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Abstract
Development of a counter-selection method for phenylalanine auxotrophy could be a useful tool in the repertoire of yeast genetics. Fluorinated and sulfurated precursors of phenylalanine were tested for toxicity in Saccharomyces cerevisiae. One such precursor, 4-fluorophenylpyruvate (FPP), was found to be toxic to several strains from the Saccharomyces and Candida genera. Toxicity was partially dependent on ARO8 and ARO9, and correlated with a strain’s ability to convert FPP into 4-fluorophenylalanine (FPA). Thus, strains with deletions in ARO8 and ARO9, having a mild phenylalanine auxotrophy, could be separated from a culture of wild-type strains using FPP. Tetrad analysis suggests FPP resistance in one strain is due to two genes. Strains resistant to FPA have previously been shown to exhibit increased phenylethanol production. However, FPP resistant isolates did not follow this trend. These results suggest that FPP could effectively be used for counter-selection but not for enhanced phenylethanol production.
Highlights
The phenylalanine biosynthetic pathway is a route to production of the high-value chemical2-phenylethanol that is used for its rosy scent in cosmetics, foods, and cleaning supplies [1].Phenylethanol is produced mainly by synthetic chemical processes and naturally by yeast, as a degradation product of phenylalanine, moving through the intermediate metabolites of phenylpyruvate phenylacetaldehyde (Figure 1)
FPP resistant isolates did not follow this trend. These results suggest that FPP could effectively be used for counter-selection but not for enhanced phenylethanol production
The use of toxic metabolites to counter-select for auxotrophies such as uracil or tryptophan, has been a boon in yeast genetics, enabling numerous techniques such as the plasmid shuffle and Synthetic Genetic
Summary
The phenylalanine biosynthetic pathway is a route to production of the high-value chemical. One such mechanism is the prohibition of toxic metabolite import, as in the case of canavanine resistance via abrogation of the arginine transporter [4] Another is to decrease endogenous production of the precursor while relying on exogenous end-product for growth, as in the case of deletion of URA3 and reliance upon external uracil for 5-fluoroorotic acid resistance. 2018, 4, x endogenous of the end-product so as to decrease exogenous nutrient dependence, such as in the case of increased phenylalanine production in response to fluorophenylalanine [3]. We We sought to identify toxic phenylalaninebiosynthetic biosynthetic pathway could sought to identify toxicprecursors precursors to to the phenylalanine pathway thatthat could be be auxotrophic counter-selection,and and that that may may lead production of phenylethanol. Number of enzymes and steps are indicated by number of arrows
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