Abstract
BackgroundProduct 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.ResultsSubstrate 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.ConclusionsOverall, 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.
Highlights
Metabolic engineering and synthetic biology efforts have been input to produce various biofuels and chemicals in microbial hosts [1, 2]
The amount of Pdr5p (1.20-fold) and Pdr10p (1.26-fold) involved in the multidrug resistance network of yeast were increased in YBX01. This is corresponding to previous results that PDR5 and PDR10 were induced in recombinant yeast to export heterologous carotenoids to reduce product toxicity [29]
The mitochondrial proteins involved in trichloroacetic acid (TCA) cycle (Sdh1p, Sdh2p Sdh3p, Lsc1p, Lsc2p, Cit2p and Cit3p) and adenosine triphosphate (ATP) synthesis (Atp7p, Atp17p, Atp19p, Atp20p and Tim11p) were up-regulated
Summary
Metabolic engineering and synthetic biology efforts have been input to produce various biofuels and chemicals in microbial hosts [1, 2]. Several studies have reported efforts to enhance efflux using exogenous ABC transporters in yeast [11, 16]. It is reasonable to believe that ABC proteins can potentially serve as the functional transporters to secret biofuel out of cells, and engineering these endogenous transporters might be an effective way to improve biofuel production in S. cerevisiae. Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-medi‐ ated 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
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