In:
Journal of Biological Engineering, Springer Science and Business Media LLC, Vol. 5, No. 1 ( 2011-12)
Abstract:
FeFe-hydrogenases are the most active class of H 2 -producing enzymes known in nature and may have important applications in clean H 2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O 2 . Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O 2 levels eliminate growth. This pathway forms the basis for a genetic selection for O 2 tolerance. Genetically selected hydrogenases did not show improved stability in O 2 and in many cases had lost H 2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H 2 metabolism. Our results also indicate a H 2 -independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H 2 -activating catalysts.
Type of Medium:
Online Resource
ISSN:
1754-1611
DOI:
10.1186/1754-1611-5-7
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2011
detail.hit.zdb_id:
2391582-1
