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Front Microbiol
2024 Jan 01;15:1376653. doi: 10.3389/fmicb.2024.1376653.
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Deorphanizing solute carriers in Saccharomyces cerevisiae for secondary uptake of xenobiotic compounds.
Møller-Hansen I
,
Sáez-Sáez J
,
van der Hoek SA
,
Dyekjær JD
,
Christensen HB
,
Wright Muelas M
,
O'Hagan S
,
Kell DB
,
Borodina I
.
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The exchange of small molecules between the cell and the environment happens through transporter proteins. Besides nutrients and native metabolic products, xenobiotic molecules are also transported, however it is not well understood which transporters are involved. In this study, by combining exo-metabolome screening in yeast with transporter characterization in Xenopus oocytes, we mapped the activity of 30 yeast transporters toward six small non-toxic substrates. Firstly, using LC-MS, we determined 385 compounds from a chemical library that were imported and exported by S. cerevisiae. Of the 385 compounds transported by yeast, we selected six compounds (viz. sn-glycero-3-phosphocholine, 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, acrylic acid, 2-benzoxazolol) for characterization against 30 S. cerevisiae xenobiotic transport proteins expressed in Xenopus oocytes. The compounds were selected to represent a diverse set of chemicals with a broad interest in applied microbiology. Twenty transporters showed activity toward one or more of the compounds. The tested transporter proteins were mostly promiscuous in equilibrative transport (i.e., facilitated diffusion). The compounds 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, and sn-glycero-3-phosphocholine were transported equilibratively by transporters that could transport up to three of the compounds. In contrast, the compounds acrylic acid and 2-benzoxazolol, were strictly transported by dedicated transporters. The prevalence of promiscuous equilibrative transporters of non-native substrates has significant implications for strain development in biotechnology and offers an explanation as to why transporter engineering has been a challenge in metabolic engineering. The method described here can be generally applied to study the transport of other small non-toxic molecules. The yeast transporter library is available at AddGene (ID 79999).
Figure 1. Experimental workflows. (A) Workflow of the uptake assay using Xenopus oocytes. Defolicated oocytes were injected with mRNA encoding the transporter of interest and mRNA encoding GFP. After 3 days of incubation at 18 °C, the fluorescence from the GFP was quantified, and oocytes with an active expression of GFP were selected for the uptake assay. Oocytes expressing the transporter of interest were exposed to a solution of the selected compounds in Kulori buffer at pH 5.0 for 3 h. After incubation, the oocytes were harvested, and the intracellular content was extracted and analyzed by LC–MS. (B) Workflow of the exometabolome assay. An overnight yeast culture was harvested by centrifugation, washed, and resuspended in human blood serum. After 30 min incubation with the human blood serum, the supernatant was harvested and subjected to analysis by LC–MS. Figure generated using BioRender.
Figure 2. Compounds transported by yeast. (A) Variation observed between the repeats. CV, coefficient of variation. Compounds with a response below a log2FC = 0.5 were excluded. The selected compounds are highlighted in red. (B) The compounds identified in human blood serum were plotted against the compounds identified in the supernatant incubated with yeast. Compounds with a log2FC below 0.5 were excluded. Selected compounds are highlighted in red. (C) UMAP (McInnes et al., 2018a, 2018b) plot of the compounds with a log2FC above 0.5. Selected compounds are indicated with names. The compounds are grouped according to their classification in DrugBank 3.0 (https://www.drugbank.ca) and Recon 2 (Thiele et al., 2013) as detailed in O’Hagan and Kell (2017).
Figure 3. Principles of the oocyte import assay. Schematic representation of the oocyte import assay. For uptake, the intracellular concentration per oocyte is compared. (A) No or very little of the compounds are measured in the control oocytes. As no endogenous import activity is detected, only no activity (Log2FC = 0) or import activity (Log2FC > 0) can be detected for the expressed transporter proteins. The import can be categorized in two groups; concentrative import, where the intracellular concentration reached is higher than the concentration in the medium, and equilibrative import, where the intracellular concentration reached is similar or lower than the concentration found in the medium. (B) Some endogenous import activity is detected. With some endogenous import activity, export activity of the expressed transporter proteins can be measured (Log2FC < 0), in addition to no activity and import activity. In this scenario, two levels of import can also be reached; concentrative import and equilibrative import.
Figure 4. Oocyte import assay. Each compound was added at an extracellular concentration of 2 mM at pH 5.0. (A) Cefadroxil, (B) 2-benzoxazolol, (C) 2,5-furandicarboxylic acid, (D) sn-glycero-3-phosphocholine, (E) acrylic acid, (F) 2-methylpyrazine. The response was calculated as fold change compared to the response of the GFP injected control oocytes. Each data point consists of data from 7 to 10 oocytes in triplicates and each experiment was repeated on several occasions. The intracellular concentration in the single oocytes was calculated depending on the number of oocytes used in the individual experiment and used for calculating the Log2 fold change compared to the GFP control. Each point is a replicate based on extraction data from 10 oocytes. Significance was tested with a two-sided Students t-test, for transporters having an intracellular concentration higher than the GFP expressing controls. Significance levels indicated in the graphs are: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 5. The mode of transport. (A) Schematic representation of the comparison with the medium concentration. A Log2FC > 0 indicates a concentrative mode of transport, as the internal concentration in the oocytes exceeds the concentration found in the medium. A Log2FC < 0 indicates an equilibrative mode of transport, as the internal concentration found in the oocyte does not exceed the concentration found in the medium. (B) The significant transporters identified in the oocyte uptake assay are plotted together with the relative concentration found in the medium. All values are calculated as Log2FC compared to the extracellular concentration found in the medium. Significance was tested with a one-sided Students t-test, for transporters having an intracellular concentration higher than the medium. Each point is a replicate based on extraction data from 10 oocytes. Significance levels indicated in the graphs are: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 6. Transporter mutant assays. Growth assay of strains with the transporter proteins identified in the screening effort. From the growth curves generated in the growth profiler, maximum OD reached (Max OD), and maximum specific growth rate (μmax) were calculated. All strains were grown in the medium composition SD –Trp, and in SD –Trp supplemented with the indicated compounds at LC50 concentration. The LC50 concentration was established in a pre-experiment using the wildtype BY4741 strain. (A) Growth assay of strains harboring deletions of transporters genes identified in the screening effort as transporter proteins having transport activity toward acrylic acid. The assay was conducted with 2.45 mM acrylic acid. (B) Growth assay of strains harboring deletions of transporters genes identified in the screening effort as transporter proteins having transport activity toward 2-benzoxazolol. The assay was conducted with 6.44 mM 2-benzoxazolol. (C) Growth assay of strains harboring deletions of transporters genes identified in the screening effort as transporter proteins having transport activity toward methylpyrazine. The assay was conducted with 155.15 mM methylpyrazine. Significance levels indicated in the graphs are: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 7. Phylogenetic tree of tested transporters with identified substrates. The tree was generated with Clustal Omega (Sievers et al., 2011) version 1.2.4 using the standard parameters. The generated phylogenetic tree was visualized with iTOL (Letunic and Bork, 2007, 2021). The transporter proteins are color-coded according to superfamily. The transporter superfamilies are abbreviated as follows: the glycerol uptake (GUP) superfamily, the auxin efflux carrier (AEC) family from the bile/arsenite/riboflavin transporter (BART) superfamily, drug/metabolite transporter (DMT) superfamily, the sideroflexin (SFXN) family, the bile acid: Na+ symporter (BASS) family, the cyclin M Mg2+ exporter (CNNM) family, the lysosomal cystine transporter (LCT) family, the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) flippase superfamily.
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