Obtainment, Selection And Characterization Of A Mutant Strain Ofkluyveromyces Marxianusthat Displays Improved Production Of 2-Phenylethanol And Enhanced Dahp Synthase Activity

Aims Yeasts produce 2-phenylethanol (2-PE) from sugars viade novosynthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to selectKluyveromyces marxianusmutant strains with improved capacity to produce 2-PE from sugars. Methods and Results Kluyveromyces marxianusCCT 7735 mutant strains were selected from UV irradiation coupled with screening ofp-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium withoutl-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced bothARO3andARO4genes of Km_PFP41 mutant and identified mutations inARO4which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. Conclusions The Km_PFP41 mutant strain presented improved 2-PE production viade novosynthesis and enhanced DAHP synthase activity. Significance and Impact of the Study The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.