Part:BBa_K5184003

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.

This part belongs to a collection used for the synthesis of 9HZ and 9H10epoZ, two sesquiterpenes with strong repellent effects towards spider mites. This part collection includes SltNPPS, Mvan4662, ShZIS, ShZPO, SlCPR2 and AtCPR1.

Essential Information

Sequences

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]

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

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.

Figure 1: (A) Biosynthesis pathway of 9HZ and 9H10epoZ, starting from IPP and DMAPP that are products of the MVA pathway (B) Integration of the two cytochrome P450 enzymes coding sequences into S. cerevisae genome: the pCRCT plasmid, encoding the endonuclease Cas9 and sgRNA for His integration locus leads to restriction at the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, leading to integration of the cytochrome P450 enzyme genes into the S. cerevisae genome

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.[figure 2]

Figure 2: (A) Colony PCR results of ShZPO-SlCPR2A in His locus, ShZPO-SlCPR2B in His locus, ShZPO-tCPR1A in His locus, and ShZPO-AtCPR1B in His locus in S. cerevisae (B) Alignments of sequencing results of colony CPR products against designed locus

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 3]

Figure 3: (A) Gas-phase chromatography results for culture of ShZPO-SlCPR2 in His locus with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample