Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad

Abstract

Background 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. Results 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. Conclusions 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.

Promising Application, Efficient Production, and Genetic Basis of Mannosylerythritol Lipids

“…Extensive clustering of functionally co-regulated gene families exists in closely related Basidiomycota fungi and allows for the stabilization of expression patterns [174]. The gene cluster encoding MELs consists of five genes: MAT1, MMF1, MAC1, EMT1, and MAC2 [155,175] (Figure 5). As the number of Basidiomycetes and some other fungal lineages within a sequenced genome expand, thorough and systematic comparisons of their genomic data will illuminate insights throughout this phylum and will help to better understand the mechanisms by which these clusters form and function and ultimately to genetically manipulate these gene clusters.…”

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

“…42,43 Yeast strains of the genus Pseudozyma (current Moesziomyces) and Ustilago abundantly produce MELs. 22,23,[35][36][37]42,[44][45][46][47][48][49][50][51][52] These basidiomycetes produce the glycolipid primarily when the culture reaches the stationary phase, which is usual for secondary metabolites.…”

“…This discovery suggests that the sugar moiety's biosynthetic pathway is more conserved than the hydrophobic moiety. 48 Consequently, the following section describes these key production parameters.…”