Pathway construction and cell factory engineering for the production of natural food additives

Modern industrial food production relies on natural food colors and aromas to achieve products with the desired appearance and flavor. However, the natural host producing said aroma or color compounds do not necessarily does so to a degree, which satisfies the market demand. To this end, the establishment of microbial cell factories for the production of these compounds can help satisfy the demand, by enabling a fermentation-based production, which to greater degree allows for scaling to satisfy the demand. In the current project, we have worked towards the establishment of a cell factory for the biosynthesis of the natural color carminic acid naturally produced by the insect Dactylopius coccus.

A semi natural pathway towards carminic acid has previously been established in the filamentous fungi Aspergillus nidulans using an Aloe arborescence octaketide synthase, two Streptomyces polyketide cyclases and a D. coccus glycosyl-transferase. However, the pathway misses a monooxygenase to catalyze one of the pathway steps necessary for the formation of carminic acid. To address the issue of the missing monooxygenase we have heterologously expressed candidate monooxygenases in a Saccharomyces cerevisiae strain engineered to produce flavokermesic acid. In these efforts, we have tested candidate monooxygenases from the natural host of carminic acid biosynthesis D. coccus, mammalian cytochrome P450 monooxygenases with broad substrate specificity, and flavin-dependent monooxygenase from filamentous fungi and Streptomyces, predicted to hydroxylate anthraquinone structures similar to that of flavokermesic acid.

Type II polyketide cyclases represents an important class of enzymes in the biosynthesis of aromatic type II polyketides, and has found use as components in the establishment of artificial biosynthetic pathways, such as the one established for carminic acid. To improve biosynthesis of flavokermesic acid in S. cerevisiae we have tested alternative first ring cyclase to ZhuI, which is currently used. The test identified cyclases leading to a higher production of flavokermesic acid in S. cerevisiae. Further, it increases the number experimentally tested first ring cyclases and provides hints for future constructions of artificial biosynthetic pathways involving type II polyketide cyclases.

Finally, we have explored whether sexual crossing of S. cerevisiae strain backgrounds can be used as a tool for the improvement of the natural food aroma vanillin-β-glucoside. The crossing of cultivars for the purpose of generating improved progeny is one of the oldest methods in the biotechnological toolbox and has been/is widely used in agriculture for this purpose. The technique has however not previously been applied as a tool, in yeast, for the improvement of cell factories expressing heterologous pathways. We have performed sexual crossing of the two commonly used strain backgrounds CEN.PK and S288C engineered for biosynthesis of vanillin-β-glucoside. Screening of the resulting hybrid strains resulted in the identification of strains with improved vanillin-β-glucoside biosynthesis, compared to the parental strains. The project thereby demonstrates the potential mating as a technique for improving cell factories, and as a strategy for further exploitation of the genotypic diversity of S. cerevisiae.