Multienzyme Cascade Reactions—Status and Recent Advances

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Multi-enzyme cascade reactions – status and recent advances Josef Michael Sperl, and Volker Sieber ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03440 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Multi-enzyme cascade reactions – status and recent advances Josef M. Sperl and Volker Sieber* Chair of Chemistry of Biogenic Resources, Technical University Munich, Campus Straubing for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing (Germany) *E-mail: [email protected]

ABSTRACT: Cascade reactions catalyzed by multi-enzymatic systems have strongly moved into the focus of researchers in the field of biocatalysis due to their unique potential for the environmentally benign production of chemicals and materials. Inspired by Nature´s ingenuity, considerable progress has been made in developing multi-step reactions that combine the synthetic power of several enzymes in one pot, in recent years. In addition, the combination of this biocatalytic power with the potential of chemical reactions, man-made transition metal catalysts and metalloenzymes even widened the repertoire of possible catalyzed reaction schemes. In this review, we describe recent developments in regard of major challenges and solutions in the field of multi-enzyme 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.

KEYWORDS: biocatalysis • multi-enzyme cascades • chemo-enzymatic cascades • cofactors • incompatibility • compartmentalization Introduction Cascade reactions are undoubtedly advantageous over classical step-by-step synthesis through eliminating the tedious isolation and purification of reaction intermediates, which directly reduces costs and waste of the transformations. Besides that, also higher yields can be gained and the atom economy is improved as well. Further benefits of cascade reactions include the possible handling of unstable intermediates and the control and shifting of unfavorable reaction equilibria.1-2 Enzymatic cascades account for the largest fraction of successful examples, which is attributed to the fact that enzymes are generally active in aqueous buffer systems and at similar pH and temperature.3 In addition to this general property, falling prices for DNA synthesis, the development of improved engineering and high-throughput technologies as well as improved expression strategies have facilitated the access to enzymes of any kind, in the last decade.4-5 Consequently, the diversity of enzymatic cascades and the number of manuscripts that were published in this field of research rapidly increased within the past years.2 Beside a rising diversity of cascades one would also expect a rising complexity of particular cascades based on the easier access to enzymes. In this contribution we present an overview by giving selected examples of artificial in vitro cascades with at least two enzymatic reactions in combination with a chemical step or purely enzymatic systems comprising at least three enzymatic reaction steps not counting enzymes required for the regeneration of cofactors or the removal of poisonous side products with a focus on challenges and solutions of the field. Hence, our selection of examples is not intended to be comprehensive and we direct the reader to excellent in-depth reviews that were recently published. Schrittwieser et al. for example just provided an extensive and

encyclopaedic review including most cascades using two or more enzymes of the last years.2 Our review will solely cover multi-enzyme cascades with three and more enzymes and will be structured according to recent topics of the field. Enzymatic cascades and cofactors The majority of enzymatic cascades incorporate reactions that require stoichiometric amounts of expensive cosubstrates such as nicotinamide cofactors or nucleoside triphosphates. Achieving efficient regeneration of these cosubstrates is critical when aiming at economically viable processes. Many two-enzyme cascades have therefore been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. Using such systems, the second enzyme reaction shifts unfavorable reaction equilibria and drives reactions to the desired products.5 The necessity of sacrificial co-substrates as well as an additional enzyme makes these approaches unfavorable. As a consequence, especially for redox-cofactor depending systems self-sufficient one-pot reactions that show high atom economy and high molecular selectivity are pursued. For example, hydrogen borrowing cascades, also termed ‘closed loop reactions’ have been developed, in which both oxido-reduction reactions were used along the substrate-toproduct pathway.6-11 The concept was also integrated into a four-enzyme cascade for the conversion of a lignin model substrate by Reiter et al., who were able to simultaneously regenerate NAD+ and glutathione (Scheme 1).12 Typical regeneration methods must further be able to recycle the cofactor at least 100 to 106 times in order to be economically feasible.13 To reach these values, one of the most important prerequisite is the long-term stability of the cofactors, even if a complete cascade is cofactor neutral and regenerates the cofactor 1

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during the course of its reactions (as the hydrogen borrowing concept, for example).14

carbon flux, be balanced? To this end, the purge valve module was created that balanced the production and consumption of NADPH and NADH by using two different pyruvate dehydrogenases that selectively accept either NADP+ or NAD+ in combination with an NADH oxidase that does not oxidize NADPH (Scheme 3). The system worked in a self-regulatory manner and high NADPH concentrations were maintained for reduction purposes while simultaneously allowing an independent carbon flux from pyruvate to PHB or isoprene by purging of excess NADH.

Scheme 1. ‘Closed loop reaction’ sequence of the LigDFG enzyme system with a glutathione reductase for simultaneous regeneration of glutathione and NAD+.

Scheme 3. Design of a molecular purge valve using two different pyruvate dehydrogenases (PDH) and an NADH oxidase (NoxE).

Buffers, salts, pH-values, and the reaction temperature influence the stability of cofactors.13 With the increasing tendency to apply thermostable enzymes and run cascades at 50 °C and above this issue has become more pressing in the last years.15 Solutions to this problem could either be the use of novel artificial (biomimetic) cofactors, but their general application still requires huge efforts in engineering to adopt the enzymes to binding and converting them.16 A very interesting approach was recently described by Honda and coworkers, who combined eight different thermophilic enzymes to construct an artificial metabolic pathway for the salvage synthesis of NAD+ from its degradation products. Using this approach, they could keep NAD+ concentrations constant for almost 15 h at a temperature of 60° C (Scheme 2).17

Next to the redox cofactors the nucleoside triphosphates, (i.e. ATP) are mostly used because of their general phosphorylating property. Many available ATP regeneration systems rely on kinases that transfer a phosphate group of another phosphorylating agent to ADP. Recently, especially polyphosphate kinases have been used due to the availability and the relatively low price of polyphosphate.19 An extension of an ATP regeneration system based on polyphosphate kinases was shown by Mordhorst et al. who designed an SAM regeneration cycle for driving SAM-dependent alkylation reactions.20 The cyclic cascade consisted of the enzymes adenosine kinase, two polyphosphate kinases (PPK2-II and PPK2-I), methionine adenosyltransferase (MAT), selected methyltransferases (MT) and the methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). The first three enzymes regenerated ATP from adenosine. Having established the adenosine-to-ATP regeneration cascade, it was then linked to the three latter enzymes, forming a methylation cascade that had been developed before in the same laboratory.21 Overall, conversions of up to 25 % could be achieved for all tested methylation reactions, which corresponds to a more than 10-fold regeneration of SAM (Scheme 4).

Scheme 2. Enzymatic cascade for the salvage synthesis of NAD+ (simplified; NADase nicotinamidase, NaPRT nicotinate phosphoribosyltransferase, NaMAT nicotinate mononucleotide adenylyltransferase, NADS NAD+ synthase). While this enzyme cascade replenishes the NAD+ cofactor pool that is diminished by unwanted side reactions, there is still the problem that even perfectly cofactor balanced pathways lose reducing equivalents (e.g. NADH) by spontaneous oxidation.18 As a consequence, NADH equivalents get lost over time and conversion is reduced or stops. Opgenorth et al. recently described a molecular purge valve module as a strategy to overcome this issue.18 If the amount of NADH generated in cofactor balanced systems is not sufficient, why not design pathways that produce an excess of cofactors? But how can the cofactor generation, which is then decoupled from the

Scheme 4. SAM regeneration cycle consisting of an ATP production module and an SAM supply/methylation/SAH degradation cascade. Most recently Bowie and coworkers showed a molecular rheostat that maintained ATP levels to drive a synthetic biochemistry system.22 Aiming at the conversion of glucose to

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Scheme 5. Modularized cascades for the production of pyruvate, ethanol and isobutanol from glucose.

isobutanol, they first designed a pathway that was stoichiometrically balanced with respect to production and consumption of NADPH and also ATP. Facing ATPase contamination and also spontaneous ATP hydrolysis they redesigned the pathway and complemented the non-phosphorylation branch for the transformation of glyceraldehyde-3 phosphate into 3phosphoglycerate by a second, phosphorylating branch. The flow through each of these branches was regulated by the availability of free phosphate. Only upon hydrolysis of ATP, free phosphate built up and the phosphorylating branch was used. Overall, Bowie and coworkers built a molecular rheostat that is able to replenish ATP levels that suffer from unwanted ATP hydrolysis. Modularisation of multi-enzyme cascades – central intermediates, simple combination With the advances in enzyme production the number of enzymes per cascade to be reasonably handled will increase and the availability of a large toolbox of modules linking different compounds and types of conversion will highly accelerate the development of novel multi-enzyme cascades. Guterl et al. for example, reported two modularized cascades for the production of ethanol and isobutanol that both incorporated the same enzyme module for the production of pyruvate from glucose via a five-step four-enzyme cascade. Pyruvate as central intermediate could then either be decarboxylated and reduced to ethanol by the addition of a two enzyme module or it was condensed to acetolactic acid which was then further converted to isobutanol via four additional enzymatic reaction steps (Scheme 5). 25 mM D-glucose could be transformed to 28.7 mM ethanol or 10.3 mM Isobutanol within 19 to 23 hours.23 This example also demonstrated the power of connecting enzymes artificially for non-natural cascades. Instead of the ten enzymes utilized by yeast cells for the conversion of glucose to pyruvate here only four enzymes were required. This is possible since the intermediates are not required for the complex cellular metabolic network that is necessary for whole cell approaches. Parts of this cascade were recently reused and

transferred to the conversion of glycerol into (R)-Acetoin via a four enzyme cascade.24 Similar to the work of Guterl et al., Gao et al. used the dihydroxyacid dehydratase from S. solfataricus and the acetolactate synthase to convert D-glyceric acid to pyruvate, but extended this reaction to a novel cascade by coupling the oxidation of glycerol in a two-step approach to the production of D-glyceric acid via an alditol oxidase from S. coelicolor. Pyruvic acid was then further converted to Racetoin via acetolactic acid through decarboxylating steps catalyzed by acetolactate synthase and acetolactate decarboxylase (Scheme 6). Starting with 10.4 mM of glycerol, (R)acetoin was formed in 86 % analytical yield. Honda and coworkers also presented modularized in vitro pathways, e.g. for the synthesis of n-butanol from glucose.25 Here, the first module again transformed glucose to pyruvate, which was then further converted to acetyl-CoA and n-butanol in two consecutive modules. Overall, a direct conversion of glucose to n-butanol was achieved with a molar yield of 82 %. Three consecutive modules were described by Liu et al. who reconstituted the entire natural pathway to convert glucose into fatty acid.26 This pathway was separated into a glycolytic part, the pyruvate dehydrogenase complex and an acetyl-CoA carboxylase and fatty acid synthase module. After successful production of fatty acid from glucose, the glycolytic and the PDC branches were individually optimized using enzyme titration studies. Further optimization led to the replacement of the glycolytic branch by a pentose phosphate/glycolysis hybrid pathway and to a 6-fold increase in fatty acid production thus demonstrating the power of a modularized pathway design. A similar combination of the pentose phosphate pathway and a glycolysis part had earlier been shown by Opgenorth et al. who included a bifido shunt pathway for the production of Acetyl-CoA and also purge valves modules for the regulation of NADPH concentrations.27 After determination of enzymatic bottlenecks and subsequent optimization, a maximum productivity of 0.7 g/L/h of PHB was reached.

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Scheme 6. Synthesis of (3R)-acetoin from glycerol via pyruvate.

Scheme 7. Overview of modularized multi-enzymatic cascades for the conversion of various carbohydrate substrates to hydrogen and other products. PPi indicates an activation module by applying polyphosphate and a kinase. The combination of even up to four different modules is typical for the pioneering multi-enzymatic cascades of Zhang and coworkers.28-31 Their synthetic pathway biotransformations32 usually contain a first module for the production of glucose-1phosphate from different substrates. Each substrate has its own module, or even more than one as shown with starch, where one module is simply consisting of glycogen phosphorylase, yielding incomplete conversion. The more advanced module for this step combines four enzymes (isoamylase (IA), αglucan phosphorylase (αGP), phosphoglucomutase (PGM), and 4-α-Glucanotransferase (4GT)), which lead to almost complete phosphorylation of each glucose moiety. From G1P on further modules are applied depending on the target product. For example, one module for the conversion of G1P to G6P, one module containing pentose phosphate pathway enzymes to produce NADPH and regenerate (in part) G6P and one module for the generation of hydrogen from NADPH resulted in the almost stoichiometric formation of hydrogen from starch. Other modules were used for the production of amylose, inositol and fructose bisphosphate from the various starting materials (Scheme 7). Modularization, i.e. the design of cascades comprising a limited number of enzymes that are compatible with other modules of different enzymes is a great challenge, but offers even greater opportunities. Having a larger number of modules that are readily combined will enable us to convert virtually any starting material into any desired product, as long as required

energy can be incorporated (see ATP demand). Still the number of compatible modules is limited and the value is most often only utilized within the laboratories that designed the modules. Larger numbers of commonly or even commercially available modules would be desirable for faster research. Optimizing the productivity of systems To work optimally modules have to be tuned within and to each other and be compatible in regard of their conditions. Different approaches have been pursued to achieve optimal reactivity. Recently Zhang and his co-workers described a four enzyme cascade for which they systematically investigated the coexpression of the four thermophilic enzymes α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6phosphate dehydrogenase (G6PDH), and 6- phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignments, and comparing one or two plasmid systems.33 They found that the addition of a T7 terminator after each gene was useful to decrease the influence of the upstream gene. The best four-gene co-expression system for the demonstration of the generation of two NADPH molecules from one glucose unit of maltodextrin, whereby NADPH was oxidized to convert xylose to xylitol, was based on two plasmids (pET and pACYC) which each harbored two genes. As a result, apparent enzymatic activities of the four enzymes 4

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ACS Catalysis

were regulated to be at similar levels and the overall fourenzyme activity was the highest based on the formation of xylitol. This study thus provides useful information for the precise control of multi-enzyme-coordinated expression and for a simplified access to multi-enzyme approaches. The use of different promoters, different RBS sites, and the introduction of different codon usages could even optimize the access to well-coordinated multiple enzyme co-expression. Dudley et al. also performed the coexpression of several genes to yield Escherichia coli extracts for the synthesis of mevalonate.34 Differing from the previous approach, they first generated selectively enriched lysates containing singly overexpressed enzymes that were mixed in different ratios and combinatorically analyzed for mevalonate yields. Subsequently, a strain expressing all enzymes was generated using the information from the combinatorial analysis. After further optimization regarding small molecule cofactors, a mevalonate productivity of 0.88 g /L/h was reached. Another optimization of an enzyme cascade was recently reported by Beer et al. who performed a classical analysis of single enzyme activities in combination with the determination of inhibition constants. Their synthetic metabolic engineering cascade for the synthesis of α-ketoglutarate from Dglucuronate comprised four recombinantly expressed and purified enzymes in the linear sequence (Scheme 8).35 The reaction conditions were set based on an analysis of pH/buffer preferences of the enzymes and depending on the stability of the cofactor NAD+ in the buffer systems. Potassium phosphate buffer with a pH of 8 turned out to be the best buffer with regard to enzymatic activity, but was replaced by ammonium bicarbonate at a pH of 7.9 due to higher cofactor stability under these conditions. The reaction temperature of the cascade was set to 25 °C to account for the limited stability of the cofactor recycling NADH oxidase. A low concentration of NAD+/NADH but efficient recycling was the key to eliminate inhibitory effects and to improve the overall productivity of the cascade. As a result, 10 g/L of glucuronate were converted to α-ketoglutarate within 5 hours with a yield of 92 % and a maximum productivity of 2.8 g/L h.

Scheme 8. Synthesis of α-ketoglutarate from Dglucuronate via a four-enzyme cascade. Multi-enzymatic cascades are most often set up by preparing and characterizing the individual catalysts followed by putting them together in one pot to catalyze the desired reaction pathway. In general distinct amounts of each enzyme are used, based on specific activities or concentrations of the biocatalysts. In cases where this approach does not lead to satisfactory results, due to poor activities or incompatibility or inhibi-

tion effects, engineering of bottleneck enzymes, as well as compartmentalization and sequential approaches are pursued and succeed in most published examples. Besides these measures, the efficiency of multi-enzyme processes can also be enhanced by an optimization of the stoichiometry of the biocatalysts. A simple, but very efficient strategy to reach this goal are titration studies of optimal enzyme ratios.26-27 Yu et al. reconstituted the Escherichia coli fatty acid synthase complex using purified components.36 In order to find optimal ratios of the individual subunits, ten proteins were individually titrated whilst leaving the concentrations of all other components constant and the production of fatty acid was analyzed. The results of the titration studies revealed an optimal molar ratio of the fatty acid synthase complex subunits and led to the synthesis of 46 µM palmitic acid equivalents per minute. Zhu et al. performed a similar experiment for the production of farnesene by reconstitution of the mevalonate pathway.37 Again, the concentration of single proteins was varied while the concentrations of the other eight enzymes were kept constant. After these first experiments, a combinatorial pairwise variation of selected enzymes that had led to an increased farnesene synthesis when enhancing their concentrations was performed to unveal synergistic effects. Overall, the titration experiments led to an optimized molar enzyme ratio and to a sixfold enhancement in farnesene production. Damborsky and co-workers presented a workflow for the optimization of a five-step chemical conversion of 1,2,3trichloropropane to glycerol catalyzed by a three-enzyme system.38 This workflow was based among others on the determination of enzyme kinetics, the development of robust kinetic models and an in silico enzyme stoichiometry modelling. Sixteen steady-state kinetic parameters were determined based on Michealis-Menten kinetics and used to validate the proposed kinetic model by using experimental data for different enzyme mixtures. Finally, the model was used to minimize the enzyme load without compromising the productivity of the cascade. As a result the total biocatalyst load for 95 % productivity could be reduced by 56 % relative to the non-optimized system. Panke and co-workers recently showed a forward design approach for a ten-enzyme cascade system by combining online mass spectrometry and continuous system operation.39 They applied standard system theory input functions and used their detailed dynamic system responses to yield a model for forward design that allowed the optimization of a ten-enzyme cascade reaction for fine chemical production. The generation of high quality system response data in combination with sophisticated modeling might soon be generally applicable for the reduction of enzyme loads and an optimization of product yields. Besides the optimization of production rate and product yield, further parameters as cofactor balancing and side reaction suppression need to be considered in order to optimize multienzyme cascades. Schwander et al. encountered several issues during the design of a CO2 fixation pathway termed CETCH cycle.40 In addition to traditional approaches as changing reaction sequences and use of enzyme engineering to render promiscuous enzymes more specific, the authors introduced a proofreading enzyme to transform the dead-end metabolite malyl-CoA to the final product malate. This CO2-fixation multi enzyme cascade is further described in the section regarding CO2-related transformations. 5

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Bowie and coworkers developed purge valve modules18, 41 and a molecular rheostat22 that allowed for the maintenance of high NADPH and ATP levels which have been described in the cofactor section. Besides that they further introduced a salvage module to account for the side reaction of the promiscuous xylulose-5-phosphate phosphoketolase enzyme.27 Instead of performing laborious enzyme engineering to make this enzyme more specific, they added three further enzymes as salvage path to convert the dead-end intermediate erythrose-4phosphate back to on-pathway intermediates. Another approach to use synergies for the optimization of multi-enzyme cascades is based on enzyme coimmobilization. Immobilization is well known to yield reusability of enzymes and also to have stabilizing effects, which gives further advantages. Especially spatial proximity is often believed to impose substrate channeling which might increase reaction rates and reduce side reactions as well as loss of material (e.g. cofactors). Many substrates and also cofactors are more stable when they are bound to enzymes as they are protected from other reactants that might undergo wrong reactions. Increased reaction efficiencies and yields can be reached when intermediates between enzymatic steps are not in equilibrium with the bulk solution, but directly transferred via substrate channeling.42 Recent examples were again described by Zhang and coworkers who coupled dockerin domains to three consecutive enzymes of the glycolysis and gluconeogenesis pathways to enable co-immobilization onto a synthetic mini-scaffoldin. Interestingly, the catalytic efficiency of their three-enzyme complex was 33-fold of that of the free enzyme mixture. Even more interestingly, subsequent work showed that this effect, which was attributed to substrate channeling, was greatly influenced by the enzyme choice. When they replaced the rate limiting aldolase enzyme by another aldolase with higher activity the inferred degree of substrate channeling was largely reduced. Another three-enzyme dockerin-complex was reported by Chen and coworkers for the production of NADH via methanol oxidation.43 Here, they used cohesins displayed on yeast surfaces to bind the expressed dockerintagged dehydrogenases as multi-enzyme complexes and determined a five times higher NADH production rate compared to the non-complexed enzymes. Despite recent positive examples, substrate channeling is influenced by many different parameters and by now difficult to design. Nevertheless, the application of substrate channeling in multi-enzyme cascade reactions will enhance catalysis rates through the control of reactant diffusion as long as certain parameters are considered. To yield proper channeling, proximity alone is insufficient, but channeling can be achieved by electrostatic guidance, bounded surface diffusion and compartmentalization by tunneling.42 Compartmented approaches are also valuable for the removal of inhibiting effects and for special control of reactions parameters like buffers, salts, etc. These will be discussed in a separate part, after discussing general problems of incompatibility. Strategies to overcome incompatibility issues The development of multi-enzyme reaction processes often suffers from incompatibility problems that arise when different catalysts are combined together in one pot. These range from inadequate pH values, reaction temperatures and reaction

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media (buffer) to inhibition effects and side reactions caused by the increased number of intermediates and reaction products present in multi enzyme reactions. In the following part we describe recent approaches to these problems. Kroutil and co-workers recently developed three-enzyme cascades for the synthesis of different phenol derivatives.44-45 The reaction sequences were in both cases initiated by the action of a tyrosine phenol lyase (TPL) that performed an initial C-C coupling step of phenol with pyruvate and ammonia. The resulting L-tyrosine derivatives were subsequently deaminated and decarboxylated by the enzymes tyrosine ammonia lyase (TAL) and ferulic acid decarboxylase (FAD) to give para vinylated phenols 44 or underwent oxidative deamination (L-AAD) coupled to subsequent reduction (D/L-Hic) to give hydroyxphenyl lactic acids (Scheme 9).45 The first transformations to para vinylated phenols were performed with the enzymes TPL, TAL and FAD that showed different pH optima. Thus, to identify a suitable pH value for the three-step cascade process, the pH ranges of the three individual catalysts were analyzed and compared. The TPL as well as the TAL achieved the highest conversions at more alkaline pH values (pH 8–10 for TPL and pH 10 for TAL) whereas the decarboxylase preferred a pH range of 6–8. A medium pH (8 to 9) turned out to be suitable to facilitate the enzyme cascade. Further optimization was achieved by analyzing the use of cosolvents and different ammonium chloride concentrations. Finally, a variety of 2-, 3- and 2,3-substituted phenols was synthesized in high yield. The second cascade to hydroxyphenyl lactic acids had to be performed in sequential way as a two-stage protocol to overcome limitations through product that inhibited the TPL reaction. To circumvent the reduced activity of this first step in the presence of the final product, the oxidative deamination and the subsequent reduction were just started once the C-C bond formation was finished. Different phenol substrate could be transformed to both lactic acid enantiomers with high conversions up to >99 % and ee values >97 % (by HPLC). The two module three-step cascade was also performed at a preparative scale yielding enantiopure (S)3-(p-hydroxyphenyl) lactic acid with 77 % isolated yield and an ee value >97 %. The application of a two-stage protocol is one solution to extend a functional two-step cascade to a three enzyme cascade, if one of the reaction products is inhibiting previous enzymatic steps. Bornscheuer, Mihovilovic and others have devised a synthetic route from cyclohexenols to caprolactones using enzyme preparations and crude cell extracts by means of a three-step biocatalytic cascade using enoate reductases (Scheme 10). 46-49 Enoate reductases are versatile enzymes for the enantio- and regioselective addition of hydrogen to double bonds. For the first step of this cascade, they could identify three different enzyme variants from Pseudomonas putida ATCC 17453 through a sequence motif search (XenA, XenB, NemA). In addition to cloning, functional expression, and biochemical characterization of these enzymes, the enoate reductases were also applied in enzyme cascade reactions in combination with a Baeyer–Villiger monooxygenase and an alcohol dehydrogenase to subsequently produce the desired lactones as final products. One problem identified was the fact that the product of the cascade led to inhibiting effects.

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Scheme 9. Cascades for the synthesis of para vinylated phenols (A) and hydroxyphenyl lactic acids (B).

Scheme 11. Three-enzyme one-pot cascade for the synthesis of cyclohexylamine derivatives. Scheme 10. Conversion of cyclohexenols to caprolactones via three-step cascades. However, further extension of the cascade with a lipase to catalyze the ring-opening polymerization of the lactone products eliminated the inhibiting effects. In addition to eliminating the inhibiting effect by the product oligomerization, also the stability of the CHMO was increased by using a double mutant that showed higher oxidative and long-term stability.50 Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. Most recently, the above presented cascades were further investigated to yield a fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to again enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates.51 The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h 40% more dihydrocarvone lactone could be detected in in vivo reactions with the fusion protein and ADH than with the single enzymes. The authors wished to create fusion proteins of ADH/ ERED/BVMO aiming to improve the reaction rates by improving channeling of intermediates and to avoid undesired ADH side reaction. In summary, they could establish the first successful fusion of an ERED and a BVMO and could further improve the performance of the fusion protein by a introducing point mutations in the CHMO that had turned out to be beneficial in earlier experiments but the effects of potential substrate channeling were low.50 A threeenzyme one-pot cascade containing a lipase was reported by Siirola et al. for the synthesis of 3-substituted cyclohexylamine derivatives (Scheme 11).52

Here, the hydrolytic cleavage of a diketone was combined with a lipase-catalyzed esterification to get a substrate suitable for the subsequent transamination step to finally yield the desired cyclohexylamine compounds. At a first glance the reaction conditions for these two steps look incompatible, since water is required for the hydrolysis, while it is detrimental for the esterification. This cascade was nevertheless realized by using an organic system with a limited amount of water to promote the hydrolysis and retain enzymatic activity but also push the esterification towards the ester side. Unfortunately the following transamination was not performed simultaneously under the same conditions, but the organic phase could at least be directly subjected to a reductive amination in organic media to yield a sequential three-step one-pot biotransformation. Depending on the used transaminase different (1S, 3S)- or (1S, 3R)-methyl-2-(3-aminocyclohexyl) acetates could be produced from prochiral bicyclic beta diketones with excellent stereocontrol (ee >99 %, de from 97 to >99 %). Combination of enzymes with chemical steps As outlined in the introduction part, this review will not only present selected examples of multi-enzymatic reaction cascades that comprise solely enzymes as catalysts, but also chemo-enzymatic cascades that combine the world of biocatalysis with the world of chemical reactions and chemical catalysis. Recently, several reviews were published that covered this particular field.7, 53-54 Here the focus is on those examples that use at least one chemical and one enzymatic step in linear sequence. Moreover to fit to the special topic on multi-enzyme cascades, we present only those manuscripts that use at least one second enzyme independent of its use within a linear synthesis route or as a recycling or detoxifying catalyst. Parmeggiani et al. recently designed a deracemization cascade involving borane reduction for the synthesis of optically enriched D-amino acids.55 Their cascade process involved a phenylalanine ammonia lyase catalyzed amination of cinnamic acids, which was coupled to a subsequent enzymatic oxidation into the corresponding imino acid and the nonselective chemical back-reduction of the latter. In this case, a borane–ammonia complex was the option of choice for the re7

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duction due to its compatibility with proteins and its stability in water at high pH values and high ammonia concentrations. Overall, several D- and L-phenylalanine derivatives could be synthesized by this two-enzyme/chemical reduction cascade with conversions ranging from 62 to 80 % and great enantiomeric excess (97->99 %) (Scheme 12).

Scheme 12. Deracemization cascade involving borane reduction for the synthesis of D-amino acids. Recent work by Schrittwieser et al. demonstrated the construction of a cascade for the formation of berbines based on a deracemization of benzylisoquinolines (Scheme 13).56

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not lead to better results. Another deracemization cascade comprising the MAO was recently described by Ward and coworkers.57 Deviating from the two previous approaches of borane reduction they engineered an artificial transfer hydrogenase based on streptavidin (Sav) and identified mutant K121R based on screening tests. They also found that a compartmentalization of the cofactor within the Sav protein was crucial to achieve high yield and excellent conversion rates. The complete cascade was then used to transform racemic cyclic amines, which were first reduced by this artificial transfer hydrogenase using NADPH as cofactor which was in situ recycled by a traditional glucose dehydrogenase regeneration system (Scheme 14). Wrongly reduced (S)-amine was directly reoxidized by MAO to yield a deracemization cascade which produces enantiopure (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in full conversion requiring only two equivalents of glucose. H2O2 which is generated through the oxidase reaction was converted to water by the action of a catalase.

Scheme 14. Combination of a monoamine oxidase with an artificial transfer hydrogenase for the synthesis of (R)Methyl—1,2,3,4-tetrahydroisoquinoline. Scheme 13. Chemo-enzymatic synthesis of optically pure (S)-Berbines. The (R)-forms of the benzylisoquinolines were first enzymatically oxidized under loss of the stereogenic center through the action of a mono amine oxidase (MAO). Subsequent chemical reduction with again borane reagents then reintroduced the stereocenter in racemic fashion. The combination of MAO and borane lead to a specific racemization of the (R)benzylisoquinolines. A comparison of the Me3N-BH3 and morpholine-BH3 complexes with NH3-BH3 in the stereoinversion revealed, that the former two actually performed better than the commonly employed reducing agent NH3-BH3, whereby morpholine-BH3 worked best as it ensured compatibility of the reduction with the C-C bond forming step. After these prerequisite investigations, a stepwise one-pot procedure was tested first which also incorporated the berberine bridge enzyme (BBE) catalyzing the last step to yield the respective berbines under stereospecific ring-closure. Thus, the stereoinversion of substrates was first run to completion using the MAO-N/borane approach before BBE was added to the reaction mixture. The BBE-catalyzed ring-closure was either carried out after removal of the MAO-N biocatalyst by centrifugation, or BBE was directly added to the reaction mixture without significant changes to the transformation yields. Consequently, the two transformations were also performed simultaneously in one pot. Thus, the two enantioselective oxidation reactions catalyzed by two different flavin-dependent enzymes (MAO and BBE) and the non-stereoselective morpholine–BH3 reduction were started at the same time, yielding a kinetic resolution process for the synthesis of (S)-berbines in high conversion and excellent ee values. In this case, deviating from the previous experience of the authors, the addition of catalase to remove H2O2 and prevent oxidative inactivation did

Compartmented approaches Compartmented approaches were pioneered by the groups of Gröger and Mihovilovic who reported on this valuable strategy to overcome incompatibility issues.58-59 Mihovilovic and co-workers, for example, had described a compartmented combination of the two worlds of catalysis (Scheme 15).58

Scheme 15. Hydrogenation and enzymatic oxidation cascade under continuous flow. In their case study, they could successfully demonstrate the combination of the two different classes of catalysis and two reaction modes by integrating a heterogeneous chemical reduction and a subsequent biocatalytic oxidation in a compartmented continuous flow setting, thus reducing a multi-step synthesis protocol of at least seven steps to a single-operation protocol. Recently, further compartmentalization approaches have been described. Gröger, Liese, Bornscheuer and coworkers reported an alternative synthetic approach towards poly-ε-caprolactone (PCL) starting from phenol and based on the combination of four synthetic key steps comprising metal catalysis as well as enzymatic reactions.60 Their process concept is based on a metal-catalyzed hydrogenation of phenol to cyclohexanol and the subsequent coupling to the bi-enzymatic transformation into ε-caprolactone as described above. Again, cyclohexanol is first oxidized by means of a dehydrogenase to 8

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Scheme 16. Compartmented chemo-enzymatic synthesis of poly-ε-caprolactone. cyclohexanone under consumption of NADP+. In the second enzymatic step NADPH is converted back into NADP+ through the oxidation of cyclohexanone to ε-CL with the help of a monooxygenase. The final step then comprises an in situ removal of the built ε-CL without work-up and direct polymerization towards PCL. The goal of this work was to establish subsequent enzymatic steps without the need for isolation and purification of any of the intermediates. To reach this, the hydrogenation step needed to be compatible with the subsequent enzymatic double oxidation and the earlier developed product polymerization to reduce inhibiting effects. This could be achieved by removing the product from the aqueous compartment across the PDMS-membrane by the application of a proper solvent (Scheme 16). The PDMS-membrane was impermeable to methylcyclohexane, so this solvent was used to protect the enzymes in the aqueous phase. In a final step, the cascade could be extended to the production of PCL by integrating the lipase catalyzed polymerization in the organic phase. Unfortunately, the total conversion dropped from 97 to 81 % when adding this additional step due to the blocking of the PDMS-membrane by the newly synthesized polymer. Again Gröger and coworkers recently described another compartmentalization approach using similar PDMS-thimbles for the formal asymmetric hydroamination of non-activated alkenes.61 This process enabled direct access to enantiomerically pure amines by conversion of the corresponding styrenes based on a combination of the chemical Wacker-oxidation and an enzymatic transamination in one pot. The key feature to realize this process was again the compartmentalization of the chemo- and biocatalyst components in PDMS-thimbles, which keep water-soluble metal salts and transaminase and cofactor separated. Overall (S)methoxyphenylethylamine could be produced in 92 % yield with excellent enantiomeric excess (Scheme 17).

Scheme 17. Hydroamination of non-activated alkenes using a compartmented chemo-enzymatic approach. Klermund et al. also used PDMS to overcome incompatibilities, but instead of using thimbles, they prepared PMOXAPDMS-PMOXA polymersomes which they used to encapsulate or attach their enzymes.62 Studying the three-enzyme conversion of N-acetylglucosamine (GlcNAc) to CMP-Nacetylneuraminic acid (CMP-Neu5Ac) the authors wanted to

overcome the limitation that CTP, a substrate of the third step, inhibits the first enzymatic step catalyzed by an Nacetylneuraminate lyase (NAL). Similarly to the production of hydroxyphenyl lactic acids (Scheme 9), a sequential process with adding CTP as soon as the second reaction finished, was not feasible here, as the reaction equilibria of the first and second step lie on the substrate side and this equilibrium is just shifted by the third step. So again a compartmentalization approach was required to separate the incompatible reactions of the N-acyl-D-glucosamine 2-epimerase (AGE) and the CMP-sialic acid synthetase. This was reached by immobilizing the NAL and the CSS on the outer surface of the polymersomes whereas the AGE was encapsulated within the lumen. To overcome mass-transport limitations, Castiglione and co-workers reconstituted highly selective channel proteins which resulted in an overall 2.2-fold improvement compared to the non-compartmentalized reaction. Sperl et al.63 recently reported a compartmented chemoenzymatic approach using a heterogeneous catalyst. They started with the synthetic aim of producing 2-keto-3-deoxy sugar acids directly from the corresponding sugars. To reach this, they first studied the gold catalyzed oxidation of sugars to the corresponding sugar acids according to previously published procedures. Subsequently, they analyzed the dehydration of these sugar acids by the dihydroxyacid dehydratase from Sulfolobus solfataricus. To yield a one-pot cascade type approach the concurrent combination of both catalysts was analyzed. Several incompatibility problems were encountered such as mutual inactivation and differing pH optima that could only be resolved by a spatial separation of the gold catalyst and the SsDHAD. Furthermore, also H2O2 which is built during the chemical catalysis step needed to be removed by the action of a catalase in order to retain the activity of the SsDHAD. In total they were able to perform the direct synthesis of 2-keto-3-deoxy sugar acids under continuous flow. Compartmentalization was crucial to achieve the combination of chemical and enzymatic catalysis, in this case. Another interesting strategy for the immobilization and colocalization of enzymes was described by Mann and coworkers who showed a method for the hierarchical selfassembly of protein–polymer surfactant bioconjugates.64 They did not show a real synthetic cascade, but they could set up three-enzyme cascades using β-glucosidase, glucose oxidase and horse radish peroxidase. In conclusion, they could present that self-supporting multi-enzyme films can be fabricated via hierarchical assembly of protein–polymer surfactant nanoconjugates and used to conduct a three-step cascade reaction.

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Selected examples in regard of products The synthesis of fine chemicals is the major playing field for the development of multi-enzyme cascades, although the desire to produce a certain molecule needs to be complemented by the availability and suitability of certain enzymes. In the last part of the review some recent developments for specific desirable substrates or products are presented. C1/CO2-related transformations Gradually increasing atmospheric concentrations of carbon dioxide promote global warming but also pose the opportunity as an important future carbon feedstock. To reach efficient strategies, several approaches have been pursued for the fixation of CO2 into methanol as C1 compound during the last years. Most recently, Erb and coworkers also worked on the fixation of CO2 but aiming at its conversion into multicarbon compounds.40 To this end, they developed a synthetic cycle comprising 17 different enzymes that convert CO2 to malate at a rate of 5 nmol of CO2 per minute and mg of protein (Scheme 18). Interestingly, several rounds of enzyme engineering had to be applied to reach this value and a concept of metabolic proofreading was included by adding enzymes that convert dead end intermediates back to on-pathway molecules.

Scheme 18. CO2 fixation cascade for conversion into multicarbon compounds (simplified). Classical approaches for the fixation of CO2 to MeOH as well as for the oxidation of MeOH to yield reduction equivalents have recently been accompanied by sophisticated coimmobilization and substrate channeling approaches. Sets of three different dehydrogenase enzymes (alcohol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase) were used by Luo et al. 65 as well as Wang et al.66 based on immobilization strategies. Stable co- and sequential immobilization of the cascade enzymes was achieved on flat-sheet polymeric membranes or within ultrathin hybrid microcapsules. A three-enzyme-cascade for the generation of NADH by methanol oxidation was described by Liu et al. by using a similar set of enzymes.43 At a first glance, enzymatic cascades of this type seem to be of rather academic interest than of industrial or applicative relevance, but these cascades can be used to provide NADH for subsequent processes. The three dehydrogenases could be tagged with dockerin domains, thus enabling the immobilization on yeast cell surfaces. The sequential assembly resulted in a more than five times higher NADH production rate compared to free enzymes, which was attributed to efficient substrate channeling. Recent work by Hollmann and co-workers also demonstrated the construction of three-enzyme cascades for the conversion of methanol to CO2.67-68 The combined use of an alcohol dehydrogenase, a formate dehydrogenase and a formaldehyde dismutase for example led to the production of three equivalents of NADH per molecule of methanol that is oxidized to CO2 (Scheme 19).

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This NADH production cascade was then used as NADH regeneration system for the hydroxylation of 3hydroxybenzoate which was catalyzed by a 3hydroxybenzoate-6-hydroxylase or for promoting the reduction of conjugated C=C-double bonds by an enoate reductase. The poor activity of the alcohol dehydrogenase was described as the major limitation for this cascade leading to significantly higher methanol concentrations that were needed in order to reach good conversion.

Scheme 19. Oxidation of methanol to CO2 for the production of NADH / H2O2. Shortly after this contribution, the methanol to CO2 cascade was adopted to the production of H2O2 (Scheme 19). To this end, the alcohol dehydrogenase was replaced by alcohol oxidases of which the Pichia pastoris enzyme proved to be superior compared to the enzyme from Candida boidinii. A further extension of the cascade by using formate oxidases was impaired by their pH optima at relatively acidic conditions ( pH 3 to 4), so the authors used again the formate dehydrogenase of the previous cascade and coupled the generation of NADH to H2O2 production via aerobically reoxidizing it through the 3hydroxybenzoate-6-hydroxylase from Rhodococcus jostii RHA1, which is also able to act as NADH oxidase. Finally, hydroxylation of ethylbenzene was shown to be promoted by H2O2 that was generated by the oxidation of methanol to CO2. When methanol was the limiting reagent, the complete system formed 3 equivalents of product, while only two equivalents were formed using the alcohol oxidase and formaldehyde dismutase and only one equivalent using the alcohol oxidase alone. Synthesis and conversion of carbohydrates The use of aldolases could provide direct stereoselective access to carbohydrate molecules without the need for tedious protecting group manipulations. One important prerequisite for the utilization of the full potential of existing aldolases in C–C bond formation is an easy access to their phosphorylated substrates. A simple procedure has been developed for the synthesis of enantio- and diastereomerically pure carbohydrate analogues from glycerol and a variety of aldehydes in one pot using a four-enzyme cascade reaction.69 The fourth enzyme in this cascade is a catalase that is required to remove H2O2 so in the linear sequence there are still only three enzymes. As a proof of concept, the naturally occurring azasugar D-fagomine was synthesized in a two-step synthesis in 69 % yield, thus highlighting the value of the developed cascade to selectively form complex products that by previous traditional organic chemistry could only be obtained via repeated isolation and purification of intermediates. Another type of cascade reactions for the synthesis of carbohydrates involving C-C bond formation was recently reported by Lemaire and co-workers (Scheme 20).70 Again, in the first stage DHAP was prepared but this time with a set of two kinases that phosphorylated DHA to DHAP by using PEP as Pi 10

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donating agent. The further reaction sequence included an isomerization of DHAP to GAP and the subsequent synthesis of F6P by the FSA aldolase. In this case, again purified enzymes were used but not in their soluble form. The authors coimmobilized the enzymes in so-called layered double hydroxides, an inorganic layered matrix. In this case, the biohybrid catalyst, a term the authors gave to their enzyme aggregates, showed the same reaction rate as cascades containing free enzymes and it was also reusable. 66.6 mM dihydroxy acetone was successfully converted to D-fructose-6-phosphate within 1 h by testing different matrix compositions and enzyme to nanomaterial ratios.

Scheme 20. Synthesis of fructose-6-phosphate by an enzymatic cascade immobilized in layered double hydroxides. A strategy for the synthesis of carbohydrates of a more complex type was recently shown by Flitsch and co-workers who developed the syntheses of glycopeptides containing the Omannosyl glycan NeuNAc α2-3Galβ1-4GlcNAcβ1-2Manα.71 Three consecutive enzymatic glycosylations were employed to yield tetrasaccharide fragments through a biomimetic stepwise assembly from the reducing end in a highly efficient manner. Besides the demonstration in a one-pot solution, the authors could also synthesize the carbohydrates on solid support, providing rapid access to the desired structures and their intermediates which are currently used to investigate the role of this unusual glycan in the binding of α-DG to its various receptors. A highly efficient enzymatic modular assembly (EMA) strategy for the diversity oriented parallel synthesis of all 15 naturally occurring histo-blood group ABH antigens was described by Ye et al. 72 They provided an operationally simple and elegant access in only three steps from 5 readily available disaccharide acceptors and 3 monosaccharides as donor precursors by using recombinant enzymes that were grouped into enzyme modules. The enzyme modules could be combined in a combinatorial way and each consisted of a sugar transferase coupled to one or two other enzymes. As an example, five H antigens could be converted to A antigens in one pot on preparative scales with up to 90 % yield after purification. Current strategies and promising developments Numerous excellent reviews on enzymatic cascades have been published within the last years.1-5, 32, 73 Most of them have not only summarized the development in the field and judged the progress, usually those reviews have also described roadmaps for future research and endeavors researchers should pursue within their work. These tasks range from classical goals as the optimization of the availability of enzymes through engineering and the development of novel expression strategies and purification methods to an optimization of the handling of

enzymes by immobilization and stabilization approaches. More recently sophisticated techniques that have not been analyzed in the past attract more and more attention. Among these are compartmentalization, co-immobilization and colocalization strategies as well as improved modeling of cascades to reduce enzyme loads and improve yields. Interestingly, when evaluating some of the most recent articles in the field, e.g. the compartmentalization approaches of Gröger and co-workers, these reviews read as a manual for the preparation of manuscripts. Thus, this last part of our review seems to us like a wish-list. What would be desirable developments for the coming years? Whole-cell based approaches still outperform in vitro cascades in many cases due to low product titers and high costs of enzymes and cofactors.74 Optimization of enzymes using enzyme engineering could enhance the activity and stability and thus lead to optimized product titers and reduced costs. However, regarding multi-enzyme cascades, engineering of a multitude of enzymes is extremely laborious and may not reach satisfactory goals in a reasonable amount of time. So definitely more and better strategies are still needed for the immobilization, compartmentalization and colocalization as these aforementioned tasks have despite impressive progress not been reached in a satisfactory way. Efficient immobilization techniques that can be applied to many different enzymes and that lead to higher stability and higher enzyme concentrations would reduce the need for enzyme engineering and speed up the optimization process. Novel compartmentalization methods would reduce incompatibility issues and effective co-localization could reduce the formation of dead-end metabolites and thus either reduce the number of enzymes (for e.g. metabolite proofreading) or again engineering efforts to make promiscuous enzymes more specific. In addition such newly developed strategies need to be readily transferable to other kinds of cascades via modularization approaches in order to unveil their complete power to the field. Sophisticated modelling approaches will help to combine different modules and optimize enzyme loadings and in vitro metabolite flux. Besides that, we believe that orchestration, sequestration, regulation and control are important parameters that have been overlooked so far.5 Research should be aimed at outperforming Nature´s great cascades within our laboratories and also within industrial plants. Orchestration and compartmentalization could, through the regulation of time and space of reactions and the ingenious arrangement of different reactions, lead to a controlled use of the Gibbs potential and a wise use of chemical energy. Tunable cascades for integrated plants that can be controlled to synthesize a variety of desired compounds irrespective of the available starting material is our vision for the near future to make life on earth sustainably better by the help of man-made enzymatic reaction cascades.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +499421187301.

Author Contributions All authors have given approval to the final version of the manuscript.

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62. Klermund, L.; Poschenrieder, S. T.; Castiglione, K., ACS Catal. 2017, 7, 3900-3904. 63. Sperl, J. M.; Carsten, J. M.; Guterl, J.-K.; Lommes, P.; Sieber, V., ACS Catal. 2016, 6, 6329-6334. 64. Farrugia, T.; Perriman, A. W.; Sharma, K. P.; Mann, S., Chem. Commun. 2017, 53, 2094-2097. 65. Luo, J.; Meyer, A. S.; Mateiu, R. V.; Pinelo, M., New Biotechnol. 2015, 32, 319-27. 66. Wang, X. L.; Li, Z.; Shi, J. F.; Wu, H.; Jiang, Z. Y.; Zhang, W. Y.; Song, X. K.; Ai, Q. H., ACS Catal. 2014, 4, 962-972. 67. Kara, S.; Schrittwieser, J. H.; Gargiulo, S.; Ni, Y.; Yanase, H.; Opperman, D. J.; van Berkel, W. J. H.; Hollmann, F., Adv. Synth. Catal. 2015, 357, 1687-1691. 68. Ni, Y.; Fernandez-Fueyo, E.; Baraibar, A. G.; Ullrich, R.; Hofrichter, M.; Yanase, H.; Alcalde, M.; van Berkel, W. J. H.; Hollmann, F., Angew. Chem., Int. Edit. 2016, 55, 798-801. 69. Babich, L.; van Hemert, L. J. C.; Bury, A.; Hartog, A. F.; Falcicchio, P.; van der Oost, J.; van Herk, T.; Wever, R.; Rutjes, F. P. J. T., Green Chem. 2011, 13, 2895-2900. 70. Mahdi, R.; Guerard-Helaine, C.; Prevot, V.; de Berardinis, V.; Forano, C.; Lemaire, M., ChemCatChem 2015, 7, 3110-3115. 71. Sardzik, R.; Green, A. P.; Laurent, N.; Both, P.; Fontana, C.; Voglmeir, J.; Weissenborn, M. J.; Haddoub, R.; Grassi, P.; Haslam, S. M.; Widmalm, G.; Flitsch, S. L., J. Am. Chem. Soc. 2012, 134, 4521-4524. 72. Ye, J. F.; Liu, X. W.; Peng, P.; Yi, W.; Chen, X.; Wang, F. S.; Cao, H. Z., ACS Catal. 2016, 6, 8140-8144. 73. Dudley, Q. M.; Karim, A. S.; Jewett, M. C., Biotechnol. J. 2015, 10, 69-82. 74. Taniguchi, H.; Okano, K.; Honda, K., Synth Syst Biotechnol 2017, 2, 65-74.

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