Scalable bioreactor production of an O2‐protected [FeFe]‐ hydrogenase enables simple aerobic handling for clean chemical synthesis

The enzyme CbA5H, a [FeFe]-hydrogenase fromClostridium beijerinckii, has previously been shown to surviveexposure to oxygen, making it a promising candidate forbiotechnological applications. Thus far [NiFe]-hydrogenases aretypically considered for such applications, due to the superior O2-tolerance and therefore simplified enzyme handling. However,methods for production of [FeFe]-hydrogenases are generally moresuccessful than for other classes of hydrogenases, therefore in thiswork we focus on demonstrating scalable CbA5H production, andreport results with active enzyme prepared in bioreactors (up to 10 L)with >20-fold improvement in purified enzyme yield. We then go on toconfirm excellent H2/H+-cycling activity of the air-purified protein,highlighting that CbA5H can be prepared and isolated without theneed for complex and expensive infrastructure. Next, we demonstrategood stability of the air-purified CbA5H both in solution assays, andas a heterogenous catalyst system when immobilized on a carbonsupport. Finally, we successfully implement this enzyme withinpreviously demonstrated biotechnologies for flavin and NADHrecycling, highlighting its relevance in chemical synthesis, and wedemonstrate production of an important API precursor, 3-quinuclidinolat >0.4g scale in standard benchtop hydrogenation infrastructure, with>100,000 CbA5H turnovers over 18 operational hours.