Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Xiao Bu; Jing-Yuan Lin; Jing Cheng; Dong Yang; Chang-Qing Duan; Mattheos Koffas; Guo-Liang Yan

doi:10.1186/s13068-020-01809-6

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Biotechnol Biofuels. 2020 Oct 10:13:168. doi: 10.1186/s13068-020-01809-6. eCollection 2020.

Authors

Xiao Bu^{1

2}, Jing-Yuan Lin^{1

2}, Jing Cheng^{1

2}, Dong Yang³, Chang-Qing Duan^{1

2}, Mattheos Koffas⁴, Guo-Liang Yan^{1

2}

Affiliations

¹ Centre for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, 17 East Tsinghua Rd, Beijing, 100083 China.
² Key Laboratory of Viticulture and Enology, Ministry of Agriculture and Rural Affairs, Beijing, 100083 China.
³ Beijing Key Laboratory of Functional Food From Plant Resources, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083 China.
⁴ Center for Biotechnology and Interdisciplinary Studies and Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 USA.

Abstract

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.

Keywords: ATP; Biofuel products; Endogenous ABC transporters; Membrane fluidity; Saccharomyces cerevisiae.