Difference between revisions of "Part:BBa K814005"

Latest revision as of 14:38, 9 August 2020

shinorine biosynthetic pathway

Our research has focused on two novel biosynthetic pathways found in two distinct algal species. A pathway ending in the production of two UV-protective compounds, shinorine and mycosporine-glycine, was cloned from Anabaena varibalis. DHQS catalyzes the first step in the pathway, converting sedoheptulose-7-phosphate into dehydroquinate. Dehydroquinate-O-methyltransferase (O-MT) then generates 4-deoxygadusol, which is converted to mycosprine-glycine by ATP-grasp (ATPG). Finally, mycosporine-glycine is converted into shinorine by a non-ribosomal peptide synthase (NRPS).

This composite part contains protein generators for DHQS, O-MT, ATPG and the shinorine NRPS.

Figure 1. The shinorine biosynthetic pathway.

4-DG Pathway (Optimised for E.coli)

The following part describes the constitutive expression of the first two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobricks BBa_K3634000 (DHQS) and BBa_K3634001 (O-MT) are responsible for converting sedoheptulose 7-phosphate to 4-deoxygadusol. Once produced, 4-deoxygadusol will then be converted by BBa_K3634005 (ATPG and NRPS composite, present on 'plasmid A') to the final product of the pathway, shinorine. The shinorine production pathway is separated in this way as a biosafety mechanism so that UV resistance is not conferred in a bacteria which has lost or gained plasmid A/B alone. As NRPS is determined to be 'rate-limiting' with respect to the pathway, plasmid A will ideally be placed at a higher copy number to plasmid B to ensure 1:1 stoichiometry of reactants.

ATPG and NRPS Composite (Optimised for E.coli)

The following part describes the constitutive expression of the final two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobrick BBa_K3634002 (ATPG) and BBa_K3634003 (NRPS) are responsible for converting 4-deoxygadusol (4-DG) to the final product shinorine. Once produced, this mycosporine-like amino acid can absorb UV radiation of wavelength ~333nm. The composite part described will be present on 'plasmid A', separate to the previous enzymes responsible for 4-deoxygadusol production which are maintained on 'plasmid B'. By this arrangement, biosafety of our gene circuit will be ensured such that UV resistance is not conferred in a bacteria which has lost or gained plasmid A/B alone. As NRPS is determined to be 'rate-limiting' with respect to the pathway, plasmid A will ideally be placed at a higher copy number to plasmid B to ensure 1:1 stoichiometry of reactants.

Sequence and Features