Abstract

Synthesis of beta-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a beta-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by beta-ionone quantification via RP-HPLC. Two single-point mutants increased beta-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed beta-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of beta-ionone synthesized by metabolically engineered yeast.