Deficiency of biodegradable plastic-degrading enzyme production in a gene-deletion mutant of phyllosphere yeast, Pseudozyma antarctica defective in mannosylerythritol lipid biosynthesis

Azusa Saika,Hiroko Kitamoto,Tohru Yarimizu,Tomotake Morita,Hideaki Koike,Takashi Watanabe

doi:10.1186/s13568-019-0825-2

Abstract

The basidiomycetous yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) produces extracellular enzymes and glycolipids, including mannosylerythritol lipids (MELs), which are biosurfactants. Strain GB-4(0) of this species was previously isolated from rice husks and produces biodegradable plastic-degrading enzyme (Pseudozyma antarctica esterase; PaE). In this study, we generated a MEL biosynthesis-deficient strain (∆PaEMT1) by deleting the gene PaEMT1, which is essential to MEL biosynthesis in strain GB-4(0). The resulting ∆PaEMT1 strain showed deficient PaE activity, and the corresponding signal was hardly detected in its culture supernatant through western blotting analysis using rabbit anti-PaE serum. On the other hand, the relative expression of the gene PaCLE1, encoding PaE, was identical between GB-4(0) and ∆PaEMT1 based on quantitative real-time PCR. When strain ∆PaEMT1 was grown in culture media supplemented with various surfactants, i.e., Tween20, BRIJ35 and TritonX-100, and MELs, PaE activity and secretion recovered. We also attempted to detect intracellular PaE using cell-free extract, but observed no signal in the soluble or insoluble fractions of ∆PaEMT1. This result suggested that the PaCLE1 gene was not translated to PaE, or that expressed PaE was degraded immediately in ∆PaEMT1. Based on these results, MEL biosynthesis is an important contributor to PaE production.

Highlights

Pseudozyma antarctica has the ability to produce some materials, including enzymes and glycolipids (Boekhout and Fell 1998)
These results indicate that PaEMT1 was replaced with natMX4, and that mannosylerythritol lipids (MELs) biosynthesis was defective in the transformants, showing that PaEMT1 is essential for MEL biosynthesis in GB-4(0)
In this study, we found that the biosynthesis of extracellular glycolipids known as MELs contributes to production of an extracellular esterase, PaE, in P. antarctica GB-4(0)

Summary

Introduction

Pseudozyma antarctica (currently designated Moesziomyces antarcticus) has the ability to produce some materials, including enzymes and glycolipids (Boekhout and Fell 1998). We have focused on an esterase (Pseudozyma antarctica esterase; PaE) that degrades biodegradable plastics (BP), including poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate). P. antarctica strains isolated from rice husks secrete relatively high amounts of PaE into the culture supernatant, and PaE production is strongly enhanced when these strains are cultivated with xylose (Watanabe et al 2014). PBSA, PBS and commercially available BP mulch films submerged in the culture supernatant of P. antarctica are rapidly degraded (Watanabe et al 2014). To accelerate PaE utilization, we constructed a recombinant strain that is able to produce large amounts of PaE (13.4-fold higher amount than that of the wild-type strain) (Watanabe et al 2016). A targeted gene manipulation method has been developed recently

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Conclusion