Cascade reactions catalyzed by multienzymatic systems have strongly moved into the focus of researchers in the field of biocatalysis because of their unique potential for the environmentally benign production of chemicals and materials. Inspired by Nature's ingenuity, considerable progress has been made in recent years to develop multistep reactions that combine the synthetic power of several enzymes in one pot. In addition, the combination of this biocatalytic power with the potential of chemical reactions, man-made transition-metal catalysts, and metalloenzymes has even further widened the repertoire of possible catalyzed reaction schemes. In this Review, we describe recent developments with respect to major challenges and solutions in the field of multienzyme cascade reactions, covering recent concurrent and sequential approaches with three or more enzymes in linear sequence as well as chemo-enzymatic reactions that combine a chemical step with at least two different enzymes over the last six years.

In order to provide parameters that can be used to tailor the crystalline and supramolecular structures of pure polyhydroxybutyrate, we synthesized polymers with fractions of meso groups in the range 0.5−1. We confirmed the random polymerization of R and S enantiomers by the catalyst. From Xray diffractograms, the lattice parameters were determined; they remained constant for the observed range of fractions. We also traced the directional crystallite sizes over tacticity, which change significantly for one unit cell direction. The respective crystalline phase atom fractions were quantified by iteratively fitting amorphous phase diffraction patterns. We found that the crystalline contents of small-crystallite polyhydroxybutyrates have so far been underestimated. X-ray diffraction and transmission electron microscopical observations from polymers with meso group fractions of 0.5 are discussed. To facilitate the quantification of crystalline atom fractions, we refined two accessible infrared absorption spectroscopy-based indices. These indices, and the fundamental correlations between chemical and crystallite structuring reported herein, allow to tune structure-dependent properties, e.g., melting point and toughness, of mixedtacticity polyhydroxybutyrates over wide ranges.

The combination of a heterogeneously catalyzed reaction with a biotransformation as a one-pot cascade process is an important strategy to reduce costs, time, and labor efforts in the production of chemicals from biogenic resources. Although one-pot cascade-type approaches generally result in more efficient chemical processes by reducing the number of workup operations needed and time consumed, the combination of different types of catalysts, both chemical and enzymatic, into a single reaction vessel often remains challenging. During our study, aimed at the direct synthesis of 2-keto-3-deoxy sugar acids as one intermediate toward biobased building blocks starting from the corresponding sugars by combining heterogeneous inorganic catalysis with enzyme catalysis, we encountered several incompatibility problems. These were overcome by a chemoenzymatic method in different compartments, which involves the gold-catalyzed direct oxidation by molecular oxygen and the subsequent conversion of the sugar acids through an enzymatic dehydration step. The described procedure represents an efficient synthesis route toward four different 2-keto-3-deoxy sugar acids and serves as a proof of concept for the combination of one-pot-incompatible catalysts under continuous flow.

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

The (βα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (βα)(8)-barrel proteins have evolved from an ancestral (βα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (βα)(8)-barrel proteins were constructed by the duplication of a (βα)(4) element of a modern (βα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (βα)(8)-barrels show the same refolding mechanism as HisF and other modern (βα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (βα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.

Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)8-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)8-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago.

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cellfree reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP + to NAD + , we identified selectivity-determining residues with the CSR-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced K M for the non-native cofactor NAD + (from 18 to 0.6 mM) and an increased activity for the desired substrate D-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.

We investigated the effect of including S enantiomers on the mechanical and thermal properties of predominantly (R)-β-polyhydroxybutyrate (PHB). From tensile testing, we determined resulting ratios of meso to racemo diads, for which elastic modulus, strength, and fracture strain combine to provide maximized fracture energies. We found that these coincide with an inversion of the respective elastic moduli of the amorphous and crystalline phases. From thermocalorimetric analyses, we determined the glass-transition temperatures and enthalpic relaxations, the heat capacities of the materials and their constituent phases, the directional crystallization rates and melting points, as well as the melting enthalpies for the α-PHB phase as functions of tacticity. We present a unifying characteristic, accounting for tacticity mismatches, based on the previously determined random polymerization action of the catalyst ethylzinc β-diketiminate and 4-methoxybenzyl alcohol. This characteristic provides a qualitative indication of the transition points in nonlinear correlations encountered between the ratios of meso to racemo diads and mixed-tacticity polyhydroxybutyrates’ fracture energies, amorphous and crystalline phase elastic moduli, melting enthalpies, and lattice vibrational frequencies.