Part:BBa_K5184059

st-t76SlCPR2

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 9epiZ. However, since the reductase SlCPR2 was originally found in eukaryotic organisms. They were originally immobilized on the ER membrane. However, since E. coli does not have this structure, the 76 amino acid N-terminus ER transit peptide of the reductase is truncated to enhance solubility and expression rate. Also, a SpyTag is added, which will form an isopeptide bond with the SpyCatcher, thus imitating the colocalization of the two enzymes in eukaryotes. Our usage of st-t76SlCPR2 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

t76SlCPR2 is the truncated version a cytochrome P450 reductase found in Solanum habrochaites. Consisting of two domains, one with a binding site for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) and another with a binding site for flavin mononucleotide (FMN), SlCPR2 functions through transferring electrons to cytochrome P450 oxidases, in our context ShZPO. To perform its functions, SlCPR2 requires the presence of NADPH and the cofactors FAD and FMN, which are two falvin priteins existing in various redox forms and able to control electron movement. The electrons provided by NADPH are transferred to FAD and FMN in order, and finally, the electrons required for the reduction reaction are transferred. 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

To increase the expression level and functionality of oxidase and reductases in E. coli, we introduced the spytag-spycatcher system in addition to truncating the N-terminal anchor regions [figure 9A]. Through the formation of an isopeptide bond between the tag and the catcher, introduction of this system links the oxidase and reductases together, thus facilitating efficient electron transfer. To test whether the enzymes can be expressed successfully in E. coli, we constructed the plasmids, plasmid pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, aiming to test whether the enzymes can maintain their functions after expression and whether the SpyTag can be linked to the SpyCatcher [figure 9B]. Also, we incorporated the plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2 and pET28a-sc t55AtCPR1 to test whether the enzymes can be successfully expressed in E. coli.[figure 1C]

Figure 1: A. Attachment of SpyTag and SpyCatcher to two cytochrome P450 enzymes; the formation of isopeptide bond between lysine on SpyCatcher and aspartic acid on SpyTag can link the two enzymes together, this proximity allows electron transfer and thus catalysis of the CYP (t25ShZPO) enzyme to occur B. Plasmid construct of pW1-p-ZIS-Mvan4662-NPPS-T-P-SpyTag-t25ShZPO-SpyCatcher-t76SlCPR2-t C. Plasmid constructs of pET28a-SpyTag-t76SlCPR2, pET28a-SpyCatcher-t25ShZPO, and pET28a-SpyCatcher-t55AtCPR1

We employed GoldenGate Assembly to build the plasmids pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1[figure 2A&B] and then transformed the built plasmids into the E. coli strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[figure2C&D]

Figure 2: A. Colony PCR results of pET28a-sc t55AtCPR1, pET28a-st t76SlCPR2, and pET28a-sc t25ShZPO B. Alignment of the sequencing results in Fig2A against designed plasmids C. Colony PCR results of pW1-ZIS-Mvan4662-NPPS-st t25ShZPO-sc t76SlCPR2; the insert region is amplified using two sets of primers and their products designated SCIE22E and SCIE22F D. Alignment of the sequencing results of colony PCR products against designed plasmids

Fermentation of pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2 in DH5α was induced by IPTG and lasted 48 hours using dodecane as solvent. After the products were collected and underwent GC-MS analysis, we discovered that still, 9HZ and 9H10epoZ were not detected. Instead, only 7epiZ was produced.[figure 3A&B] Plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1 were transformed into E.coli strain BL21(DE3), fermented and induced by IPTG. The SDS-PAGE result shows that only reductases linked with the SpyTag or SpyCatcher could be expressed successfully, and the oxidase was expressed in inclusion bodies.[figure 4]

Figure 3: A. Gas-phase chromatography results for the pET28a-t25ShZPO-t76SlCPR2 culture with dodecane as solvent B. Mass spectrometry and structure elucidation results of the sample Figure 4: Liquid culture of pET28a-CYP/CPR in BL21(DE3) was harvested, supernatant and precipitate was collected after centrifugation. S: Supernatant, P: Precipitate