Background Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae. Results Substrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01. Conclusions Overall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

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

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