Biochemistry Research Laboratory (Rm216), Department of Life Sciences, Faculty of Science and Technology, The University of the West Indies, St. Augustine, West Indies, Trinidad and Tobago.
BMC Microbiol. 2022 Feb 4;22(1):43. doi: 10.1186/s12866-021-02419-4.
Mannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment.
M. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species.
Sequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.
甘露糖基赤藓糖醇脂质（MELs）属于糖脂生物表面活性剂类，由 Ustilago 和 Moesziomyces 属的成员产生。MEL 的生产受生物合成基因簇（MEL BGC）调控。细胞外脂肪酶活性也与 MEL 的产生有关。大多数微生物糖脂产生菌是从含油污染环境中分离出来的。能够代谢原油的产 MEL 酵母研究较少，热带气候来源的土著菌株的数据非常有限。采用基因靶向方法分析特立尼达岛南极酵母（M. antarcticus）菌株的 MEL BGC 和脂肪酶基因，发现其内在降解原油的能力与其适应长期污染陆地环境的能力之间存在相关性。
M. antarcticus 从特立尼达岛自然发生的原油渗出物和沥青泥火山中分离出来；这些是以前未报道过该物种存在的栖息地。通过大亚基（LSU）和小亚基（SSU）序列比较确认了属的鉴定，通过 ITS 序列比较和系统发育推断确认了种的鉴定。使用基因特异性引物检测到 MEL BGC 的必需基因（Emt1、Mac1、Mac2、Mmf1）。Emt1p、Mac1p 和 Mmf1p 序列分析证实，特立尼达岛菌株携带新的同义氨基酸（aa）取代，结构比较显示不同区域的无序，特别是 Emt1p 序列。通过基序挖掘和突变预测确认了每个蛋白质序列的功能。对 Emt1p、Mac1p 和 Mmf1p 序列进行了系统发育关系推断。特立尼达岛菌株与其他 M. antarcticus 序列聚类，但代表特立尼达岛 M. antarcticus 的序列始终为每个蛋白形成一个单独的、高度支持的分支。LipA 和 LipB 核苷酸和蛋白质序列的相似系统发育位置表明，根据基因的不同，存在早期和晚期的复制事件，随后在 Ustilaginaceae 内发生了多次物种形成事件。特立尼达岛菌株在培养中表现出脂肪酶活性，能够利用不同的碳源。MEL BGC 和 LipA 基因的比较进化表明，根据基因的不同，存在早期和晚期的复制事件，随后在 Ustilaginaceae 内发生了多次物种形成事件。特立尼达岛菌株在培养中表现出脂肪酶活性，能够利用不同的碳源。MEL BGC 和 LipA 基因的比较进化表明，根据基因的不同，存在早期和晚期的复制事件，随后在 Ustilaginaceae 内发生了多次物种形成事件。特立尼达岛菌株在培养中表现出脂肪酶活性，能够利用不同的碳源。MEL BGC 和 LipA 基因的比较进化表明，早期和晚期的复制事件取决于基因，随后在 Ustilaginaceae 内发生了多次物种形成事件。M. antarcticus 和 M. aphidis 与 Ustilaginaceae 的所有其他成员分离，检测到两个基因同源物，每个物种一个。
本研究基于一种新的基因靶向方法，分析了特立尼达岛南极酵母（M. antarcticus）菌株的 MEL BGC 必需基因和 LipA、LipB 基因的序列。研究结果表明，这些菌株积累的核苷酸突变达到了不会影响 MEL BGC 和 LipA、LipB 基因编码的特定蛋白质功能的阈值水平。这些特立尼达岛南极酵母菌株分泌的生物表面活性剂和脂肪酶有助于它们在受原油污染的陆地环境中生存。这些发现表明，这些菌株应该作为商业生产 MEL 生物表面活性剂和脂肪酶的有前途的候选菌株进行探索。
