Fusion proteins, understood as those created by joining two or more genes that originally encoded independent proteins, have numerous applications in biotechnology, from analytical methods to metabolic engineering. The use of fusion enzymes in biocatalysis may be even more interesting due to the physical connection of enzymes catalyzing successive reactions into covalently linked complexes. The proximity of the active sites of two enzymes in multi-enzyme complexes can make a significant contribution to the catalytic efficiency of the reaction. However, the physical proximity of the active sites does not guarantee this result. Other aspects, such as the nature and length of the linker used for the fusion or the order in which the enzymes are fused, must be considered and optimized to achieve the expected increase in catalytic efficiency. In this review, we will relate the new advances in the design, creation, and use of fused enzymes with those achieved in biocatalysis over the past 20 years. Thus, we will discuss some examples of genetically fused enzymes and their application in carbon‑carbon bond formation and oxidative reactions, generation of chiral amines, synthesis of carbohydrates, biodegradation of plant biomass and plastics, and in the preparation of other high-value products.

In its natural reaction, the fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) catalyzes the light-driven, redox-neutral decarboxylation of fatty acids via carbon-centered radical intermediates. Here, we systematically repurpose this radical pathway to unlock new reactivities, establishing CvFAP as a versatile platform for photobiocatalytic radical transformations. We expand its photocatalytic reaction portfolio by four new reactions: (i) the intermolecular Giese-type coupling of fatty acids with cycloalkenones, (ii) the intramolecular decarboxylative radical cyclization via nucleophilic radicals and isolated C=C bonds of the same philicity, (iii) a cysteine-mediated (Z)→(E) photoisomerization of unsaturated fatty acids, and (iv) a radical carbohydroxylation reaction that demonstrates the enzyme’s capability of olefin bis-functionalization. Enzyme Engineering, using computational modelling as basis, enhanced cyclization efficiency, demonstrating the evolvability of CvFAP towards new reactivities. The study expands the enzymatic repertoire of radical chemistry and demonstrates how photobiocatalysis can channel reactive intermediates to selective, new-to-nature bond-forming transformations, and pave the way toward enzyme-controlled radical couplings using abundant carboxylic acid feedstocks.

Reductive amination catalysed by imine reductase (IRED) and reductive aminase (RedAm) enzymes has recently been established as a powerful method for the asymmetric synthesis of chiral amines. While this biocatalytic technology has rapidly progressed from proof of concept to initial industrial applications, its scope and limitations remain to be fully explored. In this work, we report a broad and systematic profiling of reductive amination performance in the sequence space of IREDs and RedAms. This investigation employed an iterative strategy for activity and stereoselectivity screening, guided by chemo- and bioinformatic modelling as well as machine learning. By evaluating the catalytic performance of 175 IREDs against structurally diverse panels of 36 carbonyl compounds and 24 amines, we show that the majority of these enzymes is capable of asymmetric reductive amination at equimolar concentrations of the two substrates (50 mM each). The most effective enzymes identified in this study display sequence characteristics of RedAms, are active on 29–42% of the analysed substrate combinations, and combine high specific activities for the most favourable substrate pair (1.7−27.7 U/mgIRED) with excellent stereoselectivity. Beyond assembling this high-performance enzyme panel, we demonstrate extrapolation from our collected screening data to new substrate combinations by deep learning and the scale-up of selected reactions to a preparative batch size (10 mmol substrate, 200 mL reaction volume), delivering gram amounts of reductive amination products in high yield (63–89%) and optical purity (98% to >99% ee).