Part:BBa_K5115020

hox and hyp operon

The Ni-Fe hydrogenase we use is an enzyme that functions in vivo bidirectionally for NAD⁺ reduction and NADH oxidation coupled to H₂ uptake and H₂ production, respectively^[1]. 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.

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 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⁺ 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⁺ is reduced to NADH on the surface of the hoxF subunit. The electron transferring is contrary to former ^[2]. The hoxU houses a 2Fe-2S cluster and is responsible for the role of conducting electrons between hoxH and hoxF.

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. The most important site in hoxH is the [Ni-Fe]-hydrogenase active site, which is composed of Ni and Fe particles coordinated with cysteine residues, cyanide and carbon monoxide ^[3]. 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.

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 ^[4].

Through the synergistic integration of the hox and hyp operon, our system effectively enhances hydrogen production and enables the reduction of nickel ions into nanoparticles, thereby maximizing the efficiency of nickel recovery from industrial wastewater.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony.

M: DNA Marker. Lanes 1-8: Corresponding bands for hoxF, hoxU, hoxI, hoxH, hoxW, hoxY, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp operon as designed. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 577
Illegal BglII site found at 1395
Illegal BglII site found at 1688
Illegal BglII site found at 2230
Illegal BglII site found at 2308
Illegal BamHI site found at 2596
Illegal XhoI site found at 22
Illegal XhoI site found at 2238
Illegal XhoI site found at 2430
Illegal XhoI site found at 2803
Illegal XhoI site found at 4857
Illegal XhoI site found at 5760
Illegal XhoI site found at 6171
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 456
Illegal NgoMIV site found at 994
Illegal NgoMIV site found at 1204
Illegal NgoMIV site found at 1516
Illegal NgoMIV site found at 2896
Illegal NgoMIV site found at 3496
Illegal NgoMIV site found at 5860
Illegal AgeI site found at 2191
Illegal AgeI site found at 6658
Illegal AgeI site found at 7680
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2012
Illegal BsaI site found at 2174
Illegal BsaI site found at 4616
Illegal BsaI.rc site found at 625
Illegal BsaI.rc site found at 1129
Illegal SapI.rc site found at 2123

References

↑ 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.
↑ 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.
↑ 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.
↑ Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.

[1] 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.

[2] 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.

[3] 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.

[4] Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.

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