Abstract

beta-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment beta-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeastSaccharomyces cerevisiae. However, current beta-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimizedS. cerevisiaefor the accumulation of high amounts of beta-carotene and its subsequent conversion to beta-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) fromXanthophyllomyces dendrorhoususing markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on beta-carotene accumulation, and later, on beta-ionone production. A single-copy insertion of the carotenogenic genes in high expressionlociof the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date inS. cerevisiaefor a constitutive expression system. For beta-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene fromPetunia hybrida(PhCCD1) fused with a membrane destination peptide were expressed in the highest beta-carotene-producing strains, reaching up to 33 mg/L of beta-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed toPhCCD1 unspecific cleavage activity as a potentially limiting factor reducing beta-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.