茭白筍黑穗菌(Ustilago esculenta Henning)可以感染茭白(Zizania latifolia Turcz.),造成寄主植物莖基部膨大。而該膨大的部分,在亞洲地區廣泛地被人們食用,因此為極具高經濟價值的蔬菜作物。本團隊培養茭白筍黑穗菌於特定培養基上,意外發現其具有抑菌活性推測該抑菌活性可能與特定二次代謝物的產生有關。透過基因體序列的分析與篩選,鑑定出一橫跨約47 kb染色體區域,包含13個基因的基因簇(gene cluster)。而其中11個基因,與兩個相近物種U. maydis及Pseudozyma flocculosa基因體中,生合成纖維二糖脂(cellobiose lipid)基因簇的同源基因序列高度相似。這些基因除了在茭白筍黑穗菌基因體的位置相鄰外,啟動子(promoter)中亦可鑑定出共有的短片段保守序列(consensus motif);配合基因表現實驗的驗證,顯示該基因簇的基因在轉錄表現上受到共同調控。除此之外,利用HPLC分離純化及NMR和質譜分析, 解析茭白筍黑穗菌所產生之纖維二糖脂化合物的結構。在純化產物中,至少有 四種結構類似物存在,其中一主要產物中包含2,15,16-三羥基棕櫚酸,以糖苷鍵與纖維二糖相連,而糖基上有乙醯基及3-羥基辛酸,結構 與黑穗菌酸B(ustilagic acid B)相同。而副產物中包括未報導的新化合物,長碳鏈為2,5,16-三羥基棕櫚酸甲酯,而較短鏈脂肪酸則為無羥基修飾之己酸。活性測試顯示,茭白筍黑穗菌所產生的纖維二糖脂,不僅具界面活性 ,並有廣泛地抗菌活性,可抑制Magnaporthe oryzae、Sclerotrium rolfsii、Rhizopus microsporus、Phytophthora parasitica、Candida albicans、Cryptococcus neoformans、Staphylococcus aureus等病原菌。未來在工業、醫藥與生農相關領域,皆有相當應用之潛力。本研究首次報導茭白筍黑穗菌為一新發現纖維二糖脂之產生菌株 ,可合成此類具有二極性及抗菌活性的罕見糖脂。
The smut fungus Ustilago esculenta Henning can parasitize on Zizania latifolia Turcz., one kind of wild rice species, to cause smut disease. The infection leads to production of swollen tissues at host stem base; however, the swollen galls are edible and have been a favorable vegetable crop, called Jiaobai in Taiwan, in Asia regions. Studies in our laboratory found that U. esculenta showed antimicrobial activity on certain media, suggesting that secondary metabolite(s) may be produced by U. esculenta for this inhibitory activity. With genome analysis and screening, a 13-gene cluster spanning 47 kb genomic region was identified. Eleven genes inside the cluster showed high similarities with the homologues of two gene clusters, responsible for the production of cellobiose lipids, in two closely related fungi, U. maydis and Psuedozyma flocculosa. Besides physical co-localization in the genome, consensus motifs in the promoter regions of all these genes were identified, and expression studies confirmed that the genes in the cluster were transcriptionally co-regulated. Furthermore, chemical compounds were fractioned by HPLC and structurally determined by NMR and mass spectrometry. Cellobiose lipids produced by U. esculenta were composed of at least four related derivatives. The major product contained 2,15,16-trihydroxypalmitic acid glycosidically linked to cellobiose which the sugar moieties were acetylated and acylated with 3-hydroxyoctanoic acid, known as ustilagic acid B. Among the minor products, a novel compound containing 1-methyl-2,15,16-trihydroxypalmitate was identified. Surface activity and antimicrobial activities against most fungi and Gram positive bacteria showed promising potential of these cellobiose lipids for industrial, medical, and agricultural application. In this study, we report U. esculenta as a novel cellobiose lipid producer synthesizing rare amphipathic glycolipids with antimicrobial activity.