Difference between revisions of "Part:BBa K5184018"
 
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<partinfo>BBa_K5184018 short</partinfo>
 
<partinfo>BBa_K5184018 short</partinfo>
  
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SltNPPS (part number), an neryl diphosphate synthase with its N-termial 45 amino acid-trucated. Originally found in Solanum Lycopersium, it is used for transformation of glucose into IPP and DMAPP.                       Mvan4662 (part number), a farnesyl diphosphate synthase facilitating the formation of longer-chain isoprenoid diphosphates. In our context, it is included for synthesis of Z,Z-FPP from NPP.                       ShZIS, a zingiberene synthase derived from Solanum habrochaites, catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Specifically, we us ShZIS to produce 7-epi-zingiberene from its substrate Z,Z-FPP.                                                                                                                          ShZPO, a cytochrome P450 oxygenase found in Solanum habrochaites, carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the susbtrate. In our context, ShZPO will act as a zingiberene oxidase, working with its redox partner SlCPR2, a NADPH-cytochrome P450 reductase found in Solanum lycopersicum, to produce the zingiberenoids 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene from 7-epi-zingiberene.                                              In addition, a spytag-spycatcher system was incorporated, with spytag linked to ShZPO forming an isopeptide bond with the spycathcer linked to SlCPR2. This allows more efficient electron transfer between the two cytochrome P450 enzymes through imitating the colocalization of them on the ER membrane in eukaryotic plants.
+
SltNPPS (part number), an neryl diphosphate synthase with its N-termial 45 amino acid-trucated. Originally found in <i>Solanum lycopersium</i>, it is used for transformation of glucose into IPP and DMAPP.<br>Mvan4662 (BBa_K5184001), a farnesyl diphosphate synthase facilitating the formation of longer-chain isoprenoid diphosphates. In our context, it is included for synthesis of Z,Z-FPP from NPP.<br>ShZIS, a zingiberene synthase derived from <i>Solanum habrochaites</i>, catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Specifically, we us ShZIS to produce 7-epi-zingiberene from its substrate Z,Z-FPP.                                                                                                                          ShZPO, a cytochrome P450 oxygenase found in <i>Solanum habrochaites</i>, carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the susbtrate. In our context, ShZPO will act as a zingiberene oxidase, working with its redox partner SlCPR2, a NADPH-cytochrome P450 reductase found in Solanum lycopersicum, to produce the zingiberenoids 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene from 7-epi-zingiberene.                                              In addition, a spytag-spycatcher system was incorporated, with spytag linked to ShZPO forming an isopeptide bond with the spycathcer linked to SlCPR2. This allows more efficient electron transfer between the two cytochrome P450 enzymes through imitating the colocalization of them on the ER membrane in eukaryotic plants.
  
 
===Abstract===
 
===Abstract===
  
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SltNPPS-Mvan4662-ShZIS-st-t25ShZPO-sc-t76SlCPR2 is a pathway of enzymes used for the synthesis of 9HZ and 9H10epoZ in E. coli. SltNPPS transforms IPP and DMAPP from the MVA pathway to NPP. Mvan4662 catalyzes the formation of Z,Z-FPP from NPP. ShZIS produces 7epiZ from Z,Z-FPP. Then, st t25ShZPO is linked to sc t76SlCPR2 via an isopeptide bond formed through the SpyTag-SpyCatcher system for efficient electron transfer and production of 9HZ and 9H10epoZ from 7epiZ.
+
SltNPPS-Mvan4662-ShZIS-st-t25ShZPO-sc-t76SlCPR2 is a pathway of enzymes used for the synthesis of 9HZ and 9H10epoZ in <i>E. coli</i>. SltNPPS transforms IPP and DMAPP from the MVA pathway to NPP. Mvan4662 catalyzes the formation of Z,Z-FPP from NPP. ShZIS produces 7epiZ from Z,Z-FPP. Then, st t25ShZPO is linked to sc t76SlCPR2 via an isopeptide bond formed through the SpyTag-SpyCatcher system for efficient electron transfer and production of 9HZ and 9H10epoZ from 7epiZ.
  
 
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===Usage and Biology===
 
===Usage and Biology===
  
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SltNPPS is a cis-prenyl transferase from Solanum Lycopersium with its N-terminal 45 amino acid truncated that catalyzes the conversion of geranyl diphosphate into neryl diphosphate. Mvan4662 is a cis-farnesyl diphosphate synthase that catalyzes the transfer of an isopentenyl group from isopentenyl pyrophosphate (IPP) to prenyl diphosphates, facilitating the formation of longer-chain isoprenoid diphosphates. ShZIS is a sesquiterpene synthase derived from Solanum habrochaites, and is present and expressed in the tomato's glandular trichomes. By utilizing three isoprenoid units, ShZIS catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Intermediates that are commonly used include isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and (E,E)-α-farnesyl diphosphate (FPP). t25ShZPO is the truncated version of the plant P450 monooxidase, ShZPO, which is responsible for the two sequential oxidations of 7epiZ to produce 9H10,11epoZ via first a hydroxylation at C9 and then a epoxidation and C10 and C11. It is used collaboratively with t76SlCPR2, which 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).
+
SltNPPS is a cis-prenyl transferase from <i>Solanum lycopersium</i> with its N-terminal 45 amino acid truncated that catalyzes the conversion of geranyl diphosphate into neryl diphosphate. Mvan4662 is a cis-farnesyl diphosphate synthase that catalyzes the transfer of an isopentenyl group from isopentenyl pyrophosphate (IPP) to prenyl diphosphates, facilitating the formation of longer-chain isoprenoid diphosphates. ShZIS is a sesquiterpene synthase derived from Solanum habrochaites, and is present and expressed in the tomato's glandular trichomes. By utilizing three isoprenoid units, ShZIS catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Intermediates that are commonly used include isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and (E,E)-α-farnesyl diphosphate (FPP). t25ShZPO is the truncated version of the plant P450 monooxidase, ShZPO, which is responsible for the two sequential oxidations of 7epiZ to produce 9H10,11epoZ via first a hydroxylation at C9 and then a epoxidation and C10 and C11. It is used collaboratively with t76SlCPR2, which 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).
 
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.
 
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===
 
===Characterization===
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To increase the expression level and functionality of oxidase and reductases in <i>E. coli</i>, 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.  
+
 
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To test whether the enzymes can be expressed successfully in <i>E. coli</i>, we incorporated the plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2 and pET28a-sc t55AtCPR1 to test whether the enzymes can be successfully expressed in <i>E. coli</i>.[figure 1C]
+
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 1A]. 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.[1]
 +
 
 +
We constructed the 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 1B].
  
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie88.webp" width="600"/></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie88.webp" width="600"/></html></center>
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<center><b>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 </b></center>
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<center><b>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 </b></center>
  
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We employed GoldenGate Assembly to build the plasmid pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1[figure 2A&B] and then transformed the built plasmids into the <i>E. coli</i> strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[figure2C&D]  
+
We employed GoldenGate Assembly to build the plasmid pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2 and then transformed the built plasmids into the <i>E. coli</i> strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[figure2C&D]  
  
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie89.webp" width="600"/></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie89.webp" width="600"/></html></center>
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<center><b>Figure 2: A. Colony PCR results of pET28a-sc t55AtCPR1, pET28a-st t76SlCPR2, and pET28a-sc t25ShZPO B. Alignment of the sequencing results in Fig10A 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</b></center>
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<center><b>Figure 2: (A) Colony PCR results of pET28a-sc t55AtCPR1, pET28a-st t76SlCPR2, and pET28a-sc t25ShZPO (B) Alignment of the sequencing results in Fig10A 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</b></center>
  
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Plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1 were transformed into <i>E.coli</i> 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]
+
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 3]
  
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie810.webp" width="600"/></html></center>
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie811.webp" width="600"/></html></center>
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<center><b>Figure 4: Liquid culture of pET28a-CYP/CPR in BL21(DE3) was harvested, supernatant and precipitate was collected after centrifugation. S: Supernatant, P: Precipitate</b></center>
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<center><b>Figure 3: (A) Gas-phase chromatography results for the t25ShZPO-t76SlCPR2 culture with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample </b></center>
  
 +
===Reference===
 +
[1]Chen, Y., Tan, S., Yang, F., Chen, Z., Wu, Z., & Huang, J. (2017). Soluble expression and purification of a functional harpin protein in Escherichia coli. Process Biochemistry, 57, 200–206. https://doi.org/10.1016/j.procbio.2017.03.010
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 20:34, 1 October 2024


SltNPPS-Mvan4662-ShZIS-st-t25ShZPO-sc-t76SlCPR2

SltNPPS (part number), an neryl diphosphate synthase with its N-termial 45 amino acid-trucated. Originally found in Solanum lycopersium, it is used for transformation of glucose into IPP and DMAPP.
Mvan4662 (BBa_K5184001), a farnesyl diphosphate synthase facilitating the formation of longer-chain isoprenoid diphosphates. In our context, it is included for synthesis of Z,Z-FPP from NPP.
ShZIS, a zingiberene synthase derived from Solanum habrochaites, catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Specifically, we us ShZIS to produce 7-epi-zingiberene from its substrate Z,Z-FPP. ShZPO, a cytochrome P450 oxygenase found in Solanum habrochaites, carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the susbtrate. In our context, ShZPO will act as a zingiberene oxidase, working with its redox partner SlCPR2, a NADPH-cytochrome P450 reductase found in Solanum lycopersicum, to produce the zingiberenoids 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene from 7-epi-zingiberene. In addition, a spytag-spycatcher system was incorporated, with spytag linked to ShZPO forming an isopeptide bond with the spycathcer linked to SlCPR2. This allows more efficient electron transfer between the two cytochrome P450 enzymes through imitating the colocalization of them on the ER membrane in eukaryotic plants.

Abstract

SltNPPS-Mvan4662-ShZIS-st-t25ShZPO-sc-t76SlCPR2 is a pathway of enzymes used for the synthesis of 9HZ and 9H10epoZ in E. coli. SltNPPS transforms IPP and DMAPP from the MVA pathway to NPP. Mvan4662 catalyzes the formation of Z,Z-FPP from NPP. ShZIS produces 7epiZ from Z,Z-FPP. Then, st t25ShZPO is linked to sc t76SlCPR2 via an isopeptide bond formed through the SpyTag-SpyCatcher system for efficient electron transfer and production of 9HZ and 9H10epoZ from 7epiZ.

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

SltNPPS is a cis-prenyl transferase from Solanum lycopersium with its N-terminal 45 amino acid truncated that catalyzes the conversion of geranyl diphosphate into neryl diphosphate. Mvan4662 is a cis-farnesyl diphosphate synthase that catalyzes the transfer of an isopentenyl group from isopentenyl pyrophosphate (IPP) to prenyl diphosphates, facilitating the formation of longer-chain isoprenoid diphosphates. ShZIS is a sesquiterpene synthase derived from Solanum habrochaites, and is present and expressed in the tomato's glandular trichomes. By utilizing three isoprenoid units, ShZIS catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Intermediates that are commonly used include isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and (E,E)-α-farnesyl diphosphate (FPP). t25ShZPO is the truncated version of the plant P450 monooxidase, ShZPO, which is responsible for the two sequential oxidations of 7epiZ to produce 9H10,11epoZ via first a hydroxylation at C9 and then a epoxidation and C10 and C11. It is used collaboratively with t76SlCPR2, which 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). 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 1A]. 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.[1]

We constructed the 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 1B].

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 plasmid pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2 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 Fig10A 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 3]

Figure 3: (A) Gas-phase chromatography results for the t25ShZPO-t76SlCPR2 culture with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample

Reference

[1]Chen, Y., Tan, S., Yang, F., Chen, Z., Wu, Z., & Huang, J. (2017). Soluble expression and purification of a functional harpin protein in Escherichia coli. Process Biochemistry, 57, 200–206. https://doi.org/10.1016/j.procbio.2017.03.010