Engineering Saccharomyces cerevisiae for the overproduction of ?-ionone and its precursor ?-carotene

Abstract

?-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment ?-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 yeast Saccharomyces cerevisiae. However, current ?-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of ?-carotene and its subsequent conversion to ?-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on ?-carotene accumulation, and later, on ?-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of 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 in S. cerevisiae for a constitutive expression system. For ?-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest ?-carotene-producing strains, reaching up to 33 mg/L of ?-ionone in the culture medium after 72-hour cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing ?-ionone production. Overall, the results of this work constitute a step towards the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.