Part:BBa_K5184020

pTDH3-ShZPO-tTDH1-pPGK1-AtCPR1-tPGK1

In order to achieve enhanced effects, we aimed to produce 9HZ and 9H10epoZ also, which have better repellent effects than 7epiZ. Due to difficulties of expressing the oxidase and reductases in E. coli, we decide to change the chassis to S. cerevisiae. This part presents the collection of promoters, genes coding for the oxidase and reductase and terminator. This enzyme collection may provide future iGEM teams with an insight into the expression of an oxidase and a reductase in S. cerevisiae.

Abstract

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 susbtrate. AtCPR1 is a cytochrome P450 reductase found in Arabidopsis thaliana. AtCPR1 is co-localized with ShZPO on the ER membrane, ensuring efficient electron transfer. We used pTDH3 and tTDH3 for the expression of ShZPO; pPGK1 and tPGK1 for the expression of AtCPR1.

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 3525
  Illegal XhoI site found at 2177
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2423
  Illegal BsaI.rc site found at 3535
  

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 susbtrate. 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. AtCPR1 functions through transferring electrons to ShZPO.

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