Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 7002 mirrors the protein overproduction phenotype from E. coli BL21(DE3)

Christopher M. Jones, Travis C. Korosh, David R. Nielsen, Brian F. Pfleger

Research output: Contribution to journalArticlepeer-review

Abstract

Abstract: With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. Key Points: • Promoter driving T7-RNA polymerase was optimized. • Up to 60-fold dynamic range in expression, depending on CO2conditions. • Two-thirds of total protein is T7-RNA polymerase dependent.

Original languageEnglish (US)
Pages (from-to)1147-1158
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume105
Issue number3
DOIs
StatePublished - Feb 2021

Keywords

  • Cyanobacteria
  • Gene circuits
  • Promoters
  • Synthetic biology
  • T7-RNA polymerase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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