Production of the Fragrance Geraniol in Peroxisomes of a Product-Tolerant Baker's Yeast.

Jennifer Gerke,Anja Poehlein,Nigel S Scrutton,Dominik Schneider,Thomas Hofmeister,Ziga Zebec,Eriko Takano,Gerhard H Braus,Holm Frauendorf,Maxim Wintergoller

doi:10.3389/fbioe.2020.582052

Abstract

Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes of Saccharomyces cerevisiae as production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes.

Highlights

The metabolic engineering of microorganisms such as Saccharomyces cerevisiae is a promising tool for the eco-friendly production of aromas, fragrances, biofuels, and pharmaceutics (Zebec et al, 2016)
We evaluated two approaches for their suitability to be integrated into the engineering toolbox to increase geraniol product titers in S. cerevisiae cells for industrial biomanufacturers: increasing the tolerance of yeast cells to geraniol and compartmentalizing the geraniol producing enzymes into peroxisomes as production sites (Figure 1)
In order to obtain strains with a constant high number of peroxisomes, the genes for the peroxisome number controlling proteins Pex30, Pex31, and Pex32, as well as for the pexophagy receptor Atg36 were deleted in all possible combinations in LW2591Y, resulting in 15 deletion strains (Supplementary Table S1)

Summary

Introduction

The metabolic engineering of microorganisms such as Saccharomyces cerevisiae is a promising tool for the eco-friendly production of aromas, fragrances, biofuels, and pharmaceutics (Zebec et al, 2016). The following protein-encoding genes were integrated under the control of strong, constitutive promoters into the S. cerevisiae chromosomal genome of pex30 /pex and pex30 /pex31 /atg (Supplementary Figure S6A): an N-terminally truncated 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase 1 without feedback-regulation (trHMG1) (Donald et al, 1997; Rico et al, 2010); the isopentenyl diphosphate (IPP) isomerase Idi (Ignea et al, 2011); the negative regulator of RNA polymerase III Maf, which represses tRNA biosynthesis from IPP (Liu et al, 2013); and the FPP-synthase from Gallus gallus carrying a point mutation for the N144W amino acid substitution (mFPSN144W ), which produces high amounts of GPP (Stanley Fernandez et al, 2000). The resulting strains were named Sen (“Sensitive,” in pex30 /pex31 /atg with low geraniol tolerance) and Tol (“Tolerant,” in pex30 /pex with high geraniol tolerance) We have integrated these genes on the chromosomes to enhance genetic stability. Whereas for trHMG1, MAF1, and mFPSN144W the gene expression was increased in Tol and Sen compared to LW2591Y, the expression level of IDI1 was comparable to LW2591Y in both strains

Objectives

Methods

Results