We isolated an aromatic strain of yeast (M2013310) from chili sauce. Assembly, annotation, and phylogenetic analysis based on genome sequencing, identified M2013310 as an allodiploid yeast that was closely related to
Zygosaccharomyces rouxii
. During fermentation, M2013310, produced an aromatic alcohol with a rose-honey scent; gas chromatography tandem mass spectrometry identified this alcohol as 2-phenylethanol. The concentration of 2-phenylethanol reached 3.8 mg/L, 1.79 g/L, and 3.58 g/L, in M3 (NH
4
+
), M3 (NH
4
+
+ Phe), and M3 (Phe) culture media, after 72 h of fermentation, respectively. The mRNA expression levels of
ARO8
encoding aromatic aminotransferases I and
ARO10
encoding phenylpyruvate decarboxylase by M2013310 in M3 (Phe) were the lowest of the three different forms of media tested. These results indicated that M2013310 can synthesize 2-phenylethanol via the Shikimate or Ehrlich pathways and the production of 2-phenylethanol may be significantly improved by the over-expression of these two genes. Our research identified a promising strain of yeast (M2013310) that could be used to improve the production of 2-phenylethanol.
2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for “natural” 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.
Background
2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design.
Results
To improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19–2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%–101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher).
Conclusions
In the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.
Zygosaccharomyces rouxii is an osmotolerant and halotolerant yeast that can participate in fermentation. To understand the mechanisms of salt and sugar tolerance, the transcription levels of Z. rouxii M 2013310 under 180 g/L NaCl stress and 600 g/L glucose stress were measured. The transcriptome analysis showed that 2227 differentially expressed genes (DEGs) were identified under 180 g/L NaCl stress, 1530 DEGs were identified under 600 g/L glucose stress, and 1278 DEGs were identified under both stress conditions. Then, KEGG enrichment analyses of these genes indicated that 53.3% of the upregulated genes were involved in the ergosterol synthesis pathway. Subsequently, quantitative PCR was used to verify the results, which showed that the genes of the ergosterol synthesis pathway were significantly upregulated under 180 g/L NaCl stress. Finally, further quantitative testing of ergosterol and spotting assays revealed that Z. rouxii M 2013310 increased the amount of ergosterol in response to high salt stress. These results highlighted the functional differences in ergosterol under sugar stress and salt stress, which contributes to our understanding of the tolerance mechanisms of salt and sugar in Z. rouxii.
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