Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance

Xin Xu,Thomas C Williams,Ian T Paulsen,Christina Divne,Isak S Pretorius

doi:10.1186/s13068-019-1427-6

Abstract

BackgroundPropionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.ResultsAdaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR–Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.ConclusionOur study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.

Highlights

Propionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries
The growth assay was conducted in liquid medium, which showed the same trend of improved growth with potassium supplementation under PA stress, and with TRK1 mutation (Additional file 1: Fig. S5). These results indicate the function of TRK1, which encodes the high-affinity potassium transporter, is essential for the tolerance to PA in yeast, and that the evolved TRK1 mutations enable a higher uptake of potassium under PA stress
This study demonstrates Adaptive laboratory evolution (ALE) combined with whole genome re-sequencing and functional analysis can be used to unveil non-intuitive mechanisms of PA tolerance in yeast

Summary

Introduction

Propionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. Propionic acid (PA), a key building-block chemical, has been widely used as a food preservative and a chemical intermediate in plastics, pharmaceutical, cosmetic, paint, and herbicide industries [1,2,3,4]. Yeast cells increase proton export via plasma membrane and vacuolar H+-ATPases to maintain pH homeostasis in response to multiple organic acids [20,21,22,23]. Several transcriptional regulators have been identified that mediate the response to organic acid stress in yeast. SPI1, encoding a glycosylphosphatidylinositol (GPI)-anchored cell wall protein, was identified to play a key role in yeast response to 2,4-dichlorophenoxyacetic acid [27], octanoic acid and benzoic acid [28]

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Results

Discussion

Conclusion