Part:BBa_K3634003

Nonribosomal Peptide Synthetase (NRPS) (Codon Optimised for E.coli)

Codon optimised nonribosomal peptide synthetase (NRPS) CDS for use in E.coli as the final part of the shinorine synthesis gene cluster. The original gene for NRPS (Ava_3855) can be taken from the cyanobacteria species Anabaena variabilis ATCC 29413. The enzyme catalyses the conversion of mycosporine glycine to the final product shinorine. NRPS has three known domains: an adenylation domain, a thiolation domain and a thioesterase domain. Initially, a serine amino acid is converted into an acyl adenylate species which is then later attacked by a serine residue present on the thiolation domain of NRPS. Adenosine monophosphate is then lost from the intermediate followed by imine formation, thought to occur through an enol ester intermediate and an O-N rearrangement by conjugate addition of the serine nitrogen to the cyclohexenimine ring.

This part has been previously categorised by the Minnesota iGEM team of 2012 who also sought to express the enzyme in E.coli (BBa_K814003). Here, by using the IDT codon optimisation tool, we have optimised the GC content of the sequence taken from A.variabilis to improve production efficiency of the final product shinorine. We have further made the part biobrick assembly standard RFC[10] & RFC[1000] compatible by removing XbaI, SapI, EcoR1 and PstI restriction sites introduced by this optimisation step.

The part should be used alongside the additional optimised parts (BBa_K3634000, BBa_K3634001 and BBa_K3634002) responsible for the ultimate conversion of the substrate sedoheptulose 7-phosphate to the final product shinorine. Shinorine is a UV absorbing compound which absorbs light of wavelength 333nm. It is produced by aquatic bacteria and algae species as a source of protection in regions of high UV intensity. Shinorine is just one of a whole selection of mycosporine-like amino acids (MAAs) which provide UV photoprotection across a range of different wavelengths in the UV portion of the EM spectrum.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 458
  Illegal BamHI site found at 2511
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]