Introduction

Polyynes or polyacetylenes contain a conformationally rigid rod-like architecture and an electron-rich consecutive acetylene moiety. Hundreds of polyynes have been discovered, out of which compounds have mainly been isolated from terrestrial plants and marine organisms1,2. In contrast to polyynes from plant sources, bacterial polyynes contain a distinct terminal alkyne with conjugated systems, which causes bacterial polyynes to be more unstable. This instability has discouraged surveys of bacterial polyynes using a bioactivity-guided isolation approach. Nevertheless, more than ten bacterial polyynes have been recorded in a few species. Notably, most polyynes have been reported to have a broad spectrum of antimicrobial effects. For instance, cepacins, isolated from Pseudomonas cepacian SC 11783 (taxonomically reclassified as a Burkholderia diffusa), was reported to have antibacterial activity against the majority Gram-negative bacteria and Staphylococci spp., and anti-oomycetal activity against Pythium ultimum1,2,3,4. Caryoynencin, isolated from Pseudomonas caryophylli (taxonomically reclassified as Burkholderia caryophylli), was reported to have antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococci aureus, and Bacillus subtilis5. In addition, collimonins isolated from Collimonas fungivorans Ter3316,7 and Sch 31828 isolated from Microbispora sp. SCC14388 were reported to have antifungal activity against Aspergillus niger and Candida spp., respectively. Other polyynes, such as ergoynes isolated Gynuella sunshinyii and fischerellins from Fischerella muscicola, were found to have potential cytotoxic/ antibacterial and allelopathic ability9,10,11. Recently, protegenins (including protegencin) produced by Pseudomonas protegens were reported to have antifungal, antioomycete, and anti-algae activities12,13,14,15. Despite the apparent bioactivity of these compounds, the active target and mechanism(s) remain unclear.

Here, we used a multi-omic approach and genetic engineering to identify the bioactive polyynes of Massilia sp. YMA4, characterized their biosynthesis gene cluster (BGC) named massilin (mas) BGC, and delineated the antifungal mechanism of bacterial polyynes. Furthermore, heterologous co-expression of mas BGC further illustrated the conserved genes critical to the electron-rich consecutive alkyne moiety and an additional gene responsible for structure derivatives modification of the polyynes. Furthermore, a comparative genomic analysis uncovered the self-resistance gene and identified bacterial polyynes as antifungal agents that target the first enzyme of ergosterol biosynthesis, acetyl-CoA acetyltransferase. Finally, crystallographic analysis unveiled the detailed binding model of polyynes to the acetyl-CoA acetyltransferase.

Results and discussion

Transcriptomics analysis reveals polyynes as antifungal agents and their encoding BGC in Massilia sp. YMA4

An antagonism assay of Massilia sp. YMA4 against C. albicans revealed a distinct phenotype showing that the antifungal metabolites were produced in potato dextrose agar (PDA) medium but not in yeast malt agar (YMA) medium (Supplementary Fig. 1). In addition, the antifungal metabolites were unstable in the extract and laborious to purify for bioassays using the classic bioactivity-guided isolation approach, which includes purification with click chemistry, exclusion of oxygen and light, or careful optimization7,12,16. Therefore, to reveal the antifungal metabolites and their BGCs, we used a combined transcriptomics and metabolomics approach to explore the compounds produced in the two different media (PDA and YMA). First, the BGCs in the genome of Massilia sp. YMA4 were characterized via DeepBGC17 with default settings and integrated with the criteria: DeepBGC score > 0.7 to characterize 13 BGCs in Massilia sp. YMA4 (Supplementary Data 1). Then, the differentially expressed genes (192 upregulated and 226 downregulated with P-value < 0.05 and |fold change | > 2) from transcriptomics analysis of PDA vs. YMA cultured cells were mapped into the 13 characterized BGCs. Among them, only one BGC was upregulated in PDA compared to YMA (Supplementary Data 1), and we named this gene cluster massilin (mas) BGC with 12 transcribed genes (masA to masL) (Fig. 1a).

Fig. 1: Polyynes and polyyne biosynthesis gene cluster of Massilia sp. YMA4.
figure 1

a Gene expression profile around the mas BGC under polyyne production (PDA) versus non-production (YMA) medium. The expressions of additional and core biosynthesis genes of mas BGC are framed in the gray area. b Structure of polyynes collimonin C/D 1, 2 and massilin A/B 3, 4. c Extraction ion chromatography (EIC) of collimonin C/D 1, 2 (m/z 273.1132), massilin A 3 (m/z 257.1183), and massilin B 4 (m/z 275.1289) in Massilia sp. YMA4 wild type (black) and the biosynthesis null mutant strains (YMA4::masH) with a 10 ppm mass window.

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To characterize the metabolites produced from mas BGC, we constructed mutant strains through insertion mutation at the masH (encoding fatty acid desaturase) gene locus in Massilia sp. YMA4 (Supplementary Fig. 2). The result of the antagonism assay illustrated that YMA4::masH lost antifungal activity against C. albicans (Supplementary Fig. 3), which demonstrated that mas BGC was crucial for the antifungal activity of Massilia sp. YMA4. Furthermore, a study of polyyne BGC (pgn BGC) in P. protegens had determined that the mutation of homolog (pgnH) of masH and masI in pgn BGC can effectively disrupt the production of protegencin (also known as protegenin A)12. We then conducted mass spectrometry-based untargeted metabolomics to analyze the differential features in wild type and biosynthesis-deficient mutant strain YMA4::masH. Via target isolation of differential features, we purified four polyynes (collimonin C/D 1, 2, and massilin A/B 3, 4) from ethyl acetate extract of Massilia sp. YMA4 (Fig. 1b, c). Their structures were elucidated using high-resolution mass spectrometry (Supplementary Figs. 4 and 5) and nuclear magnetic resonance (Supplementary Tables 1–4 and Supplementary Figs. 6–21; the detailed isolation procedure and structure elucidation are described in Supplementary Methods). Among the four polyynes identified from Massilia sp. YMA4, collimonin C 1 and collimonin D 2 were initially isolated from C. fungivorans Ter3317. A new compound with an ene-triyne moiety was named massilin A 3 and identified as a racemate at C(6)OH of an unsaturated hexadecanoic acid. Another new ene-diyne-ene compound, massilin B 4, was presumed to be the precursor of collimonin C 1 or collimonin D 2. Notably, massilin B 4 was more chemically stable than other polyynes with a terminal alkyne.

In antifungal activity assay (Supplementary Fig. 22), polyynes with a terminal alkyne showed potent inhibition of C. albicans with minimum inhibitory concentrations (MIC): 69.73 μM (collimonin C 1), 35.24 μM (collimonin D 2), and 2.40 μM (massilin A 3). However, massilin B 4 with a terminal alkene moiety had no antifungal activity with MIC > 500 μM. These results suggest that the terminal alkyne is essential for polyyne antifungal activity and stability.

Phylogenetic analysis of polyyne BGCs and mas heterologous co-expression revealed the core components for polyyne biosynthesis

The in-silico mining and comparative studies of polyynes/ terminal alkyne containing BGCs indicated that jamABC homologs (fatty acyl-AMP ligase (FAAL), fatty acid desaturase (FAD), and acyl carrier protein (ACP)) in jamaicamides (jam) BGC would be the minimal module for polyynes biosynthesis18,19. In addition, the gene cluster K in C. fungivorans Ter331 (collimonin BGC, which is abbreviated to col BGC) recruited not only FAAL, FAD, and ACP but also hydrolase/thioesterase (H/TE) and rubredoxin (Rd)6,7. Therefore, we further explored the architecture of 56 bacterial polyynes BGCs with mas BGC as a reference to confirm the conserved composition of polyyne biosynthetic associated enzymes. It contains the FAAL–2x FAD–ACP–FAD–H/TE–Rd (Supplementary Fig. 23).

The essential proteins, including FAAL, FAD, and H/TE in the conserved region of polyyne BGCs, have been confirmed in the biosynthesis of caryoynencin in B. caryophylli16, cepacins in Burkholderia ambifaria4, and protegenins in P. protegens12,13. To further verify the function of other core biosynthetic genes, we use plasmid-derived insertion mutagenesis to gain serial mutants of mas BGC from masD (encoded FAAL) to masL (Supplementary Figs. 2 and 3). YMA4::masD, YMA4::masE, YMA4::masF, YMA4::masH, and YMA4::masI exhibited completely disrupted polyyne biosynthesis. YMA4::masJ exhibited significantly decreased production of polyynes. However, YMA4::masK and YMA4::masL did not affect the biosynthesis process.

To complement the mutagenesis results, we further established an E. coli heterologous co-expression system of mas genes to determine whether the genes participate in polyyne biosynthesis. The engineered E. coli mas– (heterologous co-expression with masD to masJ) produced a relatively nonpolar polyyne (Fig. 2a). This new ene-triyne polyyne was named massilin C 5 (Supplementary Table 5 and Supplementary Figs. 24–28). The results demonstrated that masD to masJ are the core genes in the biosynthesis of the ene-triyne moiety. On the other hand, the expression of masB in Massilia sp. YMA4 was upregulated when cultured in PDA medium (Supplementary Data 1), like the core genes masD to masJ. Therefore, we proposed masB, a dioxygenase gene, as a candidate for the hydroxylation of polyynes since cytochrome P450s or dioxygenases can execute similar reactions20,21. The masB incorporated strain, mas + , produced collimonin C/D 1, 2 (Fig. 2b and Supplementary Fig. 24), which suggested massilin C 5 is a biosynthetic intermediate and further hydroxylated by MasB to generate polyynes 1–4. Furthermore, the absence of masJ significantly decreased the accumulation of polyynes in mas expressed E. coli (mas–' and mas + ', Fig. 2), indicating the crucial role of rubredoxin in polyyne biosynthesis. As an electron transporter, rubredoxin can shuttle reducing molecules from NAD(P)H to membrane-bound alkane hydroxylases22, which is likely crucial to activation/regeneration of acetylenase/desaturase for the poly-dehydrogenation process in polyyne biosynthesis.

Fig. 2: Heterologous co-expression of mas genes in E. coli.
figure 2

a The EIC (m/z 241.1234) of engineered E. coli strain, which carried masD, masE, masF, masG, Bv4687 (homolog of masH from Burkholderia vietnamiensis LMG 10929) and masI when co-expressed with (mas–, top) or without (mas–', bottom) masJ. b The EIC (m/z 273.1132) of E. coli strain mas– and mas–' harboring additional dioxygenase (masB) were renamed as mas + and mas + '. The structures of products are illustrated on top of each chromatogram and were verified by LC-HRMS/MS analysis (Supplementary Fig. 24).

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MasL serves as a polyyne direct target with a protective function

“Antibiotic resistome” refers to the notion that not only antibiotic resistance genes present in resistant pathogens but also those in the antibiotic producers often harbor resistance genes for self-protection23,24. Sometimes the resistance genes are located within the BGC of antibiotics25. To reveal the target protein of the bacterial polyynes of interest, we adopted the concept of the antibiotic resistome. We conducted phylogenetic analysis of bacterial polyyne BGCs and classified the BGCs into two chemotaxonomic groups: a palmitate-derived (C16) and a stearate-derived (C18) group (Fig. 3a and Supplementary Fig. 29). In the palmitate-derived group BGCs, cepacin (ccn) BGC branched out before the most recent common ancestor of mas/col BGCs.

Fig. 3: Comparative analysis of polyyne biosynthetic gene clusters and the structures of the bacterial polyynes.
figure 3

a Phylogenetical analysis of polyyne biosynthetic gene clusters (BGCs) in bacteria (Supplementary Figs. 23 and 29). Species in the red box have been reported to produce palmitate-derived polyynes (C16), and species in the blue box have been reported to produce stearate-derived polyynes (C18). The number of BGCs in each group is shown in parentheses. b Comparison of the polyyne BGC architectures of mas BGC in Massilia sp. YMA4 (i), ccn BGC in B. ambifaria BCC0191(ii)4, and col BGC in C. fungivorans Ter331(iii)7. Genes conserved in polyyne BGC across the phylogenetic tree are shown in blue, and those conserved in the C16 polyyne group are shown in gray. The potential protective genes in the BGC are shown in red for acetyl-CoA acetyltransferase and in yellow for MFS transporter. The corresponding homologs (over 40% identity) in BGCs between two species are shown as the light green area. Black triangles indicate the mutation sites in previous research and this study.

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Interestingly, the gene encoding the major facilitator superfamily (MFS) transporter, implicated in multidrug resistance and transports of small molecules and xenobiotics26, is preserved in col BGC but lost in mas BGC (Fig. 3b, Supplementary Table 6, and Supplementary Data 2). This implies that Massilia sp. YMA4 has an alternative protection mechanism for self-protection from polyynes. Notably, a conspicuous gene masL, an acetyl-CoA acetyltransferase gene, remains in mas BGC and ancestor ccn BGC but is absent in col BGC. We, therefore, proposed that masL is a potential self-resistance gene of polyynes.

To evaluate the protective effect of masL, we first heterologously expressed masL in polyyne-sensitive C. albicans. The expression of masL rescued fungal cell viability from polyyne inhibition under minimum inhibitory concentration (Fig. 4 and Supplementary Table 7). Furthermore, an in vitro acetyl-CoA acetyltransferase (MasL) inhibition assay showed that polyynes (collimonin C/D 1, 2, and massilin A 3) inhibited the MasL enzyme activity (Table 1 and Supplementary Fig. 30). Since the drug target identification could be achieved by gene overexpression for gain-of-function of resistance27, we suggest masL serves as a self-resistance gene in the mas BGC, and the acetyl-CoA acetyltransferase it encodes is the target of collimonin C/D 1, 2 and massilin A 3. This targeting manner might explain why the growth of mas expressed E. coli is unaffected when producing polyynes without masL because E. coli has an acetyl-CoA acetyltransferase-independent route, the methylerythritol phosphate (also called deoxyxylulose 5-phosphate) pathway, to produce isoprenoid28,29. Moreover, ccn BGC divided into mas BGC and col BGC independently, and each contains a different self-protection mechanism, which implies a deletion event after horizontal gene transfer30.

Fig. 4: C. albicans was rescued by heterologous overexpression of masL (OE masL) and ERG10 (OE ERG10) from the minimum inhibitory concentration of collimonin C/D 1, 2, massilin A 3, and massilin C 5.
figure 4

The cell viabilities were normalized to the mock treatment. The standard deviation was calculated based on three replicates and the two-tailed Student’s t-test for statistical analysis. ***P-value < 0.000001. ns: not significant. Each experiment comprised three biological replicates (N = 3). All data values were listed in Supplementary Table 7.

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Table 1 Inhibition kinetic of Massilia sp. YMA4 MasL by polyynes.
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Polyynes are covalent inhibitors of acetyl-CoA acetyltransferase

To investigate how bacterial polyynes target acetyl-CoA acetyltransferase, we solved the crystal structures of MasL in its apo and two holo (bound collimonin C 1 and collimonin D 2) forms at 1.78 Å, 1.66 Å, and 1.40 Å resolution, respectively (Table 2). The asymmetric unit (space group P1 for apo MasL and MasL-collimonin D complex; P21 for MasL-collimonin C complex) of both structures contains a tetramer of the protein (Fig. 5, Table 2 and Supplementary Fig. 31), as observed in solution. The monomer of MasL shares the general fold architecture reported in the type II biosynthetic thiolase family31. MasL consists of three domains: an N-terminal α/β domain (N-domain, residues 1–121 and 251–271), a loop domain (L-domain, residues 122–250), and a C-terminal α/β domain (C-domain, residues 272–394) (Supplementary Fig. 32). The N- and C-domains form a typical five-layered fold (α-β-α-β-α) as observed in the structures of the other type II biosynthetic thiolases including Zoogloea ramigera PhaA31, Clostridium acetobutylicum CEA_G288032, and Aspergillus fumigatus ERG10A33. The L-domain displays an α/β fold with a tetramerization loop associated with the C-domain.

Table 2 X-ray data collection and refinement statistics.
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Fig. 5: X-ray structures and polar interaction within the binding site of MasL-collimonin C and MasL-collimonin D complex.
figure 5

Overall views of the crystal structures of MasL-collimonin C complex (a) and MasL-collimonin D (b). Four subunits per asymmetric cell are shown in red, orange, light green, and cyan, respectively. Collimonin C/D 1, 2 is shown as grey space-filling balls. In the magnified view, the residues involved in collimonin C/D 1, 2 interactions are shown as sticks with sequence identities indicated in the main chain molecule shown in gray. The R135 residue of another molecule is shown in cyan. The dotted lines indicate the hydrogen bonds and salt bridges involved in collimonin C/D 1, 2 interactions within the binding pocket.

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Many high-resolution atomic structural models of the substrate-binding complex revealed the Claisen condensation reaction and binding model within the reaction pocket of acetyl-CoA acetyltransferases. In apo and holo forms of MasL, the substrate-binding pocket was located on the enzyme’s surface facing the opposite dimer of the tetrameric assembly. The pocket was a tunnel shape of ~10 Å depth with ~6–8 Å diameter for the linear pantothenic moiety of coenzyme A (CoA) extending through the reactive center. The reactive center in MasL contained reactive cysteine residues Cys90 and nucleophilic activation residues His350 and Cys380. In the MasL-collimonin C complex, the conjugated polyyne tail extended into the MasL substrate binding site and formed a covalent bond between the terminal carbon (C16) of the alkyne moiety and the reactive cysteine sulfhydryl moiety of Cys90 via nucleophilic addition (Supplementary Figs. 33 and 34). Thus, the terminal alkyne was essential for polyyne activity through covalent modification on the reactive cysteine sulfhydryl group of acetyl-CoA acetyltransferases.

Although we did not obtain a good X-ray diffraction result surrounding the reactive Cys90 in the MasL-collimonin D complex, the nucleophilic addition mechanism of polyynes to the acetyl-CoA acetyltransferase was confirmed by mass spectrometry analysis (Supplementary Fig. 35 and Supplementary Data 3). The observation of collimonin C/D (+C16H18O4)–derived adducts of enzyme reactive cysteine residue provided convincing evidence of protein irreversible S-alkylation via nucleophilic addition to the conjugated terminal alkyne. Furthermore, the observation of massilin A (+C16H18O3)–derived adducts suggest that massilin A 3 would also covalently inhibit acetyl-CoA acetyltransferase activity. In the further comparative analysis of the polar interactions in the activation pocket of MasL in MasL-collimonin C and MasL-collimonin D complex, the C(7)OH moiety of collimonin C/D 1, 2, His158 of MasL, and a water molecule formed a strong polar interaction network, including a direct hydrogen bond (3.00–3.16 Å) and a water-mediated hydrogen bond between C(7)OH and His158 (Fig. 5). Furthermore, although there was no significant induced-fit within the pocket, the collimonin C/D 1, 2 caused the Arg135 on the tetramerization loop to swap and form a hydrogen bond interaction and salt bridge, respectively, across the two subunits within the binding site (Fig. 5). These polar interacting residues for the collimonin C/D 1, 2 (inhibitor) binding were similar to CoA (product) in other thiolase models31. Therefore, the acetyl-CoA acetyltransferase inhibitions by collimonin C/D 1, 2 are competitive binding models against substrate acetyl-CoA.

The stereochemistry of hydroxyl groups on polyynes affects their binding affinity toward acetyl-CoA acetyltransferase

The C6 chiral center of collimonin C/D 1, 2 is critical to forming different polar interactions. The C(6)OH of collimonin C 1 had more flexibility concerning the spatial direction (with a dihedral angle to C(7)OH from 109˚ to 170˚) and built a sophisticated polar interaction network with the amide of Pro249 in the pantetheine loop, Arg135, and multiple water molecules (Fig. 5 and Supplementary Fig. 36). In contrast, the C(6)OH of collimonin D 2 did not form a polar interaction network at the contrasting face and showed fixed spatial direction (dihedral angle to C(7)OH from 32.9˚ to 42.6˚) (Supplementary Fig. 36). The polar interaction network derived from C(6)OH of collimonin C 1 further affected the distal carboxyl group binding model. Furthermore, the reduced binding strength from the salt bridge (in MasL-collimonin D complex) to the hydrogen bond (in MasL-collimonin C complex) corresponded to their inhibition kinetic, that collimonin D 2 has a lower KI (42.84 μM) than collimonin C 1 (297.10 μM) (Table 1). The above information suggests that the stereochemistry of the hydroxyl group on polyynes is vital for initial non-covalent complex affinity. This salt bridge/hydrogen bond formation will dominate the MasL binding affinity in dihydro-type polyynes 1 and 2.

Candida albicans ERG10 is the antifungal target of polyynes

As the bacterial polyynes target acetyl-CoA acetyltransferase MasL, we further evaluated the inhibition effect of bacterial polyynes on ERG10, an acetyl-CoA acetyltransferase homolog in C. albicans. ERG10 is the first enzyme of the ergosterol biosynthesis pathway and is crucial for fungal cell membrane formation. The enzyme activity of ERG10 was inhibited by polyynes 1–3 (Supplementary Fig. 37), and correspondingly the overexpression of ERG10 in C. albicans also rescued the cell viability from their inhibition (Fig. 4). Although we did not obtain the X-ray crystallography for the polyyne-ERG10 complex, the mass spectrometry analysis showed that polyynes 1–3 target the reactive Cys90 of ERG10 (Supplementary Fig. 38). A previous study reported that the homologous gene in Aspergillus fumigatus, erg10A is essential for survival and reduced expression leads to increased susceptibility to oxidative and cell wall-perturbing agents33. In the TEM analysis, we also observed that bacterial polyynes disrupted the cell membrane integrity in C. albicans (Supplementary Fig. 39). This result corresponded to the earlier finding that protegencin (or protegenin A) permeated algal cells and caused cell lysis14. Notably, massilin C 5, the analog of protegencin (or protegenin A), possessed antifungal activity against C. albicans (MIC 12.22 μM, Supplementary Fig. 22) and a comparable ERG10 inhibitory potency to polyynes 1–3 (Supplementary Fig. 37). However, neither the heterologous expression of MasL nor ERG10 in C. albicans was able to recover the cell viability under massilin C 5 treatment (Fig. 4). Together these results suggested that the polyynes without a hydroxy moiety would possess multiple antifungal targets.

Conclusion

The practical application of the antibiotic resistome concept with in-silico genome mining and comparative genetics analysis facilitated drug target identification. Our integrated approaches revealed that bacterial polyynes inhibit fungal growth by disrupting ergosterol biosynthesis. The crystallography analysis revealed that bacterial polyynes inhibit acetyl-CoA acetyltransferase via irreversible S-alkylation of the reactive residue with the functional terminal alkyne. In addition, the hydroxyl moiety stereochemistry affects the inhibitor affinity. We characterized the core biosynthesis module and modification genes for polyynes through genetic engineering. These findings will aid in the understanding of the structure-activity relationships of the polyynes and their production via microbial fermentation.

Methods

Bioinformatics analysis

The biosynthesis gene clusters (BGCs) in the genome of Massilia sp. YMA4 (Genbank accession number GCA_003293715.1) were characterized via the command-line program DeepBGC17 with default settings and integrated with DeepBGC score > 0.7 (Supplementary Data 2). Then, the mas BGC of Massilia sp. YMA4 was used to discover the homologous gene clusters in bacteria species using MultiGeneBlast34 (Supplementary Data 2). The database was built using a bacterial sequences database (BCT, 2020 December 01) and whole-genome sequences of polyyne-reported bacterial species from NCBI. A total of 56 bacteria with polyyne BGC (Cumulative Blast bit score > 1500) were found. The homologous protein sequences of each bacterial polyyne BGC were respectively concatenated (a total of five amino sequences, starting from masD homolog to masI homolog). The concatenated protein sequences were used for alignment (MUSCLE) and the distance (UPGMA, bootstrap 5000 times) between 56 bacteria with Massilia sp. YMA4 was identified for phylogenetic tree construction. The analysis was completed by using MEGA 10 with default parameters35. iTOL was used to present the results of phylogenetic analysis36.

The RNA sequencing and transcriptome analysis were performed using Illumina MiSeq system (Illumina, USA) and CLC genomics workbench (version 11, CLC bio, Denmark), and the 92.89–98.11% reads were successfully mapped to the Massilia sp. YMA4 genome (GenBank assembly accession: GCA_003293715.1) for RPKM quantification (see Supplementary Methods and Supplementary Data 1 for details). The differential gene expression analysis was analyzed through CLC software with default pipeline and settings. Identification of differentially expressed genes (DEGs) with the expression (|Fold change (FC) | ≥ 2 with P-value < 0.05) was based on RPKM and analyzed using the Empirical analysis method37. Two biologic replicates of each condition were analyzed.

Construction of mas co-expression platform in Escherichia coli

Individual mas genes were cloned to the pET-28b(+) (Merck, Germany) using restriction enzyme digestion and ligation (see Supplementary Methods and Supplementary Tables 8–10 for details). To obtain functional masH in the heterologous E. coli system, we recruited native and codon-optimized masH and the homolog (Bv4687) from the palmitate-derived (C16) species B. vietnamiensis LMG 10929 (see Supplementary Fig. 23, 29 and 40) into modular co-expression. The backbone of the constructed modules, including pET-22a(+), pACYCduet, and pRSFduet, were purchased from Merck (Germany). However, polyyne accumulations were observed only in combination with Bv4687. Thus, masH was replaced by Bv4687 in further co-expression assay. The artificial gene clusters were then subcloned to the destination vector and resulted in three modules for better flexibility of adjusting composition, including pET-22a(+)-masD-masE-masF-masG, pACYCduet-Bv4687 (masH homolog from B. vietnamiensis LMG 10929, Genbank accession number WP_011881350)-masI-masJ, and pRSFduet-masB. Two or three modules were co-transformed to E. coli strain C41 (Yeastern Biotech, Taiwan), and selected colonies were then inoculated in LB medium with suitable antibiotics. The expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) and cultured for two days. The extractions were carried out by adding equal volumes of EtOAc and sonicated for 30 min. Finally, the extracts were concentrated and transferred to DMSO for further analysis.

Minimum inhibitory concentration determination and genetic rescue assay

The minimum inhibitory concentration (MIC) measurement was modified from R J Lambert’s method38. Different concentrations of collimonin C 1, collimonin D 2, massilin A 3, and amphotericin B were prepared in YPD. The C. albicans cell viabilities were seeded with initial OD600 0.05 and incubated at 37˚C. After 24 h incubation, the final OD600 was recorded by Epoch 2 Microplate (BioTek Instruments, USA) for MIC calculation. The MIC of polyynes was built with cell viability (%) of different concentrations, fitting into the modified Gompertz function38.

For the genetic rescue assay, the ERG10 overexpression and masL heterologous expression strains (Supplementary Fig. 41) were seeded with OD600 0.05 in YPD treated with MIC of each polyyne at 37˚C and supplied with 40 µg/mL doxycycline for gene expression. The C. albicans cell viabilities were recorded at 24 h. The statistical results were analyzed using GraphPad Prism (Version 8, GraphPad Software, USA) with multiple t-test analyses (FDR < 0.05).

Enzymatic inhibition assays

The enzymatic inhibition assay of acetyl-CoA acetyltransferase was conducted using a modified method39 (see Supplementary Methods for details). The releasing CoA was monitored using a fluorescent probe. The fluorescence intensity of each experiment was obtained by subtracting the fluorescence intensity of the polyyne-free reaction. The measurement and calculation of polyynes inhibition kinetic refer to the previous covalent inhibitor model40. The progress curves used the hyperbolic regression model to calculate the kinetic inhibition parameters using GraphPad Prism (Version 8, GraphPad Software, USA).

Protein expression and crystallization

The recombinant MasL protein was purified from E. coli cells (see Supplementary Methods for details). For MasL-collimonins complex, 20 µM MasL was incubated with 100 µM collimonin C 1 or D 2 for further purification and crystallization. A freshly thawed aliquot of MasL, MasL-collimonin C complex, and MasL-collimonin D complex was concentrated to 20 mg/mL for an initial crystallization screening of ca. 500 conditions (Academia Sinica Protein Clinic, Academia Sinica). X-ray diffraction experiments were conducted by the National Synchrotron Radiation Research Center (Hsinchu, Taiwan) at the TLS beamline 15 A or the TPS beamline 05 A. The MasL, MasL-collimonin C, and MasL-collimonin D complex structures were solved by the molecular replacement method using the structure of thiolase from Clostridium acetobutylicum (pdb ID 4N44) as the search model. The molecular figures were produced with Maestro (Schrödinger Release 2021-1: Maestro, Schrödinger, USA).

Statistics and reproducibility

For Fig. 4, the cell viabilities were normalized to the mock treatment. The standard deviation was calculated based on three replicates and the two-tailed Student’s t-test for statistical analysis. The experiments in this study, including mas co-expression in E. coli, minimum inhibitory concentration determination, genetic rescue assay, and enzymatic inhibition assay, were performed in three biological replicates (N = 3).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.