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Article

The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra

by
Ning Ding
1,2,3,†,
Chang Liu
1,2,†,
Ping Lu
1,2,*,
Lu Bai
1,2 and
Bo Yuan
1,2
1
College of Life Science and Technology, Inner Mongolia Normal University, Hohhot 010022, China
2
Key Laboratory of Biodiversity Conservation and Sustainable Utilization in Mongolian Plateau for College and University of Inner Mongolia Autonomous Region, Hohhot 010022, China
3
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(6), 1326; https://doi.org/10.3390/microorganisms13061326
Submission received: 16 April 2025 / Revised: 2 June 2025 / Accepted: 3 June 2025 / Published: 6 June 2025
(This article belongs to the Special Issue Plant–Fungal Interactions in Biocontrol of Plant Diseases)
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Abstract

The swnR gene was cloned in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra, and the gene knockout mutant ΔswnR was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnR exhibited distinct morphological alterations in both colony and mycelial structure, a slower growth rate, and significant reductions in swainsonine (SW) levels, indicating that the function of the swnR gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnR, with P5CR, swnR, swnK, swnH2, and swnH1 downregulating, and sac upregulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-Lys, L-Glutamic acid, Saccharopine, and L-Proline upregulating, and L-PA downregulating. The results lay the foundation for the in-depth elucidation of molecular mechanisms and SW synthesis pathways in fungi, and are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems.

1. Introduction

Locoweed refers collectively to toxic plants of the genera Oxytropis and Astragalus that contain swainsonine (SW), representing one of the most detrimental poisonous plants to global animal husbandry [1,2]. Due to their strong stress tolerance and reproductive capacity, locoweeds exhibit superior competitive advantages in grassland ecosystems. These toxic plants are widely distributed across various regions worldwide, including the United States, Russia, Australia, Egypt, Mexico, Spain, Mongolia, and Iceland [3,4,5,6]. In China, locoweeds are predominantly found in northwestern areas such as Western Inner Mongolia, Ningxia, Gansu, Qinghai, and Xinjiang [3,7,8]. The consumption of locoweeds by livestock induces progressive intoxication characterized by weight loss, addictive grazing behavior, neurological dysfunction, and abortion in pregnant females, with severe cases culminating in mortality, thereby inflicting substantial economic losses on grassland livestock production systems [9,10,11,12,13].
The toxic alkaloid swainsonine (SW) was first isolated from A. lentiginosus and O. sericea, marking the first definitive identification of the causative toxin in locoweed poisoning [14]. Subsequent studies identified SW-producing endophytic fungi in O. sericea, A. mollisimus, and O. lambertii. Through combined ITS sequencing and morphological analyses, these fungi were initially classified as Embellisia species [15], later refined to the genus Undifilum [16], and ultimately revised to Alternaria species [17]. In addition to A. oxytropis, three other locoweed-associated endophytic fungi have been identified: A. cinereum, A. fulva, and A. bornmuellerii [16,17,18]. Notably, SW production has also been documented in various plant pathogens, insect pathogens, and human dermatopathogens [19,20,21]. SW is a fungal alkaloid toxin produced through secondary metabolism. Currently, the biosynthetic mechanisms of several mycotoxins, such as aflatoxins, deoxynivalenol (DON), and zearalenone (ZEN), have been well characterized, all being regulated by highly conserved gene clusters [22,23,24,25].
Our research group first isolated an SW-producing endophytic fungus from O. glabra. In vitro culture experiments confirmed SW biosynthesis by this fungal strain, while SW was undetectable in plants lacking this endophyte. These findings established that SW toxicity in O. glabra originates from its endophytic fungus. Through comprehensive characterization, we identified this organism as Alternaria oxytropis and designated it as Alternaria oxytropis OW7.8 [26,27,28].
The sac gene (saccharopine reductase gene) was knocked out in A. oxytropis OW7.8 by our research group previously, and the sac gene knockout mutant M1 was successfully screened out. The SW level of M1 cultured from 14 to 45 days was always lower than A. oxytropis OW7.8. The level of Saccharopine in M1 was lower than that of A. oxytropis OW7.8 [29]. The SW level of the sac gene complementary strain C1 was higher than that of A. oxytropis OW7.8 and M1, indicating that sac gene function promoted SW synthesis in A. oxytropis OW7.8 [30].
A comparative genomic analysis was conducted on diverse SW-producing fungi (Metarhizium robertsii, Ipomoea carnea endophyte, Arthroderma otea, Trichophyton equinum, A. oxytropis, and Pseudo-gymnoascus sp.), suggesting a conserved homologous gene cluster "SWN" comprising seven genes (swnA, swnT, swnR, swnK, swnN, swnH2, and swnH1) that are functionally associated with SW biosynthesis, with the encoded proteins and their respective functions detailed in Table 1 [31]. However, different fungi that produce SW do not necessarily all contain these seven genes. For example, there are no swnA and swnT genes in A. oxytropis and Pyrenophora semeniperda. Slafractonia leguminicola lacks the swnA gene, and Chaetothyriaceae sp. does not have the swnT gene [32].
Both swnR and swnN encode different reductases of the Rossmann-fold family and participate in the reduction reaction of two imine ions during SW synthesis. During the formation of 1-Hydroxyindolizine from 1-Oxoindolizine, the SwnN enzyme or SwnR enzyme catalyzed C3=N+4 imine ion to form 1-Hydroxyindolizine and the SwnN enzyme or SwnR enzyme catalyzed the formation of SW by C9=N+4 imine ion during the SW formation process of 1-Hydroxyindolizine [31]. The swnR gene, swnN gene, and swnR and swnN genes were knocked out in M. robertsii, and it was found that the SW level in each strain was ΔswnNR < ΔswnN < ΔswnR < WT; PA levels were significantly reduced in ΔswnR and ΔswnN, and extremely significantly decreased in ΔswnNR, suggesting that the SwnR enzyme in M. robertsii reduced P6C to PA [33].
The swnN gene of A. oxytropis OW7.8 was knocked out by our research group; however, SW was not detected in the mycelia of ΔswnN knockout strain cultured for 20 days, and the morphology and structure of ΔswnN colony and mycelia were changed and the growth rate was slower than that of A. oxytropis OW7.8. SW was detected in the mycelia of ΔswnN/swnN, a functional complementary strain of the swnN gene growing for 20 days, indicating that the swnN gene promoted SW synthesis [34]. Later, the swnH1 gene of A. oxytropis OW7.8 was knocked out, and SW was not detected in ΔswnH1 mycelia. The results showed that swnH1 gene function also promoted SW biosynthesis [35].
The SW synthesis pathway was first studied in Rhizoctonia leguminicola [36] and later in M. robertsii and A. oxytropis [31,33,37,38,39]. The SW biosynthetic pathway predicted in R. Liguminicola was [36] L-lysine → Saccharopine → α-Aminoadipic semialdehyde → P6C → PA → 1-Oxoindolizine → 1-Hydroxyindolizine → 1, 2-Dihydroxyindolizine → SW. At present, the predicted SW synthesis pathway in M. robertsii is relatively clear (Figure 1) [33]: L-Lys can be directly catalyzed by lysine cyclodeaminase (LCD) to synthesize PA, or L-Lys can be synthesized into P6C by the SwnA enzyme, and then reduced to PA by the SwnR enzyme. PA is catalyzed by non-ribopeptide-polyketo synthase (SwnK enzyme) to synthesize 1-Oxoindolizine or 1-Hydroxyindolizine. 1-Oxoindolizine is catalyzed by the SwnN enzyme to form 1-Hydroxyindolizine, followed by 1, 2-Dihydroxyindolizine catalyzed by the SwnH2 enzyme. Finally, 1, 2-Dihydroxyindolizine formed SW under the action of SwnH1.
Based on the results of previous studies, our research group speculated the biosynthetic pathway of SW in A. oxytropis OW7.8 (Figure 2) [34], starting from L-lysine artificially. The Sac enzyme catalyzed the synthesis of saccharopine from L-Lysine. Saccharopine was reduced to α-aminoadipic semialdehyde (α-aminoadipic semialdehyde was also formed from α-aminoadipic acid catalyzed by α-aminoadipate reductase). α-Aminoadipic semialdehyde was catalyzed by LysDN to form P6C (P6C was also formed from saccharopine catalyzed by saccharopine oxidase). P6C was then catalyzed by the P5CR enzyme or SwnR enzyme to form L-PA. There might be an alternative pathway synthesizing L-PA, in which L-lysine was converted to 6-amino-2-oxohexanoate catalyzed by L-lysyl-alpha-oxidase. 6-Amino-2-oxohexanoate cyclized to form P2C, which was subsequently catalyzed by the enzymes lhpD/dpkA/lhpI to produce L-PA.
L-PA was converted to (8aS)-1-Oxoindolizine, (1R,8aS)-1-Hydroxyindolizine, or (1S,8aS)-1-Hydroxyindolizine by the action of multifunctional SwnK proteins. Subse-quently, (8aS)-1-Oxoindolizine was synthesized from (8aS)-1-Hydroxyindolizine by the SwnN enzyme. (1R,2S,8aS)-1,2-Dihydroxyindolizine was synthesized from (1R,8aS)-1-Hydroxyindolizine by the action of the SwnH2 enzyme, and (1S,2S,8aS)-1,2-Dihydroxyindolizine was synthesized from (1S,8aS)-1-Hydroxyindolizine catalyzed by the SwnH2 enzyme. Finally, (1S,2S,8R,8aR)-1,2,8-octahydroindolizinetriol (SW) was synthesized from (1R,2S,8aS)-1,2-Dihydroxyindolizine or (1S,2S,8aS)-1,2-Dihydroxyindolizine catalyzed by the SwnH1 enzyme.
In this study, the swnR gene of the endophytic fungus Alternaria oxytropis OW7.8 was cloned for the first time, the ΔswnR knockout strain and overexpressed strain swnR-OE were constructed, the SW levels in mycelia of each strain were detected, and the effects of the gene function on SW synthesis were studied. Additionally, transcriptome sequencing and metabolomic analysis were performed on A. oxytropis OW7.8 and the ΔswnR mutant. The findings provide crucial foundational data for elucidating the molecular mechanisms and metabolic pathways underlying fungal SW biosynthesis, which also hold significant implications for the management and utilization of locoweed.

2. Materials and Methods

2.1. Strain Cultivation

Solid culture: The A. oxytropis OW7.8 [27] (previously isolated from O. glabra in Wushen Banner, Ordos city, Inner Mongolia, China (108°52′ E, 38°36′ N, elevation 1291 m), hereafter referred to as OW7.8) was inoculated onto potato dextrose agar (PDA) media and incubated at 25 °C. Liquid culture: OW7.8 was inoculated into potato dextrose broth (PDB) and incubated at 25 °C with shaking.

2.2. Genomic DNA Extraction and swnR Gene Cloning from A. oxytropis OW7.8

The Genomic DNA was extracted from OW7.8 (Ezup Column Fungi Genomic DNA Purification Kit Tiangen, Beijing, China). The swnR gene was amplified using primers RF/RR (5′-TCCTGCGGAGCAATCTACCT-3′ and 5′-GTTATCCACTGGGAAGCCTCT-3′) under the following PCR conditions: initial denaturation at 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 90 s; final extension at 72 °C for 10 min; and hold at 4 °C. The PCR products were detected by 1% agarose gel electrophoresis, purified (SanPrep Column PCR Product Purification Kit, Sangon Biotech, Shanghai, China), and then sequenced (Sangon Biotech, Shanghai, China).

2.3. Total RNA Extraction and swnR cDNA Cloning from A. oxytropis OW7.8

Total RNA of A. oxytropis OW 7.8 was extracted (OminiPlant RNA Kit, CWBIO, Shanghai, China) and reverse-transcribed to synthesize cDNA. The swnN cDNA was amplified with primers RcF/RcR (5′-CGCGGATCCATGCGTGTCGCGATCGCT-3′;5′-CGGCCCGGGTCAGATTATTGAATTGGGATA-3′). The PCR program was as follows: 94 °C for 3 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, repeated for 30 cycles; and a final extension at 72 °C for 10 min. The PCR products were detected by 1.0% agarose gel electrophoresis, purified (SanPrep Column PCR Product Purification Kit, Sangon Biotech, Shanghai, China), and then sequenced (Sangon Biotech, Shanghai, China).

2.4. Vectors Construction

2.4.1. Construction of the swnR Gene Knockout Vector

The upstream and downstream homologous regions of the swnR gene were amplified from A. oxytropis OW7.8 genomic DNA using primer pairs RsF/RsR (5′-CGCGGATCCCAGGCAAAGAGTTCCAAATCG-3′; 5′-GACCTCCACTAGCTCCAGCCAAGCCCATCGCATCTCGCTTCGTGA-3′) and RxF/RxR (5′-ATAGAGTAGATGCCGACCGCGGGTTCATATCCAGGCATTGCACTCAGC-3′; 5′-CGGAATTCGACTTGATTCGTGCGGTGTAA-3′). The 5′ and 3′ regions of the hpt gene were amplified from plasmid pCB1003 using primers hphsF/hphsR (5′-TCACGAAGCGAGATGCGATGGGCTTGGCTGGAGCTAGTGGAGGTC-3′; 5′-GTATTGACCGATTCCTTGCGGTCCGAA-3′) and hphxF/hphxR (5′-GATGTAGGAGGGCGTGGATATGTCCT-3′; 5′-GCTGAGTGCAATGCCTGGATATGAACCCGCGGTCGGCATCTACTCTAT-3′). The swnR knockout cassette (432 bp upstream homologous region + 1380 bp hygromycin phosphotransferase gene + 449 bp downstream homologous region) was assembled using split-marker recombination and subsequently cloned into the pMD-19-T Vector (Takara, Beijing, China) via TA cloning.

2.4.2. Construction of the swnR Gene Overexpression Vector

The plasmid pBARGPE1-Hygro and the purified swnR cDNA fragment were digested with restriction enzymes BamHI and XmaI. The digested products were purified and ligated overnight at 16 °C using T4 DNA ligase.

2.5. Hygromycin Sensitivity Assay of A. oxytropis OW7.8

The mycelia of A. oxytropis OW7.8 was cultured on PDA media supplemented with Hyg B at concentrations of 0 μg/mL, 0.5 μg/mL, 0.6 μg/mL, 0.7 μg/mL, 0.8 μg/mL, 0.9 μg/mL, 1 μg/mL, and 2 μg/mL. Colony growth was observed to determine the minimum Hyg B concentration required to inhibit OW7.8 growth, which was subsequently used for selecting knockout transformants.

2.6. Protoplast Preparation and Transformation of A. oxytropis OW7.8

Young mycelia of OW7.8 were inoculated in PDB media (50 μg/mL Amp) shaking at 25 °C, 200 rpm for 7 days. Protoplasts from mycelia of A. oxytropis OW 7.8 were prepared according to the method described by Hu et al. [40]. The swnR gene knockout vector was then transformed into protoplast of A. oxytropis OW 7.8 mediated by PEG8000.

2.7. Screening and Verification of Gene Knockout Transformants

The swnR gene transformants were cultured on TB3 regeneration media (base layer: 50 μg/mL ampicillin and 1 μg/mL hygromycin B; top layer: 50 μg/mL Amp and 2 μg/mL Hyg B) at 25 °C until single colonies appeared, which were then transferred to PDA media (2 μg/mL Hyg B) for continuous cultivation until stable growth was achieved. Genomic DNA was extracted from the transformants and subjected to PCR amplification using three primer sets: hptF/hptR (5′-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3′/5′-GAACCCGCGGTCGGCATCTACTCTAT-3′) for hpt gene sequence verification, RsF/hptsR (5′-CGCGGATCCCAGGCAAAGAGTTCCAAATCG-3′/5′-GTATTGACCGATTCCTTGCGGTCCGAA-3′) for amplification of the upstream homologous arm + hpt gene sequence, and hptxF/RxR (5′-GATGTAGGAGGGCGTGGATATGTCCT-3′/5′-CGGAATTCGACTTGATTCGTGCGGTGTAA-3′) for the hpt gene sequence combined with downstream homologous arm, with all PCR products subsequently sequenced for confirmation. Additionally, primers nRF/nRR were used to amplify the internal sequence of the swnR gene using both OW7.8 and the putative knockout transformants as templates. The successfully verified swnR gene knockout mutant of A. oxytropis OW7.8 was formally designated as ΔswnR.

2.8. Transformation of A. oxytropis OW7.8 Protoplasts with swnR Overexpression Vector

Young mycelia of OW7.8 were inoculated in PDB media (50 μg/mL Amp) shaking at 25 °C, 200 rpm for 7 days. Protoplasts from mycelia of A. oxytropis OW 7.8 were prepared according to the method described by Hu et al. [40]. The swnR overexpression vector was then transformed into protoplast of A. oxytropis OW 7.8 mediated by PEG8000.

2.9. Screening and Verification of Overexpression Transformants

The swnR-overexpressing transformants were cultured on TB3 regeneration media (base layer: 50 μg/mL ampicillin and 1 μg/mL hygromycin B; top layer: 50 μg/mL ampicillin and 2 μg/mL hygromycin B) at 25 °C until single colonies emerged, which were subsequently transferred to PDA media (50 μg/mL ampicillin and 2 μg/mL hygromycin B) for continuous cultivation until stabilization. Genomic DNA was extracted from the putative overexpression transformants, followed by PCR amplification using primer pairs hphF/hphR (5′-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3′/5′-GAACCCGCGGTCGGCATCTACTCTAT-3′) for the hpt resistance gene and ROE-F/ROE-R (5′-CTAGAGGATCCATGCGTGTCG-3′/5′-CTTGATATCGAATTCCTGCAGCC-3′) for swnR cDNA verification. The successfully identified swnR-overexpressing strains were designated as swnR-OE.

2.10. Colony and Hyphal Morphology

The colony morphology of A. oxytropis OW 7.8, ΔswnR, and swnR-OE after 20 days of cultivation was observed. The mycelia morphology of A. oxytropis OW 7.8 and ΔswnR was observed under a scanning electron microscope (SU81003.0 kV × 30, Wuhan Servicebio Technology, Wuhan, China).

2.11. Extraction and Detection of SW in Mycelia of A. oxytropis OW 7.8, ΔswnR, and swnR-OE Mycelia

Mycelia of A. oxytropis OW 7.8, ΔswnR, and swnR-OE cultured for 20 days were used for SW extraction by acetic acid–chloroform solution, purification by cation exchange resin, and elution with 1 mol/L ammonia solution. The SW levels in the mycelia were determined by HPLC-MS, with three replicates for each sample. Data were analyzed by one-way ANOVA in GraphPad Prism 9.5 software. The chromatographic conditions were as follows: mobile phase, 5% methanol and 20 mmol/L ammonium acetate; flow rate, 0.4 mL/min; column temperature, 30 °C. MS conditions were as follows: positive ion 156; negative ion 70; IonSpray voltage (IS), 30 V.

2.12. Transcriptomic and Metabolomic Sample Processing and Data Analysis

Mycelia of A. oxytropis OW7.8 and ΔswnR were cultured on PDA at 25 °C for 20 days. Single colonies (0.1–0.3 g fresh weight) were collected and flash-frozen in liquid nitrogen (1–3 min) with three biological replicates. Transcriptome sequencing was performed on the Illumina NovaSeq 6000 platform (Novogene, Beijing, China). Data quality was assessed using Agilent 2100 bioanalyzer and Qubit 2.0 Fluorometer. HISAT2 and featureCounts were used for alignment and quantification, with DESeq2 identifying differentially expressed genes (DEGs) (criteria: |log2(Fold Change)| > 1 and padj < 0.05). DEGs were annotated using GO (http://www.geneontology.org, accessed on 25 February 2025.) and KEGG (https://www.genome.jp/kegg/pathway, accessed on 25 February 2025.) databases [41,42,43].
Metabolomic samples were processed identically to transcriptomic samples, with five biological replicates. HPLC-MS/MS analysis was performed. Data quality was evaluated using orthogonal partial least squares-discriminant analysis (OPLS-DA) and variable importance in projection (VIP) scores. The metaX software 2.0 processed metabolomic data. Differential metabolites (VIP > 1, |log2(Fold Change)| > 1, and padj < 0.05) were identified using Python (Python-3.5.0, Python-2.7.6) and R (R-3.4.3, R-4.0.3), with KEGG database annotation [44,45,46].

2.13. RT-qPCR Analysis of SW Biosynthesis-Associated Genes in OW7.8, ΔswnR, and swnR-OE

RT-qPCR was performed using cDNA templates derived from OW7.8 and ΔswnR, with the actin gene serving as the endogenous reference. Gene-specific amplification was conducted using the following primer pairs: sacQF/sacQR (5′-GTCAACGATGCCGATGCTCTC-3′/5′-TGCGGATTGCGGACTTGATAAC-3′), P5CRQF/P5CRQR (5′-CAATAATGGGCGGCGTGATGTC-3′/5′-ACAGGCGATGAAGTTGGAGAGG-3′), swnRQF/swnRQR (5′-TTCTACTTTGCCACACACGAACC-3′/5′-GTCAGCCAACCAGCCAATGC-3′), swnKQF/swnKQR (5′-CTTGCTCGCCTGTGCTGATTC-3′/5′-CGTCAACTCGTCCAACACTTCC-3′), swnNQF/swnNQR (5′-ATGCCATTGCGGAATCAGGTAAG-3′/5′-CGTCTTGTTGGTTGCGGTAGTAG-3′), swnH2QF/swnH2QR (5′-CATCTGCTCCTCGCTTGCTACC-3′/5′-CAGGACAACGCCTCCATCTCTTTC-3′), and swnH1QF/swnH1QR (5′-TTTGCTTTGCGGAGATGGAACC-3′/5′-CTGGACGGAGTGTGCCTGAG-3′). The PCR program was as follows: 95 °C for 30 s; 95 °C for 5 s, 60 °C for 30 s, repeated for 29 cycles, 95 °C for 5 s, 65 °C for 1 min, 95 °C for 20 s. All experimental samples were analyzed in triplicate, with relative gene expression levels (sac, P5CR, swnR, swnK, swnN, swnH1, and swnH2) calculated using the 2−ΔΔCt method, where ΔCt = Ct (target gene) − Ct(actin) and ΔΔCt = ΔCt (∆swnR) − ΔCt (OW7.8). Statistical analysis was performed using GraphPad Prism 9.5.0 (one-way ANOVA with post hoc testing).

3. Results

3.1. Cloning and Bioinformatics Analysis of swnR Gene in A. oxytropis OW7.8

The swnR gene (GenBank: OP834145) was successfully cloned from the locoweed endophytic fungus A. oxytropis OW7.8, comprising 918 bp (ATG-TGA) without introns and encoding a predicted 305-amino acid protein with a molecular weight of 34.87 kDa and pI of 6.15. Bioinformatics analysis predicted the encoded SwnR protein to be non-transmembrane and hydrophilic. Phylogenetic analysis of SwnR amino acid sequences from 12 SWN gene cluster-containing fungi (Figure 3) revealed 100% identity with Alternaria oxytropis (AQV04226.1), while showing 72.13% identity with Slafractonia leguminicola, 69.81% with Ipomoea carnea endophyte (KY365740), 69.51% with both Metarhizium brunneum ARSEF 3297 and Metarhizium robertsii (ARSEF 23 and ARSEF 2575), and 69.18% with Metarhizium anisopliae ARSEF 549.

3.2. Hygromycin B Sensitivity of A. oxytropis OW7.8

The growth characteristics of OW7.8 colonies cultured for 30 days on PDA media containing varying Hyg B concentrations (0, 0.5, 0.6, 0.7, 0.8, 0.9, 1, and 2 μg/mL) are presented in Figure 4. The results demonstrated complete growth inhibition of A. oxytropis OW7.8 at Hyg B concentrations ≥ 2 μg/mL in PDA media, establishing this concentration as the threshold for effective selection pressure.

3.3. Screening and Verification of ΔswnR Mutants

PCR analysis confirmed successful genomic integration in the swnR knockout transformants, demonstrating amplification products for the hpt resistance gene, the upstream homologous sequence of the swnR and hpt genes, and the hpt gene and the downstream homologous sequence of swnR (Figure 5), while failing to amplify the internal swnR sequence. The sequencing of the PCR products confirmed their accuracy, resulting in the identification of the ∆swnR.

3.4. Screening and Identification of swnR-OE Transformants

A PCR analysis of the putative swnR-overexpressing transformants successfully amplified both the hpt gene and the swnR gene sequence (Figure 6), with subsequent sequencing confirming the correct amplification of the expected fragments.

3.5. Colony and Hyphal Morphology

The A. oxytropis OW7.8 exhibited circular colonies with central protrusions, characterized by white fluffy mycelia forming loosely arranged, radially distributed hyphae and distinct melanin pigmentation on the reverse side. In contrast, the ΔswnR knockout mutant displayed raised, cream-to-pale yellow colonies with irregular margins, featuring short, swollen-tip hyphae in granular arrangements without pigment accumulation, demonstrating slower growth rates and stratified colony architecture compared with the wild-type. The swnR-OE developed irregularly raised colonies with reduced mycelial density but retained reverse-side pigmentation, though exhibiting slower growth than OW7.8 (Figure 7).
The A. oxytropis OW7.8 exhibited typical fungal hyphal morphology, characterized by slender, well-developed hyphae with clearly visible septa, whereas the ΔswnR mutant displayed significantly altered mycelial architecture featuring shortened hyphae with bulbous tips and an overall densely packed structure, as illustrated in Figure 8.

3.6. SW Levels in A. oxytropis OW 7.8, ΔswnR and swnR-OE Mycelia

The standard curve for SW quantification (Figure 9A) was established by plotting peak areas against known SW concentrations, yielding a linear regression equation Y = 34.62 X + 14.03 (R2 = 0.9983). After 20 days of cultivation, HPLC analysis revealed significantly different SW levels among the strains: wild-type OW7.8 accumulated 262.6569 ± 8.1911 μg/g DW, while the ΔswnR knockout mutant showed markedly reduced production (45.3986 ± 12.8587 μg/g DW). The swnR-OE exhibited intermediate SW levels (108.4023 ± 2.7297 μg/g DW), demonstrating a significant increase compared with ΔswnR but not reaching wild-type concentrations, confirming the functional role of the swnR gene in promoting SW biosynthesis in A. oxytropis OW7.8 (Figure 9B).

3.7. Transcriptome Sequencing Analysis of A. oxytropis OW7.8 and ΔswnR

Transcriptome sequencing of A. oxytropis OW7.8 and ΔswnR generated a total of 288,360,652 clean reads, amounting to 43.26 G of data. The results revealed significant differential gene expression between A. oxytropis OW7.8 and ΔswnR. A total of 2997 differentially expressed genes (DEGs) were identified, of which 1555 (51.89% of DEGs) were upregulated and 1442 (48.11%) were downregulated. Among these, six DEGs closely associated with SW biosynthesis were identified, including the upregulated sac gene and the downregulated P5CR, swnN, swnK, swnH1, and swnH2 genes (Figure 10).
The GO (Gene Ontology) enrichment analysis of differentially expressed genes (DEGs) between A. oxytropis OW7.8 and ΔswnR (Figure 11) identified 1018 annotated DEGs, comprising 546 upregulated and 472 downregulated genes, which were functionally categorized into 280 biological process (BP) terms, 70 cellular component (CC) terms, and 180 molecular function (MF) terms. Within BP categories, carbohydrate metabolic processes showed differential expression of 28 upregulated and 53 downregulated genes, while cellular nitrogen compound catabolic processes and heterocycle catabolic processes each exhibited seven upregulated and six downregulated genes. The CC analysis revealed predominant DEG enrichment in ribosome-related terms, with 49 genes upregulating in ribosome and 52 upregulating plus two downregulating genes in the ribonucleoprotein complex. For MF categories, significant enrichment was observed for the structural constituent of ribosome (49 upregulated genes) and structural molecule activity (49 upregulated genes), demonstrating substantial transcriptional reprogramming in cellular architecture-related functions.
The KEGG pathway analysis identified 411 differentially expressed genes (DEGs) between A. oxytropis OW7.8 and the ΔswnR (159 upregulated and 252 downregulated), which were significantly enriched in 99 metabolic pathways (Figure 12). Among the most prominently altered pathways (padj ≤ 0.05), ribosomal biosynthesis showed substantial upregulation with 66 genes, while carbohydrate metabolism pathways exhibited distinct expression patterns: glycolysis/gluconeogenesis displayed 2 upregulated and 22 downregulated genes, and starch/sucrose metabolism contained 7 upregulated versus 21 downregulated genes.

3.8. Metabolomic Profiling of A. oxytropis OW7.8 and ΔswnR

Principal component analysis (PCA) of the metabolomic data revealed distinct metabolite profiles between ΔswnR and A. oxytropis OW7.8 in both positive and negative ion modes (Figure 13).
Comprehensive analysis identified 1626 significantly differential metabolites (DEMs), with 1005 upregulated and 621 downregulated in ΔswnR compared with the wild-type strain. Specifically, positive ion mode detection yielded 984 DEMs (566 upregulated and 418 downregulated), while negative ion mode analysis identified 642 DEMs (439 upregulated and 203 downregulated), as detailed in Table 2 and Figure 14.
KEGG-based cluster analysis of differential metabolites revealed distinct metabolic patterns between A. oxytropis OW7.8 and ΔswnR (Figure 15), with the positive ion mode showing significant alterations in secondary metabolite biosynthesis and histidine metabolism pathways closely associated with SW production, while the negative ion mode demonstrated marked changes in phenylalanine metabolism and the alanine/aspartate/glutamate metabolic axis. Further screening identified five key differential metabolites linked to SW biosynthesis: L-lysine, L-glutamic acid, saccharopine, and L-proline were upregulated in ΔswnR compared with A. oxytropis OW7.8, whereas L-pipecolic acid showed significant downregulation.

3.9. Expression of sac, P5CR, and SWN Cluster Genes in A. oxytropis OW 7.8 and ΔswnR

Comparative RT-qPCR analysis revealed differential expression patterns of SW biosynthetic genes among the studied strains (Figure 16). In the ΔswnR, significant upregulation of the sac gene was observed alongside marked downregulation of the P5CR, swnK, swnN, swnH2, and swnH1 genes compared with A. oxytropis OW7.8. The swnR-OE overexpression strain exhibited increased transcript levels of the sac, P5CR, swnK, and swnN gene, while showing decreased swnH1 gene expression and unaltered swnH2 gene expression levels.
Furthermore, swnR gene expression analysis demonstrated a complete absence of transcription in ΔswnR and significant overexpression in swnR-OE relative to the wild-type strain (Figure 17).

4. Discussion

4.1. Functional Role of SwnR Reductase and swnR Gene in SW Biosynthesis in A. oxytropis OW7.8

The swnR gene (OP834145) encodes a reductase that catalyzes the conversion of P6C to PA in SW-producing fungi. Comparative sequence analysis revealed 100% amino acid identity between A. oxytropis OW7.8 and A. oxytropis Raft River (KY365741.1), with both sequences encoding 305 amino acids and lacking introns, indicating a close phylogenetic relationship [31]. The homologous gene in M. robertsii showed 69.51% sequence identity while maintaining a protein with 305 amino acids and an intronless structure.
Due to the inherent challenges of working with the slow-growing endophytic fungus A. oxytropis OW7.8, which exhibits extremely low homologous recombination efficiency during in vitro culture, extensive screening efforts yielded limited transformants—specifically, four knockout mutants and five overexpression strains after prolonged experimentation. SW production in ΔswnR mycelia decreased significantly, attributable to the loss of SwnR enzymatic activity required for PA biosynthesis from P6C, thereby limiting downstream SW formation. The swnR-OE strains showed higher SW levels than ΔswnR, and lower SW levels than OW7.8, confirming swnR’s positive role in SW biosynthesis. RT-qPCR analysis of swnR-OE revealed the coordinated upregulation of key SW pathway genes (sac, P5CR, swnR, swnK, and swnN), while the downregulation of the swnH1 gene may explain the incomplete SW production recovery.
Previous whole-genome sequencing of A. oxytropis OW7.8 by our research group identified the P5CR gene, which encoded that the P5CR enzyme alternatively catalyzes P6C reduction to PA [47]. Consistent with our current findings, ΔP5CR similarly exhibited significantly reduced SW production compared with the wild-type. Molecular docking predictions revealed stronger binding affinity between SwnR and P6C (binding free energy: −4.56 kcal/mol) than the P5CR–P6C interaction (−4.0 kcal/mol), suggesting preferential P6C binding by SwnR.
These results align with studies in other fungal systems: Luo et al. demonstrated in M. robertsii that SW production followed ΔswnNR < ΔswnN < ΔswnR < WT, with parallel PA level measurements showing significant reduction in ΔswnR and extreme depletion in ΔswnNR, while ΔswnN accumulated PA, collectively supporting SwnR’s role in P6C → PA conversion [33]. Similarly, Sun et al. reported markedly decreased SW production in M. anisopliae ΔswnR, and that the SW level was partially restored in the gene function complementary strain relative to the wild-type [48]. Our conclusions regarding swnR gene function in SW biosynthesis thus showed cross-species consistency with these independent investigations in Metarhizium species.

4.2. Colony Morphology and SW Extraction and Purification

The A. oxytropis OW7.8 exhibited circular, white, and fluffy colonies with relatively dense hyphae, whereas the ΔswnR displayed pale yellow, loosely structured colonies with irregular margins, lacking pigment accumulation and showing reduced growth rates. These observations indicated that swnR gene knockout leads to abnormal hyphal morphology and impaired cellular proliferation. Electron microscopy further revealed shortened hyphae with swollen tips and an overall compact, stacked architecture. The swnR-OE formed irregularly raised white colonies with pigmented undersides and similarly exhibited slower growth.
For SW extraction from mycelia of A. oxytropis OW7.8, ΔswnR, and swnR-OE, freeze-dried hyphae were rapidly ground into a fine powder to minimize SW loss. Optimal amounts of glass fibers and cation-exchange resin were added, followed by elution with 1 mol/L ammonia, and the resulting SW-containing eluate was aliquoted into 1.5 mL centrifuge tubes while avoiding overfilling to prevent leakage during centrifugation, prior to drying using a rotary evaporator with limited exposure time. Samples intended for delayed SW quantification were sealed and stored at −20 °C to prevent moisture evaporation and ensure measurement accuracy.

4.3. Transcriptomic and Metabolomic Profiling of A. oxytropis OW7.8 and ΔswnR

Comparative transcriptomic analysis between A. oxytropis OW7.8 and the ΔswnR mutant revealed the significant downregulation of key SW biosynthetic genes including P5CR, swnK, swnN, swnH2, and swnH1, while the sac gene showed marked upregulation. Corresponding metabolomic profiling demonstrated elevated levels of L-Lys and saccharopine, consistent with the increased expression of the Sac enzyme responsible for saccharopine-to-L-lysine conversion. The knockout of the swnR gene resulted in reduced accumulation of both PA and SW, accompanied by the downregulation of the P5CR gene. Notably, the enzymatic reactions catalyzed by the swnK, swnN, swnH2, and swnH1 genes all operate downstream of SwnR-mediated catalysis, and all four genes exhibited decreased expression patterns. These transcriptional changes were subsequently validated by RT-qPCR analysis, confirming the reliability of the transcriptome data. However, current transcriptomic platforms showed technical limitations in detecting several other potentially relevant genes, including α-aminoadipate reductase, saccharopine oxidase, L-lysyl-alpha-oxidase, lysDH, and dpkA/lhpD/lhpI, which were not successfully enriched in our analysis.
Metabolomic analysis identified five differentially accumulated metabolites closely associated with SW biosynthesis in ΔswnR. Notably, L-PA was significantly downregulated, while L-Lys, L-glutamic acid, L-proline, and saccharopine showed marked upregulation. Among these metabolites, L-Lys, saccharopine, and L-PA served as crucial precursors in the SW biosynthetic pathway. The observed reduction in L-PA levels likely results from the knockout of swnR gene, which abolished the SwnR enzyme activity required for the reduction of P6C to PA, though alternative biosynthetic routes may partially compensate for PA production. The accumulation of L-Lys could be attributed to increased saccharopine levels coupled with the upregulation of the sac gene, leading to enhanced conversion of saccharopine to L-Lys catalyzed by the Sac enzyme. Concurrent transcriptomic analysis revealed the elevated expression of the proline iminopeptidase gene, whose encoded enzyme may catalyze the conversion of PA to L-proline, thereby contributing to the observed L-proline accumulation. It should be noted that current metabolomic databases have certain limitations, as evidenced by the failure to detect several key intermediates including 6-amino-2-oxohexanoate, P2C, P6C, 1-oxoindolizine, and 1-hydroxyindolizine in our analysis.

4.4. SW Biosynthetic Pathway

Based on integrated transcriptomic and metabolomic analyses of A. oxytropis OW7.8 and ΔswnR, the predicted SW biosynthetic pathway in this study is consistent with our recently proposed model [34]. As more SW biosynthetic genes and their functions continue to be characterized, additional details of the SW biosynthetic pathway in locoweed endophytic fungi will be further elucidated.

5. Conclusions

In this study, we successfully cloned and knocked out the swnR gene in the locoweed endophytic fungus Alternaria oxytropis OW7.8 for the first time, generating the swnR knockout mutant ΔswnR. Compared with A. oxytropis OW7.8, ΔswnR exhibited altered colony and mycelial morphology, along with slower growth. After 20 days of cultivation, SW levels in ΔswnR were significantly lower than those in A. oxytropis OW7.8. In contrast, swnR-OE showed a marked increase in SW production relative to ΔswnR, confirming that the swnR gene functionally promotes SW biosynthesis.
Transcriptomic analysis of ΔswnR and A. oxytropis OW7.8 identified 2997 DEGs, including 1555 upregulated and 1442 downregulated genes. SW synthesized closely related genes (P5CR, swnN, swnK, swnH2, and swnH1) that were downregulated, while the sac gene was upregulated, consistent with RT-qPCR validation. Metabolomic profiling revealed 1626 differential metabolites between A. oxytropis OW7.8 and ΔswnR, with five metabolites closely linked to SW biosynthesis: L-Lys, L-Glutamic acid, Saccharopine, and L-Proline were upregulated, and L-PA was downregulated.

Author Contributions

Conceptualization, P.L.; methodology, P.L., N.D., and C.L.; investigation, P.L., N.D., C.L., L.B., and B.Y.; data curation, N.D., C.L., and P.L.; writing—original draft preparation, N.D. and C.L.; writing—review and editing, P.L., N.D., and C.L., visualization, N.D. and C.L.; supervision, P.L.; project administration, P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31460235; 31960130; 30860049), Natural Science Foundation of Inner Mongolia Autonomous Region of China (2022MS03014), and Fundamental Research Funds for the Inner Mongolia Normal University (2022JBTD010).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on reasonable request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. SW synthesis pathway in M. robertsii [33].
Figure 1. SW synthesis pathway in M. robertsii [33].
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Figure 2. SW synthesis pathway in A. oxytropis OW 7.8 [34].
Figure 2. SW synthesis pathway in A. oxytropis OW 7.8 [34].
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Figure 3. Neighbor-joining phylogenetic tree of SwnR amino acid sequences.
Figure 3. Neighbor-joining phylogenetic tree of SwnR amino acid sequences.
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Figure 4. Colony morphology of A. oxytropis OW7.8 following 30-day cultivation on PDA media containing different Hyg B concentrations. Note: (A): 0 μg/mL, (B): 0.5 μg/mL, (C): 0.6 μg/mL, (D): 0.7 μg/mL, (E): 0.8 μg/mL, (F): 0.9 μg/mL, (G): 1 μg/mL, (H): 2 μg/mL.
Figure 4. Colony morphology of A. oxytropis OW7.8 following 30-day cultivation on PDA media containing different Hyg B concentrations. Note: (A): 0 μg/mL, (B): 0.5 μg/mL, (C): 0.6 μg/mL, (D): 0.7 μg/mL, (E): 0.8 μg/mL, (F): 0.9 μg/mL, (G): 1 μg/mL, (H): 2 μg/mL.
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Figure 5. Electrophoretic analysis of PCR products from ΔswnR genomic DNA. M: 1 kb Plus DNA Ladder. (A): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the hpt gene (expected 1388 bp). (B): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the swnR upstream homologous sequence + hpt gene (expected 1205 bp). (C): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the swnR downstream homologous sequence + hpt gene (expected 1388 bp). (D): Lane 1: Positive control (swnR internal sequence, expected 261 bp); Lane 2: PCR product using ΔswnR genomic DNA as template.
Figure 5. Electrophoretic analysis of PCR products from ΔswnR genomic DNA. M: 1 kb Plus DNA Ladder. (A): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the hpt gene (expected 1388 bp). (B): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the swnR upstream homologous sequence + hpt gene (expected 1205 bp). (C): Lane 1: Negative control; Lane 2: Positive control; Lane 3 shows a band of the swnR downstream homologous sequence + hpt gene (expected 1388 bp). (D): Lane 1: Positive control (swnR internal sequence, expected 261 bp); Lane 2: PCR product using ΔswnR genomic DNA as template.
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Figure 6. Electrophoretic analysis of PCR products from swnR-overexpressing transformant. M: 1 kb plus DNA ladder. (A) Lane 1–3: Negative control; Lanes 4,5: hpt gene fragment (expected 1380 bp). (B) Lane 1: Negative control; Lanes 2: swnR gene sequence (expected 956 bp).
Figure 6. Electrophoretic analysis of PCR products from swnR-overexpressing transformant. M: 1 kb plus DNA ladder. (A) Lane 1–3: Negative control; Lanes 4,5: hpt gene fragment (expected 1380 bp). (B) Lane 1: Negative control; Lanes 2: swnR gene sequence (expected 956 bp).
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Figure 7. Comparison of A. oxytropis OW7.8, ΔswnR, and swnR-OE colony morphology. Note: (A): A. oxytropis OW7.8; (B): ΔswnR; (C): swnR-OE.
Figure 7. Comparison of A. oxytropis OW7.8, ΔswnR, and swnR-OE colony morphology. Note: (A): A. oxytropis OW7.8; (B): ΔswnR; (C): swnR-OE.
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Figure 8. Comparative scanning electron micrographs of OW7.8 and ΔswnR hyphal morphology. Note: (A): A.oxytropis OW 7.8 mycelia magnified 1000×, (B): A.oxytropis OW 7.8 mycelia magnified 5000×, (C): ΔswnR mycelia magnified 1000×, (D): ΔswnR mycelia magnified 5000×.
Figure 8. Comparative scanning electron micrographs of OW7.8 and ΔswnR hyphal morphology. Note: (A): A.oxytropis OW 7.8 mycelia magnified 1000×, (B): A.oxytropis OW 7.8 mycelia magnified 5000×, (C): ΔswnR mycelia magnified 1000×, (D): ΔswnR mycelia magnified 5000×.
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Figure 9. SW level detection in OW7.8, ΔswnR, and swnR-OE in 20 d. (A): SW standard curve. (B): SW content in OW7.8, ΔswnR, and swnR-OE in 20th d. The error bar represents the standard error of the mean (n = 3), with (****) p < 0.0001.
Figure 9. SW level detection in OW7.8, ΔswnR, and swnR-OE in 20 d. (A): SW standard curve. (B): SW content in OW7.8, ΔswnR, and swnR-OE in 20th d. The error bar represents the standard error of the mean (n = 3), with (****) p < 0.0001.
Microorganisms 13 01326 g009
Microorganisms 13 01326 g010
Figure 10. Volcano map of A. oxytropis OW7.8 and ΔswnR DEGs. Note: x: log2FoldChange value, y: −log10 (padj). Red represents upregulated genes, green represents downregulated genes, and blue represents genes with no significant differential expression.
Figure 10. Volcano map of A. oxytropis OW7.8 and ΔswnR DEGs. Note: x: log2FoldChange value, y: −log10 (padj). Red represents upregulated genes, green represents downregulated genes, and blue represents genes with no significant differential expression.
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Microorganisms 13 01326 g011
Figure 11. GO enrichment of DEGs in A. oxytropis OW 7.8 and ∆swnR. Note: BP: Biological Process, CC: Cellular Component, MF: Molecular Function.
Figure 11. GO enrichment of DEGs in A. oxytropis OW 7.8 and ∆swnR. Note: BP: Biological Process, CC: Cellular Component, MF: Molecular Function.
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Microorganisms 13 01326 g012
Figure 12. KEGG enrichment of DEGs in A. oxytropis OW 7.8 and ∆swnR.
Figure 12. KEGG enrichment of DEGs in A. oxytropis OW 7.8 and ∆swnR.
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Microorganisms 13 01326 g013
Figure 13. PCA analysis of total samples based on different model subsets. (A). Positive ion mode (B). Negative ion mode.
Figure 13. PCA analysis of total samples based on different model subsets. (A). Positive ion mode (B). Negative ion mode.
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Microorganisms 13 01326 g014
Figure 14. A. oxytropis OW 7.8 and ΔswnR differential metabolite volcano plot. (A). Positive ion mode (B). Negative ion mode.
Figure 14. A. oxytropis OW 7.8 and ΔswnR differential metabolite volcano plot. (A). Positive ion mode (B). Negative ion mode.
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Microorganisms 13 01326 g015
Figure 15. Bubble chart of KEGG enrichment analysis of differential metabolites between A. oxytropis OW 7.8 and ΔswnR strains in positive and negative ion modes. (A). Positive ion mode (B). Negative ion mode.
Figure 15. Bubble chart of KEGG enrichment analysis of differential metabolites between A. oxytropis OW 7.8 and ΔswnR strains in positive and negative ion modes. (A). Positive ion mode (B). Negative ion mode.
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Microorganisms 13 01326 g016
Figure 16. RT-qPCR detection of SW synthesis-related gene expression levels in A. oxytropis OW 7.8, ΔswnR, and swnR-OE. Note: The x-axis represents genes; the y-axis represents relative expression levels. Error bars indicate the standard error of the mean (n = 3), with (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.
Figure 16. RT-qPCR detection of SW synthesis-related gene expression levels in A. oxytropis OW 7.8, ΔswnR, and swnR-OE. Note: The x-axis represents genes; the y-axis represents relative expression levels. Error bars indicate the standard error of the mean (n = 3), with (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.
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Microorganisms 13 01326 g017
Figure 17. RT-qPCR detection of swnR gene expression levels in A. oxytropis OW 7.8, ΔswnR, and swnR-OE. Note: The x-axis represents genes; the y-axis represents relative expression levels. Error bars indicate the standard error of the mean (n = 3), with (**) p < 0.01, (****) p < 0.0001.
Figure 17. RT-qPCR detection of swnR gene expression levels in A. oxytropis OW 7.8, ΔswnR, and swnR-OE. Note: The x-axis represents genes; the y-axis represents relative expression levels. Error bars indicate the standard error of the mean (n = 3), with (**) p < 0.01, (****) p < 0.0001.
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Table 1. Members of the SWN gene cluster and their predicted functions [31].
Table 1. Members of the SWN gene cluster and their predicted functions [31].
GeneEncoding ProductFunction Prediction
swnAAminotransferaseCatalyzing the synthesis of Pyrroline-6-carboxylate (P6C) from L-Lysine
swnRDehydrogenase or reductaseCatalyzing the synthesis of L-PA from P6C
swnKMultifunctional proteinCatalyzing the synthesis of 1-Oxoindolizidine (or 1-Hydroxyindolizine) from L-PA
swnNDehydrogenase or reductaseCatalyzing the synthesis of 1-Hydroxyindolizine from 1-Oxoindolizidine
swnH1Fe (II)/α-Ketoglutarate-dependent dioxygenaseCatalyzing the synthesis of SW from 1,2-Dihydroxyindolizine
swnH2Fe (II)/α-Ketoglutarate-dependent dioxygenaseCatalyzing the synthesis of 1,2-Dihydroxyindolizine form 1-Hydroxyindolizine
swnTTransmembrane transporterTransport of SW
Table 2. Identification results of differential metabolites between positive and negative ions.
Table 2. Identification results of differential metabolites between positive and negative ions.
Screening ModeTotal of
Metabolites		Total of DEMsUpregulatedDownregulated
Positive1878984566418
Negative1124642439203
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MDPI and ACS Style

Ding, N.; Liu, C.; Lu, P.; Bai, L.; Yuan, B. The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra. Microorganisms 2025, 13, 1326. https://doi.org/10.3390/microorganisms13061326

AMA Style

Ding N, Liu C, Lu P, Bai L, Yuan B. The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra. Microorganisms. 2025; 13(6):1326. https://doi.org/10.3390/microorganisms13061326

Chicago/Turabian Style

Ding, Ning, Chang Liu, Ping Lu, Lu Bai, and Bo Yuan. 2025. "The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra" Microorganisms 13, no. 6: 1326. https://doi.org/10.3390/microorganisms13061326

APA Style

Ding, N., Liu, C., Lu, P., Bai, L., & Yuan, B. (2025). The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra. Microorganisms, 13(6), 1326. https://doi.org/10.3390/microorganisms13061326

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