A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism
메타 데이터
바이오화학분류
바이오정밀화학
연료
논문
A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism
학술지
Journal of biological engineering
저자명
Barstow, Buz; Agapakis, Christina M; Boyle, Patrick M; Grandl, Gerald; Silver, Pamela A; Wintermute, Edwin H
초록
<P><B>Background</B></P><P>FeFe-hydrogenases are the most active class of H<SUB>2</SUB>-producing enzymes known in nature and may have important applications in clean H<SUB>2 </SUB>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<SUB>2</SUB>.</P><P><B>Results</B></P><P>We have developed a synthetic metabolic pathway in <I>E. coli </I>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<SUB>2 </SUB>levels eliminate growth. This pathway forms the basis for a genetic selection for O<SUB>2 </SUB>tolerance. Genetically selected hydrogenases did not show improved stability in O<SUB>2 </SUB>and in many cases had lost H<SUB>2 </SUB>production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.</P><P><B>Conclusions</B></P><P>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 <I>in vivo </I>assays to aid in the engineering of synthetic H<SUB>2 </SUB>metabolism. Our results also indicate a H<SUB>2</SUB>-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H<SUB>2</SUB>-activating catalysts.</P>