Citrate as Cost-Efficient NADPH Regenerating Agent

The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase

Reaction and process-optimization of modular synthetic enzyme cascades towards diols and hydroxy ketones meeting industrial demands

Hydroxy ketones and diols are chiral building blocks of interest in several active pharmaceutical ingredients. While classic chemical synthesis is able to provide high product concentrations, it oftentimes lacks the required regio- and stereoselectivity and encompasses ecologically problematic conditions or waste. In both regards, enzymes could provide a valuable synthesis alternative. Especially carboligases and NADPH dependent alcohol dehydrogenases would allow a synthesis of these valuable building blocks from inexpensive bulk chemicals. Unfortunately, these enzymes operate preferentially under diluted aqueous conditions, which are unsuitable to provide economically sustainable product concentrations. Hence, this thesis aims to explore methods of process intensification to lift the enzymatic synthesis of hydroxy ketones and diols to industrially relevant levels that are economically feasible. As such, the three subjects (i) NADPH regeneration, (ii) unconventional reaction media, and (iii) process design are intensively studied. In NADPH regeneration two highly atom efficient substrate coupled methods are explored: coproduct recycling and smart cosubstrates. While in the first method recycles coproduct into the main synthesis to gain the final product, the other method generates a value added coproduct, which makes both methods profitable regeneration methods. Alternatively, also whole cell NADPH regeneration from citrate was explored, which revealed promising cost reduction features besides easy applicability. A second aim is the increase of substrate concentrations by the employment of an unconventional hydrophobic reaction environment. Here, a microaqueous reaction system (MARS) proves to be beneficial in increasing substrate concentrations to 500 mM beyond their solubility limit in aqueous conditions. The importance of buffer amounts in this system to ensure catalytic activity as well as the impact of green solvent selection on the system are reflected. Beyond the synthesis application and setup characteristics of MARS for artificial enzyme cascades, it also facilitates product isolation, which makes it a highly interesting reaction environment. As third optimization step, batch and continuous process designs are evaluated to enhance space time yields. Here, in both systems the issue of possible substrate toxicity, which may limit yields, is circumvent by employing and optimizing technical methods. This allowed space time yields of up to 165 g L 1 d-1 in batch and 7296 g L 1 d-1 in continuous application. These three methods are combined, applied and compared in their profitability to fulfil industrial benchmarks. Notably, all these methods are well suited to lift the multi-step biocatalytic synthesis of diols and hydroxy ketones into industrial scale. A catalyst cost analysis reveals enzyme stability and, thereby, the catalyst selection as key factor for a profitable enzymatic process layout. In addition to economic profitability also ecologic sustainability of enzymatic syntheses is assessed. This identifies an application of MARS as indeed highly environmentally sustainable with E factors ranking between 12 to 45