Construction, characterization and application of a genome-wide promoter library in Saccharomyces cerevisiae

Abstract

Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein (YFP) reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.

Key words： synthetic biology; yeast; promoter activity; metabolic engineering; xylose utilization

Acknowledgements

We thank Jianhuo Fang at DNA sequencing facility in Tsinghua University for providing the sequencing service. This work was supported by the National Natural Science Foundation of China (Grant No. 31471254), Chinese Ministry of Science and Technology grant 2012CB725201 and Tsinghua University Initiative grant 20161080088.

References

1 Lee M E, Aswani A, Han A S, Tomlin C J, Dueber J E. Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Research, 2013, 41(22): 10668–10678

2 Paddon C J, Westfall P J, Pitera D J, Benjamin K, Fisher K, McPhee D, Leavell M D, Tai A, Main A, Eng D, High-level semi-synthetic production of the potent antimalarial artemisinin. Nature, 2013, 496(7446): 528–532

3 Smanski M J, Bhatia S, Zhao D, Park Y, Woodruff L B A, Giannoukos G, Ciulla D, Busby M, Calderon J, Nicol R, Functional optimization of gene clusters by combinatorial design and assembly. Nature Biotechnology, 2014, 32(12): 1241–1249

4 Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A III, Smith H O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 2009, 6(5): 343–345

5 Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One, 2008, 3(11): e3647

6 Casini A, MacDonald J T, De Jonghe J, Christodoulou G, Freemont P S, Baldwin G S, Ellis T. One-pot DNA construction for synthetic biology: The modular overlap-directed assembly with linkers (MODAL) strategy. Nucleic Acids Research, 2014, 42(1): e7

7 Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, Wang L, Shen Y, Luo Y, Zheng Y, YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Research, 2015, 43(13): e88

8 Lam F H, Ghaderi A, Fink G R, Stephanopoulos G. Biofuels. Engineering alcohol tolerance in yeast. Science, 2014, 346(6205): 71–75

9 Diao L, Liu Y, Qian F, Yang J, Jiang Y, Yang S. Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution. BMC Biotechnology, 2013, 13(1): 110–118

10 Caspeta L, Castillo T, Nielsen J. Modifying yeast tolerance to inhibitory conditions of ethanol production processes. Frontiers in Bioengineering and Biotechnology, 2015, 3: 184–198

11 Demeke M M, Dietz H, Li Y, Foulquie-Moreno M R, Mutturi S, Deprez S, Den Abt T, Bonini B M, Liden G, Dumortier F, Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnology for Biofuels, 2013, 6(1): 89–112

12 Zhang M M, Zhao X Q, Cheng C, Bai F W. Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1. Biotechnology Journal, 2015, 10(12): 1903–1911

13 Cao L, Tang X, Zhang X, Zhang J, Tian X, Wang J, Xiong M, Xiao W. Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metabolic Engineering, 2014, 24: 150–159

14 Smolke C D. Building outside of the box: iGEM and the biobricks foundation. Nature Biotechnology, 2009, 27(12): 1099–1102

15 Zucca S, Pasotti L, Politi N, Cusella De Angelis M G, Magni P. A standard vector for the chromosomal integration and characterization of biobrick parts in Escherichia coli. Journal of Biological Engineering, 2013, 7(1): 12–24

16 Sharon E, Kalma Y, Sharp A, Raveh-Sadka T, Levo M, Zeevi D, Keren L, Yakhini Z, Weinberger A, Segal E. Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nature Biotechnology, 2012, 30(6): 521–530

17 Solis-Escalante D, Kuijpers N G, van der Linden F H, Pronk J T, Daran J M, Daran-Lapujade P. Efficient simultaneous excision of multiple selectable marker cassettes using I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae. FEMS Yeast Research, 2014, 14(5): 741–754

18 Plessis A, Perrin A, Haber J E, Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics, 1992, 130(3): 451–460

19 Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(36): 12678–12683

20 Keren L, Zackay O, Lotan-Pompan M, Barenholz U, Dekel E, Sasson V, Aidelberg G, Bren A, Zeevi D, Weinberger A, Promoters maintain their relative activity levels under different growth conditions. Molecular Systems Biology, 2013, 9(1): 701–717

21 Ho N W, Chen Z, Brainard A P, Sedlak M. Successful design and development of genetically engineered saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol. Advances in Biochemical Engineering/Biotechnology, 1999, 65: 163–192

22 Ha S J, Galazka J M, Kim S R, Choi J H, Yang X, Seo J H, Glass N L, Cate J H, Jin Y S. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(2): 504–509

23 Wei N, Oh E J, Million G, Cate J H, Jin Y S. Simultaneous utilization of cellobiose, xylose, and Acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform. ACS Synthetic Biology, 2015, 4(6): 707–713

24 Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(36): 12678–12683

25 Curran K A, Crook N C, Karim A S, Gupta A, Wagman A M, Alper H S. Design of synthetic yeast promoters via tuning of nucleosome architecture. Nature Communications, 2014, 5: 4002–4021

26 Redden H, Alper H S. The development and characterization of synthetic minimal yeast promoters. Nature Communications, 2015, 6: 7810–7818

27 Rajkumar A S, Liu G, Bergenholm D, Arsovska D, Kristensen M, Nielsen J, Jensen M K, Keasling J D. Engineering of synthetic, stress-responsive yeast promoters. Nucleic Acids Research, 2016, 44(17): e136

28 Kuyper M, Hartog M M, Toirkens M J, Almering M J, Winkler A A, van Dijken J P, Pronk J T. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Research, 2005, 5(4-5): 399–409

29 van Maris A J, Winkler A A, Kuyper M, de Laat W T, van Dijken J P, Pronk J T. Development of efficient xylose fermentation in Saccharomyces cerevisiae: Xylose isomerase as a key component. Advances in Biochemical Engineering/Biotechnology, 2007, 108: 179–204

30 Zhou H, Cheng J S, Wang B L, Fink G R, Stephanopoulos G. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metabolic Engineering, 2012, 14(6): 611–622