A Gene Cluster for Biosynthesis of Mannosylerythritol Lipids Consisted of 4-O-β-D-Mannopyranosyl-(2R,3S)-Erythritol as the Sugar Moiety in a Basidiomycetous Yeast Pseudozyma tsukubaensis.

Azusa Saika,Tokuma Fukuoka,Shuhei Yamamoto,Hideaki Koike,Takahide Kishimoto,Tomotake Morita,Arthur J Lustig

doi:10.1371/journal.pone.0157858

Abstract

Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants and are produced by various fungi. The basidiomycetous yeast Pseudozyma tsukubaensis produces diastereomer type of MEL-B, which contains 4-O-β-D-mannopyranosyl-(2R,3S)-erythritol (R-form) as the sugar moiety. In this respect it differs from conventional type of MELs, which contain 4-O-β-D-mannopyranosyl-(2S,3R)-erythritol (S-form) as the sugar moiety. While the biosynthetic gene cluster for conventional type of MELs has been previously identified in Ustilago maydis and Pseudozyma antarctica, the genetic basis for MEL biosynthesis in P. tsukubaensis is unknown. Here, we identified a gene cluster involved in MEL biosynthesis in P. tsukubaensis. Among these genes, PtEMT1, which encodes erythritol/mannose transferase, had greater than 69% identity with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. However, phylogenetic analysis placed PtEMT1p in a separate clade from the other proteins. To investigate the function of PtEMT1, we introduced the gene into a P. antarctica mutant strain, ΔPaEMT1, which lacks MEL biosynthesis ability owing to the deletion of PaEMT1. Using NMR spectroscopy, we identified the biosynthetic product as MEL-A with altered sugar conformation. These results indicate that PtEMT1p catalyzes the sugar conformation of MELs. This is the first report of a gene cluster for the biosynthesis of diastereomer type of MEL.

Highlights

Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants which consist of mannosylerythritol (ME) as the hydrophilic moiety, and fatty acids as the hydrophobic moiety
Based on BLASTP analyses, the genes involved in MEL biosynthesis were found in the draft genome of P. tsukubaensis NBRC1940 (PtEMT1, PtMAC1, PtMAC2, PtMMF1 and PtMAT1, respectively) (Fig 4)
The gene arrangement in the MEL biosynthesis cluster of P. tsukubaensis NBRC1940 is more similar to U. maydis than P. antarctica strains JCM10317 and T-34 (Fig 4)

Summary

Introduction

Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants which consist of mannosylerythritol (ME) as the hydrophilic moiety, and fatty acids as the hydrophobic moiety. A fungal plant pathogen, has been identified as a MEL producer [8, 9] and its complete genome has been sequenced and published [10]. Genome sequences of species from the genus Pseudozyma have been reported. These species include P. antarctica T-34 and JCM10317[14, 15], P. aphidis DSM70725 [16] and P. hubeiensis SY62 [17]. Gene expression vectors and transformation methods have been developed for this genus [18,19,20] Owing to their high productivity and the availability of gene manipulation techniques [21], these Pseudozyma species are promising candidates for the commercial production of MELs

Methods

Results

Conclusion