Part:BBa_K5111008
Main body of N2OR and maturation proteins
In this insert, we have added the CDS of two nos cluster genes - NosR and NosZ – and the ApbE protein. nosZ is the gene responsible for the main structure of N2OR while nosR is copper responsive and regulates the transcription of nosZ (Benett et al., 2020). N2OR is a metalloprotein that is found in the periplasm of bacteria. ApbE is the FAD+ binding flavinyl transferase protein that donates flavin to the NosR protein for maturation, which then activates nitrous oxide reductase (Zhang et al., 2017). We included ApbE as P.stutzeri does not code for the flavin donator nosX. Together with our second NosR system (BBa_K5111009), this insert can produce the nitrous oxide breaking protein N2OR. The genome for these proteins were derived directly from P. stutzeri (Clade I) ZoBell ATCC 14405 meaning we have kept the natural RBSs present in between the open reading frames of the proteins. P. stutzeri has also shown to have the greatest enzymatic activity of N2OR under anaerobic and aerobic conditions (Yoon et al., 2016). Thus, we believe these two nos inserts would perform well in both scenarios.
Sources Bennett, S.P. et al. (2020) 'nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase,' Microbiology, 166(10), pp. 909–917. https://doi.org/10.1099/mic.0.000955.
Lin Zhang, Christian Trncik, Susana L.A. Andrade, Oliver Einsle, (2017). The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction. Biochimica et Biophysica Acta (BBA) – Bioenergetics, Volume 1858, Issue 2, Pages 95-102, ISSN 0005-2728. doi: https://doi.org/10.1016/j.bbabio.2016.11.008.
Yoon, S., Nissen, S., Park, D., Sanford, R.A. and Löffler, F.E. (2016). Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ. Applied and Environmental Microbiology, 82(13), pp.3793–3800. doi:https://doi.org/10.1128/aem.00409-16.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 361
Illegal BamHI site found at 4942 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 319
Illegal NgoMIV site found at 629
Illegal NgoMIV site found at 2194
Illegal NgoMIV site found at 3968
Illegal NgoMIV site found at 4411
Illegal NgoMIV site found at 4463
Illegal AgeI site found at 2400
Illegal AgeI site found at 3051 - 1000COMPATIBLE WITH RFC[1000]
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