Part:BBa_K5184056

sc-t55AtCPR1

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 8epiZ. However, the reductase AtCPR1 was originally found in eukaryotic organisms. They were originally immobilized on the ER membrane. However, since E. coli does not have this structure, the 55 amino acid N-terminus ER transit peptide of the reductase is truncated to enhance solubility and expression rate. Also, a SpyCathcer is added, which will form an isopeptide bond with the SpyTag, thus imitating the colocalization of the two enzymes in eukaryotes. Our usage of sc-t55AtCPR1 provide future iGEM teams with a novel method to synthesize an enzyme originally found in eukaryotes through a prokaryotic chassis.

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

"t55AtCPR1 is the truncated version 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 oxidase. The 55aa N-terminus ER transit peptide of the oxidase is truncated to enhance solubility and expression rate of the enzyme in E. coli. The SpyTag-SpyCather system was originally found in Streptococcus pyogenes, with its fibronectin-binding protein FbaB containing a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes is obtained."

Characterization

After successfully producing 7epiZ, we aim to produce 9HZ and 9H10epoZ, two terpenes that are even more efficient than 7epiZ. [figure 1A].[1] To enable our product to achieve enhanced repellent effects, we explored further on the basis of cycle 2-1 and introduced the oxidase ShZPO and the reductases AtCPR1 and SlCPR2 as its redox partners [figure 4B]. The oxidase and reductase were originally found in eukaryotic organisms, immobilized on the endoplasmic reticulum (ER) in eukaryotic plant cells [figure 1C].

Figure 1: A. Compared to 7epiZ, 9H10epoZ shows better repellency and fecundity-reducing effects against spider mites B. Metabolic pathway for 9-hydroxy-10,11-epoxy-zingiberene, a zingiberene polyoxidase, ShZPO catalyzes the two sequential oxidation of 7-epi-zingiberene C. In plant ER, cytochrome P450 reductases (SlCPR2) and cytochrome P450 oxidases (ShZPO) cluster together; the reductase oxidizes NADPH and transfers the high energy electrons to its corresponding oxidase for the oxidase’s catalysis

We aim to synthesize 9HZ and 9H10epoZ in E.coli, a prokaryotic organism without an ER. Thus, we optimize the oxidase and reductases for production in E.coli through cutting the N-terminal anchor regions of the three enzymes. Specifically, 25 amino acids at the N-terminal of ShZPO, 76 of SlCPR2 and 55 of AtCPR1 were cut according to the tag analysis results [figure 2].

Figure 1: A. Protein transit signal peptide prediction using DeepLoc 2.1 for AtCPR1, ShZPO, and SlCPR2. Only results for the first 100 amino acids are shown, a higher Y-value of a letter denotes a higher chance of the amino acid being part of the signal peptide B. Structural prediction results of AtCPR1, ShZPO, and SlCPR2 using AlphaFold Server, the first 55, 25, and 76 amino acids are colored in gray while rest of the enzymes in blue

We employed GoldenGate Assembly to construct the plasmids pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t76SlCPR2 and pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t55AtCPR1. The colony PCR results reveal successful plasmid construction. The sequencing result confirms that the fragments are successfully linked with no mutations. [figure 3A&B]

Figure 3: A. Colony PCR results of pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t76SlCPR2 and pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t55AtCPR1 in DH5ɑ B. Alignment of sequencing results of pW1-ZIS-Mvan4662-NPPS-ShZPO-SlCPR2 (top) and pW1-ZIS-Mvan4662-NPPS-ShZPO-AtCPR1 (bottom) against the design

Fermentation of pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t76SlCPR2 and pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t55AtCPR1 in DH5α was induced by IPTG and lasted 24 hours using dodecane as solvent. After the products were collected and underwent GC-MS analysis, we discovered that 9HZ and 9H10epoZ were not detected. Instead, only 7epiZ was produced. [figure 8A&B]

Figure 4: A. Gas-phase chromatography results for the pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t76SlCPR2 culture with dodecane as solvent B. Mass spectrometry and structure elucidation results of the sample

References

[1]: Dawood, Mohammad H., and John C. Snyder. ‘The Alcohol and Epoxy Alcohol of Zingiberene, Produced in Trichomes of Wild Tomato, Are More Repellent to Spider Mites Than Zingiberene’. Frontiers in Plant Science, vol. 11, Feb. 2020, p. 35. DOI.org (Crossref), https://doi.org/10.3389/fpls.2020.00035.