Transcriptional Regulation of SugarCane Response to Sporisorium scitamineum: Insights from Time-Course Gene Coexpression and Ca2+ Signaling.

Sugarcane response to Sporisorium scitamineum is determined by multiple major genes and numerous microeffector genes. Here, time-ordered gene coexpression networks were applied to explore the interaction between sugarcane and S. scitamineum. Totally, 2459 differentially expressed genes were identified and divided into 10 levels, and several stress-related subnetworks were established. Interestingly, the Ca2+ signaling pathway was activated to establish the response to sugarcane smut disease. Accordingly, two CAX genes (ScCAX2 and ScCAX3) were cloned and characterized from sugarcane. They were significantly upregulated under ABA stress but inhibited by MeJA treatment. Furthermore, overexpression of ScCAX2 and ScCAX3 enhanced the susceptibility of transgenic plants to the pathogen infection, suggesting its negative role in disease resistance. A regulatory model for ScCAX genes in disease response was thus depicted. This work helps to clarify the transcriptional regulation of sugarcane response to S. scitamineum stress and the function of the CAX gene in disease response.

A Comprehensive Analysis of Transcriptomics and Metabolomics Revealed Key Pathways Involved in Saccharum spontaneum Defense against Sporisorium scitamineum.

Sugarcane smut, caused by Sporisorium scitamineum, poses a severe threat to sugarcane production. The genetic basis of sugarcane resistance to S. scitamineum remains elusive. A comparative transcriptomic and metabolomic study was conducted on two wild Saccharum species of S. spontaneum with contrast smut resistance. Following infection, the resistant line exhibited greater down-regulation of genes and metabolites compared to the susceptible line, indicating distinct biological processes. Lignan and lignin biosynthesis and SA signal transduction were activated in the resistant line, while flavonoid biosynthesis and auxin signal transduction were enhanced in the susceptible line. TGA2.2 and ARF14 were identified as playing positive and negative roles, respectively, in plant defense. Exogenous auxin application significantly increased the susceptibility of S. spontaneum to S. scitaminum. This study established the significant switching of defense signaling pathways in contrast-resistant S. spontaneum following S. scitamineum infection, offering a hypothetical model and candidate genes for further research into sugarcane smut disease.

Comparative genomics of smut fungi suggest the ability of meiosis and mating in asexual species of the genus Pseudozyma (Ustilaginales).

BACKGROUND: The Ustilaginales comprise hundreds of plant-parasitic fungi with a characteristic life cycle that directly links sexual reproduction and parasitism: One of the two mating-type loci codes for a transcription factor that not only facilitates mating, but also initiates the infection process. However, several species within the Ustilaginales have no described parasitic stage and were historically assigned to the genus Pseudozyma. Molecular studies have shown that the group is polyphyletic, with members being scattered in various lineages of the Ustilaginales. Together with recent findings of conserved fungal effectors in these non-parasitic species, this raises the question if parasitism has been lost recently and in multiple independent events or if there are hitherto undescribed parasitic stages of these fungi. RESULTS: In this study, we sequenced genomes of five Pseudozyma species together with six parasitic species from the Ustilaginales to compare their genomic capability to perform two central functions in sexual reproduction: mating and meiosis. While the loss of sexual capability is assumed in certain lineages and asexual species are common in Asco- and Basidiomycota, we were able to successfully annotate potentially functional mating and meiosis genes that are conserved throughout the whole group. CONCLUSION: Our data suggest that at least the key functions of a sexual lifestyle are maintained in the analyzed genomes, challenging the current understanding of the so-called asexual species with respect to their evolution and ecological role.

Engineered Sucrose Metabolism Improves the Smut Disease Suppression Potency of Pseudomonas sp. ST4.

Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.

Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other Ustilaginales.

The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.

First draft genome of Thecaphora frezii, causal agent of peanut smut disease.

OBJECTIVES: The fungal pathogen Thecaphora frezii Carranza & Lindquist causes peanut smut, a severe disease currently endemic in Argentina. To study the ecology of T. frezii and to understand the mechanisms of smut resistance in peanut plants, it is crucial to know the genetics of this pathogen. The objective of this work was to isolate the pathogen and generate the first draft genome of T. frezii that will be the basis for analyzing its potential genetic diversity and its interaction with peanut cultivars. Our research group is working to identify peanut germplasm with smut resistance and to understand the genetics of the pathogen. Knowing the genome of T. frezii will help analyze potential variants of this pathogen and contribute to develop enhanced peanut germplasm with broader and long-lasting resistance. DATA DESCRIPTION: Thecaphora frezii isolate IPAVE 0401 (here referred as T.f.B7) was obtained from a single hyphal-tip culture, its DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq6000 (Nova). Data from both sequencing platforms were combined and the de novo assembling estimated a 29.3 Mb genome size. Completeness of the genome examined using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed the assembly had 84.6% of the 758 genes in fungi_odb10.

Assessment of the rhizosphere fungi and bacteria recruited by sugarcane during smut invasion.

Whip smut is one of the most serious and widely spread sugarcane diseases. Plant-associated microbes play various roles in conferring advantages to the host plant. Understanding the microbes associated with sugarcane roots will help develop strategies for the biocontrol of smut. Therefore, the present study explored microbe-mediated sugarcane response to smut invasion via 16S rRNA and ITS metabarcoding survey of the rhizosphere soils of resistant and susceptible sugarcane varieties. The bacterial and fungal diversity in the rhizosphere soils differed between the resistant and susceptible varieties. The bacterial genera Sphingomonas, Microcoleus_Es-Yyy1400, Marmoricola, Reyranella, Promicromonospora, Iamia, Phenylobacterium, Aridibacter, Actinophytocola, and Edaphobacter and one fungal genus Cyphellophora were found associated with smut resistance in sugarcane. Detailed analysis revealed that the majority of bacteria were beneficial, including the actinomycete Marmoricola and Iamia and Reyranella with denitrification activity. Analysis of bacterial network interaction showed that three major groups interacted during smut invasion. Meanwhile, seven of these genera appeared to interact and promote each other's growth. Finally, functional annotation based on the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database predicted that the abundant bacteria are dominated by oxygenic photoautotrophy, photoautotrophy, and phototrophy functions, which may be related to smut resistance in sugarcane. The present study thus provides new insights into the dynamics of the sugarcane rhizosphere microbial community during smut invasion.

High-Quality Genome Resource of Ustilaginoidea virens (UV2_4G), Causal Agent of an Emerging False Smut Disease in Rice.

Ustilaginoidea virens is the fungal pathogen causing an emerging false smut disease that affects crop yield as well as deteriorates quality of the grains by producing mycotoxins. A high quality genome of U. virens isolate UV2_4G was sequenced using Nanopore and Illumina HiSeq 2,000 sequencing platforms. The total assembled genome of Indian isolate UV2_4G was 35.9 Mb, which comprised 89 scaffolds with N50 of 700,296 bp. A total of 358,697 variants were identified in the genome, out of which 355,173 were SNPs and 3,524 were INDELS. Further, 7,390 SSRs belonging to different repeat types were also identified in the genome. Out of 7,444 proteins predicted, 7,206 were functionally annotated. A total of 1,307 CAZymes, 501 signal peptides, 1,876 effectors, and 2,709 genes involved in host-pathogen interactions were identified. Comparative analysis revealed isolate UV2_4G is distinct with 31 unique clusters and placed distantly in phylogenetic analysis. Taken together, this high-quality genome assembly and sequence annotation resource can give an improved insight for characterizing the biological and pathogenic mechanisms of U. virens.

Kynurenine 3-Monooxygenase Gene SsCI51640 Is Required for Sporisorium scitamineum Mating/Filamentation by Regulating cAMP Pathway and Improving Sporidia Environmental Adaptability.

Sugarcane smut is a serious disease caused by Sporisorium scitamineum, which causes significant losses to the sugar industry. It is critical to reveal the molecular pathogenic mechanism of S. scitamineum to explore a new control strategy for sugarcane smut. On the basis of transcriptome sequencing data of two S. scitamineum strains with different pathogenicity, we identified the gene, SsCI51640, which was predicted to encode kynurenine 3-monooxygenase. In this study, we obtained knockout mutants and complementary mutants of this gene and identified gene function. The results showed that the sporidial growth rate and acid production ability of knockout mutants were significantly higher and stronger than those of the wild-type and complementary mutants. The growth of knockout mutants under abiotic stress (osmotic stress and cell wall stress) was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly reduced, while this phenomenon could be restored by adding exogenous cyclic adenosine monophosphate (cAMP). It is thus speculated that the SsCI51640 gene may regulate sexual mating and pathogenicity of S. scitamineum by the cAMP signaling pathway. Moreover, the SsCI51640 gene enhanced the sporidial environmental adaptability, which promoted sexual mating and development of pathogenicity. This study provides a theoretical basis for the molecular pathogenesis of S. scitamineum.

WGCNA Identifies a Comprehensive and Dynamic Gene Co-Expression Network That Associates with Smut Resistance in Sugarcane.

Sugarcane smut is a major fungal disease caused by Sporisorium scitamineum, which seriously reduces the yield and quality of sugarcane. In this study, 36 transcriptome data were collected from two sugarcane genotypes, YT93-159 (resistant) and ROC22 (susceptible) upon S. scitamineum infection. Data analysis revealed 20,273 (12,659 up-regulated and 7614 down-regulated) and 11,897 (7806 up-regulated and 4091 down-regulated) differentially expressed genes (DEGs) in YT93-159 and ROC22, respectively. A co-expression network was then constructed by weighted gene co-expression network analysis (WGCNA), which identified 5010 DEGs in 15 co-expressed gene modules. Four of the 15 modules, namely, Skyblue, Salmon, Darkorange, and Grey60, were significantly associated with smut resistance. The GO and KEGG enrichment analyses indicated that the DEGs involving in these four modules could be enriched in stress-related metabolic pathways, such as MAPK and hormone signal transduction, plant-pathogen interaction, amino acid metabolism, glutathione metabolism, and flavonoid, and phenylpropanoid biosynthesis. In total, 38 hub genes, including six from the Skyblue module, four from the Salmon module, 12 from the Darkorange module, and 16 from the Grey60 module, were screened as candidate hub genes by calculating gene connectivity in the corresponding network. Only 30 hub genes were amplifiable with RT-qPCR, of which 27 were up-regulated upon S. scitamineum infection. The results were consistent with the trend of gene expression in RNA-Seq, suggesting their positive roles in smut resistance. Interestingly, the expression levels of AOX, Cyb5, and LAC were higher in ROC22 than in YT93-159, indicating these three genes may act as negative regulators in response to S. scitamineum infection. This study revealed the transcriptome dynamics in sugarcane challenged by S. scitamineum infection and provided gene targets for smut resistance breeding in sugarcane.

Import and Export of Mannosylerythritol Lipids by Ustilago maydis.

Upon nitrogen starvation, the basidiomycete Ustilago maydis, which causes smut disease on corn, secretes amphipathic glycolipids, including mannosylerythritol lipids (MELs). MELs consist of a carbohydrate core whose mannosyl moiety is both acylated with fatty acids of different lengths and acetylated. Here, we report the transport of MELs into and out of the cell depending on the transport protein Mmf1, which belongs to the major facilitator superfamily. Analysis of mmf1 mutants and mutants lacking the acetyltransferase Mat1 revealed that Mmf1 is necessary for the export of acetylated MELs, while MELs without an acetyl group are secreted independently of this transporter. Upon deletion of mmf1, we detected novel MEL species lacking the acyl side chain at C-3'. With the help of feeding experiments, we demonstrate that MELs are taken up by U. maydis in an mmf1-independent manner. This leads to catabolism or rearrangement of acetyl and acyl side groups and subsequent secretion. The catabolism of MELs involves the presence of Mac2, an enzyme required for MEL biosynthesis. In cocultivation experiments, mutual exchange of MELs between different mutants was observed. Thus, we propose a novel function for fungal glycolipids as an external carbon storage. IMPORTANCE Fungi produce and secrete various secondary metabolites that can act as weapons against competitors, help in accessing nutrients, or assist in development and communication. One group of secondary metabolites are surface-active glycolipids, which have significant biotechnological potential as biodegradable detergents. While the biosynthesis of several fungal biosurfactants is well characterized, their biological functions and transport routes are less understood. We developed a cocultivation assay to show that a class of glycolipids from Ustilago maydis called mannosylerythritol lipids (MELs) can be exchanged between cells and modified or even degraded by recipient cells. Feeding assays with purified MELs led to similar results. These data provide insight into the surprising biological role of MELs as putative external carbon sources. Applying feeding and cocultivation experiments on MEL biosynthesis mutants turned out to be a valuable strategy for systematically studying the import routes and degradation pathways of glycolipids. By using these assays, we demonstrate the function of the transport protein Mmf1 as a specific exporter of acetylated MELs. We propose that these assays may be applied more generally, thereby opening novel areas of research.

Improvement of Oil Degradation and MEL Production in a Yeast Strain, Pseudozyma tsukubaensis, by Translation Elongation Factor 1 Promoter-driven Expression of a Lipase.

The basidiomycetous yeast Pseudozyma tsukubaensis produces a mannosylerythritol lipid (MEL) homologue, a diastereomer type of MEL-B, from olive oil. In a previous study, MEL-B production was increased by the overexpression of lipase PaLIPAp in P. tsukubaensis 1E5, through the enhancement of oil consumption. In the present study, RNA sequence analysis was used to identify a promoter able to induce high-level PaLIPA expression. The recombinant strain, expressing PaLIPA via the translation elongation factor 1 alpha/Tu promoter, showed higher lipase activity, rates of oil degradation, and MEL-B production than the strain which generated in our previous study.

Screening and Research on Skin Barrier Damage Protective Efficacy of Different Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) may prevent skin barrier damage, although their protective mechanisms and active monomeric constituents remain unclear. Here, three MELs were extracted from Candida antarctica cultures containing fermented olive oil then purified using silica gel-based column chromatography and semipreparative HPLC. All three compounds (MEL-A, MEL-B, MEL-C) were well separated and stable, and reliable materials were used for NMR and HRESIMS chemical structure determinations and for assessing MELs' protective effects against skin damage. Notably, MEL-B and MEL-C effectively protected HaCaT cells from UVB-induced damage by upregulating the contents of filaggrin (FLG) and transglutaminase-1 (TGM1), as determined via ELISA. Moreover, MEL-B treatment (20 µg/mL) of UVB-irradiated HaCaT cells led to the upregulation of both the expression of mRNA genes and the key proteins FLG, LOR, and TGM1, which are known to be decreased in damaged skin cells. Additionally, histopathological analysis results revealed a markedly reduced intracellular vacuolation and cell damage, reflecting improved skin function after MEL-B treatment. Furthermore, immunofluorescence results revealed that MEL-B protected EpiKutis® three-dimensional cultured human skin cells from sodium dodecyl sulfate-induced damage by up-regulating FLG, LOR, and TGM1 expression. Accordingly, MELs' protection against skin barrier damage depended on MEL-B monomeric constituent activities, thus highlighting their promise as beneficial ingredients for use in skin-care products.

Production of mannosylerythritol lipids: biosynthesis, multi-omics approaches, and commercial exploitation.

The production of mannosylerythritol lipids (MELs) has been reported on different smut fungi. These biomolecules possess four typical conformations to which key commercially interesting properties have been attributed. In vivo, MEL production could only be explained by the chain-shortening pathway, a new route, unlike the main three fatty acid synthesis pathways described. The production of MELs requires nitrogen starvation and a carbon source, usually a fatty acid, the principals. The first MEL biosynthetic gene cluster was elucidated in U. maydis by the combination of different methodologies, which resulted in a reference genome, on which five genes comprising the MEL cluster were annotated. Subsequently, the evolution of DNA sequencing technologies advanced genome and transcriptome assembly, allowing the annotation of more MEL producers' genomes. These, in combination with different experimental techniques, coupled to bioinformatic methods offer a plethora of genomic resources for further manipulation and commercial exploitation. In this review, we present the main findings, which unraveled MEL production and multi-omics studies, leading to molecular tools for further genomic manipulation and exploitation in smut fungi.

Uracil-auxotrophic marker recycling system for multiple gene disruption in Pseudozyma antarctica.

The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast Saccharomyces cerevisiae, and a Paura3 mutant (C867T) was obtained after 3 min of UV exposure. A uracil-auxotrophic marker (URA3) recycling system developed in ascomycetous yeasts and fungi was applied to the P. antarctica Paura3 strain. The PaLYS12 and PaADE2 loci were disrupted via site-directed homologous recombination of PaURA3 (pop-in), followed by the removal of PaURA3 (pop-out). In the obtained double auxotrophic strain (Palys12Δ, Paura3), PaADE2 was further disrupted, and PaURA3 was removed to obtain the triple auxotrophic strain PGB800 (Paura3, Palys12Δ, Paade2Δ). The whole-genome sequence of the PGB800 strain did not contain foreign genes used for genetic manipulation and disrupted PaADE2 and PaLYS12, and removed PaURA3, as planned.

Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus.

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

A putative transporter gene PtMMF1-deleted strain produces mono-acylated mannosylerythritol lipids in Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis ΔPtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by ΔPtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain ΔPtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain ΔPtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B. KEY POINTS: • P. tsukubaensis is able to secrete MELs without PtMMF1p. • Strain ΔPtMMF1 mainly produced mono-acylated MELs.

Characterization of the wheat cultivars against Tilletia controversa Kühn, causal agent of wheat dwarf bunt.

Wheat is one of the most important staple crops. Tilletia controversa Kühn is the causal agent of wheat dwarf bunt. In this study, a resistant wheat cultivar displayed significantly higher expression of pathogenesis-related genes than a susceptible cultivar at 7 days post inoculation (DPI) with T. controversa. Similarly, the expression was high in the resistant cultivar after exogenous application of phytohormones, including salicylic acid. The expression of pathogenesis-related genes, especially chitinase 4, was high in the resistant cultivar, while LPT-1 was down regulated after T. controversa infection. Callose deposition was greater in the resistant cultivar than in the susceptible cultivar at 10 DPI. Confocal microscopy was used to track the fungal hyphae in both cultivars in anther and ovary cells. The anthers and ovaries of the susceptible cultivar were infected by T. controversa at 7 and 15 DPI. There were no fungal hyphae in anther and ovary cells in the resistant cultivar until 10 and 23 DPI, respectively. Moreover, anther length and width were negatively influenced by T. controversa at 16 DPI. The plant height was also affected by fungal infection. Ultimately, resistance to T. controversa was achieved in cultivars via the regulation of the expression of defense-related and pathogenesis-related genes.

Discovery and characterization of a stable lipase with preference toward long-chain fatty acids.

OBJECTIVES: To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database. RESULTS: Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 °C, and stability between 40 and 50 °C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates. CONCLUSION: The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.