Efficient Implementation of the Carotenoid Synthesis By Enzyme-Fusion Strategies in Saccharomyces Cerevisiae

Accumulation of metabolic intermediates in engineered microorganisms is particularly detrimental to the cell, not only because of the loss of metabolites but also because of the possible consumption of those intermediates by other competitive metabolic pathways, leading to toxic compounds. Spatial proximity and appropriate disposition of sequential enzymatic domains inside the metabolic pathway are assumed to contribute critically in promoting rapid and efficient consumption of pathway intermediates (Srere, 1985). In natural enzymatic systems, spatial confinement is achieved using enzymes combining several covalently-linked catalytic domains, the recruitment of several enzymes on protein scaffolds or the colocalization of activities within lipid- or protein-based compartments. Several metabolic engineering studies have demonstrated the benefit of enzymes spatial confinement (Dueber et al., 2009; Delebecque et al., 2011, Kim et al., 2016). By using enzyme fusion strategies, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of Xanthophyllomyces dendrorhous carotenoid enzymes produced in Saccharomyces cerevisiae. Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains. Remarkably, the yield of β-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes.