Part:BBa_K3747607

Spacer targeting promoter Cyo oxidase

To reduce nitrous oxide production, we developed a CRISPRi project to downregulate terminal oxidases in Pseudomonas putida. This was done to redirect the electron flux in the bacterium towards the denitrification machinery. You can read all about it here.

Pseudomonas putida is an obligate aerobic bacterium, which is not always practical in biotechnological applications [1]. Thus far, considerable research has been made to make P. putida grow in anoxic conditions [1][2]. Unfortunately, these efforts have not been successful, which illustrates the importance of oxygen respiration for P. putida. Therefore, we decided not to knock-out P. putida’s ability to respire with oxygen, but to downregulate the responsible enzymes. Batianis et al. [3] have shown that CRISPR interference can be employed to efficiently control transcription levels in P. putida. Additionally, they showed that multiple genes can be targeted at the same time, which a useful feature given that P. putida has five terminal oxidases: Cyo oxidase, Cyanide Insensitive oxidase, Aa₃ oxidase, Ccb₃-1 oxidase, and Ccb₃-2 oxidase.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This CRISPRi spacer targets the promoter of the Cyo terminal oxidase. It has been shown that a Cyo knock-out had an effect on the cell transcriptome [4]. Moreover, the Cyo oxidase is the only oxidase active during the exponential phase. When constitutively expressed in the pSEVA231-CRISPR [3] plasmid, we found longer doubling times, and a delayed exponential phase.

References

[1] L. F. C. Kampers, J. J. Koehorst, R. J. A. van Heck, M. Suarez-Diez, A. J. M. Stams, and P. J. Schaap, “A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration,” BMC Microbiol. 2021 211, vol. 21, no. 1, pp. 1–15, Jan. 2021, doi: 10.1186/S12866-020-02058-1.

[2] A. Steen et al., “Construction and characterization of nitrate and nitrite respiring Pseudomonas putida KT2440 strains for anoxic biotechnical applications,” J. Biotechnol., vol. 163, no. 2, pp. 155–165, Jan. 2013, doi: 10.1016/J.JBIOTEC.2012.09.015

[3] Batianis et al., “An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida,” doi: 10.1111/1751-7915.13533

[4] A. Ugidos, F. Rojo, G. Morales, A. Ugidos, and F. Rojo, “Inactivation of Pseudomonas putida cyo terminal oxidaseG Inactivation of the Pseudomonas putida cytochrome o ubiquinol oxidase leads to a significant change in the transcriptome and to increased expression of the CIO and cbb3-1 terminal oxidases,” Environ. Microbiol., vol. 8, no. 10, pp. 1764–1774, 2006, doi: 10.1111/j.1462-2920.2006.01061.x.<.li>