Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved β-carotene production in Saccharomyces cerevisiae

Background: The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve beta-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. Results: The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of beta-carotene in S. cerevisiae. However, due to the competition for precursor between beta-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on beta-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in beta-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to beta-carotene pathway and achieved additional 31.7% increase in beta-carotene content without adversely affecting cell growth. By inducing an extra constitutive beta-carotene synthesis pathway for further conversion precursor farnesol to beta-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of beta-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. Conclusions: This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.

Automated summary

In this study, a strategy using exogenous oleic acid and metabolic engineering approaches was developed to efficiently accumulate beta-carotene in Saccharomyces cerevisiae. By increasing storage capacity and redirecting metabolic flux, the engineered strain achieved a significant increase in beta-carotene production compared to the parent strain.