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

The enzyme CbA5H, a [FeFe]-hydrogenase from Clostridium beijerinckii, has previously been shown to survive exposure to oxygen, making it a promising candidate for biotechnological applications. Thus far [NiFe]-hydrogenases are typically considered for such applications, due to the superior O2-tolerance and therefore simplified enzyme handling. However, methods for production of [FeFe]-hydrogenases are generally more successful than for other classes of hydrogenases, therefore in this work we focus on demonstrating scalable CbA5H production, and report results with active enzyme prepared in bioreactors (up to 10 L) with >20-fold improvement in purified enzyme yield. We then go on to confirm excellent H2/H+-cycling activity of the air-purified protein, highlighting that CbA5H can be prepared and isolated without the need for complex and expensive infrastructure. Next, we demonstrate good stability of the air-purified CbA5H both in solution assays, and as a heterogenous catalyst system when immobilized on a carbon support. Finally, we successfully implement this enzyme within previously demonstrated biotechnologies for flavin and NADH recycling, highlighting its relevance in chemical synthesis, and we demonstrate production of an important API precursor, 3-quinuclidinol at >0.4 g scale in standard benchtop hydrogenation infrastructure, with >100,000 CbA5H turnovers over 18 operational hours.