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Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree-negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg/L of ergothioneine in small-scale cultivation, with an additional copy of each gene improving the titer to 205 mg/L. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate-limited fed-batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g/L ergothioneine in 220 hours, corresponding to an overall volumetric productivity of 7.41 mg L-1 h-1 , showing that Yarrowia lipolytica is a promising host for ergothioneine production.

Graphical abstract, Highlights • Transporter proteins are essential for substrate uptake, product export, and intercompartmental metabolite exchange. • Transporter activity of a cell can be engineered to improve the production of metabolites. • Most of transporters are not functionally characterized. • Transporter characterization demands specialized techniques, using Xenopus oocytes, membrane vesicles, or electrophysiology., Engineering the transport of small molecules is an effective approach to improve the performance of microbial cell factories. Transporter engineering can improve the utilization of low-cost alternative substrates, reduce the loss of pathway intermediates, and increase the titer and production rate of the target product. However, transporters are not commonly engineered in strain development programs because the functions of most of the transport proteins are not known. In the recent years, a variety of methods have been developed for identification of transporters for specific substrates and for characterizing transport mechanisms. This review presents recent examples of successful transport engineering for cell factories and discusses the methods for transporter identification and characterization.

L-(+)-Ergothioneine is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise it, so can acquire it only from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseasesin vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker’s yeastSaccharomyces cerevisiaeto produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism ofS. cerevisiaewas altered by knock-out ofTOR1 orYIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in bioreactors. BecauseS. cerevisiaeis a GRAS (‘generally recognised as safe’) organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

Significance The export of organic acids is typically proton or sodium coupled and requires energetic expenditure. Consequently, the cell factories producing organic acids must use part of the carbon feedstock on generating the energy for export, which decreases the overall process yield. Here, we show that organic acids can be exported from yeast cells by voltage-gated anion channels without the use of proton, sodium, or ATP motive force, resulting in more efficient fermentation processes., Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion–sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.