Part:BBa_K5184005

AtCPR1

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. In order to make the oxidase ShZPO funciton efficiently, we plan to incorporate the NADPH-cytochrome P450 reductase AtCPR1 as its redox partner and electron supplier. Our exploration of the reductase 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

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

AtCPR1 is a cytochrome P450 reductase found in Arabidopsis thaliana. It contains two domains, one with binding sites for FAD, flavin adenine dinucleotide, and NADPH, nicotinamide adenine dinucleotide; the other with binding site for FMN, flavin mononucleotide. The two cofactors, FAD and FMN are flavin proteins with multiple variable oxidation states, enabling them to control electron movement. The electron from NADPH is transferred via the two flavin proteins, FAD and FMN in order, and finally transferred to where the reductive power is required, in context of our part collection, ShZPO, a cytochrome P450 reductase. 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.

Figure 1: 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 of the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, lead to integration of the cytochrome P450 enzymes 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.[figure2A&B]

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 3A&B&C]

Figure 2: 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