Difference between revisions of "Part:BBa K2244003"
(→‎Design)
 
(14 intermediate revisions by 3 users not shown)
Line 14: Line 14:
 
TorA is an E. coli twin-arginine signal peptide bearing a consensus motif of SRRxFLK. The twin-arginine system a bacterial protein export pathway. Tat signal pepetides consist of three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain. In E. coli, the Tat translocase consists of TatA, TatB, TatC proteins. TatBC is a signal peptide recognition complex, while TatA complex forms a channel for protein translocation across the cytoplasmic membrane. To express functional OPH molecules into the periplamic space, a twin-arginine signal peptide of E. coli trimethylamine N-oxide (TMAO) reductase (TorA), was added to the N-terminal of the opdA. TMAO reductase is a periplasmic enzyme that catalyzes reduction of TMAO to trimethylamine, and functions as a component of the anaerobic respiratory chain which provides energy for bacterial cell growth .  
 
TorA is an E. coli twin-arginine signal peptide bearing a consensus motif of SRRxFLK. The twin-arginine system a bacterial protein export pathway. Tat signal pepetides consist of three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain. In E. coli, the Tat translocase consists of TatA, TatB, TatC proteins. TatBC is a signal peptide recognition complex, while TatA complex forms a channel for protein translocation across the cytoplasmic membrane. To express functional OPH molecules into the periplamic space, a twin-arginine signal peptide of E. coli trimethylamine N-oxide (TMAO) reductase (TorA), was added to the N-terminal of the opdA. TMAO reductase is a periplasmic enzyme that catalyzes reduction of TMAO to trimethylamine, and functions as a component of the anaerobic respiratory chain which provides energy for bacterial cell growth .  
  
 +
===More Information (added by CCA_San_Diego 2020)===
 +
 +
<html><body>
 +
 +
<p><b> Author: </b> Ayush Agrawal, Anny Wang </p>
 +
 +
<p><b> Summary: </b> Provides specific background information on the OPH gene and includes useful information about functionality, source organisms, and potential similar improved genes from other bacteria (such as OpdA). </b> </p>
 +
 +
 +
<p> <b> Documentation: </b> </p>
 +
<p> OPH (Organophosphate Hydrolase) is a stereoselective bacterial dimer that works by pre-OPH is inserted in the membrane. After the signal peptide is removed, it becomes mOPH. In Pseudomonas sp. Ind01, the OPH must be latched onto the membrane, with a correlation between the number of mOPH. However, it was found that OPH was not found latched into the membrane, but rather in the cytoplasm, and it is unknown how the substance used (methyl parathion) was transported inside (Pinjari et al., 2013). </p>
 +
 +
<p> OPH is a dimer of two identical subunits containing 336 amino acid residues that folds into a (αβ)8-barrel motif (Singh, 2006). Each subunit contains a binuclear zinc situated at the C-terminal portion. The two zinc atoms are separated by about 3.4 Å and linked to the protein through the side chain of His 55, His 57, His 201, His 230, Asp 301 and a carboxylated Lys 169. Both the Lys 169 and the water molecule (or hydroxide ion) act to bridge the two zinc ions together (Benning, 2001). It has a molecular weight of 72 kDa. </p>
 +
 +
<p> OPH’s perform poorly with P-S bonds, however, which is a bond in Acephate, a widely used insecticide. A similar enzyme, OPDA, has been isolated from A. radiobacter and was found to have 90% homology to OPH at the amino acid level and a very similar overall secondary structure (Horne, 2002b; Yang, 2003). Despite these similarities, the two enzymes have different substrate specificities. There is about a 30-sequence difference between OPH and OPDA. </p>
 +
 +
<p> It is hypothesized that OPH were already present before the use of organophosphates in World War II, and is affirmed by the discovery of OPH’s in non-treated areas. Although OPH is similar to OPDA, with only 30-sequence difference, they perform differently, with OpdA preferring substances with shorter alkali substituents. </p>
 +
 +
</body></html>
  
 
===Design===
 
===Design===
Line 30: Line 49:
  
  
−
TorA-opdA is   bp in length. Figure 2 shows a colony PCR amplifying a section of TorA-opdA in pLEV1(408) vector.   
+
TorA-opdA is 1149 bp in length. Figure 2 shows a colony PCR amplifying a section of TorA-opdA in pLEV1(408) vector.   
  
  
 
<html>
 
<html>
 
<body>
 
<body>
−
<center><img src="https://static.igem.org/mediawiki/parts/0/07/Opda_jiaotu.png" style=" width:20%" /> </center>;
+
<center><img src="https://static.igem.org/mediawiki/parts/0/07/Opda_jiaotu.png" style=" width:20%" /> </center>
 
</body>
 
</body>
 
</html>
 
</html>
  
−
<center><b>Figure 2:</b> The agarose gel electrophoresis of TorA-opdA colony PCR product</center>;
+
<center><b>Figure 2:</b> The agarose gel electrophoresis of TorA-opdA colony PCR product</center>
−
 
+
  
 
===Improvement===
 
===Improvement===
  
−
This is an improved part of opdA (BBa_K215090), which has been codon optimized for E. coli chassis. By fusing a TorA signal peptide directly to the OPH domain, functional OPH has been exported to periplasmic space, and making the whole live cell ‘biocatalyst’. In nature, microorganisms generally evolve the ability of extracellular secretion of functional enzymes. Given that OPH is a degrading enzyme of pesticide, we believe that our improvement helps with using engineered bacteria for direct pesticide degradation in the field.  
+
This is an improved part of opdA ([https://parts.igem.org/Part:BBa_K215090 BBa_K215090]), which has been codon optimized for E. coli chassis. By fusing a TorA signal peptide directly to the OPH domain, functional OPH has been exported to periplasmic space, and making the whole live cell ‘biocatalyst’. In nature, microorganisms generally evolve the ability of extracellular secretion of functional enzymes. Given that OPH is a degrading enzyme of pesticide, we believe that our improvement helps with using engineered bacteria for direct pesticide degradation in the field.  
  
  
Line 50: Line 68:
  
 
Alami, M.; Luke, I.; Deitermann, S.; Eisner, G.; Koch, H. G.; Brunner, J. & Muller, M. 2003. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. Mol. Cell, 12, 937–946.
 
Alami, M.; Luke, I.; Deitermann, S.; Eisner, G.; Koch, H. G.; Brunner, J. & Muller, M. 2003. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. Mol. Cell, 12, 937–946.
 +
 
Kang, D. G., Lim, G-B. & Cha, H. J, 2005. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli. Journal of Biotechnology, 118, 379-385.
 
Kang, D. G., Lim, G-B. & Cha, H. J, 2005. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli. Journal of Biotechnology, 118, 379-385.
 +
 
Lee, P. A.; Tullman-Ercek, D. & Georgiou, G. 2006. The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60, 373–395.
 
Lee, P. A.; Tullman-Ercek, D. & Georgiou, G. 2006. The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60, 373–395.
 
Mulbry, W.W., Karns, J.S., 1989. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene andp rotein. J. Bacteriol. 171, 6740–6746.
 
Mulbry, W.W., Karns, J.S., 1989. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene andp rotein. J. Bacteriol. 171, 6740–6746.
 +
 +
<html><body><b> (References from CCA_San_Diego 2020): </b></body></html>
 +
 +
[1] Benning, M. M., Shim, H., Raushel, F. M., &amp; Holden, H. M. (2001, February). High Resolution X-ray Structures of Different Metal-Substituted Forms of Phosphotriesterase from Pseudomonas diminuta†,‡. Retrieved October 22, 2020, from https://pubs.acs.org/doi/10.1021/bi002661e
 +
 +
[2] Pinjari, A., Pandey, J., Kamireddy, S., & Siddavattam, D. (2013). Expression and subcellular localization of organophosphate hydrolase in acephate-degrading Pseudomonassp. strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides. Letters in Applied Microbiology, 57(1), 63-68. doi:10.1111/lam.12080
 +
 +
[3] Singh, B. K., & Walker, A. (2006). Microbial degradation of organophosphorus compounds. FEMS Microbiology Reviews, 30(3), 428-471. doi:10.1111/j.1574-6976.2006.00018.x
 +
 +
[4] Yang, H., Carr, P., Mcloughlin, S. Y., Liu, J., Horne, I., Qiu, X., . . . Ollis, D. (2003). Evolution of an organophosphate-degrading enzyme: A comparison of natural and directed evolution. Protein Engineering, Design and Selection, 16(3), 241-241. doi:10.1093/proeng/gzg030
 +
  
  

Latest revision as of 05:08, 19 October 2021

TorA-opdA


This is an improved part of opdA (BBa_K215090)

This part is a coding sequence (opdA) with a TorA signal peptide fused to its N-terminus for protein export to periplasmic space.


Biology

opdA encodes organophosphate hydrolase (OPH) which is a homodimeric organophosphate triesterase that requires metal ion as a cofactor to degrade a wide range of toxic organophosphates. OPH can hydrolyze various phosphorus-ester bonds including P-O, P-F, P-CN, and P-S bonds. Recombinant E. coli expressing OPH can degrade a variety of organophosphate, however, due to low protein solubility, OPH production is at low yields. Also, as a gram-negative bacteria, E. coli. Cell membrane can be a substrate diffusion barrier affecting whole cell biocatalytic efficiency.

TorA is an E. coli twin-arginine signal peptide bearing a consensus motif of SRRxFLK. The twin-arginine system a bacterial protein export pathway. Tat signal pepetides consist of three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain. In E. coli, the Tat translocase consists of TatA, TatB, TatC proteins. TatBC is a signal peptide recognition complex, while TatA complex forms a channel for protein translocation across the cytoplasmic membrane. To express functional OPH molecules into the periplamic space, a twin-arginine signal peptide of E. coli trimethylamine N-oxide (TMAO) reductase (TorA), was added to the N-terminal of the opdA. TMAO reductase is a periplasmic enzyme that catalyzes reduction of TMAO to trimethylamine, and functions as a component of the anaerobic respiratory chain which provides energy for bacterial cell growth .

More Information (added by CCA_San_Diego 2020)

Author: Ayush Agrawal, Anny Wang

Summary: Provides specific background information on the OPH gene and includes useful information about functionality, source organisms, and potential similar improved genes from other bacteria (such as OpdA).

Documentation:

OPH (Organophosphate Hydrolase) is a stereoselective bacterial dimer that works by pre-OPH is inserted in the membrane. After the signal peptide is removed, it becomes mOPH. In Pseudomonas sp. Ind01, the OPH must be latched onto the membrane, with a correlation between the number of mOPH. However, it was found that OPH was not found latched into the membrane, but rather in the cytoplasm, and it is unknown how the substance used (methyl parathion) was transported inside (Pinjari et al., 2013).

OPH is a dimer of two identical subunits containing 336 amino acid residues that folds into a (αβ)8-barrel motif (Singh, 2006). Each subunit contains a binuclear zinc situated at the C-terminal portion. The two zinc atoms are separated by about 3.4 Å and linked to the protein through the side chain of His 55, His 57, His 201, His 230, Asp 301 and a carboxylated Lys 169. Both the Lys 169 and the water molecule (or hydroxide ion) act to bridge the two zinc ions together (Benning, 2001). It has a molecular weight of 72 kDa.

OPH’s perform poorly with P-S bonds, however, which is a bond in Acephate, a widely used insecticide. A similar enzyme, OPDA, has been isolated from A. radiobacter and was found to have 90% homology to OPH at the amino acid level and a very similar overall secondary structure (Horne, 2002b; Yang, 2003). Despite these similarities, the two enzymes have different substrate specificities. There is about a 30-sequence difference between OPH and OPDA.

It is hypothesized that OPH were already present before the use of organophosphates in World War II, and is affirmed by the discovery of OPH’s in non-treated areas. Although OPH is similar to OPDA, with only 30-sequence difference, they perform differently, with OpdA preferring substances with shorter alkali substituents.

Design

In our project, to enable secretion of OPH (gene product of opdA) to the periplasm of E. coli for the development of live cell biocatalysts, the TorA signal peptide followed by four amino acid residues of the mature TorA protein is fused directly to the N-terminal of OPH domain. TorA signal peptide contains a twin-arginine motif of ‘SRRxFLA’, and a recognition site for type I signal peptidases (Figure 1).

;

Figure 1. diagram of TorA-opdA fusion design.
;



TorA-opdA is 1149 bp in length. Figure 2 shows a colony PCR amplifying a section of TorA-opdA in pLEV1(408) vector.


Figure 2: The agarose gel electrophoresis of TorA-opdA colony PCR product

Improvement

This is an improved part of opdA (BBa_K215090), which has been codon optimized for E. coli chassis. By fusing a TorA signal peptide directly to the OPH domain, functional OPH has been exported to periplasmic space, and making the whole live cell ‘biocatalyst’. In nature, microorganisms generally evolve the ability of extracellular secretion of functional enzymes. Given that OPH is a degrading enzyme of pesticide, we believe that our improvement helps with using engineered bacteria for direct pesticide degradation in the field.


Reference

Alami, M.; Luke, I.; Deitermann, S.; Eisner, G.; Koch, H. G.; Brunner, J. & Muller, M. 2003. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. Mol. Cell, 12, 937–946.

Kang, D. G., Lim, G-B. & Cha, H. J, 2005. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli. Journal of Biotechnology, 118, 379-385.

Lee, P. A.; Tullman-Ercek, D. & Georgiou, G. 2006. The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60, 373–395. Mulbry, W.W., Karns, J.S., 1989. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene andp rotein. J. Bacteriol. 171, 6740–6746.

(References from CCA_San_Diego 2020):

[1] Benning, M. M., Shim, H., Raushel, F. M., & Holden, H. M. (2001, February). High Resolution X-ray Structures of Different Metal-Substituted Forms of Phosphotriesterase from Pseudomonas diminuta†,‡. Retrieved October 22, 2020, from https://pubs.acs.org/doi/10.1021/bi002661e

[2] Pinjari, A., Pandey, J., Kamireddy, S., & Siddavattam, D. (2013). Expression and subcellular localization of organophosphate hydrolase in acephate-degrading Pseudomonassp. strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides. Letters in Applied Microbiology, 57(1), 63-68. doi:10.1111/lam.12080

[3] Singh, B. K., & Walker, A. (2006). Microbial degradation of organophosphorus compounds. FEMS Microbiology Reviews, 30(3), 428-471. doi:10.1111/j.1574-6976.2006.00018.x

[4] Yang, H., Carr, P., Mcloughlin, S. Y., Liu, J., Horne, I., Qiu, X., . . . Ollis, D. (2003). Evolution of an organophosphate-degrading enzyme: A comparison of natural and directed evolution. Protein Engineering, Design and Selection, 16(3), 241-241. doi:10.1093/proeng/gzg030



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1123
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 220
    Illegal AgeI site found at 415
    Illegal AgeI site found at 754
  • 1000
    COMPATIBLE WITH RFC[1000]