Fengycin A Analogues with Enhanced Chemical Stability and Antifungal Properties

Article abstract of DOI:10.1021/acs.orglett.1c01387

Fengycins are cyclic lipo-depsipeptides produced by Bacillus spp. that display potent antifungal properties but are chemically unstable. This instability has meant that no total synthesis of any fengycin has been published. Here we report the synthesis of fengycin A analogues that display enhanced antifungal properties and chemical stability under both basic and acidic conditions. The analogues prepared also demonstrate that the fengycin core structure can be modified and simplified without the loss of antifungal activity.

Full text of DOI:10.1021/acs.orglett.1c01387

pubs.acs.org/OrgLett
Letter
Fengycin A Analogues with Enhanced Chemical Stability and
Antifungal Properties
Diana Gimenez, Aoife Phelan, Cormac D. Murphy, and Steven L. Cobb*
Cite This: Org. Lett. 2021, 23, 4672−4676
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Fengycins are cyclic lipo-depsipeptides produced by
Bacillus spp. that display potent antifungal properties but are
chemically unstable. This instability has meant that no total
synthesis of any fengycin has been published. Here we report the
synthesis of fengycin A analogues that display enhanced antifungal
properties and chemical stability under both basic and acidic
conditions. The analogues prepared also demonstrate that the
fengycin core structure can be modified and simplified without the
loss of antifungal activity.
lant pathogens, and in particular fungal diseases, pose an
1−3
Pincreasing risk to global food security.
An important
part of the crop protection arsenal is Bacillus subtilis strains,
which are used as biological control agents (BCAs)
(Serenade).4−6 Bioactive Bacillus spp. are able to secrete, as
an innate response to external microbiota stimuli, potent
antimicrobial metabolites that include the cyclic lipopeptides
(CliPs) from the iturin, surfactin, and fengycin families (Figure
1).5,7−13 Fengycins are the dominant CliPs in Bacillus spp.5,14,15
and are active against a range of phytopathogenic fungi,12,16−19
causing cell lysis and leakage through binding with the plasma
membrane.
While most research on BCAs has focused on the direct
application of live bacteria, the effects of a range of
environmental factors (i.e., soil type and humidity) can
produce broad inconsistencies in their performance within
the field.20 A further disadvantage relates to the fact that
antifungal BCAs act slowly when compared to typical pests,
and therefore only give a very time-limited protection to
crops.21 In light of these factors the application of individual
bioactive CliPs, rather than the entire living organisms, would
be an attractive option for the development of new crop
protection agents. However, the problem with this strategy is
that isolation and/or chemical synthesis of certain CliPs, like
fengycin, has proven to be challenging.
Figure 1. (A) General structure of fengycin. (B) Proposed approach
for fengycin stabilization.
Received: April 25, 2021
Published: June 2, 2021
Contrary to iturins and surfactins, fengycins display a
hydrolytically susceptible aromatic depsi bond between Tyr3
and Ile10 which significantly compromises the structural
,
integrity of its cyclic core (Figure 1A).22,23 This intrinsic lack
of chemical stability means that fengycins are technically
challenging to prepare via chemical synthesis.24,25 This is
© 2021 The Authors. Published by
American Chemical Society
4672
https://doi.org/10.1021/acs.orglett.1c01387
Org. Lett. 2021, 23, 4672−4676

Organic Letters
pubs.acs.org/OrgLett
Letter
clearly highlighted by the fact that to date no synthetic strategy
has succeeded in delivering a completely natural fengycin
peptide and, only very recently, a solid phase peptide synthesis
(SPPS) approach which enabled the synthesis of several
fengycin analogues was reported.26 While this marked a
significant advance for the field, the reported approach only
afforded modest product yields and it did not address the issue
of the fengycin peptides’ innate instability.
To address the challenges associated with both the synthesis
and stability of fengycins, we proposed to prepare a series of
modified and simplified analogues (Figure 1B). To solve the
issue of its chemical stability we hypothesized that fengycin
derivatization into a lactam-bridged cyclopeptide, rather than
through its natural ester functionality, would enable access to
more stable cyclic analogues. This approach has been shown
previously in the literature to help improve the chemical
stability of a range of cyclic peptides.27−29 Second, in order to
simplify and reduce the associated cost of the synthesis we
sought to replace the D-allo-Thr residue at position R4 and also
remove the chiral hydroxyl center from the lipid tail. Herein,
we report an efficient SPPS route that allows ready access to
fengycin A analogues with enhanced antifungal and chemical
stability when compared to the natural product.
flexibility of the peptide chain, allowing for increased linear to
cyclic product conversions.
On this basis, we first designed a synthetic route for the
cyclic core, using a Fmoc/(tBu/Boc)/Dmab protection
scheme, that could take advantage of the presence of a natural
L-Gln residue in the peptide structure to install a side-chain
anchor to the resin (Figure 2). Under this strategy, Fmoc-
Glu(OH)-ODmab (1) is thus attached in the first step of the
synthesis to the Rink-amide resin. Dmab protection was
selected as it is orthogonal to base-labile Fmoc and acid-labile
tBu/Boc protecting groups and its selective removal can
proceed quantitatively in the presence of hydrazine (2−5% v/v
in DMF).30 Other options, such as allyl esters, have proven on
occasion in our hands to be difficult to deprotect.31,32
To test the suitability of this approach, we synthesized
model peptidyl-resin 5 following standard Fmoc/tBu chemistry
procedures. Peptide 5 mimics the sequence of the fengycin
3
cyclic domain but for the Tyr-10Ile depsi-bridge, which was
replaced for an amide bond linkage by using Boc-4-(Fmoc-
amino)-L-phenylalanine (3) (Figure 2A, see Supporting
Information for complete experimental details). Then, we
proceeded to evaluate the efficiency of the intramolecular
cyclization step (Figure 2B,C). For this, 5 was incubated
overnight in the presence of DIC/HOBt (3 mol equiv each; rt)
and sample aliquots of the resins before (5) and after
cyclization (6) cleaved in the presence of TFA/H2O/TIPS,
95:2.5:2.5% v/v. Satisfyingly, analysis of the crude materials by
LC/(ESI+)MS spectrometry at λ = 254 nm, characteristic of
the aromatic residues present, confirmed quantitative con-
version of the linear peptide 5 (m/z = 983.6 Da, tR = 1.1 min;
[M + H]+; Figure 2B) to the expected cyclic product 6 (m/z =
965.7 Da, tR = 1.4 min; [M + H]+; Figure 2C).
Not constrained by the limitations imposed by the natural
depsi-bridge, we sought to design possible SPPS routes that
could achieve efficient cyclization yields and enable flexible
peptide diversification on resin (Figures 2 and 3). We
hypothesized that a strategy based on an early stage peptide
cyclization, rather than a late-stage peptide macrolactamiza-
tion, would be beneficial due to the lower conformational
Next, we turned our attention to addressing the complexity
of the branched fengycin structure (Figure 3). For this,
peptidyl-resin 2 was resynthesized and the peptide sequence
extended until the key residue where the peptide bifurcates
(10, Figure 3B). At this critical point, we had anticipated the
need for a protected 4Aph derivative that could enable both
the temporal protection of the peptidyl-resin N-terminus and
the controlled propagation and ring closure of the peptide
through its aniline functionality.
We also considered that such a derivative must allow for
subsequent peptide Ct → Nt elongation by means of
conventional Fmoc amino acids, to prevent posterior
racemization and to minimize the need for custom-made
materials. Chemical orthogonality to the Rink-amide C-
terminus, side chain Boc/tBu, and Dmab protecting groups
was also needed in the protecting group approach selected.
Given the aforementioned factors we opted to synthesize the
NTrityl/NFmoc protected amino acid Trt-L-4(NFmoc)Aph-
OH (9, Figure 3A).
As described, in Figure 3A, amino acid 9 could be
synthesized in 4 steps from its commercially available Boc-4-
(Fmoc-amino)-L-phenylalanine precursor (3). First, the
carboxylic functionality in 3 was converted to its allyl ester
using K2CO3 (1 mol equiv) and allyl bromide (1.5 mol
equiv).33 Next, the Boc group was removed in TFA (20% v/v
in DCM) and the Trt protection of the N-terminus achieved
by slow addition of DIPEA (4 mol equiv) and Trt-Cl (1.5 mol
equiv) in CH3CN/DCM 2:1. Lastly, selective removal of the
allyl protecting group in 8 with Pd(PPh3)3 and PhSiH3 in
DCM afforded the expected Trt-L-4(NFmoc)Aph-OH (9)
with sufficient purity that it could be readily incorporated into
Figure 2. (A) Synthesis of cyclic peptidyl-resin 6. LC/(ESI+)MS
traces of cleaved sample aliquots of 5 before (B) and after cyclization
(C) showing quantitative conversion to the cyclic product 6 (λ= 254
nm).
4673
https://doi.org/10.1021/acs.orglett.1c01387
Org. Lett. 2021, 23, 4672−4676

Organic Letters
pubs.acs.org/OrgLett
Letter
Figure 3. (A) Chemical synthesis of Trt-L-4(NFmoc)Aph-OH (9). (B) Complete total SPPS strategy, based on a quaternary Fmoc/Trt/Dmab/
(tBu/Boc) protection scheme, developed in this study for the preparation of lactam fengycin analogues 17A−D. Product purities of the crude
materials are given as analyzed by RP-HPLC (λ= 220 nm). (C) HPLC traces of crude peptides 17A−D (λ= 220 nm).
the peptide synthesis without any further purification (see
Supporting Information for further details). Once 9 was
coupled to the growing sequence, 11 was further elongated
using standard procedures to yield target peptide 13, which
was then cyclized in situ (Figure 3B).
With the fengycin cyclic core in hand, we then proceeded to
complete the lipidated N-terminus. For this, the Trityl group in
14 was removed using a diluted solution of TFA/TIPS in
DCM (0.2/1% v/v, 1 min, 5×) and the resulting peptide
neutralized in DIPEA/DMF (5% v/v, 60 min). D-Orn (2.5
equiv) and L-Glu (5 equiv) were then sequentially incorpo-
rated via DIC/HOBt assisted couplings to complete the
synthesis of the peptide component of the target. Finally,
incorporation of the lipid tail was achieved by acylation of
Fmoc deprotected resin (16) with palmitoyl chloride in the
presence of DIPEA.
∼50%; Figure 3C). It is worth noting that, as seen in the
cyclization of test peptide 5, no significant traces of the linear
peptide or dimeric species could be found upon analysis of the
crude material.22 The only byproduct observed was a D-Orn
depleted analogue produced after cyclization due to the
modest excess of this amino acid employed during the
synthesis (21%, [M-114] Da, Figure 3; see also supporting
Figures S19−20).
Similar or improved results were obtained when this
methodology was employed to prepare 17B−17D, where we
replaced the initial D-Thr residue at position 4 by the fengycin
A naturally occurring D-allo-Thr and also their corresponding
enantiomers (Figure 3C). Previous reports have shown that
the chirality of the residues within the cyclic core of natural
fengycin affects the ease of ring formation.22,26 However, our
results in the synthesis of analogues 17B and 17D, using L-
amino acids, show that this is not the case for fengycin lactam
derivatives, as all of them could be synthesized with similar
Gratifyingly, when the corresponding peptidyl-resin was
cleaved we obtained the fengycin analogue 17A (crude yield of
4674
https://doi.org/10.1021/acs.orglett.1c01387
Org. Lett. 2021, 23, 4672−4676

Organic Letters
pubs.acs.org/OrgLett
Letter
overall efficacy (50−70% crude purities, Figure 3C and Figures
S23−29). Overall, the synthesis of 17A−D clearly demon-
strates the suitability of our synthetic approach for the
convenient SPPS of fengycin cyclic lipopeptide amide
analogues.
With peptides 17A−D prepared, we moved to investigate
the differences in their chemical stability in comparison to that
of the natural product. To this end, biosynthetic fengycin
expressed from Bacillus spp. was purified by RP-HPLC and the
major product, C16-fengycin B, was isolated and characterized
(see Figures S32−S36). Hydrolysis studies were carried out
with this compound due to the challenges in isolating enough
pure fengycin A. We then selected hydrolysis conditions for
our stability studies (50 mM NaOH for basic hydrolysis and
50% v/v TFA/H2O for acidic degradation; see Supporting
Information for details).
When natural fengycin (C16-fengycin B) was incubated at
room temperature in the presence of 50 mM NaOH, complete
peptide degradation was observed within 5 min (Figure S37).
In comparison, all of the amide-based fengycins analogues were
found to be remarkably stable under the same conditions even
for periods as long as 18−24 h (76−90% intact peptide, see
Figures S39, S41, S43, S45). Acidolytic hydrolysis in the
presence of TFA also revealed significantly different degrada-
tion profiles for the natural product (complete degradation in
12 h; Figure S38) when compared to the modified amide-
bridged analogues (75−90% intact peptide, Figures S40, S42,
S44, S46). The results obtained highlight the superior chemical
stability exhibited by all of the amide analogues of fengycin.
They also demonstrate that modification of the labile natural
depsi Tyr3-O-Ile10 ester bond is a useful route by which to
enhance the half-life of this family of bioactive molecules.
While the enhanced chemical stability of the fengycin
analogues was welcomed, it was expected that the change to a
lactam bridge would impact the biological activity; thus,
bioassays were conducted to examine the effect of 17A−D on
the growth of the fungus Fusarium graminearum. This fungus
was selected as it is known to be sensitive to the natural
lipopeptide.34 Figure 4 shows the results from growth
measurements in well-plates in the absence and presence of
the natural and synthetic fengycins.
In conclusion, we have developed an efficient synthetic route
for the preparation of lactam-containing fengycin analogues.
Given its modular approach and compatibility with readily
available Fmoc amino acids, this method can be easily adapted
to give access to a range of new fengycin derivatives. The
fengycin analogues prepared in this study displayed enhanced
antifungal properties over the naturally occurring material.
Importantly, in addition to the enhanced antifungal properties,
replacement of the natural depsi-bridge by an amide-bond
linkage was found to significantly enhance their chemical
stability under both basic and acidic conditions. Finally, this
work demonstrates that the fengycin core structure could be
modified (e.g., via amino acid substitution) and the lipid tail
structure simplified without the loss of antifungal activity. This
discovery combined with the SPPS approach reported herein
will offer new opportunities to further develop this class of
molecules as anti-infective agents for applications in both
medicine and agriculture.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c01387.
Experimental procedures, product characterization,
HPLC stability studies data, and microbiological
experimental description (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Steven L. Cobb − Department of Chemistry, Durham
University, Durham DH1 3LE, United Kingdom;
orcid.org/0000-0002-3790-7023; Email: s.l.cobb@
durham.ac.uk
Authors
Diana Gimenez − Department of Chemistry, Durham
University, Durham DH1 3LE, United Kingdom
Aoife Phelan − School of Biomolecular and Biomedical
Science, University College Dublin, Dublin 4, Ireland
Cormac D. Murphy − School of Biomolecular and Biomedical
Science, University College Dublin, Dublin 4, Ireland;
orcid.org/0000-0002-2137-3338
The results presented in Figure 4 show that the synthetic
fengycins (17A−D) inhibited growth to a much greater degree
than natural fengycin isolated from Bacillus CS93. In fact, 17D
was found to completely inhibit fungal growth.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c01387
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This publication has emanated from research conducted with
the financial support of Science Foundation Ireland under the
Grant Number SFI/17/BBSRC/3417 and the BBSRC under
Grant Reference BB/P022189/1.
REFERENCES
■
(1) Bhattacharya, S. Deadly New Wheat Disease Threatens Europe’s
Crops. Nature 2017, 542 (7640), 145.
Figure 4. Effect of natural and synthetic fengycins on the growth of
Fusarium graminearum. Fungal growth was measured in the absence
(control) or presence of 500 μg/mL fengycin. t tests confirmed that
all differences between natural and synthetic fengycins were significant
(p < 0.05).
(2) Butler, D. Fungus Threatens Top Banana. Nature 2013, 504
(7479), 195.
(3) Paini, D. R.; Sheppard, A. W.; Cook, D. C.; Barro, P. J. D.;
Worner, S. P.; Thomas, M. B. Global Threat to Agriculture from
4675
https://doi.org/10.1021/acs.orglett.1c01387
Org. Lett. 2021, 23, 4672−4676

Organic Letters
pubs.acs.org/OrgLett
Letter
Invasive Species. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (27), 7575−
7579.
(21) Butt, T. M.; Copping, L. G. Fungal Biological Control Agents.
Pestic. Outlook 2000, 11 (5), 186−191.
(22) Rosés, C.; Camó, C.; Vogels, K.; Planas, M.; Feliu, L. Solid-
Phase Synthesis of Cyclic Depsipeptides Containing a Tyrosine
Phenyl Ester Bond. Org. Lett. 2016, 18 (16), 4140−4143.
(23) Honma, M.; Tanaka, K.; Konno, K.; Tsuge, K.; Okuno, T.;
Hashimoto, M. Termination of the Structural Confusion between
Plipastatin A1 and Fengycin IX. Bioorg. Med. Chem. 2012, 20 (12),
3793−3798.
(4) Cawoy, H.; Bettiol, W.; Fickers, P.; Ongena, M. Bacillus-Based
Biological Control of Plant Diseases. In Pesticides in the Modern
WorldPesticides Use and Management; IntechOpen, 2011.
(5) Ongena, M.; Jacques, P. Bacillus Lipopeptides: Versatile
Weapons for Plant Disease Biocontrol. Trends Microbiol. 2008, 16
(3), 115−125.
(6) Jacobsen, B. J.; Zidack, N. K.; Larson, B. J. The Role of Bacillus
-Based Biological Control Agents in Integrated Pest Management
Systems: Plant Diseases. Phytopathology 2004, 94 (11), 1272−1275.
(7) Chen, H.; Wang, L.; Su, C. X.; Gong, G. H.; Wang, P.; Yu, Z. L.
Isolation and Characterization of Lipopeptide Antibiotics Produced
by Bacillus Subtilis. Lett. Appl. Microbiol. 2008, 47 (3), 180−186.
(8) Maget-Dana, R.; Thimon, L.; Peypoux, F.; Ptak, M. Surfactin/
Iturin A Interactions May Explain the Synergistic Effect of Surfactin
on the Biological Properties of Iturin A. Biochimie 1992, 74 (12),
1047−1051.
(9) Cawoy, H.; Debois, D.; Franzil, L.; Pauw, E. D.; Thonart, P.;
Ongena, M. Lipopeptides as Main Ingredients for Inhibition of Fungal
Phytopathogens by Bacillus Subtilis/Amyloliquefaciens. Microb.
Biotechnol. 2015, 8 (2), 281−295.
(10) Peypoux, F.; Bonmatin, J. M.; Wallach, J. Recent Trends in the
Biochemistry of Surfactin. Appl. Microbiol. Biotechnol. 1999, 51 (5),
553−563.
(11) Yu, G. Y.; Sinclair, J. B.; Hartman, G. L.; Bertagnolli, B. L.
Production of Iturin A by Bacillus Amyloliquefaciens Suppressing
Rhizoctonia Solani. Soil Biol. Biochem. 2002, 34 (7), 955−963.
(12) Ongena, M.; Jacques, P.; Touré, Y.; Destain, J.; Jabrane, A.;
Thonart, P. Involvement of Fengycin-Type Lipopeptides in the
Multifaceted Biocontrol Potential of Bacillus Subtilis. Appl. Microbiol.
Biotechnol. 2005, 69 (1), 29.
(13) Razafindralambo, H.; Popineau, Y.; Deleu, M.; Hbid, C.;
Jacques, P.; Thonart, P.; Paquot, M. Surface-Active Properties of
Surfactin/Iturin A Mixtures Produced by Bacillus Subtilis. Langmuir
1997, 13 (23), 6026−6031.
(14) Maget-Dana, R.; Peypoux, F. Iturins, a Special Class of Pore-
Forming Lipopeptides: Biological and Physicochemical Properties.
Toxicology 1994, 87 (1), 151−174.
(24) Sieber, S. A.; Marahiel, M. A. Learning from Nature’s Drug
Factories: Nonribosomal Synthesis of Macrocyclic Peptides. J.
Bacteriol. 2003, 185 (24), 7036−7043.
(25) Sieber, S. A.; Tao, J.; Walsh, C. T.; Marahiel, M. A. Peptidyl
Thiophenols as Substrates for Nonribosomal Peptide Cyclases. Angew.
Chem., Int. Ed. 2004, 43 (4), 493−498.
̀
(26) Rosés, C.; Camó, C.; Oliveras, A.; Moll, L.; López, N.; Feliu, L.;
Planas, M. Total Solid-Phase Synthesis of Dehydroxy Fengycin
Derivatives. J. Org. Chem. 2018, 83 (24), 15297−15311.
(27) Barrett, D.; Tanaka, A.; Fujie, A.; Shigematsu, N.; Hashimoto,
M.; Hashimoto, S. An Expedient Synthesis of the Amide Analog of the
Potent Antifungal Lipopeptidolactone FR901469. Tetrahedron Lett.
2001, 42 (4), 703−705.
(28) Bionda, N.; Stawikowski, M.; Stawikowska, R.; Cudic, M.;
López-Vallejo, F.; Treitl, D.; Medina-Franco, J.; Cudic, P. Effects of
Cyclic Lipodepsipeptide Structural Modulation on Stability, Anti-
bacterial Activity, and Human Cell Toxicity. ChemMedChem 2012, 7
(5), 871−882.
(29) Bionda, N.; Pastar, I.; Davis, S. C.; Cudic, P. In Vitro and in
Vivo Activities of Novel Cyclic Lipopeptides against Staphylococcal
Biofilms. Protein Pept. Lett. 2014, 21 (4), 352−356.
(30) Conroy, T.; Jolliffe, K. A.; Payne, R. J. Efficient Use of the
Dmab Protecting Group: Applications for the Solid-Phase Synthesis
of N-Linked Glycopeptides. Org. Biomol. Chem. 2009, 7 (11), 2255−
2258.
(31) Grieco, P.; Gitu, P. M.; Hruby, V. J. Preparation of ‘Side-Chain-
to-Side-Chain’ Cyclic Peptides by Allyl and Alloc Strategy: Potential
for Library Synthesis. J. Pept. Res. 2001, 57 (3), 250−256.
(32) Lear, S.; Munshi, T. K.; Hudson, A. S.; Hatton, C.; Clardy, J.;
Mosely, J. A.; Bull, T. J.; Sit, C. S.-W.; Cobb, S. L. Total Chemical
Synthesis of Lassomycin and Lassomycin-Amide. Org. Biomol. Chem.
2016, 14 (19), 4534−4541.
(33) Crich, D.; Sana, K. Solid Phase Synthesis of Peptidyl Thioacids
Employing a 9-Fluorenylmethyl Thioester-Based Linker in Con-
junction with Boc Chemistry. J. Org. Chem. 2009, 74 (19), 7383−
7388.
(34) Wang, J.; Liu, J.; Chen, H.; Yao, J. Characterization of Fusarium
Graminearum Inhibitory Lipopeptide from Bacillus Subtilis IB. Appl.
Microbiol. Biotechnol. 2007, 76 (4), 889−894.
(15) Zhao, H.; Shao, D.; Jiang, C.; Shi, J.; Li, Q.; Huang, Q.; Rajoka,
M. S. R.; Yang, H.; Jin, M. Biological Activity of Lipopeptides from
Bacillus. Appl. Microbiol. Biotechnol. 2017, 101 (15), 5951−5960.
(16) Vanittanakom, N.; Loeffler, W.; Koch, U.; Jung, G. Fengycin-A
Novel Antifungal Lipopeptide Antibiotic Produced by Bacillus Subtilis
F-29−3. J. Antibiot. 1986, 39 (7), 888−901.
(17) Romero, D.; de Vicente, A.; Rakotoaly, R. H.; Dufour, S. E.;
Veening, J.-W.; Arrebola, E.; Cazorla, F. M.; Kuipers, O. P.; Paquot,
M.; Pérez-García, A. The Iturin and Fengycin Families of Lip-
opeptides Are Key Factors in Antagonism of Bacillus Subtilis toward
Podosphaera Fusca. Mol. Plant-Microbe Interact. 2007, 20 (4), 430−
440.
(18) Ali, G. S.; El-Sayed, A. S. A.; Patel, J. S.; Green, K. B.; Ali, M.;
Brennan, M.; Norman, D. Ex Vivo Application of Secreted
Metabolites Produced by Soil-Inhabiting Bacillus Spp. Efficiently
Controls Foliar Diseases Caused by Alternaria Spp. Appl. Environ.
Microbiol. 2016, 82 (2), 478−490.
(19) (a) Zihalirwa Kulimushi, P.; Arguelles Arias, A.; Franzil, L.;
̈
Steels, S.; Ongena, M. Stimulation of Fengycin-Type Antifungal
Lipopeptides in Bacillus Amyloliquefaciens in the Presence of the
Maize Fungal Pathogen Rhizomucor Variabilis. Front. Microbiol. 2017,
8, 850. (b) Kim, P. I.; Ryu, J.; Kim, Y. H.; Chi, Y. T. Production of
biosurfactant lipopeptides Iturin A, fengycin and surfactin A from
Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides. J.
Microbiol. Biotechnol. 2010, 20 (1), 138−45.
(20) Weller, D. M.; Thomashow, L. S. Current Challenges in
Introducing Beneficial Microorganisms into the Rhizosphere. In
Molecular Ecology of Rhizosphere Microorganisms; John Wiley & Sons,
Ltd., 2007; pp 1−18.
4676
https://doi.org/10.1021/acs.orglett.1c01387
Org. Lett. 2021, 23, 4672−4676