Genome Mining Discovery of Protegenins A-D, Bacterial Polyynes Involved in the Antioomycete and Biocontrol Activities of Pseudomonas protegens

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Genome Mining Discovery of Protegenins A−D, Bacterial Polyynes

Involved in the Antioomycete and Biocontrol Activities of

Pseudomonas protegens

‡

,‡

Kazuya Murata, Mayuna Suenaga, and Kenji Kai*

Cite This: https://doi.org/10.1021/acschembio.1c00276

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ABSTRACT: Some bacteria uniquely produce “bacterial polyynes”,

which possess a conjugated CC bond starting with a terminal

alkyne, and use them as chemical weapons against hosts and

competitors. Pseudomonas protegens Cab57, a biocontrol agent against

plant pathogens, has an orphan biosynthetic gene cluster for bacterial

polyynes (named protegenins). In this study, the isolation, structure

elucidation, and biological characterization of protegenins A−D are

reported. The structures of protegenins A−D determined by

spectroscopic and chemical techniques were octadecanoic acid

derivatives possessing an ene−tetrayne, ene−triyne−ene, or ene−

triyne moiety. The protegenins exhibited weak to strong antioomycete

activity against Pythium ultimum OPU774. The deletion of proA, a

protegenin biosynthetic gene, resulted in the reduction of the antioomycete activity of P. protegens. The Gac/Rsm system, a quorum

sensing-like system of Pseudomonas bacteria, regulated the production of protegenins. The production proﬁle of protegenins was

dependent on the culturing conditions, suggesting a control mechanism for protegenin production selectivity. P. protegens suppressed

the damping-oﬀ of cucumber seedlings caused by P. ultimum, and this protective eﬀect was reduced in the proA-deletion mutant.

Altogether, protegenins are a new class of bacterial polyynes which contribute to the antioomycete and plant-protective eﬀects of P.

protegens.

,10

INTRODUCTION

oomycetes, bacteria, nematodes, and insects.

Substantial

■

eﬀorts have been devoted to identifying the secondary

metabolites (e.g., 2,4-diacetylphloroglucinol, pyoluteorin, and

orfamides) (Figure S1 ) and extracellular enzymes (e.g., AprA

Polyynes are a group of natural compounds with alternating

single and triple C−C bonds, reported especially from plants,

−3

basidiomycetes, and insects.

Meanwhile, some bacteria

protease) involved in the biocontrol activity of the strains Pf-5

uniquely produce “bacterial polyynes” containing a conjugated

polyyne moiety starting with a terminal alkyne (Figure 1A) and

use them as potential chemical weapons against hosts and

competitors. Because of their structural features, bacterial

polyynes are unstable and require special caution throughout

experiments. Genomic approaches have identiﬁed the bio-

synthetic gene clusters (BGCs) for several bacterial polyynes

8−10

and CHA0.

However, there are still orphan BGCs for

secondary metabolites (including possible polyynes) in the

5,9,10

genomes of P. fluorescens/protegens strains.

A continuous

chemical investigation is required to completely unveil the

antimicrobial and biocontrol activities of P. fluorescens/

protegens strains.

Previously, we reported the isolation and chemical/bio-

logical characterization of the new bacterial polyynes,

collimonins A−D (Figure S2A ), from the fungus-feeding

−7

(

e.g., cay BGC for caryoynencin) (Figure 1B).

The BGCs

characteristically contain the genes encoding a fatty acyl-ACP

ligase, an ACP (acyl carrier protein), three desaturases/

acetylenases, and a thioesterase. Our understanding regarding

the chemistry and biology of bacterial polyynes has improved

in recent years; however, it has been limited to a few

compounds that have been identiﬁed and characterized.

Various plant-commensal bacteria have been classiﬁed into

Pseudomonas bacteria, including Pseudomonas fluorescens/

protegens, which have an exceptional capacity to produce a

bacterium Collimonas fungivorans Ter331. Collimonins

(

previously called collimomycins) were unique derivatives

of polyoxygenated hexadecanoic acid containing an ene−triyne

Received: April 12, 2021

Accepted: May 12, 2021

variety of secondary metabolites. Among them, P. protegens

strains Pf-5 and CHA0 have been evaluated as model

biocontrol agents against plant pathogens, including fungi,

XXXX American Chemical Society

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(Figure 2 A). These culturing conditions were equal to those

Articles

genase (proH). This gene combination was similar to those of

caryoynencin and collimonin biosynthetic genes, except for the

proH (cay BGC: cayG encoding a P450 monooxygenase; col

−7

BGC: no corresponding gene) (Figures 1B and S2B).

Although the involvement of some additional genes in cepacin

biosynthesis was recently suggested, these genes could not be

identiﬁed in the surrounding region of the pro gene cluster.

Thus, it was plausible that protegenins are long-chain polyyne

fatty acids but diﬀerent from those of the known polyyne

compounds.

Detection and Isolation of Protegenins. Although the

secondary metabolites of P. protegens strains have been

extensively studied, the production of protegenins has not

been observed. This implied that the culturing conditions,

extraction, and/or analysis methods used were not optimal for

the detection of protegenins. The metabolic proﬁles of Cab57

cultures obtained using various media with special sample

preparation treatment were compared. When the common

media (King’s B and LB) for P. protegens were used, P.

protegens Cab57 actively produced well-known secondary

metabolites such as 2,4-diacetylphloroglucinol, pyoluteorin,

and orfamide A (Figure S3). The production of possible

polyynes 1 and 2 (because they exhibited characteristic UV

Figure 1. Bacterial polyynes and their BGCs. (A) Structures of

protegenins A−D (1−4) and caryoynencin. (B) pro and cay BGCs

from P. protegens Cab57 (Pp) and Burkholderia caryophylli DSM

0341 (Bc), respectively.

spectra for polyynes) was observed in the culture extract of

WYA plates supplemented with 2 mM N-acetyl-D-glucosamine

moiety, and a col gene cluster may be involved in their

biosynthesis (Figure S2B ). This was the ﬁrst example reported

on the elucidation of the complete structures (including

absolute conﬁgurations) of bacterial polyynes. In that study,

the stable isolation, structural analyses, and derivatization

methods were established, enabling the elucidation of the

biological activities of collimonins, such as the antifungal

activity against Aspergillus niger.

P. protegens Cab57 (deposited as MAFF 212077) was

identiﬁed as a biocontrol strain by Takeuchi et al. in Japan.

The results of genome comparison revealed that this Japanese

strain shares many biological characteristics with the Pf-5 and

CHA0 model strains. In the present study, the genome- and

MS-based discovery of protegenins A−D (1−4) (Figure 1A)

from P. protegens Cab57 and their chemical/biological

characterization are reported. Protegenins are the ﬁrst natural

compounds uniquely harboring an ene−tetrayne, ene−triyne−

ene, or ene−triyne moiety. The gene deletion and

complementation experiments conﬁrmed that the pro gene

cluster (Figure 1B) is responsible for the protegenin

biosynthesis. The detailed analysis of the biological activity

demonstrates that protegenins, mainly protegenin A (1),

contribute to the antioomycete and biocontrol activities of P.

protegens strains.

Figure 2. Comparison of the HPLC proﬁles of the P. protegens Cab57

culture extracts prepared using diﬀerent growth conditions. (A)

HPLC analysis of the Cab57 culture extracts prepared from WYA

(upper) or King’s B (lower) plates. The peaks of protegenins A−D

(

1−4) are highlighted in blue. (B) HPLC separation of protegenins C

RESULTS

(3) and D (4). The UV spectra of protegenins are shown as insets.

■

Comprehensive Analysis of BGCs for Bacterial

Polyynes. The P. protegens strains (including Cab57) whose

genomes were sequenced harbor a BGC for possible bacterial

used for collimonin production by C. fungivorans Ter331.

After the UV-detector optimization for the polyynes, two

additional polyynes (compounds 3 and 4) were identiﬁed in

the King’s B culture extract by HPLC analysis (Figure 2A).

The polyynes 1, 3, and 4 exhibited identical chromatographic

behavior under the HPLC conditions used. These compounds

were named protegenins A−D and isolated from the culture

extracts of P. protegens Cab57.

,12

polyynes (Figure 1B).

However, the structures and

biological roles of their products (named here “protegenins”)

have not been elucidated yet. The genes encoding the

enzymes/proteins for possible protegenin production were

called proABCDEFGH. In silico analysis suggested that the pro

gene cluster starts with a long-chain fatty acyl-ACP ligase

(

proA) containing two desaturases/acetylenases (proB and

The EtOAc extract of 1000 WYA plates, in which P.

protegens Cab57 was grown for 4 days, was separated on an

proC), an ACP (proD), a desaturase/thioesterase (proE), a

rubredoxin (proF), an α/β-hydrolase (proG), and a dehydro-

ODS column by eluting with a mixture of H O/MeOH. The

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Figure 3. Structure elucidation of protegenins. (A) Key H NMR, COSY, and HMBC data of protegenins A−D (1−4). The diﬀerence in the δ

values of acetylenic protons between 1 and 2 is expected to be due to either the diﬀerence in solvents, the eﬀect of residual water and triﬂuoroacetic

acid in the NMR sample of 2, or both. (B) Preparation of 6 (left) and chiral LC/MS analysis of its absolute conﬁguration (right). SIM detection

was performed at m/z 299.2 (APCI, negative).

fractions eluted with 60, 80, and 100% MeOH showed

antioomycete activity against Pythium ultimum OPU774

the ESI- and APCI-MS spectra; thus, it was diﬃcult to

−

distinguish its [M + H] /[M − H] ions from the background

ions. Probably compound 1 is too hydrophobic to be

eﬀectively ionized using these ionization methods. To solve

this, compound 1 was converted to cycloadduct 5 by

copper(I)-catalyzed azide −alkyne cycloaddition (CuAAC)

(Figure S4A). Protegenins A (1) and B (2) were detected in

00% MeOH as the HPLC peaks showed the characteristic

UV spectrum for polyynes, although 2,4-diacetylphloroglucinol

and pyoluteorin were eluted in 60 and 80% MeOH fractions.

The complete evaporation of the fraction containing

protegenins frequently caused severe polymerization into

insoluble matters and loss of the antioomycete activity. By

avoiding complete evaporation and exposure to oxygen with

temperatures higher than the ambient temperature and light,

compounds 1 (20 mg) and 2 (0.8 mg) were isolated by one-

step reversed-phase HPLC separation from the fraction eluted.

The EtOAc extract of 1500 King’s B plates, in which P.

protegens Cab57 was grown for 4 days, was separated on an

with benzyl azide (Figure S5).

Compound 5 strongly

exhibited an [M + H] ion at m/z 400 in the positive ESI-

MS. The molecular formula of 5 was determined as

C H N O by HRESIMS with m/z 400.2022 [M + H]

(calcd for C H N O , 400.2020). That is, the molecular

formula of 1 was C H O . The H NMR data (in CDCl )

accounted for 17 nonexchangeable protons, composed of one

terminal alkyne proton, two (E)-oleﬁnic protons ( J = 16.0

H,H

Hz), and seven methylene groups (Figure 3A and Table S1).

ODS column by eluting with a mixture of H O/MeOH. The

Analysis of C NMR, DEPT, and HMQC spectra accounted

for 18 carbon signals, and the presence of a carboxyl group, an

(E)-oleﬁn, a tetrayne, and seven methylene groups were

identiﬁed. The characteristic UV spectrum suggested the

fractions eluted with 60 and 80% MeOH showed antioomycete

activity against P. ultimum; however, protegenins C (3) and D

(4) were detected in 100% MeOH (only weak antioomycete

activity) as one major HPLC peak showing a characteristic UV

spectrum for polyynes (Figure S4B ). After the HPLC

separation of compounds 3 and 4 using an ODS column as

a single peak, a long-time HPLC puriﬁcation using a

naphtylethyl column allowed the separation of compounds 3

presence of an ene−tetrayne moiety (Figure 2A). Through

the COSY and HMBC correlations, the structure of 1 was

obtained as an octadecanoic acid derivative possessing an ene−

tetrayne moiety (Figure 3A).

The molecular formula of protegenin B (2) was elucidated

−

(

0.3 mg) and 4 (0.2 mg) (Figure 2B). Protegenins C (3) and

as C H O by HRESIMS with m/z 281.1183 [M − H]

−

D (4) were more stable than protegenins A (1) and B (2)

during the isolation experiments, but their production was

much lower than that of protegenin A (1).

Structure Elucidation of Protegenins A−D. Protegenin

A (1) was obtained as a white solid with a slightly yellow tinge

but occasionally changed instantly to another substance with a

brown color. Compound 1 originated only some minor ions in

(calcd for C H O , 281.1183). Moreover, the UV spectrum

18 17 3

indicated that compound 2 also contains an ene−tetrayne

moiety (Figure 2A). These results suggested that compound 2

was a hydroxylated derivative of 1. The presence of the

substructure 3-hydroxyaliphatic acid in 2 was conﬁrmed by H

NMR and COSY spectra (in DMSO-d ) (Figure 3A and Table

S2 ). The 1D and 2D NMR data analyses showed that the

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Figure 4. Analysis of the protegenin biosynthesis and its regulation. (A) HPLC comparison of protegenin productivity in the P. protegens Cab57

and its mutants. (B) Comparison of protegenin A (1) productivity between Cab57 and its mutants. The ordinate represents the HPLC peak area of

. Error bars are ±SEM (n = 4). Diﬀerent letters above the bars indicate signiﬁcant diﬀerence (p < 0.05, Tukey’s test). (C) HPLC and LC/MS

comparison of secondary metabolites produced by Cab57 (left) and ΔgacS (right).

planar structure of compound 2 is related to the (E)-3-

hydroxyoctadeca-9-en-11,13,15,17-tetraynoic acid (Figure 3A).

To elucidate its absolute conﬁguration, compound 2 was

converted to 6 by hydrogenation with Pd/C under H₂

atmosphere, and its chiral-LC retention time was compared

with that of the synthetic 3-hydroxyoctadecanoic acid (Figure

Protegenin D (4) was also puriﬁed as an amorphous white

powder, and the UV spectrum of compound 4 was similar to

those of collimonins (Figure 2B). The CuAAC reaction of

compound 4 with benzyl azide did not form any cycloadduct.

Therefore, compound 4 probably has an ene−triyne moiety,

but it does not start with a terminal alkyne. The molecular

formula of 4 was determined as C H O by HRESIMS with

B). As a result, compound 6 was determined to be a racemic

mixture. Consequently, the structure of protegenin B (2) was

m/z 271.1692 [M + H] (calcd for C H O , 271.1693). The

18 23 2

completely determined.

1D and 2D NMR data (Figure 3A and Table S4 ) analyses

revealed that compound 4 is the (E)-octadeca-9-en-11,13,15-

triynoic acid.

Protegenin C (3) was obtained as an amorphous white

powder. The UV spectrum of compound 3 was diﬀerent from

those of protegenins A (1) and B (2) (Figure 2B), indicating

that it possesses a diﬀerent ene−yne substructure. Compound

Protegenin Biosynthesis and Its Regulation by

Quorum Sensing. To evaluate if the pro gene cluster is

responsible for protegenin production, the deletion mutant of

proA (ΔproA) was obtained by double crossover recombina-

did not react with benzyl azide under CuAAC conditions,

suggesting the absence of a terminal alkyne in this compound.

The molecular formula of compound 3 was determined as

13,14

tion using the pK18mobsacB vector.

The deletion of proA

C H O by HRESIMS with m/z 269.1537 [M + H] (calcd

resulted in the protegenin production loss (Figure 4A and B).

The complementation of the proA gene into ΔproA (proA-

comp) rescued the protegenin production in the created

for C H O , 269.1536). The H NMR data (in CDCl )

accounted for 19 protons, composed of three vinyl protons,

two (E)-oleﬁnic protons ( J

methylene groups (Figure 3A and Table S3). Analysis of

= 16.0 Hz), and seven

mutant. The pro gene cluster turned out to be responsible for

H,H

the biosynthesis of protegenins in P. protegens Cab57.

NMR and HSQC spectra accounted for 18 carbon signals, and

the presence of a carboxyl group, two pairs of oleﬁnic

methines, a triyne, and seven methylene groups were identiﬁed.

The COSY and HMBC correlations shown in Figure 3A

indicated that compound 3 is a derivative of 1, in which the

terminal alkyne was hydrogenated to the corresponding alkene.

Because proH seemed to be characteristic in the protegenin

BGC, its deletion mutant was created, and the culture extract

was analyzed. The protegenin A (1) production of ΔproH on

the WYA plates was signiﬁcantly reduced compared with that

observed for wild-type Cab57 (Figure 4A and B). ΔproH

produced protegenins C (3) and D (4) on King’s B plates, but

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Articles

their production was also greatly reduced. Furthermore,

apparent accumulation of the possible intermediate and

shunt product was not observed, indicating that proH is not

essential but important for protegenin production.

The production of secondary metabolites in P. fluorescens/

protegens strains is regulated by the Gac/Rsm system, a

quorum sensing (QS)-like system of Pseudomonas bacteria.

This cascade is initiated by the GacS/GacA two-component

system. The GacS sensor kinase is known to be phosphorylated

when an unidentiﬁed signal is received and activates the

cognate GacA response regulator. GacA in turn triggers the

expression of small regulatory RNA molecules, which titrate

translational repressors and relieve the translational inhibition

of target mRNAs. The QS system participation in the

regulation of the production of protegenins was also evaluated.

In contrast to Cab57, ΔgacS (a gacS-deletion mutant of

Cab57) produced only a trace amount of protegenin A (1),

and also 2,4-diacetylphloroglucinol, pyoluteorin, and orfamide

A on the WYA plates (Figure 4C). The time-course

experiments revealed that the dynamics of protegenin A (1)

was similar to those of pyoluteorin (Figure S6). Consequently,

these results indicated that the production of protegenins is

under the Gac/Rsm system control.

Possible Control Mechanism for Protegenin Produc-

tion Selectivity. In this study, the evaluation of the regulation

mechanism involved in protegenin production selectivity is

Figure 5. Evaluation of antioomycete activities of protegenins against

P. ultimum OPU774. (A) Antioomycete activities of protegenins A−D

(

1−4). The activities were evaluated by disc diﬀusion assay, and the

17−19

important. According to previous studies,

the terminal

colony diameter (mm) was measured. Error bars are ± SEM (n = 3).

alkynes of protegenins A (1) and B (2) may be constructed

through the desaturase/acetylenase ProB. Thus, the protegenin

production shift (A/B → C/D) was expected to be derived

from the inhibition of ProB activity. If this is the case, growing

ΔproB on a WYA plate should also produce protegenins C (3)

and D (4). To verify this, ΔproB was obtained, and its

metabolite proﬁle was analyzed. However, as a result, ΔproB

did not produce protegenins C (3) and D (4) or protegenins A

p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control

(Dunnett’s test). (B) Antioomycete activities of the P. protegens

Cab57 and its mutants on WYA (left) and King’s B (right) plates. The

activities were evaluated by confrontation assay against P. ultimum,

and the colony area (mm ) was measured. Error bars are ±SEM (n =

). Diﬀerent letters above the bars indicate signiﬁcant diﬀerence (p <

.05, Tukey’s test).

(

1) and B (2) (Figure S7A ). Hence, this production shift was

not derived from the ProB enzyme disability.

Conversely, among the typical metabolites regulated by the

Gac/Rsm system, only pyoluteorin exhibited a strong

antioomycete activity (IC₅₀= 7.36 nmol/disc) (Figure S9A −

C ). 2,4-Diacetylphloroglucinol was more active against the

fungi (Figure S9A ), while orfamide A was not so e ﬀ ective

against the microbes tested in this study (Figure S9C ).

Confrontation assays of the P. protegenins strains on WYA

plates also indicated that protegenin production is highly

related to the antioomycete activity against P. ultimum and

slightly related to the antifungal activities against F. oxysporum

and A. niger (Figures 5B and S8B ). Therefore, the production

of ene−tetrayne-type protegenins is essential for the strong

antioomycete activity of P. protegens Cab57.

Protegenins C (3) and D (4) exhibited only a weak growth

inhibition against P. ultimum and A. niger and no inhibitory

activity against F. oxysporum (Figures 5A and S10A). The low

contribution of compounds 3 and 4 to the antioomycete/

antifungal activities was also observed in the confrontation

assays on King’s B agar. Under the conditions used, ΔproA

exhibited antioomycete/antifungal activities similar to those of

Cab57, while ΔgacS displayed a lower inhibition compared

with Cab57 (Figures 5B and S10B ). Furthermore, the

antagonism of the tested microbes was still observed in

ΔgacS, i.e., the antimicrobial activities observed here can be

attributed to the metabolites/exoenzymes, which include both

the QS-dependent and -independent ones, other than

protegenins. Altogether, the protegenins C (3) and D (4)

Certain metabolite(s) could aﬀect the Pro enzyme

including ProB) activity. To verify this, we prepared the

EtOAc extract from the King’s B media of Cab57 and

examined whether it aﬀects the production levels and proﬁles

of protegenins. As a result, the extract inhibited the production

of protegenin A (1) by P. protegens Cab57 on WYA plates

(

Figure S7B). Furthermore, ΔproA strongly inhibited the

accumulation of protegenin A (1), and this inhibitory eﬀect

was reduced in the ΔgacS extract. However, while the EtOAc

extracts of P. protegenins strains (King’s B media) exhibited the

inhibitory eﬀect on the protegenin A (1) production, the

production shift from protegenins A/B to C/D was not

observed in these Cab57 cultures. Collectively, certain EtOAc-

soluble metabolite(s) could aﬀect the activity or expression of

Pro enzymes/proteins and an undetermined mechanism might

be involved in changing the proﬁle of protegenins.

Biological Evaluation of Protegenins. To investigate the

biological activities of protegenins, antimicrobial assays of

puriﬁed protegenins and confrontation assays of the wild-type

Cab57, ΔproA, and ΔgacS were performed. Protegenin A (1)

showed a growth inhibition against P. ultimum (strong, IC

4.5 nmol/disc) and Fusarium oxysporum (moderate, IC₅₀

6.0 nmol/disc) but not against Aspergillus niger (Figures 5A

and S8A ). The antimicrobial activities of protegenin B (2)

were similar to those of protegenin A (1) (P. ultimum, IC₅₀

5.4 nmol/disc; F. oxysporum, IC₅₀= 87.5 nmol/disc).

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survival rate to 59.7% (Figure 6 A). The plant survival rates of

Articles

scarcely contribute to the antimicrobial eﬀects of P. protegens

Cab57.

DISCUSSION

■

Various secondary metabolites have been identiﬁed from the P.

fluorescens/protegens group, and the compounds exhibited

antimicrobial, antinematode, and/or insecticidal activities.

In these studies, rich media (such as King’s B media) were

generally used, even though the nutrient conditions were

diﬀerent from those of the plant rhizosphere. In this study, it

was revealed that P. protegens Cab57 produces a high level of

protegenin A (1) and a moderate level of protegenin B (2) on

poor WYA media and low levels of protegenins C (3) and D

Suppression of the Cucumber Damping-Oﬀ by

Protegenins. The contribution of protegenin production to

the biocontrol activity of P. protegens Cab57 against P. ultimum

OPU774 was evaluated. For this, a suppression assay of the

8−10

damping-oﬀ of cucumber seedlings was used. The inocu-

lation of P. ultimum caused the plant survival rate reduction to

2.8%, and the application of P. protegens Cab57 rescued the

(

4) on rich King’s B media. The contribution of protegenins A

1) and B (2) to the antioomycete and plant-protective

properties was demonstrated by disc diﬀusion and confronta-

tion assays. The possible pro BGCs were observed in several

data sets of the Pseudomonas genome, and the production of

protegenin A (1) was conﬁrmed in some strains of P.

fluorescens/protegens. Therefore, protegenins, especially prote-

genin A (1), are key metabolites that have been overlooked for

a long time in the biocontrol eﬀects of Pseudomonas bacteria.

Although a compound identical to protegenin A (1) was

previously identiﬁed as a possible biosynthetic intermediate of

caryoynencin from a cayG-deﬁcient mutant of Burkholderia

caryophylli, the present study is the ﬁrst to identify it as a

natural product and isolated it without chemical derivatization.

It is plausible that protegenin A (1) or the related compounds

are commonly produced by bacterial polyyne BGCs as

biosynthetic intermediates for subsequent modiﬁcations (e.g.,

oxidation). This is the second report of a natural compound

with an ene−tetrayne assembly, after caryoynencin. Although

protegenin B (2) is a 3-hydroxylated derivative of compound

, the involvement of a further enzyme in protegenin B (2)

biosynthesis seems to be excluded; rather, compound 2 might

be synthesized from (±)-3-hydorxyoctadecanoic acid instead

of octadecanoic acid. To the best of our knowledge, the

terminal ene−tetrayne−ene moiety identiﬁed in protegenin C

Figure 6. Suppression of the damping-oﬀ of cucumber seedlings. (A)

Evaluation of the damping-oﬀ suppression abilities of P. protegens

Cab57 and its mutants. Twelve seedlings were used for the trial. Error

bars are ±SEM (n = 6). Diﬀerent letters above the bars indicate

signiﬁcant diﬀerence (p < 0.05, Tukey’s test). Representative assay

images are also shown (right). (B) LC/MS detection of protegenin A

(

3) was the ﬁrst-ever discovered in natural products. The

derivatives with terminal C−C bonds ranging from single to

triple bonds seem to be extremely rare in a single species.

Additional genes were suggested to be involved in the

biosynthesis of cepacins and collimonins, though their genetic

(1) from the Cab57-applied soils (upper) and control soils (lower).

The soil extracts (from 12 wells, ca. 480 g soil) were subjected to

preparative HPLC to obtain the fractions in which protegenin A (1)

was possibly eluted. The detection of protegenin A (1) was achieved

using SIM mode (m/z 267.1).

and biochemical characterizations have not been performed.

Meanwhile, proABCDEFGH genes are enough to construct the

protegenin structures. The functions of Pro enzymes/proteins

were expected to be identical to those of the enzymes of cay

ΔproA and ΔgacS applications were 29.1 and 16.6%,

respectively. These results indicated that, although the

biocontrol eﬀect of P. protegens Cab57 is mainly exerted by

the combination of gacS-regulated metabolites/exoenzymes,

protegenins also exhibit a signiﬁcant contribution. Further-

more, protegenin A (1) from the Cab57-applied soil samples

was detected by LC/MS (Figure 6B). Taken together, our data

indicated that protegenins play important roles in the

biocontrol activity of P. protegens Cab57.

Detection of Protegenin A from P. fluorescens/

protegens. Finally, the conserved production of protegenins

in Pseudomonas bacteria was evaluated. As a result, the

production of protegenin A (1) on WYA plates was conﬁrmed

in all 11 strains examined, though the production levels of

protegenin A (1) in P. fluorescens strains were low compared to

P. protegens MAFF 550081 (Figure S11). The P. fluorescens

strains examined did not produce any protegenins on King’s B

plates. The production of protegenin A (1) may be widely

conserved in P. fluorescens/protegens strains.

gene clusters as follows: after the coupling of octadecanoic

acid with ACP (ProD) by the fatty acyl-ACP ligase ProA, the

desaturase/acetylenase ProB/C/E construct the ene−tetrayne

moiety; then, thioester linkage between protegenin and ACP is

hydrolyzed by thioesterase ProE and probably α/β-hydrolase

ProF, yielding protegenin A (1) (Figure S12). In silico analysis

indicated that ProH belongs to the family of dihydrolipoyl

dehydrogenases, which is usually involved in the recycling of

NAD /NADH. By considering the signiﬁcant reduction of

protegenin A (1) in ΔproH, the reaction using ProH might be

coupled to the biosynthetic step(s) by other Pro enzymes.

Furthermore, proF may encode a rubredoxin, which belongs to

a class of iron-containing proteins that play an important role

in the reduction of superoxide in some anaerobic bacteria and

also act as electron carriers in many biochemical processes.

However, their involvement in the secondary metabolism has

not been well investigated to date. How the substructure ene−

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

Complete contact information is available at:

Articles

tetrayne is biochemically assembled by ProB/C/E also remains

unrevealed.

AUTHOR INFORMATION

Corresponding Author

■

Like 2,4-diacetylphloroglucinol and others, protegenin

production was found to be regulated by the Gac/Rsm system

in P. protegens Cab57. The dynamics of protegenin A (1)

accumulated in the Cab57 cultures were typical for the QS-

Kenji Kai − Graduate School of Life and Environmental

Sciences, Osaka Prefecture University, Sakai, Osaka 599-

8531, Japan; orcid.org/0000-0002-4036-9959;

Email: kai@biochem.osakafu-u.ac.jp

controlled secondary metabolites. The natural ligands that

activate the Gac/Rsm system have not been identiﬁed so far.

Authors

Meanwhile, the QS signals that are received by such two-

Kazuya Murata − Graduate School of Life and Environmental

Sciences, Osaka Prefecture University, Sakai, Osaka 599-

component systems have been identiﬁed in several bacterial

4−28

8531, Japan

species.

Because protegenin production is tightly

Mayuna Suenaga − Graduate School of Life and

Environmental Sciences, Osaka Prefecture University, Sakai,

Osaka 599-8531, Japan

regulated by the QS system, it may be a good indicator for

the activity-based ligand-discovery of GacS.

The biological evaluation of protegenins A (1) and B (2)

clearly indicated that they are essential for strong antioomycete

activity of P. protegens Cab57. The antioomycete activities of

protegenins A (1) and B (2) were higher than those of 4-

diacetylphloroglucinol and orfamide A and comparable to that

of pyoluteorin. Furthermore, protegenin A (1) was detected in

the cucumber seedling rhizosphere, in which the P. protegens

Cab57 actually functioned. Conversely, the biological

importance of protegenins C (3) and D (4) has not been

determined. Considering the existence of a possible mecha-

nism that causes metabolic shifts, it is expected that these

compounds may have some biological roles. Moreover, the

possibility that these compounds are being actively metabo-

lized from protegenin A (1) because they are unnecessary

https://pubs.acs.org/10.1021/acschembio.1c00276

Author Contributions

K.M. and K.K. contributed equally to this work.

‡

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank M. Tojo (Osaka Prefecture University) and K.

Takeuchi (National Agriculture and Food Research Organ-

ization) for technical advice, M. Sogame (OPU) for technical

assistance, and S. Mori (Suntory Foundation for Life Sciences)

for NMR (800 MHz) and HRESIMS measurements. This

work was supported by JSPS KAKENHI (Grants 18H04628

and 20H04784).

cannot be denied. As in the case of collimonins, the terminal

alkyne was important for the antioomycete/antifungal activities

of protegenins.

Recently, Mullins et al. described cepacin A as a plant-

protective metabolite of Burkholderia ambifaria. Additionally,

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