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Title: Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era

Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determinemore » the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R2 values more than 0.7 and 0.9, respectively. This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ; ORCiD logo [2] ;  [2] ;  [2] ;  [2] ;  [1] ;  [1] ;  [1] ; ORCiD logo [1]
  1. Hubei University, Wuhan (China)
  2. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Publication Date:
Report Number(s):
NREL/JA-2700-73636
Journal ID: ISSN 1754-6834
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Zymomonas mobilis; systems biology; promoter; ribosomal binding site; RBS; reporter genes; dual reporter-gene system
OSTI Identifier:
1506610

Yang, Yongfu, Shen, Wei, Huang, Ju, Li, Runxia, Xiao, Yubei, Wei, Hui, Chou, Yat-Chen, Zhang, Min, Himmel, Michael E., Chen, Shouwen, Yi, Li, Ma, Lixin, and Yang, Shihui. Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era. United States: N. p., Web. doi:10.1186/s13068-019-1399-6.
Yang, Yongfu, Shen, Wei, Huang, Ju, Li, Runxia, Xiao, Yubei, Wei, Hui, Chou, Yat-Chen, Zhang, Min, Himmel, Michael E., Chen, Shouwen, Yi, Li, Ma, Lixin, & Yang, Shihui. Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era. United States. doi:10.1186/s13068-019-1399-6.
Yang, Yongfu, Shen, Wei, Huang, Ju, Li, Runxia, Xiao, Yubei, Wei, Hui, Chou, Yat-Chen, Zhang, Min, Himmel, Michael E., Chen, Shouwen, Yi, Li, Ma, Lixin, and Yang, Shihui. 2019. "Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era". United States. doi:10.1186/s13068-019-1399-6. https://www.osti.gov/servlets/purl/1506610.
@article{osti_1506610,
title = {Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era},
author = {Yang, Yongfu and Shen, Wei and Huang, Ju and Li, Runxia and Xiao, Yubei and Wei, Hui and Chou, Yat-Chen and Zhang, Min and Himmel, Michael E. and Chen, Shouwen and Yi, Li and Ma, Lixin and Yang, Shihui},
abstractNote = {Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R2 values more than 0.7 and 0.9, respectively. This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.},
doi = {10.1186/s13068-019-1399-6},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 12,
place = {United States},
year = {2019},
month = {3}
}

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