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Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2017, Vol. 11 Issue (1) : 107-116    https://doi.org/10.1007/s11705-017-1621-7
RESEARCH ARTICLE
Construction, characterization and application of a genome-wide promoter library in Saccharomyces cerevisiae
Ting Yuan1,Yakun Guo1,Junkai Dong1,Tianyi Li1,Tong Zhou1,Kaiwen Sun1,Mei Zhang2,Qingyu Wu1,Zhen Xie3,Yizhi Cai4,Limin Cao2,Junbiao Dai1()
1. MOE Key Laboratory of Bioinformatics and Centre for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
2. College of Life Sciences, Capital Normal University, Beijing 100048, China
3. MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, China
4. School of Biological Sciences, The King’s Buildings, University of Edinburgh, Edinburgh, EH9 3BF, UK
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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.

Keywords synthetic biology      yeast      promoter activity      metabolic engineering      xylose utilization     
PACS:     
Fund: 
Corresponding Author(s): Junbiao Dai   
Just Accepted Date: 12 January 2017   Online First Date: 28 February 2017    Issue Date: 17 March 2017
 Cite this article:   
Ting Yuan,Yakun Guo,Junkai Dong, et al. Construction, characterization and application of a genome-wide promoter library in Saccharomyces cerevisiae[J]. Front. Chem. Sci. Eng., 2017, 11(1): 107-116.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-017-1621-7
https://academic.hep.com.cn/fcse/EN/Y2017/V11/I1/107
Strains Transcription units (TUs) Promoter activity
YTY1 pPGK1-XI-tCYC1, pADH1-XK-tTEF2 Common
YTY2 pYHR135C-XI-tCYC1, pCYC1-XK-tTEF2 Weak
YTY3 pYHR020C-XI-tCYC1, pYIL069C-XK-tTEF2 Medium
YTY4 pYKL060C-XI-tCYC1, pYHR174W-XK-tTEF2 Strong
Tab.1  TUs of constructed strains
Fig.1  Construct genome-wide promoter libraries.

(A) The workflow to construct both the bacteria and yeast libraries. Every native promoter was amplified by PCR, arbitrarily divided into 9 pools and cloned into the reporter plasmid to drive the expression of YFP gene. Plasmids were prepared from the bacteria pools, mixed together and integrated into the yeast genome at HO locus after enzymatic digestion. (B) The composition and coverage of the 9 bacteria libraries. The assembly efficiency is calculated as the percentage of clones, which could generate a fragment at expected size after colony PCR. At least 24 clones were randomly isolated and analyzed. The library depth equals to the number of transformants divided by the number of promoters and multiply the assembly efficiency. (C) A schematic representation of the DNA fragments to be integrated. Homologous regions are DNA sequences flanking the native HO gene. The arrow represents the promoter and T represents the terminator

Bacterial library Promoter number BY4741 integration pools depth WXY15 integration pools depth
Library 1–4 2572 27.8 27.3
Library 5–9 3043 >50 24
Tab.2  Depth of yeast reporter library
Fig.2  Accurate expression measurement for all yeast native promoters under different growth conditions.

(A) 10-fold serial dilution was used to determine the suitable stress conditions. BY4741 (left) and WXY15 (right) were treated with various concentration of acetic acid or ethanol, normalized to the starting OD600 = 0.2, 10-fold diluted and spotted onto YPD medium. Pictures were taken after the plates were incubated at 30 °C for two days; (B) illustration of the cell sorting experiment; (C) fluorescence plot of yeast library in WXY15 grown in YPD medium as an example. The dots between the two red dash lines represent cells expressing mCherry, which was constitutively expressed once the reporter fragment was integrated. Different expression bins are labeled by different color. The cells were divided into 12 bins, with increased expression from 1 to 12; (D) the distribution of YFP fluorescence in WXY15 library grown in YPD as an example. The blue dash line indicates the fluorescence intensive if YFP is under the control of CYC1 promoter; (E) the distribution of YFP fluorescence of cells from the sorted bins. The sorted cells from each expression bin were cultured and analyzed separately

Fig.3  Identify promoters with high activity under stress condition.

(A) Comparison of the expression measurements obtained for two independent replicates (cultivated in YPD). For each replicate, the cells were sorted by FACS and subjected to high-throughput sequencing to identify the promoter in sorted expression bins. a.u., arbitrary unit; (B) confirm the activity of identify promoters from NGS sequencing. Independent clones were analyzed by FACS. Five promoters that frequently identified in almost all conditions from Bin 12 were examined individually in different conditions in BY4741 (D, left panel) or WXY15 (F), respectively. In BY4741, the five selected promoters (left panel) generally performed even better than some commonly used promoters (right panel); (C) expression measurement of randomly isolated clones from selected expression bins in two independent replicates. At least four clones were tested for each bin

Yeast strain YPD Ethanol stress Acetic acid stress
BY4741 pYKL081W
pYPL081W
pYGL030W
pYJL159W
pYHR174W
pYFR049W
pYJL159W
pYPL081W
pYHR174W
pYKL096W-A
pYJR105W
pYBR072W
pYJL59W
pYHR174W
pYFR049W
pYDL229W
pYBR123C
pYBR118W
pYBR072W
WXY15 pYDR381W
pYBR072W
pYGL030W
pYJL159W
pYMR116C
pYGL030W
pYBR118W
pYHR174W
pYPR145W
pYDR381W
pYPL081W
pYKL060C
Tab.3  Promoters identified from random selected clones within Bin 12
Fig.4  Construction and characterization of xylose-fermenting yeast using identified promoters.

(A) An overall scheme for glucose and xylose metabolism in engineered Saccharomyces cerevisiae. G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-BP, fructose-1,6-biphosphate; G-3-P, glyceraldehyde-3-phosphate; PPP, pentose phosphate pathway. To simplify the analysis, only the expression of genes in rectangle was modulated; (B) the yeast strains with XI and XK under the control of promoters with different activities; (C) the residual concentration of glucose during fermentation; (D) the residual concentration of xylose during fermentation; (E) the productivity of xylitol during fermentation; (F) the productivity of ethanol during fermentation

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