Prediction and Characterization of Promoters and Ribosomal Binding Sites of Zymomonas mobilis in System Biology Era

Hui Wei, Yat Chou, Min Zhang, Michael Himmel, Yongfu Yang, Wei Shen, Ju Huang, Runxia Li, Yubei Xiao, Shouwen Chen, Li Yi, Lixin Ma, Shihui Yang

Research output: Contribution to journalArticlepeer-review

31 Scopus Citations

Abstract

Background: 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. Results: 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 R 2 values more than 0.7 and 0.9, respectively. Conclusions: 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.

Original languageAmerican English
Article number52
Number of pages13
JournalBiotechnology for Biofuels
Volume12
Issue number1
DOIs
StatePublished - 2019

Bibliographical note

Publisher Copyright:
© 2019 The Author(s).

NREL Publication Number

  • NREL/JA-2700-73636

Keywords

  • Dual reporter-gene system
  • Promoter
  • Reporter genes
  • Ribosomal binding site (RBS)
  • Systems biology
  • Zymomonas mobilis

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