https://orcid.org/0000-0001-7602-2394
ABSTRACT
We identified the biosynthetic gene cluster for lucilactaene, a cell cycle inhibitor from a filamentous fungus Fusarium sp. RK 97–94. The luc1 knockout strain accumulated demethylated analogs, indicating the involvement of Luc1 methyltransferase in lucilactaene biosynthesis. Lucilactaene showed potent antimalarial activity. Our data suggested that methylation and ether ring formation are essential for its potent antimalarial activity.
A new cell cycle inhibitor lucilactaene was identified from a filamentous fungus Fusarium sp. RK97-94, from a screening program designed to identify p53-independent cell cycle inhibitors in a lung cancer cell line transfected with temperature-sensitive human p53 gene ()) [Citation1]. The unique structure of lucilactaene attracted the interest of chemists resulting in total synthesis [Citation2,Citation3]. Lucilactaene arrested the cell cycle progression of a lung cancer cell line at the G1 phase in a p53-independent manner. However, its biological activity spectrum is unknown. We also isolated a known lucilactaene-related compound NG-391 from Fusarium sp. RK97-94 ()). NG-391 was isolated as a neuronal cell-protecting molecule [Citation4].
Figure 1. Analysis of the lucilactaene biosynthetic gene cluster.
(a) Chemical structure of lucilactaene (1), NG-391 (2), fusarin C, and fusarin A. (b) The Δluc5 strain lost the ability to produce lucilactaene and NG-391. The Δluc1 strain lost the ability to produce lucilactaene (peak 1) and NG-391 (peak 2) and accumulated peak 3. The wild-type, Δluc5, and Δluc1 strains were cultured in FDY for three days and analyzed by UPLC/MS. Lucilactaene, NG-391, and peak 3 were detected by measuring absorbance at 365 nm. (c) Comparison of lucilactaene, fusarin C [Citation7], and NG-391 (partial) [Citation8] biosynthetic gene clusters. Sequencing data from the 40-kb region is available from DDBJ/EMBL/GenBank under Accession No. LC515193. d) Structural determination of the mixture of 3 and 4.
![Figure 1. Analysis of the lucilactaene biosynthetic gene cluster.(a) Chemical structure of lucilactaene (1), NG-391 (2), fusarin C, and fusarin A. (b) The Δluc5 strain lost the ability to produce lucilactaene and NG-391. The Δluc1 strain lost the ability to produce lucilactaene (peak 1) and NG-391 (peak 2) and accumulated peak 3. The wild-type, Δluc5, and Δluc1 strains were cultured in FDY for three days and analyzed by UPLC/MS. Lucilactaene, NG-391, and peak 3 were detected by measuring absorbance at 365 nm. (c) Comparison of lucilactaene, fusarin C [Citation7], and NG-391 (partial) [Citation8] biosynthetic gene clusters. Sequencing data from the 40-kb region is available from DDBJ/EMBL/GenBank under Accession No. LC515193. d) Structural determination of the mixture of 3 and 4.](/cms/asset/a049ea2e-16bf-4a2a-a841-4893e3db5a2a/tbbb_a_1725419_f0001_oc.jpg)
The identification of the biosynthetic genes enables the analysis of the biosynthetic mechanism and bioactivity. It is predicted that the same biosynthetic gene cluster biosynthesizes lucilactaene and NG-391 due to their structural similarity. The chemical structures of these compounds are similar to fusarin C, a mycotoxin produced by certain Fusarium species ()) [Citation5]. The fusarin C biosynthetic gene cluster was identified and established that the fusarin C backbone was biosynthesized by a PKS-NRPS (polyketide synthase-nonribosomal peptide synthetase) hybrid enzyme [Citation6]. In Fusarium fujikuroi, the fusarin C biosynthetic gene cluster consisted of nine co-expressed genes, and only four genes are required for the biosynthesis [Citation7]. The NG-391 biosynthetic gene cluster was identified in an insect pathogen Metarhizium robertsii [Citation8] and also included a PKS-NRPS hybrid gene essential for biosynthesis.
In this study, we identified the biosynthetic gene cluster for lucilactaene in Fusarium sp. RK97-94 and analyzed the biological activity of lucilactaene and its analogs.
To clone the PKS-NRPS hybrid gene involved in lucilactaene biosynthesis, the AT (acyltransferase) domains of PKS (polyketide synthase) genes were amplified by degenerate polymerase chain reaction (PCR) using FTGQGAQ and GHSSGE(IM)c (Table S1) from the genomic DNA of the lucilactaene producer Fusarium sp. RK97-94. The amplified 0.3-kb PCR products were cloned and sequenced. The deduced amino acid sequence of one fragment showed high similarity to fusarin C biosynthetic PKS-NRPS hybrid enzyme (identity: 71%, E-value: 4e-32). A 1.5-kb upstream and 1.5-kb downstream sequence of this partial putative lucilactaene biosynthetic PKS-NRPS gene (luc5) was cloned by genome walking to construct a gene knockout vector pBI-luc5::HPH. The luc5 gene knockout strain, created by Agrobacterium tumefaciens-mediated transformation (Figure S1(a)), lost the ability to produce lucilactaene and NG-391 ()), indicating its involvement in lucilactaene biosynthesis. A 40-kb region around the luc5 gene fragment was cloned by genome walking and sequenced. This region contained 13 putative genes (), Table S2). All 13 putative genes showed high similarity to Nectria haematococca or Fusarium spp. genes. Eight of the 13 genes showed high similarity to genes in the fusarin C biosynthetic gene cluster [Citation7], and were designated as luc1-8 (Table S2). For fusarin C biosynthesis, four genes (fus1, fus2, fus8, and fus9) are essential [Citation7], and homologs of these genes (luc5, luc6, luc2, and luc1, respectively) were identified in the lucilactaene biosynthetic gene cluster. Lucilactaene and NG-391 lack the 7-methyl group present in fusarins ()). This methyl group is inserted in fusarins by the C-methyltransferase (CMeT) domain of a PKS-NRPS hybrid enzyme (Fus1) [Citation6], thus suggesting that the CMeT domain of Luc5 cannot methylate this position.
We focused our efforts on luc1 the methyltransferase gene, for an in-depth analysis of the lucilactaene biosynthetic gene cluster, and to isolate a lucilactaene analog. The luc1 gene knockout strains, created by A. tumefaciens-mediated transformation (Figure S1(b)), lost their ability to produce lucilactaene (peak 1) and NG-391 (peak 2), and instead, new metabolites, including peak 3, accumulated in the Δluc1 strain ()). A major peak (peak 3) was purified (Figure S2) and identified as a mixture of isomers as shown below. The physicochemical properties of this mixture are summarized in Table S3. The molecular formula of the mixture was determined to be C21H25NO6 using high-resolution electrospray ionization time-of-flight MS (found m/z: 386.1599 [M-H]−, calculated for C21H24NO6 386.1609). Chemical shifts of lucilactaene and the mixture were similar (). In the mixture signals, except for two, other signals corresponded with lucilactaene signals. The C21 signal was absent, and the C20 signal shifted to a lower value, indicating that C20 is a carboxylic acid structure. Based on the nuclear magnetic resonance (NMR) spectral data, peak 3 was identified as a mixture (almost 1:1) of demethyllucilactaene (3) and (8Z)-demethyllucilactaene (4), as determined by 1H-1H COrrelated SpectroscopY (COSY) and Heteronuclear Multiple Bond Correlation (HMBC) (, ), S3(a–c)). The relative stereochemistry of the mixture of 3 and 4 at positions C13, C14, and C15 was indicated to be identical to that of lucilactaene (13S*, 14R*, 15S*), because the chemical shifts (both C-13 and H-1) at the ring (C13-C19) were almost the same as those of lucilactaene. The analog ((8Z)-demethyllucilactaene) was in cis form because C8 and C9 proton coupling constant was 11 Hz. We also tried to isolate 3 under dark conditions to prevent the 8Z isomer (4) formation. However, we failed to prevent formation of the 8Z isomer. Our data indicate that Luc1 methylates the carboxyl group of demethyllucilactaene and is essential for lucilactaene biosynthesis. This methylation step corresponds to the final step in fusarin C biosynthesis. In fusarin C, Fus9, annotated as a methyltransferase, plays a role in the carboxyl group methylation [Citation9]. Our data indicate that Luc1 is an ortholog of Fus9 ()). Interestingly, the primary metabolite accumulated in the Δluc1 strain was demethylated lucilactaene and not demethylated NG-391, although the main metabolite of the parental strain is NG-391 ()).
Table 1. 1H and 13C NMR chemical shift comparison of lucilactaene and demethylated analogs in CDCl3.
We isolated lucilactaene and showed its activity as a cell cycle inhibitor in a lung cancer cell line [Citation1]. We tested the biological activity of lucilactaene and analogs, NG-391, and the mixture of 3 and 4 (). Lucilactaene and NG-391 showed moderate growth inhibitory activity against cancer cells, whereas the mixture of 3 and 4 showed no activity. The above compounds showed no antimicrobial activity against bacteria and fungi. Lucilactaene showed significant antimalarial activity [IC50: 0.063 μg/mL (160 nM)]. In the antimalarial assay, we used a commercial antimalarial compound artemisinin as a control and found that this compound has an IC50 value of 0.0085 μg/mL (30 nM). However, NG-391 and the mixture of 3 and 4 exhibited moderate and weak antimalarial activity, respectively. These data indicate that methylation of the carboxyl group by Luc1 is essential for growth inhibitory activity against cancer cells and malarial parasites. The methylation modification might be necessary for compounds membrane permeability. These data also suggest that the presence of the ether ring is critical for antimalarial activity. Fusarin C was shown to be a mutagenic mycotoxin [Citation5]. An analog of fusarin C, fusarin A ()), has a similar chemical structure to lucilactaene and has no mutagenic activity [Citation10] and might possess lucilactaene-like activity.
Table 2. Growth inhibitory activity of lucilactaene, NG-391, and the mixture of 3 and 4 (IC50 values, μg/mL).
Lucilactaene is a cell cycle inhibitor isolated from a filamentous fungus Fusarium sp. RK 97–94 [Citation1]. In this study, we identified the lucilactaene biosynthetic gene cluster from Fusarium sp. RK 97-94, which has eight genes (luc1-luc8), including a PKS-NRPS hybrid gene (luc5) required for lucilactaene biosynthesis. Luc1 is a methyltransferase involved in lucilactaene biosynthesis, and luc1 knockout results in the accumulation of demethylated analogs. Lucilactaene showed potent growth inhibitory activity against malarial parasites, moderate growth inhibitory activity against cancer cells, and no activity against bacteria and fungi. In contrast, demethylated analogs showed no such activities, indicating that methylation of the carboxyl group is required for the biological activity. Lucilactaene and NG-391 showed a spectrum of similar growth inhibitory activities. However, the antimalarial activity of lucilactaene was nearly ten times higher than NG-391, suggesting that the ether ring is important for its antimalarial activity.
Author contributions
S. K. and A. T. isolated and analyzed the biosynthetic genes. S. K., T. H., and A. T. purified compounds. S. K., M. U., T. N., H. H., and A. T. determined chemical structures. Y. F. analyzed bioactivity. T. M., N. T.-A., T. K., and H. O. designed the research. S. K. and T. M. wrote the paper.
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We would like to thank Ms. Harumi Aono, Dr. Rachael Uson-Lopez, Ms. Emiko Sanada, and Dr. Motoko Uchida for their technical support.
Data availability statement
The data described in this article are openly available in the Open Science Framework at DOI:10.17605/OSF.IO/TPA6U.
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No potential conflict of interest was reported by the authors.
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References
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