellular concentration and resulting in antibiotic resistance [
30
,
31
]. There
was a correlation between porin expression and the level of resistance to carbapenems
within numerous Enterobacteriaceae strains exhibiting decreased susceptibility to carbapen-
ems [
32
]. LamB could compensate for the absence of other nonspecific proteins to protect
Klebsiella pneumoniae against cefoxitin [
33
]. Porin OmpN could enhance tetracycline resis-
tance in Vibrrio splendidus [
34
] and replace the function of TolC in pumping drugs with the
assistance of the protein AcrAB [35].
In this study, porins LamB and OmpN and efflux pumps EmrAB, MdtABC, and SrpB
were identified to enhance E. coli PG tolerance by inhibiting the generation of hydroxyl rad-
icals resulting from the PG–iron complex. LamB and OmpN overexpression improved the
production of phloroglucinol. Furthermore, efflux pumps and porins could enhance bacte-
Microorganisms 2025,13, 202 3 of 12
rial tolerance to various phenolic compounds, like phenol, catechol, resorcinol, pyrogallol,
and 2-naphthol.
2. Methods
2.1. Bacterial Strains and Growth Conditions
All bacterial strains used in this study are E. coli. The main flow of the experiment
was carried out according to a study by Sui et al. and optimized for the specifics of the
experiment [
10
]. Briefly, the construction of strains and plasmids was cultured with Luria–
Bertani broth (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA). All recombinant
strains and plasmids used in this study are listed in Table 1. For phenol challenge and
PG production, strains were cultured in a shake flask with MSM containing 9.8 g/L
of
K2HPO4·3H2O
, 3.0 g/L of (NH
4
)
2
SO
4
, 2.1 g/L of citrate monohydrate, 0.3 g/L of
ferric ammonium citrate, 0.24 g/L of MgSO
4
, 1 mL of trace element solution (3.7 g/L
(NH
4
)
6
Mo
7
O
24 ·
4H
2
O, 2.9 g/L ZnSO
4·
7H
2
O, 24.7 g/L H
3
BO
3
, 2.5 g/L CuSO
4·
5H
2
O,
and 15.8 g/L MnCl
2·
4H
2
O), and 20 g/L of glucose as a carbon source. When necessary,
100
µ
g/mL of ampicillin (Amp), 20
µ
g/mL of chloramphenicol (Cm), or 25
µ
g/mL of
kanamycin (Kan) was added. If not specified, the reagents were purchased from Sinopharm
Chemical Reagent Co., Ltd (Shanghai, China).
Table 1. Bacterial strains and plasmids used in this study.
Strain or Plasmid Description Source
E. coil DH5αF−supE44 ∆lacU169 (ϕ80 lacZ ∆M15)hsdR17 recA1endA1 gyrA96 thi-1 relA1Lab collection
E. coil BL21(DE3) F−ompT gal dcm lon hsdSB (rB−mB−)λ(DE3) Lab collection
E. coil W3110 F- λ- rph-1 INV (rrnD, rrnE) Lab collection
Q3595 E. coil BL21(DE3)/pA-phlD/marA/acc [36]
Q3861 E. coil BL21(DE3)/pTrcHis2B This study
Q5660 E. coil BL21(DE3)/pTRC-tolC This study
Q5661 E. coil BL21(DE3)/pTRC-ompA This study
Q5662 E. coil BL21(DE3)/pTRC-ompN This study
Q5663 E. coil BL21(DE3)/pTRC-lamB This study
Q5664 E. coil BL21(DE3)/pTRC-acrAB This study
Q5665 E. coil BL21(DE3)/pTRC-ompC This study
Q5666 E. coil BL21(DE3)/pTRC-emrAB This study
Q5667 E. coil BL21(DE3)/pTRC-mdtABC This study
Q6285 E. coil BL21(DE3)/pTRC-ompT This study
Q6286 E. coil BL21(DE3)/pTRC-srpA This study
Q6287 E. coil BL21(DE3)/pTRC-srpB This study
Q6288 E. coil BL21(DE3)/pTRC-srpC This study
Q6329 E. coil BL21(DE3)/pA-lamB This study
Q6330 E. coil BL21(DE3)/pA-ompN This study
Q6331 E. coil BL21(DE3)/pA-srpB This study
Q6332 E. coil BL21(DE3)/pA-mdtABC This study
Q6333 E. coil BL21(DE3)/pA-emrAB This study
Q6424 E. coil BL21(DE3) ∆tolC This study
Q6425 E. coil BL21(DE3) ∆tolC/ pTrcHis2B This study
Q6426 E. coil BL21(DE3) ∆tolC/ pTRC-emrAB This study
Q6427 E. coil BL21(DE3) ∆tolC/ pTRC-mdtABC This study
Q6428 E. coil BL21(DE3) ∆tolC/ pTRC-srpB This study
Plasmids
pTrcHis2B reppBR322 AmpRlacIqPtrc Invitrogen
pA-phlD/marA/acc repp15A CmRlacI PT7-phlD-marA-accADBC [36]
pCas reppSC101Ts KanRPcas-cas9 ParaB -Red lacIQPtrc-sgRNApMB1 MolecularCloud:
MC0000011 [37]
pPaper-∆tolC repp15A CmRPlacIQ-sgRNA-TetRPJ23119-sgRNA ∆tolC This study
pTRC-lamB reppBR322 ApRlacIqPtrc-lamB This study
pTRC-ompN reppBR322 ApRlacIqPtrc-ompN This study
pTRC-tolC reppBR322 ApRlacIqPtrc-tolC This study
pTRC-srpA reppBR322 ApRlacIqPtrc-srpA This study
pTRC-srpB reppBR322 ApRlacIqPtrc-srpB This study
pTRC-srpC reppBR322 ApRlacIqPtrc-emrAB This study
pTRC-emrAB reppBR322 ApRlacIqPtrc-emrAB This study
pTRC-mdtABC reppBR322 ApRlacIqPtrc-mdtABC This study
pTRC-acrAB reppBR322 ApRlacIqPtrc-acrAB This study
pTRC-ompA reppBR322 ApRlacIqPtrc-ompA This study
pTRC-ompC reppBR322 ApRlacIqPtrc-ompC This study
pTRC-ompN reppBR322 ApRlacIqPtrc-ompN This study
pTRC-ompT reppBR322 ApRlacIqPtrc-ompT This study
pA-lamB repp15A CmRlacI PT7-phlD-marA-accADBC-Ptrc -lamB This study
pA-ompN repp15A CmRlacI PT7-phlD-marA-accADBC-Ptrc -ompN This study
pA-srpB repp15A CmRlacI PT7-phlD-marA-accADBC-Ptrc -srpB This study
pA-mdtABC repp15A CmRlacI PT7-phlD-marA-accADBC-Ptrc -mdtABC This study
pA-emrAB repp15A CmRlacI PT7-phlD-marA-accADBC-Ptrc -emrAB This study
Microorganisms 2025,13, 202 4 of 12
All primers used in this study are listed in Table S1. All primers were synthesized by
Sangon Biotech (Shanghai, China), and the genes srpA,srpB, and srpC were synthesized by
GenScrip Corporation (Nanjing, China). Plasmid constructions were performed by Clon
Express Ultra One Cloning Kit (Vazyme Biotech, Nanjing, China) using E. coil DH5
α
as a
host and confirmed by DNA sequencing (Sangon Biotech). E. coil BL21(DE3)
∆
tolC was
generated using the pCas/pRPS system [38].
For tolerance assay, strains carrying plasmid were cultured overnight and re-
inoculated (1:50) into 100 mL MSM with 100
µ
g/mL Amp and induced by 0.1 mM IPTG
at an OD
600
of 0.8. When the strain was further grown to a late-exponential phase (OD
600
of 2.5), the cells were collected to determine the colony-forming unit (CFU); meanwhile,
phenolic compounds were added in MSM, and the cells were further grown. Specifically,
1.3 g/L PG (Aladdin, Shanghai, China) was used for 4 h. In addition, 3 g/L phenol (Macklin,
Shanghai, China), 3.2 g/L catechol (Aladdin, Shanghai, China), 3 g/L resorcinol (Aladdin,
Shanghai, China), 3.5 g/L pyrogallol (Aladdin, Shanghai, China), and 0.27 g/L 2-naphthol
(Aladdin, Shanghai, China) were used for 5 h. Then, the cells were collected to determine
CFU, intracellular iron, and hydroxyl radical concentrations. Survival = (CFU with phenolic
compound challenge/CFU without phenolic compound challenge). The intracellular iron
and hydroxyl radical concentrations were determined using the Iron Colorimetric Assay
Kit (Applygen Technologies, Beijing, China) and Hydroxyphenyl fluorescein (Shanghai
Maokang Biotechnology, Shanghai, China), respectively.
2.2. Protein Expression and Gel Electrophoresis Analysis
Strains were cultured in 50 mL fresh MSM. Then, 0.1 mM IPTG was added when
strains were grown to an OD
600
of 0.8 and further grown to an OD
600
of 2.5. Cells were
collected and disrupted by high pressure and centrifuged to separate the supernatant and
precipitate. Then, proteins were analyzed by 12% SDS-PAGE.
2.3. Biosynthesis of PG
PG biosynthesis was performed in 250 mL shake flasks containing 50 mL of MSM with
20
µ
g/mL Cm. After incubation to OD
600
0.8 at 37
◦
C, 0.1 mM IPTG was added and then
the temperature was decreased to 30
◦
C for further culture of 24 h. PG concentration was
detected using the colorimetric reaction between PG and cinnamaldehyde at 446 nm.
3. Results
3.1. Identification of Efflux Pumps and Porin Gene Essential to PG Tolerance
In our previous experiments, it was proven that the generation of hydroxyl radicals
(HO
·
) promoted by the PG–iron complex was the main factor of PG toxicity to E. coli
BL21(DE3) [
10
]. Based on this, reducing the intracellular PG concentration is supposed
to enhance cell tolerance. To test whether efflux pumps and porins affect PG tolerance,
genes encoding nine efflux pumps and porins of E. coli and three proteins of P. putida
were cloned into plasmid vector pTrcHis2B and overexpressed in the BL21(DE3) strain,
respectively, including acrAB operon, tolC,mdtABC operon, emrAB operon, lamB,ompN,
ompA, and ompC from E. coli BL21(DE3), ompT from E. coli W3110, and srpA,srpB, and
srpC from P. putida S12. The strains were cultured to the late-exponential phase (OD
600
2.5) in MSM and then treated with 1.3 g/L PG for 4 h. As shown in Figure 1a, almost all
strains carrying empty vectors were killed by PG, and only 0.2% cells survived. The PG
tolerance of SrpA, SrpC, OmpA, OmpT, and TolC overexpression strains was similar to
strains carrying empty vectors, whereas overexpression of MdtABC, EmrAB, LamB, OmpN,
and SrpB could improve the host tolerance against PG, especially LamB and EmrAB, with
survival rates of 80% and 115.3%, respectively. Overexpression of MdtABC, OmpN, and
Microorganisms 2025,13, 202 5 of 12
SrpB also restored bacterial survival to 17.3%, 10.9%, and 5%, respectively. In addition,
pompC and pacrAB strains became much more susceptible to PG than the wild-type strain,
and the growth of the pacrAB strain was significantly inhibited even in the absence of PG,
probably because the excessive AcrAB level affected normal growth and metabolism of this
strain. SrpABC was similar to the AcrAB-TolC efflux pump. In general, EmrAB, MdtABC,
and AcrAB in E. coli need to be together with TolC to function as efflux pumps. To further
verify that TolC in the efflux pump was not associated with PG tolerance, the tolC gene
was knocked and tested for PG toxicity. The survival rate of the
∆
tolC strain was similar to
that of wild E. coli BL21(DE3) (Figure 1b). Following this, the function of EmrAB, MdtABC,
and SrpB was confirmed by overexpression in the tolC knockout strain (Figure 1b). Both
EmrAB and MdtABC increased PG tolerance regardless of the presence or absence of the
TolC protein (Figure 1a,b). SrpB was promoted more significantly in the presence of TolC
(Figure 1a,b). The above results indicated that EmrAB and MdtABC improved PG tolerance
in E. coli independently of TolC, and SrpB probably required TolC for its role in E. coli.
Overall, five membrane proteins (MdtABC, OmpN, SrpB, LamB, and EmrAB) proved to be
instrumental in improving the PG tolerance of E. coli.
Microorganisms 2025, 13, x FOR PEER REVIEW 6 of 13
Figure 1. Identification of efflux pumps and porins related to phloroglucinol (PG) tolerance. (a) Tol-
erance of E. coli BL21(DE3) strain carrying empty vector or membrane protein overexpression vector
after treatment with PG at 1.3 g/L for 4 h. The circle represents a data (n = 3 biological independent
samples). (b) Confirmation of the role of TolC, EmrAB, MdtABC, and SrpB in PG tolerance by
knockout tolC, and EmrAB, MdtABC, and SrpB overexpression in tolC knockout strain. The circle
represents a data (n = 3 biological independent samples). (c) The protein of different strains from
samples at an OD600 of 2.5. A total of 0.1 mM IPTG was added to induce proteins when OD600 was
0.8. Lane M, pre-stained protein molecular weight marker (kDa); lane 1, soluble proteins in the su-
pernatant; lane 2, insoluble proteins in precipitation. LamB, 49.9kDa; MdtA, 44.5 kDa; MdtB, 112.1
kDa; MdtC, 111.0 kDa; EmrA, 42.7kDa; EmrB, 55.6kDa; SrpA, 41.4 kDa; SrpB, 114.1 kDa; SrpC, 51.4
kDa; OmpA, 37.2kDa; OmpC, 34.4 kDa; OmpN, 41.2 kDa; OmpT, 35.6 kDa; AcrA, 42.2 kDa; AcrB,
113.6 kDa; TolC, 53.7 kDa.
3.2. Efflux Pumps and Porins Inhibit the Generation of Hydroxyl Radical
According to our previous report, the PG–iron complex promotes the generation of
HO·. In order to verify whether overexpression of the above five proteins could inhibit the
intracellular Fenton reaction, the intracellular levels of HO· were examined with 1.3 g/L
PG for 4 h. As we expected, the higher tolerance the strain has, the lower the intracellular
HO· level that was detected. Strains carrying empty vectors presented a HO· concentra-
tion of 153 times and 49 times as high as pemrAB and plamB, which significantly restored
the viability of the E. coli BL21(DE3) strain (Figure 2a). Next, pompN and pmdtABC were
found to be at least 29 times lower than the pVector strain. In addition, the HO· content of
psrpB was only about 56% lower than that of the control strain, which slightly affected PG
susceptibility. All these results demonstrated that efflux pumps and porins protect E. coli
Figure 1. Identification of efflux pumps and porins related to phloroglucinol (PG) tolerance. (a) Toler-
ance of E. coli BL21(DE3) strain carrying empty vector or membrane protein overexpression vector
after treatment with PG at 1.3 g/L for 4 h. The circle represents a data (n= 3 biological independent
samples). (b) Confirmation of the role of TolC, EmrAB, MdtABC, and SrpB in PG tolerance by
knockout tolC, and EmrAB, MdtABC, and SrpB overexpression in tolC knockout strain. The circle
represents a data (n= 3 biological independent samples). (c) The protein of different strains from
Microorganisms 2025,13, 202 6 of 12
samples at an OD
600
of 2.5. A total of 0.1 mM IPTG was added to induce proteins when OD
600
was 0.8. Lane M, pre-stained protein molecular weight marker (kDa); lane 1, soluble proteins in
the supernatant; lane 2, insoluble proteins in precipitation. LamB, 49.9kDa; MdtA, 44.5 kDa; MdtB,
112.1 kDa; MdtC, 111.0 kDa; EmrA, 42.7kDa; EmrB, 55.6kDa; SrpA, 41.4 kDa; SrpB, 114.1 kDa; SrpC,
51.4 kDa; OmpA, 37.2kDa; OmpC, 34.4 kDa; OmpN, 41.2 kDa; OmpT, 35.6 kDa; AcrA, 42.2 kDa; AcrB,
113.6 kDa; TolC, 53.7 kDa.
Apart from that, the protein expression level was further confirmed using SDS-PAGE
and Coomassie blue staining (Figure 1c). Despite the fact that overexpression of LamB,
EmrAB, MdtABC, OmpN, and SrpB improved the bacterial PG tolerance, LamB and EmrAB
proteins were mainly present in inclusion bodies, and no obvious band of MdtABC, OmpN,
and SrpB was observed in either the cell lysate supernatant or the precipitation (Figure 1c),
suggesting that only a small amount of efflux pumps and porins were needed to play
the role of material efflux and improve PG tolerance. Although TolC, OmpA, and SrpA
were partially soluble expressed, they did not promote bacterial survival under PG stress,
implying that they are not related to the PG tolerance of E. coli. In addition, OmpC, OmpT,
and AcrAB were misfolded or aggregated to form inclusion bodies, and no SrpC band was
observed because of the low expression.
3.2. Efflux Pumps and Porins Inhibit the Generation of Hydroxyl Radical
According to our previous report, the PG–iron complex promotes the generation of
HO
·
. In order to verify whether overexpression of the above five proteins could inhibit the
intracellular Fenton reaction, the intracellular levels of HO
·
were examined with 1.3 g/L
PG for 4 h. As we expected, the higher tolerance the strain has, the lower the intracellular
HO
·
level that was detected. Strains carrying empty vectors presented a HO
·
concentration
of 153 times and 49 times as high as pemrAB and plamB, which significantly restored the
viability of the E. coli BL21(DE3) strain (Figure 2a). Next, pompN and pmdtABC were
found to be at least 29 times lower than the pVector strain. In addition, the HO
·
content of
psrpB was only about 56% lower than that of the control strain, which slightly affected PG
susceptibility. All these results demonstrated that efflux pumps and porins protect E. coli
from PG toxicity as they limit the potential for PG–iron complex-dependent HO
·
formation.
Microorganisms 2025, 13, x FOR PEER REVIEW 7 of 13
from PG toxicity as they limit the potential for PG–iron complex-dependent HO· for-
mation.
Figure 2. Efflux pumps and porins inhibited generation of hydroxy radicals (HO·). (a) Intracellular
HO· concentration determined of E. coli BL21(DE3) strain carrying empty vector or membrane pro-
tein overexpression vector grown in MSM after challenge of 1.3 g/L PG for 4 h. The circle represents
a data (n = 3 biological independent samples). (b) Intracellular iron concentration in E. coli
BL21(DE3) strain carrying empty vector or membrane protein overexpression vector after challenge
of 1.3 g/L PG for 30 min. The circle represents a data (n = 3 biological independent samples).
3.3. Comparison of Intracellular Iron Levels
Iron is known to be necessary for the toxicity of phenolic compounds [10]. Next, it
was determined whether the intracellular iron levels of membrane protein overexpression
strains changed. As expected, pemrAB had a lower iron concentration than the control
strain (pemrAB 18.65 ± 1.77 and pVector 27.28 ± 1.08). Surprisingly, intracellular iron con-
tent was significantly increased in the strain overexpressing LamB, which was 36% higher
than the pVector strain (Figure 2b). LamB has been shown to compensate for the absence
of other nonspecific proteins to improve strain tolerance [33]. We speculate that LamB
avoids ferroptosis-like death by excluding PG from the body rather than iron. RND-type
MdtABC is a drug efflux pump that can export antibiotics [39]. It has been shown that
MdtABC and AcrD cooperate with AcrB to excrete enterobactin (a siderophore) from the
cytosol to the extracellular space [26]. But in this study, there was no difference in iron
concentration (27.71 ± 0.64) compared to the control strain (Figure 2b). Similarly, pompN
and psrpB strains showed no significant changes in iron ion levels (Figure 2b). According
to the results of the intracellular levels of HO· and iron, the enhancement of PG resistance
should be due to the reduction in intracellular PG concentration.
3.4. Porin Overexpression Benefits Biosynthesis of PG
In recent years, the synthesis of phenolic compounds by microorganisms has become
a research frontier in the world. However, E. coli suffers from the toxicity of PG with
growth inhibition, limiting the maximum titer of PG accumulated in the medium [40]. To
test the influence of our screened efflux pumps and porins on PG biosynthesis, five pro-
teins that enhance PG tolerance were cloned into pA-phlD/marA/acc carrying PG biosyn-
thetic pathways and then introduced into E. coli BL21(DE3). In shake flask cultivation, the
pA-ompN strain produced 1.00 ± 0.02 g/L PG and the pA-lamB strain produced 0.82 ± 0.04
g/L PG, respectively, which is 53.85% and 26.15% higher than that of the wild-type strain
(0.65 ± 0.00 g/L) (Figure 3a). And the cell density (OD600) of pA-ompN was increased to 7.16
± 0.09, while the control strain was 6.39 ± 0.14 (Figure 3b). Although MdtABC, EmrAB,
and SrpB enhanced the survival rate in the presence of PG (Figure 1a), the PG production
Figure 2. Efflux pumps and porins inhibited generation of hydroxy radicals (HO
·
). (a) Intracellular
HO
·
concentration determined of E. coli BL21(DE3) strain carrying empty vector or membrane protein
overexpression vector grown in MSM after challenge of 1.3 g/L PG for 4 h. The circle represents a
data (n= 3 biological independent samples). (b) Intracellular iron concentration in E. coli BL21(DE3)
strain carrying empty vector or membrane protein overexpression vector after challenge of 1.3 g/L
PG for 30 min. The circle represents a data (n= 3 biological independent samples).
Microorganisms 2025,13, 202 7 of 12
3.3. Comparison of Intracellular Iron Levels
Iron is known to be necessary for the toxicity of phenolic compounds [
10
]. Next, it
was determined whether the intracellular iron levels of membrane protein overexpression
strains changed. As expected, pemrAB had a lower iron concentration than the control strain
(pemrAB 18.65
±
1.77 and pVector 27.28
±
1.08). Surprisingly, intracellular iron content
was significantly increased in the strain overexpressing LamB, which was 36% higher than
the pVector strain (Figure 2b). LamB has been shown to compensate for the absence of
other nonspecific proteins to improve strain tolerance [
33
]. We speculate that LamB avoids
ferroptosis-like death by excluding PG from the body rather than iron. RND-type MdtABC
is a drug efflux pump that can export antibiotics [
39
]. It has been shown that MdtABC and
AcrD cooperate with AcrB to excrete enterobactin (a siderophore) from the cytosol to the
extracellular space [
26
]. But in this study, there was no difference in iron concentration
(27.71
±
0.64) compared to the control strain (Figure 2b). Similarly, pompN and psrpB strains
showed no significant changes in iron ion levels (Figure 2b). According to the results of the
intracellular levels of HO
·
and iron, the enhancement of PG resistance should be due to the
reduction in intracellular PG concentration.
3.4. Porin Overexpression Benefits Biosynthesis of PG
In recent years, the synthesis of phenolic compounds by microorganisms has become a
research frontier in the world. However, E. coli suffers from the toxicity of PG with growth
inhibition, limiting the maximum titer of PG accumulated in the medium [
40
]. To test
the influence of our screened efflux pumps and porins on PG biosynthesis, five proteins
that enhance PG tolerance were cloned into pA-phlD/marA/acc carrying PG biosynthetic
pathways and then introduced into E. coli BL21(DE3). In shake flask cultivation, the pA-
ompN strain produced 1.00
±
0.02 g/L PG and the pA-lamB strain produced 0.82
±
0.04
g/L PG, respectively, which is 53.85% and 26.15% higher than that of the wild-type strain
(0.65
±
0.00 g/L) (Figure 3a). And the cell density (OD
600
) of pA-ompN was increased
to
7.16 ±0.09
, while the control strain was 6.39
±
0.14 (Figure 3b). Although MdtABC,
EmrAB, and SrpB enhanced the survival rate in the presence of PG (Figure 1a), the PG
production was instead reduced (Figure 3a), probably because the exogenous protein
expression increased the metabolic burden of the strain or affected the normal metabolism
of the strains. This demonstrated that overexpression of porins improves cellular tolerance
to phenolics and benefits phenol-related biochemical processes.
Microorganisms 2025, 13, x FOR PEER REVIEW 8 of 13
was instead reduced (Figure 3a), probably because the exogenous protein expression in-
creased the metabolic burden of the strain or affected the normal metabolism of the
strains. This demonstrated that overexpression of porins improves cellular tolerance to
phenolics and benefits phenol-related biochemical processes.
Figure 3. Biosynthesis of PG. (a) PG production of membrane protein overexpression strains. The
circle represents a data (n = 3 biological independent samples). (b) Growth of membrane protein
overexpression strains. The circle represents a data (n = 3 biological independent samples).
3.5. Efflux Pumps and Porins Improve Bacterial Tolerance to Diverse Phenolic Compounds
Some other phenolic compounds including phenol [11,41], resorcinol [42], catechol
[7,43], pyrogallol [9,44], and 2-naphthol [45] were produced or degraded by microorgan-
isms. Therefore, they were tested for cytotoxicity to confirm the abilities of efflux pumps
or porins to improve tolerance. Figure 4a showed that the viability of the plamB strain was
enhanced dramatically in the presence of all five different phenolic compounds. Similar
results were also observed in the strain pmdtABC (Figure 4b). This suggested that LamB
and MdtABC confer a generalized tolerance to phenols. However, overexpression of other
proteins showed inconsistent effects on bacterial tolerance to various phenolic com-
pounds. The pompN strain showed a significant reduction in sensitivity to the tested com-
pounds except for phenol (Figure 4c). In regard to the pemrAB strain, all cells exposed to
catechol and pyrogallol were killed, while its tolerance to resorcinol and 2-naphthol was
highly improved (Figure 4d). Interestingly, though SrpB is derived from solvent-resistant P.
putida, the psrpB strain was less resistant to phenol and resorcinol (Figure 4e). Substrate
specificity of proteins may be responsible for differences in the tolerance of strains to differ-
ent phenolics.
Figure 3. Biosynthesis of PG. (a) PG production of membrane protein overexpression strains. The
circle represents a data (n= 3 biological independent samples). (b) Growth of membrane protein
overexpression strains. The circle represents a data (n= 3 biological independent samples).
Microorganisms 2025,13, 202 8 of 12
3.5. Efflux Pumps and Porins Improve Bacterial Tolerance to Diverse Phenolic Compounds
Some other phenolic compounds including phenol [
11
,
41
], resorcinol [
42
], catechol [
7
,
43
],
pyrogallol [
9
,
44
], and 2-naphthol [
45
] were produced or degraded by microorganisms.
Therefore, they were tested for cytotoxicity to confirm the abilities of efflux pumps or
porins to improve tolerance. Figure 4a showed that the viability of the plamB strain was
enhanced dramatically in the presence of all five different phenolic compounds. Similar
results were also observed in the strain pmdtABC (Figure 4b). This suggested that LamB
and MdtABC confer a generalized tolerance to phenols. However, overexpression of other
proteins showed inconsistent effects on bacterial tolerance to various phenolic compounds.
The pompN strain showed a significant reduction in sensitivity to the tested compounds
except for phenol (Figure 4c). In regard to the pemrAB strain, all cells exposed to catechol
and pyrogallol were killed, while its tolerance to resorcinol and 2-naphthol was highly
improved (Figure 4d). Interestingly, though SrpB is derived from solvent-resistant P.
putida, the psrpB strain was less resistant to phenol and resorcinol (Figure 4e). Substrate
specificity of proteins may be responsible for differences in the tolerance of strains to
different phenolics.
Microorganisms 2025, 13, x FOR PEER REVIEW 9 of 13
Figure 4. Efflux pumps and porins improve tolerance to diverse phenolic compounds. Tolerance of
E. coli BL21(DE3) strain carrying empty vector and plamB (a), pmdtABC (b), pompN (c), pemrAB (d),
or psrpB (e) upon exposure to different phenolic compounds. The circle represents a data (n = 3
biological independent samples).
4. Discussion
Phenolic compounds play an important role in the medicine, food, cosmetics, textiles,
and chemical industries. In recent years, the synthesis of phenolic compounds by micro-
organisms has aracted more aention. However, the toxic effects of phenolics on bacteria
are a factor in its yield limitation. According to the results shown above, we discovered
that efflux pumps and porins significantly restored the viability of strains with the pres-
ence of phenolic compounds. Moreover, LamB and MdtABC had a generalized tolerance
to phenols, such as PG, phenol, catechol, resorcinol, pyrogallol, and 2-naphthol (Figures
1a and 4). LamB could compensate for the absence of other nonspecific proteins to protect
strains from harm [33]. This may explain why it can enhance phenol tolerance. MdtABC
requires TolC for its efflux function, which is associated with resistance to antibiotics, bile
salt derivatives, and SDS [39]. OmpN is a nonspecific porin and not highly expressed un-
der typical laboratory growth conditions. When overexpressed, it operates similarly to
OmpC. But due to the different substrate specificity between the two proteins [46], the PG
tolerance of pompN was significantly enhanced, whereas pompC was the same as that of
controls (Figure 1a). The srpB gene is derived from solvent-resistant P. putida and im-
proves n-butanol tolerance in E. coil [27], but in our study, the psrpB strain was less re-
sistant to phenol and resorcinol, and the tolerance to PG just slightly improved (Figures
1a and 4). AcrAB-TolC is the most important efflux pump in bacteria since it is not sub-
strate specific [23], but its response to PG was ineffective, and the pacrAB strain almost
stopped growing even in the absence of PG. To our knowledge, this is the first report of
MdtABC, OmpN, and EmrAB being associated with bacterial tolerance to phenolic com-
pounds. The phenolic compounds used in this study are basic molecular structures, each
comprising just one or two benzene rings, capable of triggering ferroptosis in a variety of
organisms. In contrast, polyphenols are a category of natural substances prevalent in ad-
vanced plant species and are usually composed of more benzene ring structures and
Figure 4. Efflux pumps and porins improve tolerance to diverse phenolic compounds. Tolerance
of E. coli BL21(DE3) strain carrying empty vector and plamB (a), pmdtABC (b), pompN (c), pemrAB
(d), or psrpB (e) upon exposure to different phenolic compounds. The circle represents a data (n= 3
biological independent samples).
4. Discussion
Phenolic compounds play an important role in the medicine, food, cosmetics, textiles,
and chemical industries. In recent years, the synthesis of phenolic compounds by microor-
ganisms has attracted more attention. However, the toxic effects of phenolics on bacteria
are a factor in its yield limitation. According to the results shown above, we discovered
that efflux pumps and porins significantly restored the viability of strains with the presence
of phenolic compounds. Moreover, LamB and MdtABC had a generalized tolerance to
phenols, such as PG, phenol, catechol, resorcinol, pyrogallol, and 2-naphthol (Figures 1a
and 4). LamB could compensate for the absence of other nonspecific proteins to protect
Microorganisms 2025,13, 202 9 of 12
strains from harm [
33
]. This may explain why it can enhance phenol tolerance. MdtABC
requires TolC for its efflux function, which is associated with resistance to antibiotics, bile
salt derivatives, and SDS [
39
]. OmpN is a nonspecific porin and not highly expressed
under typical laboratory growth conditions. When overexpressed, it operates similarly to
OmpC. But due to the different substrate specificity between the two proteins [
46
], the PG
tolerance of pompN was significantly enhanced, whereas pompC was the same as that of
controls (Figure 1a). The srpB gene is derived from solvent-resistant P. putida and improves
n-butanol tolerance in E. coil [
27
], but in our study, the psrpB strain was less resistant to
phenol and resorcinol, and the tolerance to PG just slightly improved (
Figures 1a and 4)
.
AcrAB-TolC is the most important efflux pump in bacteria since it is not substrate spe-
cific [
23
], but its response to PG was ineffective, and the pacrAB strain almost stopped
growing even in the absence of PG. To our knowledge, this is the first report of MdtABC,
OmpN, and EmrAB being associated with bacterial tolerance to phenolic compounds. The
phenolic compounds used in this study are basic molecular structures, each comprising
just one or two benzene rings, capable of triggering ferroptosis in a variety of organisms.
In contrast, polyphenols are a category of natural substances prevalent in advanced plant
species and are usually composed of more benzene ring structures and diverse functional
groups, exhibit antioxidant properties, and are effective in inhibiting ferroptosis [
47
]. The
variance in their biological activities is likely attributable to the distinct molecular structures
between simple phenols and polyphenolic compounds.
The PG–iron complex promotes the generation of HO
·
, causing ferroptosis-like cell
death. In this study, efflux pumps and porins were demonstrated to reduce intracellular
HO
·
accumulation, especially LamB and EmrAB, being 153 times and 49 times as low
as the wild strain, respectively, and effectively mitigating ferroptosis-like death of E. coli
(
Figures 2a and 5
). In addition, our research has great application potential in the biosyn-
thesis of phenolic compounds. The pA-ompN strain produced a PG titer that was 53.85%
higher than that of the wild-type strain (Figure 3a). The enhancement of bacterial tolerance
to phenolic compounds can theoretically prolong the fermentation process, but the fer-
mentation process can be affected by a variety of factors, such as enzyme activity, plasmid
stability, the medium, and culture conditions. It has been shown that an enhanced tolerance
to exogenous solvents does not inherently result in a higher yield of that solvent [
48
].
Future studies can combine multiple factors to boost the production of phenolics.
Microorganisms 2025, 13, x FOR PEER REVIEW 10 of 13
diverse functional groups, exhibit antioxidant properties, and are effective in inhibiting
ferroptosis [47]. The variance in their biological activities is likely aributable to the dis-
tinct molecular structures between simple phenols and polyphenolic compounds.
The PG–iron complex promotes the generation of HO·, causing ferroptosis-like cell
death. In this study, efflux pumps and porins were demonstrated to reduce intracellular
HO· accumulation, especially LamB and EmrAB, being 153 times and 49 times as low as
the wild strain, respectively, and effectively mitigating ferroptosis-like death of E. coli
(Figures 2a and 5). In addition, our research has great application potential in the biosyn-
thesis of phenolic compounds. The pA-ompN strain produced a PG titer that was 53.85%
higher than that of the wild-type strain (Figure 3a). The enhancement of bacterial tolerance
to phenolic compounds can theoretically prolong the fermentation process, but the fer-
mentation process can be affected by a variety of factors, such as enzyme activity, plasmid
stability, the medium, and culture conditions. It has been shown that an enhanced toler-
ance to exogenous solvents does not inherently result in a higher yield of that solvent [48].
Future studies can combine multiple factors to boost the production of phenolics.
In conclusion, this study provides an effective method for optimizing phenolic toler-
ance in E. coli and the development of PG production. Our study will provide some theo-
retical basis for the construction of phenol-tolerant strains and the biosynthesis of phenols.
Figure 5. Model illustrating the mechanism of the efflux pumps and porins that increased phenolic toler-
ance.
5. Conclusions
This study investigated the effects of various efflux pumps and porins on bacterial
phenolic compound tolerance. The results indicated that five proteins, LamB, OmpN,
EmrAB, MdtABC, and SrpB, significantly improved E. coli PG resistance. And the higher
tolerance the strain has, the lower intracellular the HO· level that was detected. The en-
hancement of tolerance is due to the efflux pumps and porins inhibiting the formation of
HO· induced by the PG–iron complex. Furthermore, the PG biosynthesis experiment
showed that overexpression of porins OmpN and LamB improved PG production, which
was beneficial for phenol-related biochemical processes. In addition, efflux pumps and
porins could significantly restore the viability of strains with the presence of various phe-
nolic compounds, including phenol, resorcinol, catechol, pyrogallol, and 2-naphthol.
These findings will provide a theoretical foundation for the development of bacterial
strains with enhanced tolerance to phenolic compounds.
Figure 5. Model illustrating the mechanism of the efflux pumps and porins that increased phenolic
tolerance.
Microorganisms 2025,13, 202 10 of 12
In conclusion, this study provides an effective method for optimizing phenolic toler-
ance in E. coli and the development of PG production. Our study will provide some
theoretical basis for the construction of phenol-tolerant strains and the biosynthesis
of phenols.
5. Conclusions
This study investigated the effects of various efflux pumps and porins on bacterial
phenolic compound tolerance. The results indicated that five proteins, LamB, OmpN,
EmrAB, MdtABC, and SrpB, significantly improved E. coli PG resistance. And the higher
tolerance the strain has, the lower intracellular the HO
·
level that was detected. The
enhancement of tolerance is due to the efflux pumps and porins inhibiting the formation
of HO
·
induced by the PG–iron complex. Furthermore, the PG biosynthesis experiment
showed that overexpression of porins OmpN and LamB improved PG production, which
was beneficial for phenol-related biochemical processes. In addition, efflux pumps and
porins could significantly restore the viability of strains with the presence of various
phenolic compounds, including phenol, resorcinol, catechol, pyrogallol, and 2-naphthol.
These findings will provide a theoretical foundation for the development of bacterial strains
with enhanced tolerance to phenolic compounds.
Supplementary Materials: The following supporting information can be downloaded at https://www.
mdpi.com/article/10.3390/microorganisms13010202/s1: Table S1: Primers used in this study.
Author Contributions: G.Z. designed the experiments. X.S. and L.G. performed the experiments.
G.Z., X.S., M.X., L.G., and Z.B. analyzed the results. G.Z. and X.S. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This study was financially supported by the NSFC (32170085, 32370068), the National
Key Research and Development Program of China (2022YFC2104700), the Taishan Scholars Program
(tstp20231208), the SKLMT Frontiers and Challenges Project (SKLMTFCP-2023-03), and the Qingdao
Natural Science Foundation (23-2-1-170-zyyd-jch).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in this study are included in the
article/Supplementary Materials. Further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflicts of interest.
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