microorganisms
Review
The Nonribosomal Peptide Valinomycin: From Discovery to
Bioactivity and Biosynthesis
Shuhui Huang 1,†, Yushi Liu 1,†, Wan-Qiu Liu 1, Peter Neubauer 2,* and Jian Li 1,*
Citation: Huang, S.; Liu, Y.; Liu,
W.-Q.; Neubauer, P.; Li, J. The
Nonribosomal Peptide Valinomycin:
From Discovery to Bioactivity and
Biosynthesis. Microorganisms 2021,9,
780. https://doi.org/10.3390/
microorganisms9040780
Academic Editor: Andreana Marino
Received: 17 March 2021
Accepted: 7 April 2021
Published: 8 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China;
[email protected] (W.-Q.L.)
2Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin,
13355 Berlin, Germany
*Correspondence: peter[email protected] (P.N.); [email protected] (J.L.)
† These authors contributed equally to this work.
Abstract:
Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955.
Over the past more than six decades, it has received continuous attention due to its special chemical
structure and broad biological activities. Although many research papers have been published on
valinomycin, there has not yet been a comprehensive review that summarizes the diverse studies
ranging from structural characterization, biogenesis, and bioactivity to the identification of biosyn-
thetic gene clusters and heterologous biosynthesis. In this review, we aim to provide an overview
of valinomycin to address this gap, covering from 1955 to 2020. First, we introduce the chemical
structure of valinomycin together with its chemical properties. Then, we summarize the broad
spectrum of bioactivities of valinomycin. Finally, we describe the valinomycin biosynthetic gene
cluster and reconstituted biosynthesis of valinomycin. With that, we discuss possible opportunities
for the future research and development of valinomycin.
Keywords:
valinomycin; nonribiosomal peptide; bioactivities; biosynthesis; Streptomyces; heterolo-
gous production; cell-free biosynthesis
1. Introduction
Nature, like a magic chemist, is able to synthesize complex compounds from simple
building blocks. These compounds are called natural products (NPs), whose diverse
chemical structures usually confer significant biological and pharmaceutical activities
to them [
1
]. Therefore, NPs have historically served as an abundant source for potent
drugs to combat human diseases such as pain, infections, and cancers [
2
]. During the
past two centuries, tens of thousands of NPs have been isolated and characterized from
various living organisms (e.g., microorganisms, plants, and animals) [
3
]. Among the large
number of NPs, nonribosomal peptides (NRPs) belong to an important class of peptide
compounds, which are synthesized through mRNA-independent assembly lines termed
nonribosomal peptide synthetases (NRPSs) [
4
,
5
]. NRPSs are giant multimodular enzymes
with molecular weights often ranging from one hundred to several hundreds of kilodaltons
(kDa). Usually, one typical NRPS module contains three core domains, which are an
adenylation (A) domain (~50 kDa) for substrate (monomer/building block) selection and
activation, a thiolation (T) domain (8–10 kDa) for substrate and growing peptidyl chain
tethering, and a condensation (C) domain (~50 kDa) for peptide bond formation. Once all
building blocks are incorporated into the final full-length peptide chain, a thioesterase (TE)
domain (~35 kDa) located furthest downstream in the last module catalyzes the release
of the final product. In addition, some optional domains, for example, epimerase (E),
ketoreductase (KR), and methyltransferase (MT), also present in NRPS modules to perform
modifications of monomeric building blocks. To date, more than 500 variant monomers for
Microorganisms 2021,9, 780. https://doi.org/10.3390/microorganisms9040780 https://www.mdpi.com/journal/microorganisms
Microorganisms 2021,9, 780 2 of 22
NRPS assembly lines have been identified including proteinogenic and nonproteinogenic
amino acids and other carboxylic acids (e.g., aryl acids) [
6
,
7
]. As a consequence, the NRP
structures are remarkably diverse and complex, which leads to a high density of functional
groups and contributes notably to the observed pharmaceutical properties [
8
,
9
]. The most
well-known examples of NRP antibiotics are the penicillin and cephalosporin families [
10
],
as well as the vancomycin [11], which are still being used daily in clinic.
Valinomycin is an NRP compound that was first isolated and characterized 65 years
ago from Streptomyces fulvissimus [
12
]. Two years later, in 1957, Brockmann and Geeren to-
tally hydrolyzed valinomycin and proposed the structure of valinomycin as a 24-membered
cyclic peptide cyclo-(D-
α
-hydroxyisovaleryl-D-valyl-L-lactyl-L-valyl)
2
[
13
]. However, sub-
sequent work indicated that the correct chemical structure of valinomycin was a 36-
membered cyclododecadepsipeptide, which consists of a triple repeating unit of D-
α
-
hydroxyisovaleryl-D-valyl-L-lactyl-L-valyl with a molecular weight of 1111.3 g mol
−1
(C
54
H
90
N
6
O
18
) [
14
,
15
]. Clearly, the building blocks of valinomycin include not only two
proteinogenic and nonproteinogenic amino acids (L-valine and D-valine), but also two
carboxylic acids (D-
α
-hydroxyisovaleric acid and L-lactic acid). As shown in the structural
formula (Figure 1A), valinomycin as a cyclodepsipeptide is composed of alternating pep-
tide and ester linkages between each residue [
16
]. This structure conformation forms a
hydrophobic surface and a polar cavity in which one potassium ion (K
+
) can be coordi-
nated with the six oxygen atoms of the interior ester carbonyls, forming a valinomycin-K
+
complex [
17
–
20
]. The size of the cavity is suitable for accommodating a K
+
ion but not
for other metal ions (e.g., Na
+
and Li
+
), which makes valinomycin a potassium-specific
ionophore and the resulting bioactivities are connected to it. Importantly, crystal structures
of valinomycin have been known for a long time [
21
,
22
]. Its polymorphism would have to
be taken into account in a possible manufacturing of drug formulations, as discussed in
a report [
23
]. Previous studies on the mechanism of action of valinomycin have demon-
strated that, due to the hydrophobic surface of valinomycin, the valinomycin-K
+
complex
can be incorporated into biological bilayer membranes and allows the transportation of
K
+
through the membrane to destroy the normal K
+
gradient across the membrane, thus
dissipating the membrane potential, and as a result kills the cells [
24
–
29
]. The working
mechanism of valinomycin as a potassium ionophore in the biological membrane is il-
lustrated in Figure 1B. Structure–activity relationships studied with valinomycin analogs
showed that the cyclic 12-residue peptide is critical for the bioactivities of valinomycin.
Alteration of the valinomycin structure by changing the ring size or amino acid residues
significantly reduces the capacity to form a stable valinomycin-K
+
complex and, conse-
quently, its antimicrobial activity [
30
–
32
]. While numerous studies have revealed that the
effect of valinomycin on cells is primarily due to the dissipation of membrane potential,
inhibition of protein synthesis at the level of elongation was proven to be another mode of
action of valinomycin [
33
–
36
]. Moreover, recent evidence also indicated that the cellular
response to valinomycin is very complex and involves an intricate network of proteins
related to mitochondria, vacuoles, and other membrane compartments [37].
Microorganisms 2021,9, 780 3 of 22
Figure 1.
(
A
) Chemical structure of valinomycin and (
B
) valinomycin acts as a potassium-
specific ionophore.
Since its discovery in 1955, continuous studies on valinomycin have been carried
out from early structure characterization, chemical synthesis, biogenesis, and bioactivity
(mechanism of action) to recent biosynthetic gene cluster identification and heterologous
in vivo
/
in vitro
production (see a research timeline of valinomycin in Figure 2). Although
many research papers of valinomycin have been published so far, a comprehensive review
with a focus on valinomycin is still lacking. Therefore, this motivates us to comprehensively
summarize valinomycin, ranging from its discovery and characterization to bioactivity
and biosynthesis. To this end, we first briefly introduce the chemical structure and related
features of valinomyicn, as demonstrated above. In the following sections, we then sum-
marize many documented bioactivities of valinomycin. Finally, we describe in detail the
progress made in characterizing the valinomycin biosynthetic gene cluster (i.e., the NRPS
valinomycin synthetase), and the production of valinomycin in native producers as well as
reconstituted biosynthesis of valinomycin in vivo and in vitro.
Figure 2. The timeline of valinomycin researches.
2. Biological Activities of Valinomycin
Valinomycin was initially isolated as an antibiotic compound, showing the antibacte-
rial activity against Mycobacterium tuberculosis [
12
]. It was also the first natural compound
recognized as an ionophore with antibiotic activity [
38
]. Later, a diverse spectrum of bio-
logical activities of valinomycin was demonstrated that ranges from antifungal, antiviral,
and insecticidal to antitumor efficacy. Recent studies even reported that valinomycin as a
mitophagy activator also plays a positive role in the treatment of Parkinson’s disease [
39
]
Microorganisms 2021,9, 780 4 of 22
and Alzheimer’s disease [
40
]. Examples of such bioactivities are introduced in this section
and representative dose-dependent activities are summarized in Table 1.
2.1. Antibacterial Activity
Valinomycin shows a wide range of antibacterial activity. The inhibition of cell growth
was extensively tested on a series of bacteria, including Gram-positive bacteria, for example,
Staphylococcus aureus,Staphylococcus faecalis,Streptococcus pyogenes,Clostridium sporogenes,
Listeria innocua,Bacillus subtilis,Enterococcus faecalis, and Micrococcus luteus, as well as
Gram-negative bacteria like Escherichia coli,Salmonella enterica,Enterobacter cloacae,Neisseria
gonorrhoeae, and Stenotrophomonas maltophilia [
29
,
41
–
44
]. These studies indicated that,
although to different levels, valinomycin showed growth inhibition against all tested Gram-
positive bacteria. However, none of the selected Gram-negative bacteria were inhibited
by valinomycin. The lack of susceptibility of Gram-negative bacteria to valinomycin is
largely attributed to their outer membrane of the cell wall, serving as a selective barrier
and limiting the intracellular access of an antibiotic [
43
,
45
]. This was also demonstrated
by a previous report that if Gram-negative E. coli cells were treated with Tris/EDTA, the
cells then became more permeable and sensitive to valinomycin [
46
]. The antibacterial
activity of valinomycin was found to be dependent on the pH value and K
+
concentration
in the cultivation medium. While valinomycin displayed activity at a relatively broad
pH range, significant inhibition of bacterial growth was observed mainly at alkaline pH
values (e.g., pH 8.5) [
29
]. Previous studies also demonstrated that, in a medium with a
low K
+
concentration, valinomycin remarkably inhibited bacterial cell growth [
41
,
44
]. For
example, when Streptococcus pyogenes was cultivated in Tryptose Broth (TB) with a low K
+
concentration (3 mM), the minimum inhibitory concentration (MIC) for S. pyogenes was
0.02
µ
g/mL. However, by switching the cultivation to Trypticase Soy Broth (TSB) with
a high concentration of K
+
(35 mM), the MIC was notably increased to 1
µ
g/mL. This
is interesting, as the inhibitory effect of valinomycin can be largely reversed by addition
of excess K
+
to the medium. The antibacterial action of valinomycin was ascribed to the
ionophore-mediated loss of K
+
from the bacterial cell, leading to the impairment of protein
synthesis [41,44].
2.2. Antifungal Activity
Valinomycin also possesses antifungal activity against different fungi including hu-
man and plant pathogens. Pettit et al. [
42
] reported that valinomycin inhibited the growth
of the two human pathogenic fungi Candida albicans and Cryptococcus neoformans in disk
diffusion assays with MICs of 0.39–0.78
µ
g/disk and 50–100
µ
g/disk, respectively. The
fungistatic spectrum of valinomycin on various plant pathogenic fungi was also examined.
For instance, valinomycin exhibited antifungal activity against Phytophthora capsici, a highly
destructive pathogen of vegetables, with a half-maximal inhibitory concentration (IC
50
)
of 15.9
µ
g/mL [
47
]. In another study, the growth of two plant pathogens, Botrytis cinerea
(gray mold) and Magnaporthe grisea (rice blast), were strongly inhibited by valinomycin [
48
].
Germination of their spores was completely inhibited at a MIC of 4
µ
g/mL. The mycelial
growth of B. cinerea and M. grisea was also inhibited by valinomycin with half-maximal
effective concentrations (EC
50
) of 5.2 and 4.3
µ
g/mL, respectively. In addition,
in vivo
control efficacy tests indicated that valinomycin can be an effective control agent for the
Botrytis disease in cucumber plants, with a comparable efficiency as the commercial fungi-
cide vinclozolin [
48
]. This suggests that valinomycin might be a potential antifungal agent
for the control of Botrytis diseases in agriculture. Furthermore, valinomycin displays anti-
fungal activity against other fungal species such as Aspergillus niger,Fusarium graminearum,
Sclerotinia minor,Penicillium verrucosum, and Rhizoctonia solani [
49
–
51
]. However, the anti-
fungal mechanism of valinomycin was not elucidated in these above-mentioned studies.
Using two representative fungi Candida albicans and Cryptococcus albidus, Makarasen and
colleagues investigated the mode of action and verified the efficacy of valinomycin alone
and in combination with an antifungal antibiotic amphotericin B (AmB) against both fungi.
Microorganisms 2021,9, 780 5 of 22
They found that valinomycin in combination with AmB can promote the permeability of
the fungal cell wall and the cell membrane and may penetrate into the cell wall and inhibit
ergosterol formation, eventually leading to cell death [52].
2.3. Antiviral Activity
In human history, virus infection is one of the most serious threats to human health and
lives. Some well-known pathogenic viruses include Variola virus, Flu virus, Ebola virus,
Zika virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Middle
East Respiratory Syndrome Coronavirus (MERS-CoV). Currently, the global pandemic of
Coronavirus Disease 2019 (COVID-19), caused by a novel coronavirus SARS-CoV-2, has
triggered enormous human casualties and serious economic losses worldwide. Therefore,
it is highly urgent to find effective therapeutic drugs to combat against COVID-19 and
save people’s lives. Valinomycin has been demonstrated as a potent antiviral agent against
a broad spectrum of viruses such as human coronaviruses, bunyaviruses, enteroviruses,
and flavivirus [
53
]. In 1999, an initial test of valinomycin on the vesicular stomatitis virus
(VSV) in Vero cells was performed [
42
]. It was found that adding 10
µ
M valinomycin to
VSV infected cells led to a 90% decrease in viral titer within 12 h after infection. After
the outbreak of SARS in 2003, Wu and colleagues extensively screened more than 10,000
compounds to identify effective anti-SARS agents using a cell-based assay with SARS virus
and Vero E6 cells [
54
]. Notably, valinomycin was found to be the most potent inhibitor
among all tested compounds with an EC
50
value of 0.85
µ
M, which is 4 times lower than that
of the second effective compound reserpine (EC
50
= 3.4
µ
M). In another antiviral study, from
a library of 502 compounds, valinomycin also exhibited the highest inhibition (IC
50
= 24 nM)
on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in
infected monkey embryonic kidney epithelial (MARC)-145 cells [
55
]. Recently, different
groups have demonstrated the inhibitory effect of valinomycin on MERS-CoV [
56
–
58
].
When Vero B4 cells were infected by MERS-CoV and treated with 5
µ
M of valinomycin, the
replication of MERS-CoV was remarkably reduced by up to 1000-fold at 48 h post-injection.
A further dose-effect relationship experiment indicated that valinomycin was highly potent
against MERS-CoV with an IC
50
value of 84 nM [
56
]. Meanwhile, another study evaluated
MERS-CoV infected Vero E6 cells and found that valinomycin was able to inhibit the
virus replication with an EC
50
of 6.07
µ
M [
58
]. A recent study further demonstrated
the anti-MERS-CoV activity of valinomycin and the IC
50
value was determined to be as
low as 5 nM using infected Vero E6 cells [
57
]. In the same study, valinomycin was also
shown to be effective against another human coronavirus (HCoV-229E, IC
50
= 67 nM),
Zika virus (IC
50
= 78 nM), and five other viruses (e.g., bunyaviruses and enteroviruses,
IC
50
values ranged from 41 to 971 nM) [
57
]. Taken together, these findings demonstrated
that valinomycin possesses broad-spectrum antiviral activity. Virus genome sequencing
and analysis has revealed that the genome sequence of SARS-CoV-2 is closely related to
SARS-CoV (79.6% identity) and MERS-CoV (50% identity) [
59
,
60
]. Notably, the amino
acid sequences of the seven conserved replicase domains in ORF1ab that are used for CoV
species classification are 94.4% identical between SARS-CoV-2 and SARS-CoV [
60
]. In
addition, a recent study reported that valinomycin shows high binding energies towards
SARS-CoV-2 proteins by molecular docking and dynamic simulations [
61
]. Therefore,
valinomycin has the potential to be developed as a potent antiviral agent to combat the
ongoing, fast-spreading global COVID-19 pandemic.
Possible mechanisms of valinomycin against viruses were also described. In the
example of anti-VSV infection, the viral envelope glycoprotein (G protein) was not fully
processed in valinomycin-treated cells, making G protein oligosaccharides sensitive to
endo-
β
-N-acetylglucosaminidase H cleavage. As a result, most of the oligosaccharides in
VSV G protein were not converted to their mature forms, leading to the failure of transport
of G protein to the cell surface and its further incorporation into budding viral particles [
42
].
In another way, valinomycin inhibited the activity of the viral ribonucleoprotein (vRNP)
that is responsible for directing viral RNA genome replication and gene transcription [
62
].
Microorganisms 2021,9, 780 6 of 22
In addition, Sandler et al. [
57
] also reported that valinomycin significantly blocked virus
replication and reduced the number of viral genomes by >90%. This is likely due to the
disruption of cellular K
+
gradient, which is a conserved and critical host factor in virus
replication [
57
]. The antiviral effect of valinomycin can be strongly attenuated if the external
medium was supplemented with excess potassium ions (KCl) [63].
2.4. Insecticidal and Antiparasitic Activity
Insects and parasites are often vectors that cause human diseases such as the two
well-known infectious illnesses malaria and dengue [
64
]. Valinomycin has been shown to
have insecticidal, nematocidal, and antiparasitic activity as well [
65
–
68
]. Angus reported
that free ingestion of valinomycin by fifth-instar larvae Bombyx mori (approximately 5
µ
g
per larva) caused cessation of feed intake followed by sluggishness and finally paralysis
of larvae [
69
]. The toxicity of valinomycin on the insects Musca domestica (house fly) and
Periplaneta americana (cockroach) was tested and the results indicated that valinomycin
was quite toxic to both insects [
70
]. Flies and cockroaches behaved abnormally at 24 h
after injection, appearing sluggish and motor paresis. Interestingly, the toxic effect of
valinomycin was found to be dependent on sex, as females of both species were more
resistant than males. The half-lethal dose (LD
50
) for male flies was 0.02
µ
g (1.18
µ
g/g of
body weight). Female flies showed a slightly lower susceptibility to valinomycin with
a LD
50
value of 0.03
µ
g (1.5
µ
g/g of body weight). In cockroaches, the LD
50
values for
males and females were 0.19
µ
g (0.25
µ
g/g of body weight) and 0.5
µ
g (0.5
µ
g/g of body
weight), respectively [
70
]. Insecticidal activity bioassays also demonstrated that valino-
mycin yielded half-lethal concentrations (LC
50
) of 2–3 pm (2–3
µ
g/mL) for mosquito larvae
(Aedes aegypti) after 36 h, 3 ppm for two-spotted spider mites (Tetranychus urticae), and
35 ppm for Mexican bean beetle larvae (Epilachna varivestis) [
65
]. Gumila et al. screened
22 ionophore compounds for their antimalarial activities and found that valinomycin was
active against the growth of Plasmodium falciparum, a malaria-causing parasite, with an
IC
50
of 5.3 ng/mL [
71
]. In addition, valinomycin exhibited significant inhibitory activity
against the parasites Leishmania major (IC
50
< 0.11
µ
M) and Trypanosoma brucei brucei (IC
50
0.0032
µ
M), which cause leishmaniasis and African sleeping sickness, respectively [
68
]. An-
other study investigated the effect and the mechanism of action of valinomycin on Babesia
gibsoni, a blood parasite that causes hemolytic anemia in dogs [
72
]. The authors found
that the antibabesial activity of valinomycin depended on the potassium concentration in
canine erythrocytes. In erythrocytes with low potassium, the IC
50
value was calculated
as 2.32 ng/mL. However, a high concentration of potassium in canine erythrocytes no-
tably impaired the antibabesial activity of valinomycin with an increasing IC
50
value of
570 ng/mL. Furthermore, the effect of valinomycin on B. gibsoni almost disappeared in
culture media containing high concentrations of potassium. The mechanism of action was
suggested that valinomycin may destroy B. gibsoni by changing the intracellular potassium
concentration, which might be maintained by an active transporter. However, the cation
(K+) transporter of B. gibsoni was not identified in the study.
2.5. Antitumor Activity
Antitumor activity is another important bioactivity of valinomycin and its antitu-
mor efficacy has been evaluated against multiple tumor cell lines [
42
,
73
–
78
]. The half-
maximal growth inhibition (GI
50
) concentrations of valinomycin against six human tumor
cells (e.g., ovary OVCAR-3, lung NCI-H460, and renal A-498) were determined to be at
nanogram levels, ranging from 0.19 to 1.9 ng/mL [
42
]. The antitumor mechanism of vali-
nomycin is primarily based on the induction of cell apoptosis/death through a couple of
different pathways. Valinomycin-treated rat ascites hepatoma cells (AH-130) underwent
several typical apoptotic events including the loss of mitochondrial membrane poten-
tial, caspase-3 activation, DNA fragmentation, cell shrinkage, and formation of pycnotic
nucleus [
76
]. Under a hypoglycemic growth condition (glucose starvation), valinomycin
strongly inhibited the transcription and translation of glucose-regulated protein 78 (GRP78),
Microorganisms 2021,9, 780 7 of 22
a molecular chaperone associated with a stress-signaling pathway in tumor cells, which
induced selective cell death of glucose-starved HT-29 human colon carcinoma cells [
77
].
Valinomycin was also found to inhibit human neuronal glioblastoma cells. Similar to non-
neuronal cells, glioblastoma cells were sensitive to K
+
-efflux mediated apoptosis, which
was induced by valinomycin, resulting in a dramatic depletion of intracellular K+, signifi-
cant depolarization of mitochondria, and obvious cell shrinkage. Meanwhile, valinomycin
increased caspase-3 activation and reduced the expression of an anti-apoptotic protein Bcl-
2. In addition, valinomycin stimulated a nuclear translocation of the apoptosis-inducing
factor (AIF), which represents a caspase-independent apoptotic pathway in glioblastoma
cells [
73
]. Despite its promising antitumor activity, valinomycin can also induce apoptosis
of human natural killer (NK) cells [
79
,
80
] and of many other mammalian cell types
[81–83]
,
making it potentially cellular toxic and consequently it is currently not approved for clin-
ical use. However, the toxic effect of valinomycin on normal cells of the host could be
significantly reduced by incorporation of valinomycin in liposomes while maintaining or
even enhancing its antitumor activity against the P388 mouse leukemia [
75
]. Moreover,
the low toxic liposomal valinomycin displayed synergistic cytotoxicity against human
ovarian carcinoma cells (CaOV-3) when used in combination with the antitumor drug
cisplatin [
74
]. Alternatively, modifying chemical structure resulted in the generation of
valinomycin derivatives with a low toxicity. For example, the isopropyl side chains of
D-
α
-hydroxyisovalerate, D-valine, and L-valine were hydroxylated, respectively, yielding
hydroxyl valinomycin analogs (Figure S1). While hydroxyl analogs showed less antitumor
activity than the parent valinomycin to different extents, they were still pharmacologically
significant [
84
]. Taken together, valinomycin can be a promising candidate for combating a
range of human tumors if its overall cytotoxicity could be reduced. Therefore, tailoring
valinomycin derivatives with well-defined properties by semi-synthesis or biosynthesis
and developing specific valinomycin delivery systems may be reasonable routes to achieve
this goal.
Table 1. Summary of valinomycin bioactivities.
Bioactivity Efficacy aReference
Antibacterial
Streptococcus pyogenes MIC 0.02 µg/mL [44]
Clostridium sporogenes MIC 8 µg/mL [44]
Enterococcus faecalis MIC 0.39–0.78 µg/disk [42]
Streptococcus pneumoniae MIC 0.39–0.78 µg/disk [42]
Micrococcus luteus MIC 25–50 µg/disk [42]
Antifungal
Candida albicans MIC 0.39–0.78 µg/disk [42]
Cryptococcus neoformans MIC 50–100 µg/disk [42]
Phytophthora capsici IC50 15.9 µg/mL [47]
Botrytis cinerea MIC 4 µg/mL [48]
Magnaporthe grisea MIC 4 µg/mL [48]
Candida albicans MIC 32 µg/mL [48]
Colletotrichum gloeosporioides MIC 256 µg/mL [48]
Rhizoctonia solani MIC 256 µg/mL [48]
Penicillium verrucosum IC50 0.005 ng/mL [51]
Antiviral
Vesicular stomatitis virus (VSV) GI90 10 µM [42]
Severe acute respiratory syndrome coronavirus (SARS-CoV) EC50 0.85 µM [54]
Porcine reproductive and respiratory syndrome virus (PRRSV) IC50 24 nM [55]
Respiratory syncytial virus (RSV) IC50 0.0015 µM [63]
Middle East respiratory syndrome coronavirus (MERS-CoV) IC50 84 nM [56]
MERS-CoV EC50 6.07 µM [58]
MERS-CoV IC50 5 nM [57]
Human coronavirus OC43 (HCoV-OC43) EC50 4.43 µM [58]
Human coronavirus NL63 (HCoV-NL63) EC50 1.89 µM [58]
Microorganisms 2021,9, 780 8 of 22
Table 1. Cont.
Bioactivity Efficacy aReference
Mouse hepatitis virus A59 (MHV-A59) EC50 6.78 µM [58]
La Crosse virus (LACV) IC50 588 nM [57]
Rift Valley fever virus MP12 (RVFV MP-12) IC50 41 nM [57]
Human rhinovirus 2 (HRV2) IC50 610 nM [57]
Coxsackievirus B3 (CVB3) IC50 971 nM [57]
Zika virus (ZIKV) IC50 78 nM [57]
Keystone virus (KEYV) IC50 156 nM [57]
Human coronavirus 229E (HCoV-229E) IC50 67 nM [57]
Lassa virus (LASV) EC50 0.61 µM [62]
Lymphocytic choriomeningitis virus (LCMV) EC50 0.15 µM [62]
Insecticidal
Musca domestica (male) LD50 0.02 µg [70]
Musca domestica (female) LD50 0.03 µg [70]
Periplaneta americana (male) LD50 0.19 µg [70]
Periplaneta americana (female) LD50 0.5 µg [70]
Aedes aegypti LC50 2–3 µg/mL [65]
Tetranychus urticae LC50 3 ppm [65]
Epilachna varivestis LC50 35 ppm [65]
Plasmodium falciparum IC50 5.3 ng/mL [71]
Babesia gibsoni (in low potassium erythrocytes) IC50 2.32 ng/mL [72]
Babesia gibsoni (in high potassium erythrocytes) IC50 570 ng/mL [72]
Leishmania major IC50 <0.11 µM [68]
Trypanosoma brucei brucei IC50 0.0032 µM [68]
Antitumor
Human ovarian tumor cells CaOV-3 IC50 0.1 nM [74]
Murine P388 leukemia cancer cells GI50 0.019 µg/mL [42]
Human ovary OVCAR-3 tumor cells GI50 1.9 ×10−4µg/mL [42]
Brain SF-295 tumor cells GI50 3.5 ×10−4µg/mL [42]
Renal A-498 carcinoma cells GI50 1.9 ×10−3µg/mL [42]
Lung NCI-H460 cancer cells GI50 2.1 ×10−4µg/mL [42]
Colon KM20L2 carcinoma cells GI50 2.7 ×10−4µg/mL [42]
Melanoma SK-MEL-5 cancer cells GI50 2.6 ×10−4µg/mL [42]
Rat C6 glioma cells IC50 0.0004 µM [84]
Human A2780 ovarian carcinoma cells IC50 2.18 µM [84]
Human MCF-7 breast carcinoma cells IC50 1.77 µM [84]
Human HepG2 liver hepatocellular carcinoma cells IC50 0.0008 µM [84]
Human U251 glioma cells IC50 7.6 nM [85]
a
The values of efficacy are as reported in the references. Note that the molecular weight of valinomycin is 1111.3 g/mol and 1
µ
g/mL of
valinomycin equals to 0.9
µ
M of valinomycin. MIC, minimum inhibitory concentration; IC
50
, half-maximal inhibitory concentration, EC
50
,
half-maximal effective concentration; LD
50
, half-lethal dose; LC
50
, half-lethal concentration; GI
90
, 90% growth inhibition concentration;
GI50, half-maximal growth inhibition concentration.
3. Biogenesis of Valinomycin
After valinomycin was discovered from S.fulvissimus in 1955, researchers attempted
to study the biogenesis of valinomycin. As a first step for this purpose, MacDonald initially
investigated possible precursors for valinomycin biosynthesis [
86
]. To do this, three isotopic
compounds (i.e., DL-valine-1-
14
C, D-valine-1-
14
C, and L-valine-1-
14
C) were individually
added to the cultivation medium. The results indicated that both D- and L-valyl portions
of valinomycin were equally labelled from L-valine-1-
14
C rather than D-valine-1-
14
C in the
medium. A lower radioactivity was also found from the D-
α
-hydroxyisovaleryl portion,
however, no activity was detected in the L-lactyl portion of valinomycin. Later, MacDonald
and Slater [
87
] further reported that D-
α
-hydroxyisovaleric-1-
14
C directly supplied in the
medium was incorporated specifically into the corresponding portion of valinomycin but
the L-isomer was not incorporated. The authors also pointed out that
α
-ketoisovaleric acid
was not a necessary precursor, however, may be converted to D-
α
-hydroxyisovaleric acid
Microorganisms 2021,9, 780 9 of 22
prior to incorporation. In order to figure out the precursor for the L-lactyl portion of valino-
mycin, different groups purified responsible enzymes and tested their activities towards
possible precursors. Ristow and coworkers [
88
] found that a partially purified enzyme
fraction was able to synthesize valinomycin from L-valine using L-alanine or L-threonine
as a precursor of lactic acid. While both
14
C-labeled amino acids were identified from
the L-lactyl moieties of valinomycin, L-threonine was incorporated more efficiently than
L-alanine into the L-lactyl portion. The authors proposed that lactic acid could be formed
from L-alanine via deamination and reduction, however, the mechanism for transforming
L-threonine to lactic acid remained unknown. They also tested if the enzyme complex
could directly activate lactic acid, but no activation was observed. By contrast, Anke
and Lipmann [
89
] reported that lactic acid was easily, but L-alanine poorly, incorporated
into the L-lactyl moieties of valinomycin. Meanwhile, both groups showed that pyruvate
was not able to be activated by their own purified enzyme fractions. Taken together,
early studies all agreed that L-valine was a precursor for both L- and D-valyl portions in
valinomycin. However, it remained unclear whether the hydroxyl acids like lactic acid
were direct precursors or transformed from upstream amino acids (e.g., L-alanine) for the
synthesis of valinomycin. The results from early studies thus were ambiguous and partially
contradicted with each other.
In 1990, the genetic loci involved in valinomycin biosynthesis were identified within
a 120 kb region of chromosomal DNA from Streptomyces levoris A-9; however, the exact
boundary of each necessary gene was not determined [
90
]. After more than a decade,
the complete gene cluster responsible for valinomycin biosynthesis (termed as vlm gene
cluster) in Streptomyces tsusimaensis ATCC 15141 was cloned, sequenced, and partially
characterized by Cheng [
91
]. Sequence analysis indicated that the vlm gene cluster (>23 kb)
encompassed seven open reading frames (ORFs), of which vlm1 (10,287 bp) and vlm2
(7968 bp) encode two distinct NRPSs Vlm1 and Vlm2, respectively. Vlm1 and Vlm2 to-
gether constituted valinomycin synthetase, which is classified as an iterative type B NRPS
for valinomycin biosynthesis. According to the protein sequence analysis, Cheng proposed
that valinomycin synthetase was a four-module NRPS system with sixteen distinctive
domains [
91
]. Vlm1 and Vlm2 comprised modules 1/2 and modules 3/4, respectively.
This modular organization was consistent with the assembly of the tetradepsipeptide
basic unit D-
α
-hydroxyisovaleryl-D-valyl-L-lactyl-L-valyl. Module 1 was predicted to be
responsible for D-
α
-hydroxyisovaleric acid (D-Hiv) incorporation containing two hypo-
thetical domains transaminase (TA) and dehydrogenase (DH
2
). D-Hiv was postulated to
be transformed from L-valine (L-Val) by sequential transamination (catalyzed by TA to
form
α
-ketoisovalerate (Kiv)) and dehydrogenation (catalyzed by DH
2
to yield D-Hiv). In
module 2, an epimerase (E) domain was identified for the conversion of L-Val to D-Val.
A hypothetical DH
2
domain was also suggested to embed in module 3 that acts as a L-
lactate (L-Lac) dehydrogenase to convert pyruvate (Pyr) to L-Lac. The final module 4 was
proposed being responsible for L-Val incorporation. A thioesterase (TE) domain located
at the end of module 4 catalyzing a head-to-tail cyclization to form the mature product
valinomycin. Soon after, Magarvey et al. [
92
] independently sequenced the vlm gene cluster
from S. levoris A-9 but no TA domain was found. Instead, reductase domains were found
within Vlm1 and Vlm2. As a result, the domain organization of valinomycin synthetase
was rationally modified based on the above proposed model by Cheng [
91
]. Specifically,
modules 1 and 3 each contained a ketoreductase (KR) domain, but not TA/DH
2
, being
responsible for the reduction of Kiv to D-Hiv and Pyr to L-Lac, respectively. However, this
assumption solely depended on sequence homologies to similar NRPS clusters and was
not proven by further experiments in the study [92].
Until 2014, Jaitzig and colleagues provided the first experimental proof for the previ-
ous proposed model of valinomycin synthetase [
93
]. The authors purified Vlm1 and Vlm2
from a heterologous expression host E. coli and performed an
in vitro
enzymatic activity
assay towards possible substrates. The results indicated that both Vlm1 and Vlm2 activated
L-Val, which has been demonstrated as a substrate for valinomycin biosynthesis in the early
Microorganisms 2021,9, 780 10 of 22
studies. By contrast, the activation of D-Val was not observed. Importantly, two keto acids
Kiv and Pyr were selectively activated by Vlm1 and Vlm2, respectively. However, other
tested hydroxyl acids L/D-Hiv and L/D-Lac were not activated by both NRPSs. Therefore,
the direct substrates of valinomycin were concluded to be L-Val, Pyr, and Kiv. This also
confirmed the correctness of the proposed valinomycin synthetase model by Magarvey
et al. [
92
]. As shown in Figure 3, module 1 activates Kiv, which is subsequently reduced
to D-Hiv by a dedicated KR domain; L-Val is activated by module 2 and an E domain in
this module converts L-Val to D-Val; another KR domain in module 3 reduces activated Pyr
to L-Lac; and the fourth module activates L-Val. Collectively, the four modules of valino-
mycin synthetase are iteratively reused to assemble three tetradepsipeptide basic units of
D-
α
-hydroxyisovaleryl-D-valyl-L-lactyl-L-valyl, which are finally oligomerized and macro-
lactonized by a C-terminal TE domain to form the 36-membered cyclododecadepsipeptide
valinomycin. Recently, the structure of the TE domain was solved and the mechanism how
TE oligomerizes and cyclizes a linear full-length dodecadepsipeptidyl intermediate was
elucidated [
94
]. However, the structure of the whole NRPS valinomycin synthetase (Vlm1
and Vlm2) has not been reported up to now.
Figure 3.
Proposed domain organization of valinomycin synthetase and valinomycin biosynthesis.
Kiv, ketoisovalerate; D-Hiv, D-hydroxyisovalerate; L-Val, L-valine; D-Val, D-valine; Pyr, pyruvate;
L-Lac, L-lactate; A, adenylation domain; KR, ketoreductase domain; T, thiolation domain; C, conden-
sation domain; E, epimerase domain; TE, thioesterase domain.
Microorganisms 2021,9, 780 11 of 22
4. Biosynthesis of Valinomycin in Native Producers
Native producers of valinomycin are mostly Streptomyces species that have been
isolated from various environments including soil, desert, food, plants, feces, indoor air,
and marine sponges [
42
,
47
,
48
,
51
,
65
,
68
,
90
,
91
,
95
,
96
]. Interestingly, several Bacillus strains
isolated from Brassica seeds and plants were also found to produce valinomycin. Notably,
12 out of 14 Bacillus pumilus isolates were able to synthesize valinomycin. In addition,
valinomycin was detected from the culture extracts of Bacillus amyloliquefaciens (3 out of 18
isolates) and Bacillus subtilis (1 out of 19 isolates) as well [
97
]. More interestingly, the gene
cluster for valinomycin biosynthesis was also identified from a Rothia nasimurium strain
isolated from a porcine tonsil [
98
]. The occurrence of valinomycin in many Streptomyces
strains is interesting. Matter et al. [
99
] studied the conservation, ecology, and evolution of
vlm gene clusters from eight valinomycin-producing strains originated from North America,
Europe, and Asia. They found that the DNA sequences of vlm gene cluster were highly
conserved among all eight strains. According to the phylogenetic relationships of these
strains, the evolution of vlm gene cluster represented a vertical transmission (VT) pattern
rather than horizontal gene transfer (HGT), which often happens in the transmission of
natural product biosynthetic gene clusters [100].
Currently, more than 20 Streptomyces strains have been reported to produce valino-
mycin, albeit their productivities are very different as summarized in Table 2. Production
of valinomycin using native producers is still limited because, for example, the cultiva-
tion of Streptomyces microorganisms is time-consuming (several days) and the genetic
manipulation of Streptomyces is complex and laborious. Only a few studies optimized
different conditions for enhanced production of valinomycin using native Streptomyces
strains. An orchard soil isolated strain Streptomyces sp. M10 was capable of synthesizing
valinomycin yet with a relative low yield (about 3.83 mg/L) [
48
]. A later study discovered
that, in addition to valinomycin, this M10 strain also produced bafilomycin (Figure S2),
which shares a common precursor (
α
-ketoisovaleric acid, Kiv) with valinomycin [
101
]. By
disrupting the bafilomycin biosynthetic genes, the yield of valinomycin was increased up
to 6 mg/L, which is 1.5-fold higher than the productivity of the parental strain. Further dis-
ruption of the branched-chain
α
-keto acid dehydrogenase (BCDH) gene clusters, encoding
a BCDH enzyme complex that can convert Kiv to isobutyric acid, valinomycin production
in the mutant was enhanced to 16 mg/L. Another optimization process was performed
with a psychrotrophic strain Streptomyces lavendulae ACR-DA1, isolated from the soil of
a high altitude cold desert [
96
]. Production of valinomycin was optimized for different
fermentation conditions like medium, temperature, and addition of precursors. Finally,
the maximum yield of valinomycin (84 mg/L) was obtained with a synthetic mineral base
starch (MBS) medium and precursor addition (L-valine) at a low temperature of 10
◦
C
for a period of eight-day cultivation in shake flasks. However, the yield was reduced to
19.4 mg/L when the fermentation was scaled up to a 5 L bioreactor level [102].
5. Reconstituted Biosynthesis of Valinomycin In Vivo and In Vitro
5.1. Heterologous Production of Valinomycin in Escherichia coli
Although valinomycin is a Streptomyces originated natural compound, there is so far
no report on reconstituted biosynthesis of valinomycin in Streptomyces hosts, which have
been well-developed for heterologous expression of natural product gene clusters [
103
,
104
].
During the past decades, E. coli has emerged as a powerful surrogate host for heterologous
production of intricate natural products by recombinant expression of their entire gene
clusters in the host cells [
105
–
108
]. The choice of E. coli as a heterologous producer is due
to its remarkable advantages: simple cultivation conditions, fast growth rate, extensive
genetic tools, well-understood native metabolic networks, and easy scale-up fermentation
processes. To establish a robust cell factory for heterologous production of valinomycin,
the Neubauer group at Technische Universität Berlin intensively utilized E. coli to achieve
this goal with multiple strategies from strain improvement to bioprocess engineering. In
Microorganisms 2021,9, 780 12 of 22
this section, we thus summarize recent reports on heterologous production of valinomycin
in E. coli (valinomycin yields are listed in Table 2).
Prior to reconstituted biosynthesis of valinomycin in E. coli, at least two challenging
questions have to be answered. First, is the heterologous host capable of expressing cor-
rectly folded and posttranslationally modified NRPSs (i.e., Vlm1 and Vlm2)? Both enzymes
Vlm1 and Vlm2 are large multimodular NRPSs with molecular weights of 370 and 284 kDa,
respectively. Therefore, heterologous expression of such megaenzymes individually or
together would be a heavy metabolic burden on host cells. In addition, a prerequisite for
the synthesis of valinomycin is posttranslational phosphopantetheinylation of apo-T (thio-
lation) domains in Vlm1 and Vlm2 by a phosphopantetheinyl transferase (PPTase) [
109
].
This modification of apo-T domains generates functional, holo-T domains and consequently
leads to active holo-NRPS [
5
]. Second, what are precursors of valinomycin and is the host
chassis able to provide the building blocks for valinomycin biosynthesis? Early studies in
1960s and 1970s did not unambiguously confirm all precursors of valinomycin except the
one L-valine [
86
–
89
]. Although in 2006 a rational model of valinomycin synthetase was
proposed and possible substrates were predicted, no experimental evidence to prove this
assumption [91,92].
As a first step towards this goal, Jaitzig and colleagues [
93
] initially cloned two valino-
mycin NRPS genes vlm1 (~10 kb) and vlm2 (~8 kb) from the native producer S. tsusimaensis
genomic DNA. Soluble expression of Vlm1 or Vlm2 in E. coli was successful, enabling the
purification of both NRPSs to perform
in vitro
enzymatic activity and substrate specificity
testing. Like other NRPSs, adenylation (A) domains in Vlm1 and Vlm2 are responsible
for the selection and activation of dedicated substrates. The substrate activation catalyzed
by A domains does not depend on holo-T domains and, therefore, the reaction can be as-
sayed with apo-NRPSs. With purified Vlm1 and Vlm2, the substrates of valinomycin were
experimentally confirmed to be Pyr, Kiv, and L-Val (for details, see the description in the
section of “3. Biogenesis of Valinomycin”). However, apo-NRPSs have to be functionalized
by PPTase to catalyze valinomycin formation. To this end, the sfp gene from Bacillus subtilis
encoding a promiscuous PPTase was integrated into the E. coli genome. Finally, coexpres-
sion of Vlm1 and Vlm2 in the engineered E. coli host, which can directly provide three
substrates from its own primary metabolism (i.e., the glycolysis and the branched chain
amino acid L-valine biosynthesis pathway; see Figure 4), allowed autonomous formation
of valinomycin without substrate feeding. While recombinant production of valinomycin
in E. coli was successful, only a relatively low yield was obtained (0.3 mg/L) from a batch
cultivation using the Terrific Broth (TB) medium. This motivates further optimization to
achieve high valinomycin yields.
Figure 4.
The biosynthetic pathway of valinomycin precursors in E. coli. IlvBN, acetohydroxy
acid synthase I; IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase; IlvE,
branched chain amino acid aminotransferase; Pyr, pyruvate; Kiv, ketoisovalerate; L-Val, L-valine.
Microorganisms 2021,9, 780 13 of 22
The initial low yield of valinomycin was ascribed to the batch cultivation format [
93
].
Once the nutrients in TB medium were depleted, the growth of E. coli gradually ceased
that limited the supply of valinomycin substrates. To overcome this limitation, E. coli
cultivations were subsequently switched from a batch to an enzyme-controlled fed-batch-
like glucose delivery (EnBase
®
) system in shake flasks [
110
]. This fed-batch-like cultivation
system enabled a prolonged cell growth phase and high cell densities by a controlled
continuous internal supply of glucose, which is gradually released by a biocatalyst from the
dissolved glucose polymer [
111
,
112
]. With this fed-batch cultivation mode, valinomycin
yields were significantly increased from 0.3 to 2.4 mg/L. A subsequent design of experiment
(DoE)-driven optimization further improved the yield up to 6.4 mg/L. Notably, repeated
glucose polymer feeding to the cultivation system not only resulted in flask-scale high
cell densities (a final OD
600
of 55) but also enhanced valinomycin production to a yield
of 10 mg/L, which is a 33-fold increase as compared to the initial yield obtained with TB
batch cultivations [110].
While two core NRPS genes in the valinomycin biosynthetic gene cluster are vlm1 and
vlm2, other five genes are also identified in the gene cluster and one of them encodes a
discrete type II thioesterase (TEII) [
91
]. In general, TEII in NRPS gene clusters serves as a
repair (editing) enzyme to restore the functionality (activity) of NRPS through hydrolysis
of either misacylated thiol groups or incorrectly loaded substrates on the T domains
(Figure 5) [
113
–
115
]. However, whether this editing enzyme TEII could keep the activity of
valinomycin synthetase in a heterologous host remained unknown. To test its dedicated
function, the cognate TEII was coexpressed with Vlm1 and Vlm2 in E. coli for valinomycin
production [
116
]. By doing this, valinomycin yields were significantly improved from 0.5
(without TEII coexpression) to 3.3 mg/L, demonstrating the reconstitutive function of TEII
in heterologous valinomycin biosynthesis. A following enzyme-based fed-batch cultivation
system in shake flasks finally gave rise to the highest production of valinomycin (13 mg/L).
Figure 5.
Regeneration of the functionality of T domains catalyzed by type II thioesterase (TEII). T,
thiolation domain; PPTase, phosphopantetheinyl transferase; NRP, nonribosomal peptide.
Since fed-batch high cell density cultivations benefit valinomycin production in E. coli,
this cultivation mode was scaled up from 1 mL in 24-well plates to a benchtop bioreac-
tor [
117
]. An initial cultivation was performed with 2 L culture volume. Although the cell
density at the end of the fermentation reached 120 (OD
600
), the valinomycin yield was
modest (2 mg/L). Protein expression analysis indicated that Vlm2 was not well expressed
in this bioreactor fermentation, which is likely due to plasmid instability. Additionally,
a 10 L laboratory scale-down two-compartment reactor (TCR), which can mimic similar
situations in large industrial bioreactors, was used to produce valinomycin. The produc-
Microorganisms 2021,9, 780 14 of 22
tion was not affected by oscillating conditions (i.e., high glucose and oxygen limitation
in a feeding zone) in the TCR bioreactor, suggesting that this scale-up strategy based on
consistent fed-batch cultivations for bioprocess development may be robust and feasible
even for larger production scales. However, there is still space for further optimization to
achieve mass production of valinomycin in large-scale fermentations to meet the industrial
demand. Such possibilities might arise from different levels, for example, constructing a
more stable coexpression system (Vlm1, Vlm2, and TEII), engineering the metabolic path-
ways of host cells to provide abundant substrates (Pyr, Kiv, and L-Val), and optimizing key
large-scale fermentation parameters (e.g., glucose feeding, cell growth rate, and dissolved
oxygen, etc.).
5.2. In Vitro Total Biosynthesis of Valinomycin
In recent years,
in vitro
cell-free systems are becoming a promising complementary
platform to
in vivo
cell-based systems for biomanufacturing of various products [
118
–
122
].
In this context, cell-free protein synthesis (CFPS), cell-free metabolic engineering (CFME),
and coupled CFPS-ME have been used to synthesize value-added chemicals, cellular toxic
compounds, and complex natural products, among others [
123
–
129
]. The first example of
using CFPS for NRP biosynthesis was a diketopiperazine (DKP) D-Phe-L-Pro, which is a
shunt product of gramicidin S, by coexpression of two NRPS enzymes GrsA and GrsB1
in vitro
[
124
]. This work represented a proof-of-concept for how one could apply CFPS to
synthesize complex natural products like NRPs.
Recently, total
in vitro
biosynthesis of valinomycin was achieved by using E. coli
cell-free systems [
130
]. The first challenge was active expression of the two large NRPSs
Vlm1 (370 kDa) and Vlm2 (284 kDa), although the E. coli-based CFPS system is robust
to express many types of proteins with molecular weights often less than one hundred
kDa [
120
,
121
,
131
]. Initially, individual plasmids harboring the genes vlm1 and vlm2 were
used as DNA templates to express the two enzymes in CFPS reactions. With cell extracts
prepared from E. coli BL21 Star (DE3), both enzymes Vlm1 and Vlm2 were separately
expressed in full-length as high soluble fractions. Importantly, they could be coexpressed
solubly in a single-pot cell-free reaction. Then, the PPTase protein Sfp was coexpressed
with Vlm1 and Vlm2, and this cell-free expressed Sfp was able to activate both enzymes
for valinomycin biosynthesis. Finally, the target product valinomycin was successfully
detected by LC-MS analysis, however, with a low yield of 9.76
µ
g/L. Next, the repair
enzyme TEII, which was earlier found to be able to regenerate the activity of Vlm1 and
Vlm2 and enhance valinomycin production in E. coli cells [
116
], was coexpressed with
the aforementioned three proteins (Vlm1, Vlm2, and Sfp). Notably, all four proteins were
actively expressed in a one-pot CFPS reaction and TEII indeed also improved valinomycin
biosynthesis in the
in vitro
reaction environment. The final yield from this reaction reached
37
µ
g/L. Taken together, this work demonstrated the robustness of the E. coli-based CFPS
system that can express the entire valinomycin biosynthetic gene cluster (>19 kb), which
contains two large NRPSs (Vlm1 and Vlm2) and one associated editing enzyme (TEII), as
well as a heterologous modification enzyme (Sfp). While this was remarkable, valinomycin
yields in CFPS reactions were about three orders of magnitude lower than previous
in vivo
production in E. coli [116].
One of the reasons for low valinomycin yields was possibly resource limitations in
CFPS caused by coexperssion of the four enzymes. To bypass this constraint, Vlm1 and
Vlm2 were first produced
in vivo
, followed by preparation of two cell lysates each enriched
with Vlm1 or Vlm2. Afterwards, valinomycin was synthesized through an approach called
cell-free metabolic engineering (CFME) by simple mixing of two cell lysates. However
also here, an unexpected low yield of valinomycin was obtained (5.59
µ
g/L), even if Vlm1
and Vlm2 were enriched in the cell-free biosynthesis reaction. Therefore, other efforts
were carried out to optimize the CFME system for enhanced valinomycin production. By
investigating the effect of supplemental cofactors (CoA, NAD, and ATP) and the mass
ratio of two lysates on valinomycin synthesis, the yield was increased up to 76.9
µ
g/L at
Microorganisms 2021,9, 780 15 of 22
a mass ratio of 3:1 (cell lysate-Vlm1:cell lysate-Vlm2) without cofactor supplementation.
Finally, a coupled CFPS-ME system was used to perform a two-phase biosynthesis. In this
approach, TEII was expressed by CFPS in the first reaction phase that could be used to
restore the activity of Vlm1 and Vlm2 during the second CFME phase. In addition, when
enough TEII enzyme was expressed (3 h), glucose (200 mM) was added to the reaction to
fuel the CFME process for another 12 h. This strategy dramatically improved valinomycin
biosynthesis with a yield of nearly 30 mg/L, which is more than 5000 times higher than
the initial CFME yield (5.59
µ
g/L). This result suggested that (i) CFPS expressed TEII is
active to regenerate the activity of
in vivo
heterologously expressed Vlm1 and Vlm2 and
(ii) native enzymes in cell lysates are active to convert glucose to valinomycin substrates
(Pyr, Kiv, and L-Val) through the glycolysis and the branched chain amino acid L-valine
biosynthesis pathway (Figure 4).
In sum, this work shows an example to use the E. coli-based cell-free system for
rapid synthesis of valinomycin and production improvement. It should also be noted
that valinomycin originates from Streptomyces species with a high GC-content vlm gene
cluster. Therefore, one might wonder if recently developed Streptomyces-based CFPS
systems
[132–134]
are more suitable to express vlm gene cluster than the E. coli CFPS
system. Especially, with the improvement of expressed protein yields [
135
], valinomycin
biosynthesis in Streptomyces CFPS systems is possible and deserves future studies. Given
the robustness and flexibility of cell-free systems, expression of the engineered vlm gene
cluster using CFPS might be easy and fast for the synthesis of valinomycin analogs with
potential novel bioactivities, but low cellular toxicity.
6. Conclusions and Outlook
Since the discovery of valinomycin in 1955, it has always received broad attentions
from biologists, chemists, engineers, and pharmacologists because of its outstanding bio-
logical features and functions. Notably, valinomycin, which was initially identified as an
antibiotic, also shows a diverse spectrum of bioactivities including antifungal, antiviral, in-
secticidal, and antitumor activity. In particular, valinomycin exhibits remarkable inhibitory
effects on the viruses SARS-CoV and MERS-CoV, which share high genome similarity with
SARS-CoV-2 that causes the current global pandemic of COVID-19. Moreover, molecular
docking and dynamic simulations have shown that valinomycin shows high binding en-
ergies to SARS-CoV-2 proteins [
61
]. Therefore, valinomycin is a potential compound to
be developed as a potent anti-SARS-CoV-2 agent to combat the ongoing, fast-spreading
COVID-19 pandemic. In this context, at least mass production of valinomycin is highly
desirable for further structure modification to reduce its overall cellular toxicity.
Although many Streptomyces strains have been reported to produce valinomycin,
these native hosts are not ideal producers due to, for example, time-consuming cultivations
(around 10 days) and laborious genetic manipulations. Alternatively, a heterologous
host E. coli has been engineered for valinomycin production, followed by a bioprocess
development to enhance valinomycin yields (from 0.3 to 13 mg/L) [
110
,
116
]. In addition,
total
in vitro
biosynthesis of valinomycin has also been established based on an E. coli
CFPS system. This approach allows for easy control and optimization without the use
of living cells, giving rise to a valinomycin yield as high as 30 mg/L [
130
]. Of note, the
valinomycin synthesis rate (mg/L/h) in
in vitro
systems was the highest as compared to
in vivo
production systems using both native producers and the heterologous E. coli host
(Table 2). Previous work has shown that CFPS reaction volumes could be scaled up linearly
over a range of six orders of magnitude to 100 L [
136
]. Moreover, the costs of cell-free
systems can be reduced by numerous opportunities like removing expensive reagents
(e.g., exogenous tRNAs) and decreasing substrate concentrations (e.g., amino acids) [
121
].
These technological progresses will make industrialization of cell-free biotechnology with
a great potential to produce target products such as, for example, valinomycin.
Microorganisms 2021,9, 780 16 of 22
Table 2. Production levels of valinomycin with native and heterologous hosts.
Production Yield aNote Reference
Native host
Streptomyces fulvissimus 17 mg/L Static cultivation in flask, 20 d [12]
Streptomyces sp. PRL1642 50–58 mg/L Shake-flask, 3–5 d [86]
Streptomyces griseus var. flexipertum N.R. Bioreactor, 5 d [65]
Streptomyces levoris A-9 N.R. Shake-flask, 5 d [90]
Streptomyces griseus strains 2/ppi,
8/ppi, 10/ppi, and 1/k 0.6–1.4 mg/mg wet biomass Cultivation on agar plates, 10–12 d [95]
Streptomyces anulatus (Montana) 4.65 mg/L Shake-flask, 3 d [42]
Streptomyces anulatus (Malaysian) 1.5 mg/L Shake-flask, 3 d [42]
Streptomyces exfoliatus (Malaysian) 3.9 mg/L Shake-flask, 3 d [42]
Streptomyces padanus TH-04 70 mg/L Shake-flask, 7 d [47]
Streptomyces sp. M10 3. 83 mg/L Shake-flask, 2 d [48]
Streptomyces sp. M10 16 mg/L
Shake-flask, 3 d, engineered M10 strain
[101]
Streptomyces tsusimaensis 8.45 mg/L Shake-flask, 6 d [99]
Streptomyces griseus 1/k 10.19 mg/L Shake-flask, 6 d [99]
Streptomyces griseus 10/ppi 22.08 mg/L Shake-flask, 6 d [99]
Streptomyces sp. 22 N.R. Cultivation on agar plates, 7 d [68]
Streptomyces sp. 34 N.R. Cultivation on agar plates, 7 d [68]
Streptomyces sp. P11–23B 0.74 mg/L Shake-flask, 14 d [85]
Streptomyces lavendulae ACR-DA1 84 mg/L Shake-flask, 8 d, L-valine feeding [96]
Streptomyces lavendulae ACR-DA1 19.4 mg/L Bioreactor, 8 d [102]
Streptomyces sp. S8 N.R. Shake-flask, 14 d [49]
Streptomyces parvus 0.15 mg/g dry biomass Shake-flask, 5 d [51]
Bacillus subtilis N.R. Shake-flask, 3 d [97]
Bacillus pumilus N.R. Shake-flask, 3 d [97]
Bacillus amyloliquefaciens N.R. Shake-flask, 3 d [97]
Rothia nasimurium N.R. Genome sequencing and prediction [98]
Heterologous host
Escherichia coli 0.3 mg/L Shake-flask, 36 h [93]
Escherichia coli 6.4 mg/L 24-well plate, 48 h [110]
Escherichia coli 10 mg/L Shake-flask, fed-batch cultivation, 48 h [110]
Escherichia coli 2 mg/L Bioreactor, 48 h [117]
Escherichia coli 13 mg/L Shake-flask, 48 h, coexpression of TEII [116]
In vitro system
Escherichia coli cell-free system 37 µg/L CFPS, 20 h [130]
Escherichia coli cell-free system 77 µg/L CFME, 12 h [130]
Escherichia coli cell-free system 30 mg/L CFPS-ME, 15 h [130]
aYields are as reported or calculated from reported data. N.R., not reported.
Looking forward, more researches and developments of valinomycin will continue to
expand in the years to come due to its specific properties and bioactivities. Such directions
might include, but are not limited to, the generation of valinomycin analogs/derivatives
with low cellular toxicity, development of efficient valinomycin delivery system for pre-
cisely targeted treatments, and high-yielding production of valinomycin using an easy,
inexpensive, and sustainable way.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/microorganisms9040780/s1. Figure S1: Valinomycin analogs with a hydroxyl group (OH)
at the isopropyl side chain of D-Hiv (left), D-Val (middle), and L-Val (right), respectively. Figure S2:
Chemical structure of bafilomycin.
Author Contributions:
Conceptualization, J.L.; writing—original draft preparation, S.H. and Y.L.;
writing—review and editing, J.L., P.N. and W.-Q.L.; supervision, J.L.; funding acquisition, J.L. All
authors have read and agreed to the published version of the manuscript.
Microorganisms 2021,9, 780 17 of 22
Funding:
This work was supported by grants from the National Natural Science Foundation of China
(No. 31971348 and No. 31800720) and the Natural Science Foundation of Shanghai (No. 19ZR1477200).
J.L. also acknowledges the starting grant of ShanghaiTech University.
Acknowledgments: This review article is dedicated to the memory of Ju-E Kang (1955–2020).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Koehn, F.E.; Carter, G.T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov.
2005
,4, 206–220.
[CrossRef] [PubMed]
2. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J.
Nat. Prod. 2020,83, 770–803. [CrossRef] [PubMed]
3.
Walsh, C.T.; Tang, Y. Natural Product Biosynthesis: Chemical Logic and Enzymatic Machinery; Royal Society of Chemistry Publishing:
London, UK, 2017.
4.
Finking, R.; Marahiel, M.A. Biosynthesis of nonribosomal peptides. Annu. Rev. Microbiol.
2004
,58, 453–488. [CrossRef] [PubMed]
5.
Fischbach, M.A.; Walsh, C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery,
and mechanisms. Chem. Rev. 2006,106, 3468–3496. [CrossRef]
6.
Caboche, S.; Leclere, V.; Pupin, M.; Kucherov, G.; Jacques, P. Diversity of monomers in nonribosomal peptides: Towards the
prediction of origin and biological activity. J. Bacteriol. 2010,192, 5143–5150. [CrossRef]
7.
Walsh, C.T.; O’Brien, R.V.; Khosla, C. Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid
polyketide scaffolds. Angew. Chem. Int. Ed. 2013,52, 7098–7124. [CrossRef]
8.
Felnagle, E.A.; Jackson, E.E.; Chan, Y.A.; Podevels, A.M.; Berti, A.D.; McMahon, M.D.; Thomas, M.G. Nonribosomal peptide
synthetases involved in the production of medically relevant natural products. Mol. Pharm. 2008,5, 191–211. [CrossRef]
9.
Marahiel, M.A. Working outside the protein-synthesis rules: Insights into non-ribosomal peptide synthesis. J. Pept. Sci.
2009
,15,
799–807. [CrossRef]
10.
Martín, J.F. New aspects of genes and enzymes for
β
-lactam antibiotic biosynthesis. Appl. Microbiol. Biotechnol.
1998
,50, 1–15.
[CrossRef]
11. Hubbard, B.K.; Walsh, C.T. Vancomycin assembly: Nature’s way. Angew. Chem. Int. Ed. 2003,42, 730–765. [CrossRef]
12.
Brockmann, H.; Schmidt-Kastner, G. Valinomycin I, XXVII. Mitteil. über Antibiotica aus Actinomyceten. Chem. Ber.
1955
,88,
57–61. [CrossRef]
13.
Brockmann, H.; Geeren, H. Valinomycin II. Antibiotika aus Actinomyceten XXXVII. Die konstitution des Valinomycins. Justus
Liebigs Ann. Chem. 1957,603, 216–232. [CrossRef]
14.
Brockmann, H.; Springorum, M.; Träxler, G.; Höfer, I. Molekulargewicht des Valinomycins. Naturwissenschaften
1963
,50, 689.
[CrossRef]
15.
Shemyakin, M.M.; Aldanova, N.A.; Vinogradova, E.I.; Feigina, M.Y. The structure and total synthesis of valinomycin. Tetrahedron
Lett. 1963,4, 1921–1925. [CrossRef]
16.
Sivanathan, S.; Scherkenbeck, J. Cyclodepsipeptides: A rich source of biologically active compounds for drug research. Molecules
2014,19, 12368–12420. [CrossRef]
17.
Asher, I.M.; Rothschild, K.J.; Stanley, H.E. Raman spectroscopic study of the valinomycin-KSCN complex. J. Mol. Biol.
1974
,89,
205–222. [CrossRef]
18.
Hamilton, J.A.; Sabesan, M.N.; Steinrauf, L.K. Crystal structure of valinomycin potassium picrate: Anion effects on valinomycin
cation complexes. J. Am. Chem. Soc. 1981,103, 5880–5885. [CrossRef]
19.
Neupert-Laves, K.; Dobler, M. The crystal structure of a K
+
complex of valinomycin. Helv. Chim. Acta
1975
,58, 432–442. [CrossRef]
20.
Stillwell, W. Membrane transport. In An Introduction to Biological Membranes, 2nd ed.; Stillwell, W., Ed.; Academic Press:
Cambridge, MA, USA, 2016; pp. 440–442.
21. Karle, I.L. Conformation of valinomycin in a triclinic crystal form. J. Am. Chem. Soc. 1975,97, 4379–4386. [CrossRef]
22.
Karle, I.L.; Flippen-Anderson, J.L. A new conformation exhibiting near-threefold symmetry for uncomplexed valinomycin in
crystals from dimethyl sulfoxide. J. Am. Chem. Soc. 1988,110, 3253–3257. [CrossRef]
23.
Czernek, J.; Brus, J. Polymorphic forms of valinomycin investigated by NMR crystallography. Int. J. Mol. Sci.
2020
,21, 4907.
[CrossRef]
24.
Andreoli, T.E.; Tieffenberg, M.; Tosteson, D.C. The effect of valinomycin on the ionic permeability of thin lipid membranes. J. Gen.
Physiol. 1967,50, 2527–2545. [CrossRef]
25.
Junge, W.; Schmid, R. The mechanism of action of valinomycin on the thylakoid membrane: Characterization of the electric
current density. J. Membr. Biol. 1971,4, 179–192. [CrossRef]
26. Pressman, B. Mechanism of action of transport-mediating antibiotics. Ann. N. Y. Acad. Sci. 1969,147, 829–841. [CrossRef]
27.
Shemyakin, M.M.; Ovchinnikov, Y.A.; Ivanov, V.T.; Antonov, V.K.; Vinogradova, E.I.; Shkrob, A.M.; Malenkov, G.G.; Evstratov,
A.V.; Laine, I.A.; Melnik, E.I.; et al. Cyclodepsipeptides as chemical tools for studying ionic transport through membranes. J.
Membr. Biol. 1969,1, 402–430. [CrossRef]
Microorganisms 2021,9, 780 18 of 22
28.
Su, Z.F.; Ran, X.Q.; Leitch, J.J.; Schwan, A.L.; Faragher, R.; Lipkowski, J. How valinomycin ionophores enter and transport K
+
across model lipid bilayer membranes. Langmuir 2019,35, 16935–16943. [CrossRef]
29.
Tempelaars, M.H.; Rodrigues, S.; Abee, T. Comparative analysis of antimicrobial activities of valinomycin and cereulide, the
Bacillus cereus emetic toxin. Appl. Environ. Microbiol. 2011,77, 2755–2762. [CrossRef]
30.
Ovchinnikov, Y.A. Second FEBS-Ferdinand Springer lecture: Membrane active complexones. Chemistry and biological function.
FEBS Lett. 1974,44, 1–21. [CrossRef]
31.
Shemyakin, M.M.; Vinogradova, E.I.; Feigina, M.Y.; Aldanova, N.A.; Loginova, N.F.; Ryabova, I.D.; Pavlenko, I.A. The structure-
antimicrobial relation for valinomycin depsipeptides. Experientia 1965,21, 548–552. [CrossRef]
32.
Shemyakin, M.M.; Vinogradova, E.I.; Ryabova, I.D.; Fonina, L.A.; Sanasaryan, A.A. Relationship between structure, stability
of potassium complexes, and antimicrobial activity in a series of analogs of valinomycin. Chem. Nat. Compd.
1973
,9, 229–234.
[CrossRef]
33.
Breitbart, H.; Herzberg, M. Membrane mediated inhibition of protein synthesis by valinomycin in reticulocytes. FEBS Lett.
1973
,
32, 15–18. [CrossRef]
34.
Breitbart, H.; Herzberg, M. Changes in energy charge and block of protein synthesis in rabbit reticulocytes under the action of
valinomycin. Mol. Biol. Rep. 1980,6, 195–198. [CrossRef]
35.
Breitbart, H.; Atlan, H.; Eltes, F.; Herzberg, M. Interaction between membrane properties and proteins synthesis in reticulocytes—
A two step inhibition of protein synthesis by valinomycin. Mol. Biol. Rep. 1975,2, 167–173. [CrossRef]
36.
Herzberg, M.; Breitbart, H. Block in the elongation of protein synthesis in rabbit reticulocyte by action of the ionophore
valinomycin. Mol. Biol. Rep. 1980,6, 163–167. [CrossRef] [PubMed]
37.
Jakubkova, M.; Dzugasova, V.; Truban, D.; Abelovska, L.; Bhatia-Kissova, I.; Valachovic, M.; Klobucnikova, V.; Zeiselova, L.;
Griac, P.; Nosek, J.; et al. Identification of yeast mutants exhibiting altered sensitivity to valinomycin and nigericin demonstrate
pleiotropic effects of ionophores on cellular processes. PLoS ONE 2016,11, e0164175. [CrossRef]
38.
Wieland, T. The History of Peptide Chemistry. In Peptide: Synthesis, Structure, and Applications; Gutte, B., Ed.; Academic Press:
Cambridge, MA, USA, 1995; pp. 1–38.
39.
Rakovic, A.; Ziegler, J.; Mårtensson, C.U.; Prasuhn, J.; Shurkewitsch, K.; König, P.; Paulson, H.L.; Klein, C. PINK1-dependent
mitophagy is driven by the UPS and can occur independently of LC3 conversion. Cell Death. Differ.
2019
,26, 1428–1441. [CrossRef]
[PubMed]
40.
Xiong, X.; Li, S.; Han, T.L.; Zhou, F.; Zhang, X.; Tian, M.; Tang, L.; Li, Y. Study of mitophagy and ATP-related metabolomics based
on β-amyloid levels in Alzheimer’s disease. Exp. Cell. Res. 2020,396, 112266. [CrossRef] [PubMed]
41.
Harold, F.M.; Baarda, J.R. Gramicidin, valinomycin, and cation permeability of Streptococcus faecalis.J. Bacteriol.
1967
,94, 53–60.
[CrossRef]
42.
Pettit, G.R.; Tan, R.; Melody, N.; Kielty, J.M.; Pettit, R.K.; Herald, D.L.; Tucker, B.E.; Mallavia, L.P.; Doubek, D.L.; Schmidt, J.M.
Antineoplastic agents. Part 409: Isolation and structure of montanastatin from a terrestrial actinomycete. Bioorg. Med. Chem.
1999
,
7, 895–899. [CrossRef]
43.
Ryabova, I.D.; Gorneva, G.A.; Ovchinnikov, Y.A. Effect of valinomycin on ion transport in bacterial cells and on bacterial growth.
Biochim. Biophys. Acta 1975,401, 109–118. [CrossRef]
44.
Seshachalam, D.; Frahm, D.H.; Ferraro, F.M. Cation reversal of inhibition of growth by valinomycin in Streptococcus pyogenes and
Clostridium sporogenes.Antimicrob. Agents Chemother. 1973,3, 63–67. [CrossRef]
45.
Pagès, J.; James, C.; Winterhalter, M. The porin and the permeating antibiotic: A selective diffusion barrier in Gram-negative
bacteria. Nat. Rev. Microbiol. 2008,6, 893–903. [CrossRef]
46.
Pavlasova, E.; Harold, F.M. Energy coupling in the transport of beta-galactosides by Escherichia coli: Effect of proton conductors. J.
Bacteriol. 1969,98, 198–204. [CrossRef]
47.
Lim, T.H.; Oh, H.C.; Kwon, S.Y.; Kim, J.H.; Seo, H.W.; Lee, J.H.; Kim, J.C.; Lim, C.H.; Cha, B.J.; Min, B.S. Antifungal activity of
valinomycin, a cyclodepsipeptide from Streptomyces padanus TH-04. Nat. Prod. Sci. 2007,13, 144–147.
48.
Park, C.N.; Lee, J.M.; Lee, D.; Kim, B.S. Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp.
strain M10 antagonistic to Botrytis cinerea.J. Microbiol. Biotechnol. 2008,18, 880–884.
49.
Jeon, C.W.; Kim, D.R.; Kwak, Y.S. Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch
disease suppressiveness. World J. Microbiol. Biotechnol. 2019,35, 128. [CrossRef]
50.
Ladeuze, S.; Lentz, N.; Delbrassinne, L.; Hu, X.; Mahillon, J. Antifungal activity displayed by cereulide, the emetic toxin produced
by Bacillus cereus.Appl. Environ. Microbiol. 2011,77, 2555–2558. [CrossRef]
51.
Mohd Danial, A.; Medina, A.; Sulyok, M.; Magan, N. Efficacy of metabolites of a Streptomyces strain (AS1) to control growth and
mycotoxin production by Penicillium verrucosum,Fusarium verticillioides and Aspergillus fumigatus in culture. Mycotoxin Res.
2020
,
36, 225–234. [CrossRef]
52.
Makarasen, A.; Reukngam, N.; Khlaychan, P.; Chuysinuan, P.; Isobe, M.; Techasakul, S. Mode of action and synergistic effect of
valinomycin and cereulide with amphotericin B against Candida albicans and Cryptococcus albidus.J. Mycol. Med.
2018
,28, 112–121.
[CrossRef]
53.
Zhang, D.; Ma, Z.; Chen, H.; Lu, Y.; Chen, X. Valinomycin as a potential antiviral agent against coronaviruses: A review. Biomed. J.
2020,43, 414–423. [CrossRef]
Microorganisms 2021,9, 780 19 of 22
54.
Wu, C.Y.; Jan, J.T.; Ma, S.H.; Kuo, C.J.; Juan, H.F.; Cheng, Y.S.; Hsu, H.H.; Huang, H.C.; Wu, D.; Brik, A.; et al. Small molecules
targeting severe acute respiratory syndrome human coronavirus. Proc. Natl. Acad. Sci. USA
2004
,101, 10012–10017. [CrossRef]
[PubMed]
55.
Karuppannan, A.K.; Wu, K.X.; Qiang, J.; Chu, J.J.; Kwang, J. Natural compounds inhibiting the replication of Porcine reproductive
and respiratory syndrome virus. Antiviral Res. 2012,94, 188–194. [CrossRef] [PubMed]
56.
Gassen, N.C.; Niemeyer, D.; Muth, D.; Corman, V.M.; Martinelli, S.; Gassen, A.; Hafner, K.; Papies, J.; Mösbauer, K.; Zellner, A.;
et al. SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection. Nat.
Commun. 2019,10, 5770. [CrossRef] [PubMed]
57.
Sandler, Z.J.; Firpo, M.R.; Omoba, O.S.; Vu, M.N.; Menachery, V.D.; Mounce, B.C. Novel ionophores active against La Crosse virus
identified through rapid antiviral screening. Antimicrob. Agents Chemother. 2020,64, e00086-20. [CrossRef]
58.
Shen, L.; Niu, J.; Wang, C.; Huang, B.; Wang, W.; Zhu, N.; Deng, Y.; Wang, H.; Ye, F.; Cen, S.; et al. High-throughput screening and
identification of potent broad-spectrum inhibitors of coronaviruses. J. Virol. 2019,93, e00023-19. [CrossRef]
59.
Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N.; et al. Genomic characterisation and
epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet
2020
,395, 565–574. [CrossRef]
60.
Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. A pneumonia outbreak
associated with a new coronavirus of probable bat origin. Nature 2020,579, 270–273. [CrossRef]
61.
Fatoki, T.H.; Ibraheem, O.; Ogunyemi, I.O.; Akinmoladun, A.C.; Ugboko, H.U.; Adeseko, C.J.; Awofisayo, O.A.; Olusegun,
S.J.; Enibukun, J.M. Network analysis, sequence and structure dynamics of key proteins of coronavirus and human host, and
molecular docking of selected phytochemicals of nine medicinal plants. J. Biomol. Struct. Dyn. 2020,20, 1–23. [CrossRef]
62. Cubitt, B.; Ortiz-Riano, E.; Cheng, B.Y.; Kim, Y.J.; Yeh, C.D.; Chen, C.Z.; Southall, N.O.E.; Zheng, W.; Martinez-Sobrido, L.; de la
Torre, J.C. A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity. Antivir.
Res. 2020,173, 104667. [CrossRef]
63.
Norris, M.J.; Malhi, M.; Duan, W.; Ouyang, H.; Granados, A.; Cen, Y.; Tseng, Y.C.; Gubbay, J.; Maynes, J.; Moraes, T.J. Targeting
intracellular ion homeostasis for the control of respiratory syncytial virus. Am. J. Respir. Cell Mol. Biol.
2018
,59, 733–744.
[CrossRef]
64.
Wooding, M.; Naudé, Y.; Rohwer, E.; Bouwer, M. Controlling mosquitoes with semiochemicals: A review. Parasit Vectors
2020
,13, 80.
[CrossRef]
65.
Heisey, R.M.; Huang, J.; Mishra, S.K.; Keller, J.E.; Miller, J.R.; Putnam, A.R.; D’Silva, T.D.J. Production of valinomycin, an
insecticidal antibiotic, by Streptomyces griseus var. flexipertum var. nov.J. Agric. Food Chem. 1988,36, 1283–1286. [CrossRef]
66.
Mishra, S.K.; Keller, J.E.; Miller, J.R.; Heisey, R.M.; Nair, M.G.; Putnam, A.R. Insecticidal and nematicidal properties of microbial
metabolites. J. Ind. Microbiol. 1987,2, 267–276. [CrossRef]
67.
Patterson, E.L.; Wright, D.P. Process for Controlling Insects, Nematodes and Mites Using Valinomycin. U.S. Patent No. 3,520,973,
21 July 1970.
68.
Pimentel-Elardo, S.M.; Kozytska, S.; Bugni, T.S.; Ireland, C.M.; Moll, H.; Hentschel, U. Anti-parasitic compounds from Streptomyces
sp. strains isolated from Mediterranean sponges. Mar. Drugs 2010,8, 373–380. [CrossRef]
69.
Angus, T.A. Similarity of effect of valinomycin and Bacillus thuringiensis parasporal protein in larvae of Bombyx mori.J. Invertebr.
Pathol. 1968,11, 145–146. [CrossRef]
70. Pansa, M.C.; Natalizi, G.M.; Bettini, S. Toxicity of valinomycin on insects. J. Invertebr. Pathol. 1973,22, 148–152. [CrossRef]
71.
Gumila, C.; Ancelin, M.L.; Jeminet, G.; Delort, A.M.; Miquel, G.; Vial, H.J. Differential
in vitro
activities of ionophore compounds
against Plasmodium falciparum and mammalian cells. Antimicrob. Agents. Chemother. 1996,40, 602–608. [CrossRef]
72.
Yamasaki, M.; Nakamura, K.; Tamura, N.; Hwang, S.J.; Yoshikawa, M.; Sasaki, N.; Ohta, H.; Yamato, O.; Maede, Y.; Takiguchi,
M. Effects and mechanisms of action of ionophorous antibiotics valinomycin and salinomycin-Na on Babesia gibsoni
in vitro
.J.
Parasitol. 2009,95, 1532–1538. [CrossRef]
73.
Chen, D.; Song, M.; Mohamad, O.; Yu, S.P. Inhibition of Na
+
/K
+
-ATPase induces hybrid cell death and enhanced sensitivity to
chemotherapy in human glioblastoma cells. BMC Cancer 2014,14, 716. [CrossRef]
74.
Daoud, S.S.; Forde, N. Synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells.
Cancer Chemother. Pharmacol. 1991,28, 370–376. [CrossRef]
75.
Daoud, S.S.; Juliano, R.L. Reduced toxicity and enhanced antitumor effects in mice of the ionophoric drug valinomycin when
incorporated in liposomes. Cancer Res. 1986,46, 5518–5523. [PubMed]
76.
Inai, Y.; Yabuki, M.; Kanno, T.; Akiyama, J.; Yasuda, T.; Utsumi, K. Valinomycin induces apoptosis of ascites hepatoma cells
(AH-130) in relation to mitochondrial membrane potential. Cell Struct. Funct. 1997,22, 555–563. [CrossRef] [PubMed]
77.
Ryoo, I.J.; Park, H.R.; Choo, S.J.; Hwang, J.H.; Park, Y.M.; Bae, K.H.; Shin-Ya, K.; Yoo, I.D. Selective cytotoxic activity of
valinomycin against HT-29 human colon carcinoma cells via down-regulation of GRP78. Biol. Pharm. Bull.
2006
,29, 817–820.
[CrossRef] [PubMed]
78.
Smith, T.A.D.; Blaylock, M.G. Treatment of breast tumor cells
in vitro
with the mitochondrial membrane potential dissipater
valinomycin increases 18F-FDG incorporation. J. Nucl. Med. 2007,48, 1308–1312. [CrossRef] [PubMed]
79.
Paananen, A.; Mikkola, R.; Sareneva, T.; Matikainen, S.; Andersson, M.; Julkunen, I.; Salkinoja-Salonen, M.S.; Timonen, T.
Inhibition of human NK cell function by valinomycin, a toxin from Streptomyces griseus in indoor air. Infect. Immun.
2000
,68,
165–169. [CrossRef]
Microorganisms 2021,9, 780 20 of 22
80.
Paananen, A.; Järvinen, K.; Sareneva, T.; Salkinoja-Salonen, M.S.; Timonen, T.; Hölttä, E. Valinomycin-induced apoptosis of
human NK cells is predominantly caspase independent. Toxicology 2005,212, 37–45. [CrossRef]
81.
Abdalah, R.; Wei, L.; Francis, K.; Yu, S.P. Valinomycin-induced apoptosis in Chinese hamster ovary cells. Neurosci. Lett.
2006
,405,
68–73. [CrossRef]
82.
Deckers, C.L.P.; Lyons, A.B.; Samuel, K.; Sanderson, A.; Maddy, A.H. Alternative pathways of apoptosis induced by methylpred-
nisolone and valinomycin analyzed by flow cytometry. Exp. Cell Res. 1993,208, 362–370. [CrossRef]
83.
Furlong, I.J.; Lopez Mediavilla, C.; Ascaso, R.; Lopez Rivas, A.; Collins, M.K. Induction of apoptosis by valinomycin: Mitochon-
drial permeability transition causes intracellular acidification. Cell Death. Differ. 1998,5, 214–221. [CrossRef]
84.
Iacobazzi, R.M.; Annese, C.; Azzariti, A.; D’Accolti, L.; Franco, M.; Fusco, C.; La Piana, G.; Laquintana, V.; Denora, N. Antitumor
potential of conjugable valinomycins bearing hydroxyl sites:
In vitro
studies. ACS Med. Chem. Lett.
2013
,4, 1189–1192. [CrossRef]
85.
Ye, X.; Anjum, K.; Song, T.; Wang, W.; Liang, Y.; Chen, M.; Huang, H.; Lian, X.Y.; Zhang, Z. Antiproliferative cyclodepsipep-
tides from the marine actinomycete Streptomyces sp. P11-23B downregulating the tumor metabolic enzymes of glycolysis,
glutaminolysis, and lipogenesis. Phytochemistry 2017,135, 151–159. [CrossRef]
86. MacDonald, J.C. Biosynthesis of valinomycin. Can. J. Microbiol. 1960,6, 27–34. [CrossRef]
87. MacDonald, J.C.; Slater, G.P. Biosynthesis of valinomycin. Can. J. Biochem. 1968,46, 573–578. [CrossRef]
88. Ristow, H.; Salnikow, J.; Kleinkauf, H. Biosynthesis of valinomycin. FEBS Lett. 1974,42, 127–130. [CrossRef]
89. Anke, T.; Lipmann, F. Studies on the biosynthesis of valinomycin. FEBS Lett. 1977,82, 337–340. [CrossRef]
90.
Perkins, J.B.; Guterman, S.K.; Howitt, C.L.; Williams, V.E.; Pero, J. Streptomyces genes involved in biosynthesis of the peptide
antibiotic valinomycin. J. Bacteriol. 1990,172, 3108–3116. [CrossRef]
91.
Cheng, Y.Q. Deciphering the biosynthetic codes for the potent anti-SARS-CoV cyclodepsipeptide valinomycin in Streptomyces
tsusimaensis ATCC 15141. ChemBioChem 2006,7, 471–477. [CrossRef]
92.
Magarvey, N.A.; Ehling-Schulz, M.; Walsh, C.T. Characterization of the cereulide NRPS
α
-hydroxy acid specifying modules:
Activation of α-keto acids and chiral reduction on the assembly line. J. Am. Chem. Soc. 2006,128, 10698–10699. [CrossRef]
93.
Jaitzig, J.; Li, J.; Süssmuth, R.D.; Neubauer, P. Reconstituted biosynthesis of the nonribosomal macrolactone antibiotic valinomycin
in Escherichia coli.ACS Synth. Biol. 2014,3, 432–438. [CrossRef]
94.
Huguenin-Dezot, N.; Alonzo, D.A.; Heberlig, G.W.; Mahesh, M.; Nguyen, D.P.; Dornan, M.H.; Boddy, C.N.; Schmeing, T.M.;
Chin, J.W. Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid. Nature
2019
,565, 112–117.
[CrossRef]
95.
Andersson, M.A.; Mikkola, R.; Kroppenstedt, R.M.; Rainey, F.A.; Salkinoja-Salonen, M.S. The mitochondrial toxin produced by
Streptomyces griseus strains isolated from an indoor environment is valinomycin. Appl. Environ. Microbiol.
1998
,64, 4767–4773.
[CrossRef]
96.
Sharma, R.; Jamwal, V.; Singh, V.P.; Wazir, P.; Awasthi, P.; Singh, D.; Vishwakarma, R.A.; Gandhi, S.G.; Chaubey, A. Revelation
and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different
fermentation and elicitation conditions. J. Biotechnol. 2017,253, 40–47. [CrossRef] [PubMed]
97.
Wulff, E.G.; Mguni, C.M.; Mansfeld-Giese, K.; Fels, J.; Lübeck, M.; Hockenhull, J. Biochemical and molecular characterization of
Bacillus amyloliquefaciens,B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv.
campestris.Plant Pathol. 2002,51, 574–584. [CrossRef]
98.
Gaiser, R.A.; Medema, M.H.; Kleerebezem, M.; van Baarlen, P.; Wells, J.M. Draft Genome sequence of a porcine commensal, Rothia
nasimurium, encoding a nonribosomal peptide synthetase predicted to produce the ionophore antibiotic valinomycin. Genome
Announc. 2017,5, e00453-17. [CrossRef] [PubMed]
99.
Matter, A.M.; Hoot, S.B.; Anderson, P.D.; Neves, S.S.; Cheng, Y.Q. Valinomycin biosynthetic gene cluster in Streptomyces:
Conservation, ecology and evolution. PLoS ONE 2009,4, e7194. [CrossRef] [PubMed]
100. Jensen, P.R. Natural products and the gene cluster revolution. Trends Microbiol. 2016,24, 968–977. [CrossRef]
101.
Lee, D.W.; Ng, B.G.; Kim, B.S. Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin
biosynthesis and branched-chain
α
-keto acid dehydrogenase complex expression. J. Ind. Microbiol. Biotechnol.
2015
,42, 1507–1517.
[CrossRef]
102.
Singh, V.P.; Sharma, R.; Sharma, V.; Raina, C.; Kapoor, K.K.; Kumar, A.; Chaubey, A.; Singh, D.; Vishwakarma, R.A. Isolation of
depsipeptides and optimization for enhanced production of valinomycin from the North-Western Himalayan cold desert strain
Streptomyces lavendulae.J. Antibiot. 2019,72, 617–624. [CrossRef]
103.
Myronovskyi, M.; Luzhetskyy, A. Heterologous production of small molecules in the optimized Streptomyces hosts. Nat. Prod.
Rep. 2019,36, 1281–1294. [CrossRef]
104.
Nepal, K.K.; Wang, G. Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production
of natural products. Biotechnol. Adv. 2019,37, 1–20. [CrossRef]
105.
Li, J.; Neubauer, P. Escherichia coli as a cell factory for heterologous production of nonribosomal peptides and polyketides. N.
Biotechnol. 2014,31, 579–585. [CrossRef]
106.
Luo, Y.; Li, B.Z.; Liu, D.; Zhang, L.; Chen, Y.; Jia, B.; Zeng, B.X.; Zhao, H.; Yuan, Y.J. Engineered biosynthesis of natural products in
heterologous hosts. Chem. Soc. Rev. 2015,44, 5265–5290. [CrossRef]
107.
Zhang, H.; Boghigian, B.A.; Armando, J.; Pfeifer, B.A. Methods and options for the heterologous production of complex natural
products. Nat. Prod. Rep. 2011,28, 125–151. [CrossRef]
Microorganisms 2021,9, 780 21 of 22
108.
Huo, L.; Hug, J.J.; Fu, C.; Bian, X.; Zhang, Y.; Müller, R. Heterologous expression of bacterial natural product biosynthetic
pathways. Nat. Prod. Rep. 2019,36, 1412–1436. [CrossRef]
109.
Lambalot, R.H.; Gehring, A.M.; Flugel, R.S.; Zuber, P.; LaCelle, M.; Marahiel, M.A.; Reid, R.; Khosla, C.; Walsh, C.T. A new
enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 1996,3, 923–936. [CrossRef]
110.
Li, J.; Jaitzig, J.; Hillig, F.; Süssmuth, R.; Neubauer, P. Enhanced production of the nonribosomal peptide antibiotic valinomycin in
Escherichia coli through small-scale high cell density fed-batch cultivation. Appl. Microbiol. Biotechnol.
2014
,98, 591–601. [CrossRef]
111.
Krause, M.; Ukkonen, K.; Haataja, T.; Ruottinen, M.; Glumoff, T.; Neubauer, A.; Neubauer, P.; Vasala, A. A novel fed-batch based
cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb.
Cell Fact. 2010,9, 11. [CrossRef]
112.
Panula-Perälä, J.; Siurkus, J.; Vasala, A.; Wilmanowski, R.; Casteleijn, M.G.; Neubauer, P. Enzyme controlled glucose auto-delivery
for high cell density cultivations in microplates and shake flasks. Microb. Cell Fact. 2008,7, 31. [CrossRef]
113.
Kotowska, M.; Pawlik, K. Roles of type II thioesterases and their application for secondary metabolite yield improvement. Appl.
Microbiol. Biotechnol. 2014,98, 7735–7746. [CrossRef]
114.
Schwarzer, D.; Mootz, H.D.; Linne, U.; Marahiel, M.A. Regeneration of misprimed nonribosomal peptide synthetases by type II
thioesterases. Proc. Natl. Acad. Sci. USA 2002,99, 14083–14088. [CrossRef]
115.
Yeh, E.; Kohli, R.M.; Bruner, S.D.; Walsh, C.T. Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-
enzyme that cannot be elongated. ChemBioChem 2004,5, 1290–1293. [CrossRef] [PubMed]
116.
Li, J.; Jaitzig, J.; Theuer, L.; Legala, O.E.; Süssmuth, R.D.; Neubauer, P. Type II thioesterase improves heterologous biosynthesis of
valinomycin in Escherichia coli.J. Biotechnol. 2015,193, 16–22. [CrossRef] [PubMed]
117.
Li, J.; Jaitzig, J.; Lu, P.; Süssmuth, R.D.; Neubauer, P. Scale-up bioprocess development for production of the antibiotic valinomycin
in Escherichia coli based on consistent fed-batch cultivations. Microb. Cell Fact. 2015,14, 83. [CrossRef] [PubMed]
118.
Bundy, B.C.; Hunt, J.P.; Jewett, M.C.; Swartz, J.R.; Wood, D.W.; Frey, D.D.; Rao, G. Cell-free biomanufacturing. Curr. Opin. Chem.
Eng. 2018,22, 177–183. [CrossRef]
119.
Li, J.; Zhang, L.; Liu, W. Cell-free synthetic biology for
in vitro
biosynthesis of pharmaceutical natural products. Synth. Syst.
Biotechnol. 2018,3, 83–89. [CrossRef] [PubMed]
120.
Liu, W.Q.; Zhang, L.; Chen, M.; Li, J. Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded
applications. Biochem. Eng. J. 2019,141, 182–189. [CrossRef]
121.
Silverman, A.D.; Karim, A.S.; Jewett, M.C. Cell-free gene expression systems: An expanding repertoire of applications. Nat. Rev.
Genet. 2020,21, 151–170. [CrossRef]
122.
Swartz, J.R. Expanding biological applications using cell-free metabolic engineering: An overview. Metab. Eng.
2018
,50, 156–172.
[CrossRef]
123.
Dudley, Q.M.; Karim, A.S.; Nash, C.J.; Jewett, M.C.
In vitro
prototyping of limonene biosynthesis using cell-free protein synthesis.
Metab. Eng. 2020,61, 251–260. [CrossRef]
124.
Goering, A.W.; Li, J.; McClure, R.A.; Thomson, R.J.; Jewett, M.C.; Kelleher, N.L.
In vitro
reconstruction of nonribosomal peptide
biosynthesis directly from DNA using cell-free protein synthesis. ACS Synth. Biol. 2017,6, 39–44. [CrossRef]
125.
Grubbe, W.S.; Rasor, B.J.; Krüger, A.; Jewett, M.C.; Karim, A.S. Cell-free styrene biosynthesis at high titers. Metab. Eng.
2020
,61,
89–95. [CrossRef]
126.
Karim, A.S.; Jewett, M.C. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab. Eng.
2016,36, 116–126. [CrossRef]
127.
Karim, A.S.; Dudley, Q.M.; Juminga, A.; Yuan, Y.; Crowe, S.A.; Heggestad, J.T.; Garg, S.; Abdalla, T.; Grubbe, W.S.; Rasor, B.J.; et al.
In vitro
prototyping and rapid optimization of biosynthetic enzymes for cellular design. Nat. Chem. Biol.
2020
,16, 912–919.
[CrossRef]
128.
Feng, J.; Yang, C.; Zhao, Z.; Xu, J.; Li, J.; Li, P. Application of cell-free protein synthesis system for the biosynthesis of L-theanine.
ACS Synth. Biol. 2021,10, 620–631. [CrossRef]
129.
Liu, W.Q.; Wu, C.; Jewett, M.C.; Li, J. Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic
biphasic system. Biotechnol. Bioeng. 2020,117, 4001–4008. [CrossRef]
130.
Zhuang, L.; Huang, S.; Liu, W.Q.; Karim, A.S.; Jewett, M.C.; Li, J. Total
in vitro
biosynthesis of the nonribosomal macrolactone
peptide valinomycin. Metab. Eng. 2020,60, 37–44. [CrossRef]
131.
Li, J.; Lawton, T.J.; Kostecki, J.S.; Nisthal, A.; Fang, J.; Mayo, S.L.; Rosenzweig, A.C.; Jewett, M.C. Cell-free protein synthesis
enables high yielding synthesis of an active multicopper oxidase. Biotechnol. J. 2016,11, 212–218. [CrossRef]
132.
Li, J.; Wang, H.; Kwon, Y.C.; Jewett, M.C. Establishing a high yielding Streptomyces-based cell-free protein synthesis system.
Biotechnol. Bioeng. 2017,114, 1343–1353. [CrossRef]
133.
Li, J.; Wang, H.; Jewett, M.C. Expanding the palette of Streptomyces-based cell-free protein synthesis systems with enhanced
yields. Biochem. Eng. J. 2018,130, 29–33. [CrossRef]
134.
Moore, S.J.; Lai, H.E.; Needham, H.; Polizzi, K.M.; Freemont, P.S. Streptomyces venezuelae TX-TL—A next generation cell-free
synthetic biology tool. Biotechnol. J. 2017,12, 1600678. [CrossRef]
Microorganisms 2021,9, 780 22 of 22
135.
Xu, H.; Liu, W.Q.; Li, J. Translation related factors improve the productivity of a Streptomyces-based cell-free protein synthesis
system. ACS Synth. Biol. 2020,9, 1221–1224. [CrossRef]
136.
Zawada, J.F.; Yin, G.; Steiner, A.R.; Yang, J.; Naresh, A.; Roy, S.M.; Gold, D.S.; Heinsohn, H.G.; Murray, C.J. Microscale to
manufacturing scale-up of cell-free cytokine production—A new approach for shortening protein production development
timelines. Biotechnol. Bioeng. 2011,108, 1570–1578. [CrossRef]
