Difference between revisions of "Part:BBa K4808008"
 
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In our project, we increased the copy number of thrABC in E. coli to increase the production of a-KB.
 
In our project, we increased the copy number of thrABC in E. coli to increase the production of a-KB.
  
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===Usage and Biology===
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    <h2><b>Characterization</b></h2>
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    <p> We tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids, p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition of the thrABC gene in the E.coli genome was inflicted by the externally transformed thrABC gene that expresses via plasmid, in the end leading to an overall decrease in production of a-kb.
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https://static.igem.wiki/teams/4808/wiki/thrabc.png
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    <p> Figure : (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321 and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb production of AIS-2 with plasmid.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4808008 parameters</partinfo>
 
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<b>References：</b>
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<p >Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058
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Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036
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<br/>
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<br/>
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Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023
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<br/>
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<br/>
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Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z
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<br/>
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<br/>
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Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959
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<br/>
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<br/>
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Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a
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Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948</p >

Latest revision as of 15:00, 12 October 2023

thrABC

In Escherichia coli, thrA, thrB and thrC are arranged adjacently on the chromosome to form the thrABC operon. The thrABC operon controls several key enzymes, from aspartate to L-threonine synthesis. Research on thrABC expression and regulation is important for L-threonine synthesis and the downstream products, L-isoleucine and L-glycine. Overexpression of the thrABC operon in a bacterial strain increased L-threonine production significantly.

In our project, we increased the copy number of thrABC in E. coli to increase the production of a-KB.


Characterization

We tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids, p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition of the thrABC gene in the E.coli genome was inflicted by the externally transformed thrABC gene that expresses via plasmid, in the end leading to an overall decrease in production of a-kb. 

thrabc.png

Figure : (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321 and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb production of AIS-2 with plasmid.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3506
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3506
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3506
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3506
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3506
    Illegal NgoMIV site found at 403
    Illegal NgoMIV site found at 3357
    Illegal AgeI site found at 912
    Illegal AgeI site found at 1839
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 3614


References：

Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058

Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948