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

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.

Keywords: Saccharomyces cerevisiae; compartmentalization; geraniol; monoterpenoids; peroxisomes; tolerance.

FIGURE 1

Engineering of tolerant S. cerevisiae cells for compartmentalized geraniol production in peroxisomes. Peroxisome number is increased by deleting the genes for the peroxins Pex30 and Pex32. A truncated Bul1 α-arrestin-like adaptor protein, caused by a point-mutation in the corresponding gene, increases geraniol tolerance. Introduction of the geraniol biosynthetic enzymes into the peroxisomes leads to geraniol production.

FIGURE 2

Strains with all possible combinations of PEX30, PEX31, PEX32, and ATG36 deletions show increased peroxisome numbers. (A) Fluorescence microscopy of the reference S. cerevisiae strain LW2591Y, pex30Δ/pex32Δ, and pex30Δ/pex31Δ/atg36Δ with integrated mCherry-SKL under a constitutive promoter. Yeast strains were grown overnight in liquid YPD medium at 30°C, diluted and used for microscopy. Due to the reiterative recombination system site in the parental strain, all strains carry gfp integrated into the genome. Scale bar = 10 μm. (B) Quantification of the number of peroxisomes per cell detected via fluorescence microscopy in the parental strain LW2591Y and all PEX30, PEX31, PEX32, and ATG36 deletion combinations with introduced mCherry-SKL in YPD medium. For counting of the cells and peroxisomes in total 76 fluorescence images from two biological replicates were taken and the peroxisome/cell ratios were calculated. The 76 ratios are depicted in a Tukey’s box whisker plot. The p-value for each strain was calculated in comparison to LW2591Y (two-sample T-test). ***p ≤ 0.01; **p ≤ 0.05.

FIGURE 3

The S. cerevisiae strains pex30Δ, pex32Δ, and pex30Δ/pex32Δ with a truncated Bul1 protein are highly tolerant to geraniol. (A) Spot-tests of the reference strain LW2591Y and all single, double, triple, and quadruple deletion mutants of PEX30, PEX31, PEX32, and ATG36 on a synthetic complete medium (SC), a yeast extract peptone medium with glucose (YPD) or with oleate (YPO) as sole carbon-source and on YPD with 200 mg/l geraniol. A serial dilution was used (OD = 10^–1, 10^–2, 10^–3, 10^–4). The plates were incubated at 30°C for 3 days for SC, YPD, and YPD + geraniol and for 6 days for YPO medium. (B) Schematic representation of the truncated Bul1 proteins present in the tolerant strains pex30Δ, pex32Δ, and pex30Δ/pex32Δ. The wildtype Bul1 harbors an N-terminal conserved domain (Bul1_N) with the ubiquitin ligase binding motif PPSY and a C-terminal conserved domain (Bul1_C). pex30Δ carries a SNP from G to T at position 1579 bp of the BUL1 gene, leading to a stop-codon and a truncated Bul1 protein with 526 aa (Bul1^E527*). pex32Δ carries an additional adenine and pex30Δ/pex32Δ an additional thymine at position 2125 bp of the BUL1 gene, leading to a truncated Bul1 protein with 713 aa (Bul1^1–708+5aa). The nucleotide insertions induce a frameshift, leading to the insertion of an early stop-codon in addition to 5 mutated amino acids at the end of the truncated protein.

FIGURE 4

Schematic representation of the engineered biosynthetic pathway for geraniol in S. cerevisiae. Geranyl pyrophosphate (GPP) is produced in wildtype S. cerevisiae via the mevalonate pathway (dark gray box). One copy of the genes encoding the blue enzymes were introduced into the yeast genome. The genes for the green enzymes were introduced on a high-copy plasmid. For peroxisomal localization of the green enzymes, the peroxisomal target sequence 1 (PTS1), consisting of the amino acids SKL, was attached to the C-terminus. All genes were expressed under constitutive promoters. The genes encoding the red, crossed enzymes have been deleted from the genome.

FIGURE 5

Tolerance to geraniol combined with peroxisomal enzyme localization in yeast strains with high peroxisome numbers yields highest geraniol titer in this study. (A) Growth curves for indicated S. cerevisiae mutants. Data give the means with standard deviation from two biological with each three technical replicates. (B) Relative geraniol titer calculated from absolute values in Table 1. Data give the means with percentage difference from two biological with each two technical GCMS replicates.