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

ABSTRACT

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 ß-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 ß-carotene in S. cerevisiae. However, due to the competition for precursor between ß-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on ß-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in ß-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 ß-carotene pathway and achieved additional 31.7% increase in ß-carotene content without adversely affecting cell growth. By inducing an extra constitutive ß-carotene synthesis pathway for further conversion precursor farnesol to ß-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of ß-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.