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 CO 2 conditions.
• Two-thirds of total protein is T7-RNA polymerase dependent.
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All data generated or analyzed during this study are included in this published article and its supplementary information files.
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Funding
CMJ, TCK, and BFP were supported by the US National Science Foundation (EFRI-1240268).
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C.M.J. conceived the original idea, designed and carried out experiments, and wrote and edited the manuscript. T.K. provided technical support. D.R.N. provided critical analysis and resources and wrote and edited the manuscript. B.F.P. conceived experiments, provided critical analysis and resources, and wrote and edited the manuscript.
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Jones, C.M., Korosh, T.C., Nielsen, D.R. et al. Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 7002 mirrors the protein overproduction phenotype from E. coli BL21(DE3). Appl Microbiol Biotechnol 105, 1147–1158 (2021). https://doi.org/10.1007/s00253-020-11085-x
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DOI: https://doi.org/10.1007/s00253-020-11085-x