Publication Details |
| Category | Text Publication |
| Reference Category | Journals |
| DOI | 10.1021/acssynbio.5c00494 |
| Title (Primary) | Engineering O2-tolerant chimeric hydrogenases optimized for ferredoxin coupling in Synechocystis sp. PCC 6803 |
| Author | Lettau, E.; Till, J.; Toepel, J.; Appel, J.; Boehm, M.; Sacco, D.; Lorent, C.; Teutloff, C.; Mach, R.L.; Gutekunst, K.; Bühler, B.; Lauterbach, L. |
| Source Titel | ACS Synthetic Biology |
| Year | 2025 |
| Department | MIBITECH; AME |
| Volume | 14 |
| Issue | 11 |
| Page From | 4478 |
| Page To | 4495 |
| Language | englisch |
| Topic | T7 Bioeconomy |
| Keywords | hydrogenases; protein design; chimeric enzymes; H-2 production; Synechocystis sp. PCC 6803; Cupriavidusnecator H16 |
| Abstract | The development of hydrogenases capable of operating under oxygenic photosynthetic conditions remains a key challenge for sustainable biohydrogen production. In this study, we developed a series of chimeric NAD(+)-reducing [NiFe]-hydrogenases (SH) combining structural elements from the O-2-tolerant SH of Cupriavidus necator (CnSH) and the ferredoxin-interacting SH of Synechocystis sp. PCC6803 (SynSH). By engineering chimeric HoxU and HoxF subunits, we developed constructs-MixSH, Ch-HoxEF(Syn)+U-Cn, and Ch-HoxU(swapCTD)-that successfully couple the CnHoxYH hydrogenase module to the SynHoxEFU reductase module while retaining O-2 tolerance and enhancing interaction with reduced ferredoxin. The lithoautotrophic growth of C. necator confirmed the tolerance of these variants to O-2, while activity assays in Synechocystis demonstrated partial hydrogenase function, including H-2 consumption and fermentative H-2 production. Notably, Ch-HoxEF(Syn)+U-Cn retained ferredoxin interaction despite lacking the [4Fe4S](U4) cluster, showing [2Fe2S](F2) in HoxF as a functional ferredoxin-binding site. Moreover, we achieved the artificial integration of a [2Fe2S] cluster into CnHoxF and identified the CnHoxF N-terminal domain as structurally and functionally analogous to SynHoxE. Although electron transfer efficiency and activity in Synechocystis remained limited, this work validates the modular engineering of [NiFe]-hydrogenases, uniting O-2-tolerance with ferredoxin interaction and offering a foundational step toward photosynthesis-coupled H-2 production. |
| Persistent UFZ Identifier | https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=31721 |
| Lettau, E., Till, J., Toepel, J., Appel, J., Boehm, M., Sacco, D., Lorent, C., Teutloff, C., Mach, R.L., Gutekunst, K., Bühler, B., Lauterbach, L. (2025): Engineering O2-tolerant chimeric hydrogenases optimized for ferredoxin coupling in Synechocystis sp. PCC 6803 ACS Synth. Biol. 14 (11), 4478 - 4495 10.1021/acssynbio.5c00494 |
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