Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

Abstract

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.

Contributions of Adaptive Laboratory Evolution towards the Enhancement of the Biotechnological Potential of Non-Conventional Yeast Species

“…With this thermal study, Dallinger showed that all evolved organisms were able to survive at 70 °C, in opposition to the initial ones. Reports of ALE employing S. cerevisiae and Escherichia coli , the most well-known species within the yeast and bacteria groups, respectively, alongside microalgae, viruses, and mammalian cell lines, have grown exponentially in recent years [ 3 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].…”

“…During the evolutionary process, aliquots of the evolving populations are usually stocked in glycerol (within 10% to 40%, v/v ) and maintained at −80 °C for further phenotypic analysis [ 35 , 36 , 37 ]. The primary advantages are that this strategy is very easy to set up and use, requires low-cost equipment, can usually be executed by a single operator, and can be adapted to massive parallel cultures [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ], as summarized in Table 1 . However, batch cultures are more susceptible to random drift due to certain shortfalls, which include variations in the population density, growth rates, and different nutrient supplementations, together with fluctuations in environmental conditions [ 29 ].…”

Hybrid adaptation is hampered by Haldane’s sieve

“…We used ChimeraX v1.5 153 to visualize Pdr1p amino acid changes we found throughout the experimental evolution and other mutations found in the literature [75][76][77][78][79] (Source Data 2) on the AlphaFold 154,155 generated structure for Pdr1p (AF-P12383-F1, Source Data 3). Subsequently, we analyzed Pdr1 Nakaseomyces glabratus protein by superimposing structures and amino acid changes 80,83,84 across species (Source Data 4; AF-B9VI40-F1, Source Data 5).…”

Engineering the synthesis of unsaturated fatty acids by introducing desaturase improved the stress tolerance of yeast

“…17 The transcription factor Pdr1p mutation (C862R) was reported to increase unsaturated fatty acid/saturated fatty acid ratio by 42%, decrease cell membrane damage by 81% and increase the 2-phenylethanol tolerance of S. cerevisiae. 18 The study reported here investigated the effect of engineering unsaturated fatty acid synthesis on the stress tolerance in yeast. Two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed singly or simultaneously in S. cerevisiae to construct recombinant cells.…”

“…In addition, antioxidant dipeptides have also been reported to improve the cell membrane integrity of Saccharomyces pastorianus by increasing the proportion of unsaturated fatty acids and regulating the expression of key genes in fatty acid synthesis, such as the expression of genes ACC1 and HFA1 17 . The transcription factor Pdr1p mutation (C862R) was reported to increase unsaturated fatty acid/saturated fatty acid ratio by 42%, decrease cell membrane damage by 81% and increase the 2‐phenylethanol tolerance of S. cerevisiae 18 …”