A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Bo Wang, Carrie Eckert, Pin Ching Maness, Jianping Yu

Research output: Contribution to journalArticlepeer-review

74 Scopus Citations

Abstract

Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5′-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced through these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. These systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.

Original languageAmerican English
Pages (from-to)276-286
Number of pages11
JournalACS Synthetic Biology
Volume7
Issue number1
DOIs
StatePublished - 2018

Bibliographical note

Publisher Copyright:
© 2017 American Chemical Society.

NREL Publication Number

  • NREL/JA-2700-70604

Keywords

  • cyanobacteria
  • ethylene-forming enzyme
  • promoter
  • protein expression
  • RBS
  • synthetic biology

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