Difference between revisions of "Part:BBa K5184003"

Revision as of 07:30, 30 September 2024

ShZPO

To equip our insecticide with enhanced prevention efficacy against spider mites, we also decide to synthesize 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), two oxidized products of the monocyclic sesquiterpene 7epiZ. This requires the involvement of ShZPO, a zingiberene oxidase, which is able to produce 9HZ and 9H10epoZ from 7epiZ in E. coli. It will be collectively used with the reductase SlCPR2 or AtCPR1, which acts as its redox partner and electron supplier. Our exploration of the oxidase provide future iGEM team with a novel way of generating sesquiterpenes from a monocyclic sesquiterpene through oxidation carried out by the collaboration of an oxidase and a reductase.

Essential Information

Sequences

Sequence and Features

Usage and Biology

ShZPO is a cytochrome P450 oxygenase found in Solanum habrochaites. It carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the substrate. ShZPO requires a cytochrome P450 reductase, in our case SlCPR2 or AtCPR1 as its redox partner and electron supplier. The enzyme was found to be predominantly localized on the ER membrane. The co-localization of oxidase ShZPO and reductase SlCPR2 on the ER membrane ensures efficient electron transfer. We constructed a novel sesquiterpene synthesis pathway in E. coli. Using glucose as our raw material, we introduce the MVA pathway, which transforms glucose into dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Afterwards, SltNPPS, a neryl diphosphate synthase catalyze the production of NPP from IPP and DMAPP. Mvan4662 is then introduced to catalyze the formation of Z,Z-FPP. Then, ShZIS transforms Z,Z-FPP into 7epiZ. In the end, ShZPO works collaboratively with SlCPR2 or AtCPR1.

Characterization

Due to the fact that our oxidase cannot be successfully expressed in E. coli, we decide to change the chassis for our terpene synthesis. After communication with Dr. Su from Earlham Institute, we opted for the yeast S. cerevisiae (strain CEN.PK2-1C).[figure 1] As an eukaryote with ER membranes, S. cerevisiae enables co-localization of the oxidase and reductases while also ensures efficient enzyme expression, reducing the chance of the proteins folding incorrectly.

We inserted DNA fragments to site His of CEN.PK2-1C using lithium acetate transformation. Afterwards, yeast colony PCR was conducted, which shows the target strands were integrated into the genome successfully. The sequencing result also shows that the fragments are integrated into the yeast genome with no mutation. The constructed strains are named ShZPO-SlCPR2 and ShZPO-AtCPR1 respectively.[figure2A&B]

ShZPO-SlCPR2 and ShZPO-AtCPR1 were cocultured with pW1-ZIS-NPPS-Mvan4662+pMVA in E. coli strain DH5α respectively. Fermentation of the coculture was carried out, which is induced by IPTG and lasted 48 hours at 28°C 200 rpm using dodecane as solvent. After the products were collected and underwent GC-MS analysis, 9HZ, a mid-product of the redox reaction of 7epiZ to 9H10epoZ was detected from the co-culture using ShZPO-SlCPR2 only.[figure 3A&B&C]