Difference between revisions of "Part:BBa K5115063"

Revision as of 12:48, 2 October 2024

hox and hyp, with EP targeted hoxF

Introduction

This composite part combines BBa_K5115052(ribozyme connected hox and hyp, without hoxF),BBa_K5115061(ribozyme+RBS+hoxF-GS-EP+stem-loop). We introduced this ribozyme-assisted polycistronic co-expression system from 2022. By inserting ribozyme sequences between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.

With this design, we achieve co-expression of all the subunits of hydrogenase and EP at similar level. These parts make up the Ni-Fe hydrogenase with the hoxF targeted by EP.

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. 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. ^[3]

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.^[4] 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. ^[5] 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.^[6]

Through the synergistic integration of the hox and hyp subunits, 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.

The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes. EP supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes. This targeting mechanism is essential for protein encapsulation within these structures, aiding in the assembly of a functional, proteinaceous shell that sequesters enzymes or other proteins, ensuring efficient catalysis or protection from environmental stress^[7].

Usage and Biology

With hoxF targeted by EP, the hydrogenase will be finally directed into the carboxysomes, obtaining a stable environment to wokr in. This part will finally be exhibited in BBa_K5115067(mineral, F module), please visit this part for more details on our experiment.

Sequence and Features

References

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. ↑ 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.
4. ↑ 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.
5. ↑ 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.
6. ↑ 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.
7. ↑ 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.