Difference between revisions of "Part:BBa K5115034"
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__TOC__
 
__TOC__
 
===Introduction===
 
===Introduction===
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The Ni-Fe hydrogenase we use is an enzyme that functions in vivo bidirectionally for NAD<sup>+</sup> reduction and NADH oxidation coupled to H<sub>2</sub> uptake and H<sub>2</sub> production, respectively. <ref>Teramoto, H., Shimizu, T., Suda, M., & Inui, M. (2022). Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains. International Journal of Hydrogen Energy, 47(52), 22010–22021. </ref> In our design, the Ni-Fe hydrogenase works mainly to restore the nickel to a zero valence, which can help reduce nickel toxicity and collect nickel particles.
+
The csoS operon, originating from the ''Halothiobacillus neapolitanus'', encodes a series of proteins essential for the assembly of α-carboxysomes, a type of microcompartment that facilitates the sequestration and concentration of enzymes involved in carbon fixation, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In literature, α-carboxysomes have been extensively studied and successfully utilized in ''Escherichia coli'' for enhancing carbon fixation efficiency and optimizing metabolic pathways. The csoS operon includes key structural proteins such as CsoS1A, CsoS1B, and CsoS2, which play crucial roles in forming the shell and encapsulating cargo enzymes, including those required for hydrogen production. The operon serves as a model for synthetic biology applications, particularly in constructing nanoreactors capable of enhancing catalytic functions through encapsulation of heterologous enzymes. The successful expression of this operon in ''E. coli'' demonstrates its potential for industrial and biotechnological applications, enabling the creation of efficient microbial systems for sustainable bioprocessing<ref>Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448. https://doi.org/10.1038/s41467-020-19280-0.
  
 
===Usage and Biology===
 
===Usage and Biology===
−
The Ni-Fe hydrogenase is made up of six major and three auxiliary subunits. The former includes hoxF, hoxU, hoxY, hoxH, hoxW and hoxI, while the latter includes hypA, hypB and hypF.
+
The csoS operon composite part is designed for use in ''E. coli'' to facilitate the expression and assembly of α-carboxysomes. These microcompartments are advantageous for engineering metabolic pathways, especially in enhancing the efficiency of carbon fixation and enzyme activity. The operon includes genes that encode shell proteins, such as CsoS1A and CsoS1B, which form the structural framework of the carboxysome. Additionally, the CsoS2 protein is vital for organizing Rubisco within the carboxysome, playing a role analogous to that of CcmM in β-carboxysomes, where it links cargo and shell assemblies.
  
−
The hoxF and the hoxU form the module of NADH dehydrogenase. The hoxF is a hydrogenase subunit responsible for electron transport. The most important group in hoxF is FMN-b, which has the ability of switching electron. Under anaerobic conditions, NADH is oxidized to NAD<sup>+</sup> on the surface of hoxF subunit. In the meanwhile, the electrons generated in this reaction travel through a series of processes to the hoxH, completing the reduction of the hydrogen ion. Under aerobic conditions, NAD<sup>+</sup> is reduced to NADH on the surface of the hoxF subunit. The electron transferring is contrary to former. <ref>Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.</ref> The hoxU houses a 2Fe-2S cluster and is responsible for the role of conducting electrons between hoxH and hoxF. <ref>Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.</ref>
+
Research indicates that the CsoS2 protein exists in two isoforms—CsoS2A and CsoS2B—differing in size and functionality, with the longer form being particularly important for the proper assembly of the empty α-carboxysome shells. Experimental results have shown that deletion of CsoS2 results in the failure to form shell structures in E. coli, underscoring its necessity in carboxysome assembly. The C-terminus of CsoS2 has been identified as an encapsulation peptide (EP) that facilitates the incorporation of cargo enzymes into the shell, allowing for the construction of functional nanoreactors.
  
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The hoxY and the hoxH form the module of catalytic center.The hoxY houses a [4Fe-4S] cluster of the site, and an FMN group (FMN-a) near the Ni-Fe site in the hoxH. It is also responsible for the role of conducting electrons between hoxH and hoxF.<ref>Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.</ref> The most important site in hoxH is the [NiFe] -hydrogenase active site, which is composed of Ni and Fe particles coordinated with cysteine residues, cyanide and carbon monoxide. <ref>Chan, K.-H., Lee, K.-M., & Wong, K.-B. (2012). Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation. PLOS ONE, 7(2), e32592.</ref> It is the most central component of our intracellular conversion of nickel ions. On its surface, oxidation and reduction of hydrogen gas happens alternately according to different oxygen status.
+
By employing this composite part in ''E. coli'', researchers can harness the natural assembly mechanisms of carboxysomes to create efficient systems for producing biofuels and other valuable bioproducts. The incorporation of enzymes such as [FeFe]-hydrogenase into the α-carboxysome shell offers a promising strategy for enhancing hydrogen production while protecting these sensitive enzymes from oxygen, thereby optimizing catalytic activities within a controlled microenvironment. This operon not only serves as a tool for synthetic biology but also represents a significant advancement in the engineering of microbial cell factories for sustainable bioproduction.
  
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The rest of the subunits may work together to ensure that the hydrogenase can assemble and function well. It's worth noting that hypA and hypB can cooperate to precisely guide and insert the nickel ions into the hydrogenase catalytic center.
 
 
===Characterization===
 
===Characterization===
  

Revision as of 11:58, 24 September 2024


csoS operon

contributed by Fudan iGEM 2023

Contents

Introduction

The csoS operon, originating from the Halothiobacillus neapolitanus, encodes a series of proteins essential for the assembly of α-carboxysomes, a type of microcompartment that facilitates the sequestration and concentration of enzymes involved in carbon fixation, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In literature, α-carboxysomes have been extensively studied and successfully utilized in Escherichia coli for enhancing carbon fixation efficiency and optimizing metabolic pathways. The csoS operon includes key structural proteins such as CsoS1A, CsoS1B, and CsoS2, which play crucial roles in forming the shell and encapsulating cargo enzymes, including those required for hydrogen production. The operon serves as a model for synthetic biology applications, particularly in constructing nanoreactors capable of enhancing catalytic functions through encapsulation of heterologous enzymes. The successful expression of this operon in E. coli demonstrates its potential for industrial and biotechnological applications, enabling the creation of efficient microbial systems for sustainable bioprocessing[1]
  1. ↑ Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448. https://doi.org/10.1038/s41467-020-19280-0.

    Usage and Biology

    The csoS operon composite part is designed for use in E. coli to facilitate the expression and assembly of α-carboxysomes. These microcompartments are advantageous for engineering metabolic pathways, especially in enhancing the efficiency of carbon fixation and enzyme activity. The operon includes genes that encode shell proteins, such as CsoS1A and CsoS1B, which form the structural framework of the carboxysome. Additionally, the CsoS2 protein is vital for organizing Rubisco within the carboxysome, playing a role analogous to that of CcmM in β-carboxysomes, where it links cargo and shell assemblies.

    Research indicates that the CsoS2 protein exists in two isoforms—CsoS2A and CsoS2B—differing in size and functionality, with the longer form being particularly important for the proper assembly of the empty α-carboxysome shells. Experimental results have shown that deletion of CsoS2 results in the failure to form shell structures in E. coli, underscoring its necessity in carboxysome assembly. The C-terminus of CsoS2 has been identified as an encapsulation peptide (EP) that facilitates the incorporation of cargo enzymes into the shell, allowing for the construction of functional nanoreactors.

    By employing this composite part in E. coli, researchers can harness the natural assembly mechanisms of carboxysomes to create efficient systems for producing biofuels and other valuable bioproducts. The incorporation of enzymes such as [FeFe]-hydrogenase into the α-carboxysome shell offers a promising strategy for enhancing hydrogen production while protecting these sensitive enzymes from oxygen, thereby optimizing catalytic activities within a controlled microenvironment. This operon not only serves as a tool for synthetic biology but also represents a significant advancement in the engineering of microbial cell factories for sustainable bioproduction.

    Characterization

    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal NheI site found at 133
      Illegal NotI site found at 6599
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 291
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 2805
      Illegal AgeI site found at 799
      Illegal AgeI site found at 1750
      Illegal AgeI site found at 2431
      Illegal AgeI site found at 4933
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal SapI site found at 191


    References

    1. ↑ Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448. https://doi.org/10.1038/s41467-020-19280-0.

      Usage and Biology

      The csoS operon composite part is designed for use in E. coli to facilitate the expression and assembly of α-carboxysomes. These microcompartments are advantageous for engineering metabolic pathways, especially in enhancing the efficiency of carbon fixation and enzyme activity. The operon includes genes that encode shell proteins, such as CsoS1A and CsoS1B, which form the structural framework of the carboxysome. Additionally, the CsoS2 protein is vital for organizing Rubisco within the carboxysome, playing a role analogous to that of CcmM in β-carboxysomes, where it links cargo and shell assemblies.

      Research indicates that the CsoS2 protein exists in two isoforms—CsoS2A and CsoS2B—differing in size and functionality, with the longer form being particularly important for the proper assembly of the empty α-carboxysome shells. Experimental results have shown that deletion of CsoS2 results in the failure to form shell structures in E. coli, underscoring its necessity in carboxysome assembly. The C-terminus of CsoS2 has been identified as an encapsulation peptide (EP) that facilitates the incorporation of cargo enzymes into the shell, allowing for the construction of functional nanoreactors.

      By employing this composite part in E. coli, researchers can harness the natural assembly mechanisms of carboxysomes to create efficient systems for producing biofuels and other valuable bioproducts. The incorporation of enzymes such as [FeFe]-hydrogenase into the α-carboxysome shell offers a promising strategy for enhancing hydrogen production while protecting these sensitive enzymes from oxygen, thereby optimizing catalytic activities within a controlled microenvironment. This operon not only serves as a tool for synthetic biology but also represents a significant advancement in the engineering of microbial cell factories for sustainable bioproduction.

      Characterization

      Sequence and Features


      Assembly Compatibility:
      • 10
        COMPATIBLE WITH RFC[10]
      • 12
        INCOMPATIBLE WITH RFC[12]
        Illegal NheI site found at 133
        Illegal NotI site found at 6599
      • 21
        INCOMPATIBLE WITH RFC[21]
        Illegal BglII site found at 291
      • 23
        COMPATIBLE WITH RFC[23]
      • 25
        INCOMPATIBLE WITH RFC[25]
        Illegal NgoMIV site found at 2805
        Illegal AgeI site found at 799
        Illegal AgeI site found at 1750
        Illegal AgeI site found at 2431
        Illegal AgeI site found at 4933
      • 1000
        INCOMPATIBLE WITH RFC[1000]
        Illegal SapI site found at 191


      References

      <references/>