Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

Abstract

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surfaceactive properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA 004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA 004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained PaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed PaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.

Genome and Transcriptome Analysis of the Basidiomycetous Yeast Pseudozyma antarctica Producing Extracellular Glycolipids, Mannosylerythritol Lipids

“…MELs are possibly secreted by the putative transporter ( PaMMF1 ). Of the 5 genes, PaEMT1 of P. antarctica has been characterized by developing the gene deletion mutant, lacking the MEL production property [59]. This result supports the conclusion that the gene cluster of P. antarctica works in the same way as that of U. maydis .…”

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

“…For example, the hygromycin resistance gene consisting of an E. coli hygromycin phosphotransferase gene and Ustilago maydis HSP70 promoter and terminator has been used to introduce recombinant green florescent protein genes into P. flocculosa and P. antarctica (Cheng et al ., ). The construct was also used to disrupt a gene essential for MEL synthesis (Pa EMT1 ) in P. antarctica T‐34 using homologous recombination (Morita et al ., ). Then a Pa emt1 Δ clone was obtained among tested over 160 hygromycin resistant clones (unpublished data).…”

“…Pseudozyma yeasts also produce mannosylethritol lipids (MELs), which are valuable biosurfactants used in cosmetics (Morita et al ., ). A mutant strain deficient in MEL production was generated from P. antarctica T‐34 by replacement of a gene essential for MEL synthesis (Pa emt1 Δ) using homologous recombination with the hygromycin resistance gene as a selection marker (Morita et al ., ). When grown on a plant surface, the wild strain changed morphology from single cells to elongated ones for a short period and colonized a large area, but the Pa emt1 Δ strain did not (Yoshida et al ., ).…”

Production and characterization of novel self-assembling biosurfactants from Aspergillus flavus

“…Decanted both layers to different containers. Ethyl acetate fraction was dried at 65°C on a hotplate (Irmeco GmBH, MSH00195, Geesthacht, Germany) until ethyl acetate was evaporated and mud like thick paste remained at the bottom of container (Morita et al 2010). The biosurfactant paste (10 ml) extracted by ethyl acetate was re-suspended in 100 ml methanol and centrifuged at 2697 g for 3Á0 min to sediment the biosurfactant.…”