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

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.

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

“…After 12 h of induction, the volatile compounds in the culture supernatant produced were confirmed to be ethyl hexanoate (C 6 ), ethyl octanoate (C 8 ) and ethyl decanoate (C 10 ). Production by P. pastoris EHT1 was at 2, 10, and 11 %, respectively, and by EEB1 at 1, 7, and 13 %, respectively …”

Study of the Enzymatic Capacity of <b><i>Kluyveromyces marxianus</i></b> for the Synthesis of Esters

“…The synthesis of ethyl esters in yeast is produced by the condensation of medium chain fatty acids (C6-C14) with ethanol [Kruis et al, 2017;Zhuang et al, 2015]. This reaction requires the catalytic action of esterases, which are proteins with dual nature, since they can act as ethanol Oacyltransferases and as thioesterases (Eht1p, Eeb1p) [Ichikawa et al, 1991;Yin et al, 2019;Zelner et al, 2013].…”

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

“…The effectiveness of P. pastoris as a whole cell biocatalyst has been investigated for ester biosynthesis, acetophenone reduction . d ‐Amino acid oxidation and kinetic resolution of racemic α‐methylbenzylamine to 99% ee ( R )‐α‐methylbenzylamine .…”

“…Methylotrophic yeasts have also been observed to tolerate extreme overexpression of the alcohol oxygenase 1 (Aox1p) enzyme essential for methanol utilization, to a level where 30% of cell biomass can be taken up with this enzyme during growth phase. 26 The effectiveness of P. pastoris as a whole cell biocatalyst has been investigated for ester biosynthesis, 27 acetophenone reduction. 28 D-Amino acid oxidation 29 and kinetic resolution of racemic a-methylbenzylamine to 99% ee (R)-a-methylbenzylamine.…”