Production of medium-chain volatile flavour esters in Pichia pastoris whole-cell biocatalysts with extracellular expression of Saccharomyces cerevisiae acyl-CoA:ethanol O-acyltransferase Eht1 or Eeb1.

Shiwen Zhuang,Chris Powell,Yihe Xia,Ruixiang Yan,Jinhai Huang,Junshu Fu

doi:10.1186/s40064-015-1195-0

Shiwen Zhuang, Chris Powell + Show 4 more

Open Access

https://doi.org/10.1186/s40064-015-1195-0

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Abstract

Medium-chain volatile flavour esters are important molecules since they have extensive applications in food, fragrance, cosmetic, paint and coating industries, which determine different characteristics of aroma or taste in commercial products. Biosynthesis of these compounds by alcoholysis is catalyzed by acyl-CoA:ethanol O-acyltransferases Eht1 or Eeb1 in Saccharomyces cerevisiae. In this study, these two yeast enzymes were selected to explore their preparations as the form of whole cell biocatalysts for the production of volatile flavour esters. Here, the novel whole cell biocatalysts Pichia pastoris yeasts with functional extracellular expression of Eht1 or Eeb1 were constructed. Flavour production was established through an integrated process with coupled enzyme formation and ester biosynthesis in the recombinant yeasts in one pot, leading to the formation of volatile C6–C14 methyl and ethyl esters from wort medium. Interestingly, there is no significant difference between P. pastoris-EHT1 and P. pastoris-EEB1 in substrate preference during flavour biosynthesis, indicating a similar role of Eht1 and Eeb1 in P. pastoris cells, in contradiction with previous findings in S. cerevisiae to some extent. Consequently the study not only provides a greater understanding of these two enzymes in a heterogeneous host, but also demonstrated the positive effect of the recombinant Eht1 and Eeb1 in ester formation by P. pastoris live cells, potentially paving the way towards achieving efficient production of volatile flavour by an integrated biocatalytic system composed of recombinant enzyme production and flavour biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1195-0) contains supplementary material, which is available to authorized users.

Highlights

Flavour compounds have a wide range of applications in food, beverage, cosmetic and pharmaceutical fields (Noguerol-Pato et al 2012; Cheong et al 2013; Descours et al 2013; Madruga et al 2013; Zamzuri and Abd-Aziz, 2013)
Gene cloning of sequence alignment of EHT1 and EEB1 The genes EHT1 and EEB1 were cloned from a wildtype S. cerevisiae A1 (Huang et al 2010a, b) and gene sequences are available in NCBI database
In order to expand the biosynthesis of volatile flavours, we investigated the whole-cell biocatalysis model of Eht1 and Eeb1 by using recombinant P. pastoris live cells

Summary

Introduction

Flavour compounds have a wide range of applications in food, beverage, cosmetic and pharmaceutical fields (Noguerol-Pato et al 2012; Cheong et al 2013; Descours et al 2013; Madruga et al 2013; Zamzuri and Abd-Aziz, 2013). In the global consumer-driven market, one of the challenges for manufacturers is to create products faster and cheaper while continuing to offer consumers. One development was the introduction of enzymatic and whole-cell biocatalysis. The enzymes isolated from organisms provide several benefits, such as simpler reaction apparatus, higher productivity owing to higher catalyst concentration, and simpler product purification. An enzyme-based catalysis always relies on enzyme reaction kinetics and enzyme stability in aqueous solutions. Only enzymes that were abundantly produced by cells could be used in industrial applications

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