Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to

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Constructing Biocatalytic Cascades: In Vitro and In Vivo Approaches to De Novo Multi-Enzyme Pathways Scott P. France, Lorna J Hepworth, Nicholas J Turner, and Sabine L. Flitsch ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b02979 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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Constructing Biocatalytic Cascades: In Vitro and In Vivo Approaches to De Novo Multi-Enzyme Pathways Scott P. France,‡ Lorna J. Hepworth,‡ Nicholas J. Turner* and Sabine L. Flitsch*.

School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. ‡ These authors contributed equally to the manuscript.

ABSTRACT The combination of sequential biocatalytic reactions, via non-natural synthetic cascades, is a rapidly developing field and leads to the generation of complex valuable chemicals from simple precursors. As the toolbox of available biocatalysts continues to expand, so do the options for biocatalytic retrosynthesis of a target molecule, leading to new routes employing enzymatic transformations. The implementation of such cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed in vitro or in vivo. In this Perspective, we describe the relative merits of in vitro, in vivo or hybrid approaches to building biocatalytic cascades and showcase recent developments in the area. We also highlight the factors that influence the design and implementation of purely enzymatic or chemo-enzymatic, one-pot, multi-step pathways.

KEYWORDS: biocatalysis, enzymatic cascades, in vitro biotransformations, in vivo. biotransformations, biocatalytic retrosynthesis, whole-cell biocatalysis, enzymes, cofactors.

INTRODUCTION The repertoire of organic reactions that can be mediated by biocatalysts is rapidly increasing, driven by improved methods for enzyme discovery and screening together with faster and cheaper gene synthesis.1–9 The outcome is an ever-expanding biocatalytic toolbox consisting of natural enzymes, engineered or evolved enzymes10–16 and artificial enzymes17–19 which are capable of catalyzing an increasing range of chemical transformations. The availability of a broader range of biocatalysts also means that it is increasingly possible to build entirely de novo enzymatic pathways, both in vitro and in vivo, conceived and designed through a biocatalytic retrosynthetic approach (Figure 1).20–22

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Figure 1. Design-Implement-Analyze Cycle of Multi-Enzyme Cascade Development.

The term ‘enzymatic cascades’ has been used to describe (chemo)enzymatic processes consisting of two or more steps for the production of valuable chemical compounds.23–35 These cascades are distinct from natural biosynthetic pathways (not discussed in this Perspective) that have evolved over time to ensure often exquisite flow of simple metabolites to complex natural products. However, an analysis of natural biosynthetic pathways reveals some of the hallmarks and important features of efficient enzymatic cascades: (1) The overall thermodynamic parameters of the cascade are favorable (ΔGcascade < 0). (2) Selectivity: Enzymes catalyze reactions with high reaction specificity and functional group orthogonality in order to avoid unwanted cross-reactivity between different substrates. (3) The overall kinetic reaction parameters are controlled by enzyme activities to ensure reaction flux. In this Perspective we review recently published examples of synthetically useful non-natural enzyme cascades to illustrate the design principles involved and also how these important features of cascade processes are addressed in practice. We discuss these cascades in terms of their operation as either cell-free systems (in vitro) or in whole-cells (in vivo), and focus on concurrent cascade reactions rather than sequential processes. The selection of examples is not intended to be comprehensive, but rather didactic in nature, and hence we direct the reader to several recent excellent in-depth reviews.36–43 Although not covered in this Perspective, considerable effort in recent years has been made regarding the engineering of native biosynthetic pathways, and a variety of valuable natural products, such as vanillin44 and artemisinin,45 have been successfully


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obtained in considerable yields through a synthetic biology approach. This area has been extensively reviewed elsewhere in many informative articles.46–52

IN VITRO VS IN VIVO Enzyme cascades can be broadly classified as either (i) in vitro, (ii) in vivo or (iii) hybrid as shown in Figure 2. The selection amongst these three options for a particular application typically depends upon a number of factors, including availability of gene sequences and heterologous enzymes, cofactor requirements, substrate and product uptake and release, and the metabolic stability of substrates and product.

Figure 2. Comparison of (i) in vitro, (ii) in vivo and (iii) hybrid cascade reactions. Product D is generated from starting material A using highly selective biocatalysts cat i-iii.

IN VITRO CASCADES In vitro cascades based on enzymatic or chemo-enzymatic reactions have been developed for a number of years, with an early example reported by Fessner and co-workers using aldolases for the synthesis of branched-chain saccharides.53 These cascade reactions use purified enzymes, cell lysates, cell-free extracts or freeze-dried whole-cells, and offer advantages such as the relative ease of fine-tuning the amounts of each (bio)catalyst present in the system to maximize the overall flux and yield of products. The application of purified enzymes avoids any complications that arise from the complex metabolic pathways operating in living whole-cells, although the use of crude enzyme preparations does not completely eliminate these potential side reactions. Additionally, enzyme purification is an expensive and time-consuming process, especially for scale-up and potential industrial application. Likewise, if biocatalysts are cofactor dependent, expensive exogenous cofactors need to be added in stoichiometric amounts or alternatively the cascade needs to be supplemented with a recycling system.


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Scheme 1. In Vitro Hydrogen-Borrowing Cascade for the Production of Chiral Amines from Alcohols.

A solution to the issue of cofactor requirements was recently reported for the production of chiral amines from the corresponding alcohols via an intermediary ketone compound (Scheme 1).54 In this example an alcohol dehydrogenase (ADH) was employed to oxidize the alcohol starting material 1 to the ketone 2. The NADH generated during the first step was used in the subsequent step in which an amine dehydrogenase (AmDH) catalyzed a reductive amination of the ketone with ammonia. Overall, the cascade is redox neutral and the NAD(H) cofactor was recycled by a ‘hydrogenborrowing’ process55,56 in which the hydrogen abstracted in the first step was reinstalled in the second. Ultimately, an in vitro approach using purified enzymes was required due to competing reactions catalyzed by endogenous proteins present when a crude cell preparation of the ADH was used. These side reactions sequestered and oxidized the NADH cofactor required for the AmDH step and thus disrupted the hydrogen-borrowing nature of the cascade. High conversions were achieved (85%, >99% ee) and preparative-scale reactions (100 - 126 mg of starting material 1) were demonstrated to afford moderate to excellent isolated yields (30 to 91%) and high ee values (82 to >99%).

Scheme 2. In Vitro Cascade for the Production of para-Vinylphenols 8 Starting from Phenols 5 and Pyruvate 4.

Kroutil and coworkers developed a highly selective biocatalytic route to perform the formal paravinylation of phenols via a three enzyme in vitro cascade (Scheme 2).57 The important initial C-C bond forming step was catalyzed by a tyrosine phenol lyase (TPL) that coupled the phenol substrate 5 with pyruvate 4 and ammonia to generate an L-tyrosine intermediate 6. This compound was then


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deaminated by a tyrosine ammonia lyase (TAL) to afford a coumaric acid derivate 7 that was finally decarboxylated by a ferulic acid decarboxylase (FAD). The ammonia by-product generated as part of the TAL transformation could be reused in the first TPL-catalyzed step and all reactions were run in a one-pot concurrent fashion. A wild-type or engineered TPL was used as a cell-free extract and the TAL and FAD were recombinantly expressed separately in E. coli and employed as freeze-dried cell preparations. A range of 2-, 3- and 2,3-substituted phenols could be successfully vinylated in high conversion (>99%) and the synthetic utility of the cascade was demonstrated by high yielding preparative-scale examples (65 to 83%, on approximately 50 mg of starting material 5).

Scheme 3. In Vitro Cascade for the Production of (1R,2R)-Norpseudoephedrine (1R,2R)-11 and (1R,2S)-Norephedrine (1R,2S)-11 from Benzaldehyde 9 and Pyruvate 4 with an Optional Recycling Mode for Pyruvate.

Rother et al. have devised a synthetic route to (1R,2R)-norpseudoephedrine (1R,2R)-11, or (1R,2S)norephedrine (1R,2S)-11 by means of a two-step biocatalytic cascade (Scheme 3).58 A thiamine diphosphate (ThDP)-dependent acetohydroxyacid synthase I (AHAS-I) was employed in the first step to decarboxylate pyruvate 4 and perform a ligation to benzaldehyde 9, affording the intermediate (R)-phenylacetylcarbinol 10 with high stereoselectivity (>98%). Subsequently an (R)- or (S)-selective transaminase converted 10 into the final products (1R,2R)-11 and (1R,2S)-11, respectively, using alanine as a co-substrate. Interestingly, the cascade could be operated in a ‘recycling’ mode in which the pyruvate by-product generated by the transaminase step could then re-enter the cascade, either directly as a substrate in the first step or via the reversible formation of an acetolactate intermediate 12. One problem identified when operating the cascade as a concurrent process was the fact that benzaldehyde 9 was also a substrate for the transaminase biocatalyst. Indeed, using an (S)-selective transaminase gave only 2% product (1R,2S)-11 with the major product being undesired benzylamine. However, a commercially available (R)-selective transaminase was identified that possessed initial rate activities for benzaldehyde 9 that were approximately ten times lower than the AHAS-I step. This enabled greater conversion to 10 by the AHAS-I before 9 could be intercepted by the transaminase, and allowed the cascade to be run in a concurrent fashion to afford product (1R,2R)-


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11 with 85% conversion. In comparison, when run in the concurrent ‘recycling’ mode, only 70% conversions could be achieved. To overcome the problems associated with the cross-reactivity between 9 and the transaminases, a sequential process was developed in which the transaminase was added after complete conversion in the first step. This enabled higher conversions to be achieved compared to the concurrent process: (1R,2S)-11 was obtained with 80% conversion and (1R,2R)-11 with >96% conversion with high de and ee (>98%). The temporal separation of the two biocatalytic cascade steps in the sequential process meant that the ‘recycling’ mode was not possible directly; however, more 9 could be added after the complete sequential cascade had been run to achieve further conversion.

Scheme 4. In Vitro ‘Triangular’ Cascade for the Formation of Benzylisoquinoline Alkaloids 15 from Phenylethylamines 13.

Recent work by Hailes, Ward and coworkers demonstrated the construction of a ‘triangular’ cascade for the formation of benzylisoquinoline alkaloids (Scheme 4).59 The reaction sequence was initiated by the addition of amine 13, which generated the aldehyde 14 by means of a transaminase (ω-TA) step with pyruvate as the amine acceptor. Subsequently, a norcoclaurine synthase (NCS) catalyzed the asymmetric Pictet-Spengler reaction between 13 and 14 to give the benzylisoquinoline 15. Control of the number of equivalents of pyruvate in the transaminase step enabled the correct stoichiometry of 13 and 14 in the system and problems often associated with transaminase equilibria were avoided by the subsequent removal of equal quantities of the amine and aldehyde by the NCS. It was important to optimize the reaction parameters to ensure the rates of the two biocatalytic steps were sufficiently matched to circumvent aldehyde 14 accumulation that would lead to a competing, non-enzyme catalyzed, Pictet-Spengler reaction and formation of racemic product. The cascade was operated with a purified NCS and a transaminase employed as a lysate. Optimization of the reaction parameters resulted in a system capable of converting 20 mM starting material in 87% conversion and in 99% ee. Tetrahydroprotoberberine alkaloids could also be accessed by incorporating an additional step after the biocatalytic cascade in which formaldehyde


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was added after 3 h reaction time. This initiated a non-enzymatic Pictet-Spengler condensation to afford the alkaloid 16 as the major regioisomer. The cascade was also utilized for the preparativescale synthesis (using 94.5 mg of 13) of 15 and 16 in 62% and 42% isolated yield, respectively, and both with an enantiomeric excess of >95%. Scheme 5. Complementary In Vitro Cascades for the Production of Enantiopure D- or L-Arylalanines 18 from Cinnamic Acids 17.

A cascade route to both enantiomers of phenylalanine derivatives has been developed by the Turner group (Scheme 5).60 Phenylalanine ammonia lyases (PALs) have previously been used for the amination of cinnamic acid derivatives 17 using free ammonia to generate L-arylalanines L-18, although not always with perfect enantioselectivity. To access the more challenging D-enantiomers, an amination/racemization cascade system was constructed, in which the first step was a PALmediated amination of the cinnamic acid employing an engineered variant to generate the arylalanine with imperfect enantioselectivity. The L-enantiomer was then oxidized by an L-amino acid deaminase (LAAD) to an imine 19 which was then reduced by the non-selective chemical reducing agent ammonia borane. In this way, all of the L-enantiomer was converted to the D-enantiomer which was not a substrate for the LAAD, and therefore accumulated in the system. This approach enabled the synthesis of a range of D-arylalanines in good conversion and excellent ee (62 to 80% conv., 98 to >99% ee). Some α-keto acid by-product 20 was observed as a result of the interception and hydrolysis of the imine before it could be reduced by ammonia borane. Furthermore, the simple


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substitution of the engineered PAL variant with the wild-type PAL enzyme, and of the LAAD with a Damino acid oxidase (DAAO), generated a new cascade to upgrade the ee of nitro- and cyano-Larylalanines (>99% ee), known to be produced in lower enantiopurity by PAL alone (0 to >99% ee).

Scheme 6. In Vitro Cascade for the Production of Tetrasaccharide 24.

Carbohydrates are complex targets for chemical synthesis and biocatalysis has become a very attractive strategy to assemble both natural and unnatural glycosides. A recent example is the biomimetic route to O-mannosyl glycans which are important biomarkers on cell surfaces (Scheme 6).61 The synthetic strategy employed purified glycosyltransferases to sequentially attach sugar monomer units to the glycan chain. A mannosyl unit was first attached to a peptide chain of interest using solid phase peptide synthesis techniques to form glycopeptide 21. Sequentially, N-acetylglucosamine (GlcNAc), galactose (Gal) and sialic acid moieties were then attached, catalyzed by human protein O-mannose β-1,2-N-acetyl-glucosaminyltransferase 1 (POMGnT1), bovine β-1,4galactosyltransferase (β-1,4-GalT) and Trypanozoma cruzi trans-sialidase (TcTS) in 85% (22), 87% (23) and 47% (24) yield after HPLC, respectively. The TcTS enzyme used the sialoprotein fetuin as the sugar donor for this step and required careful reaction monitoring to avoid hydrolysis of the newly created sialosidic linkage, making this step difficult to drive to completion. Due to the very high selectivity of all three biocatalysts in this synthetic route, a one-pot cascade was implemented which significantly shortened the amount of time needed to generate the desired tetrasaccharide, and removed all intermediate purification processes. Using this cascade approach, the tri- 23 and


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tetrasaccharides 24 were formed as a 1:1 mixture with only trace amounts of mono- 21 and disaccharide 22 observed. Scheme 7. In Vitro Isomerization/Reduction Cascade for the Production of Chiral Saturated Alcohols 27 from Racemic Allylic Alcohols 25.

The simultaneous application of a chemocatalyst and a biocatalyst has been thoroughly studied in the context of dynamic kinetic resolutions. Pioneering work by Bäckvall employed a chemocatalyst to equilibrate two enantiomers, followed by a successive enzymatic reaction catalyzed by a lipase to achieve enantiopure products.62–64 A recent development in the field of chemoenzymatic reactions was a concurrent cascade reported by González-Sabín, García-Álvarez and coworkers for the formal asymmetric reduction of allylic alcohols (Scheme 7).65 Their approach was to combine a bis(allyl)ruthenium (IV) complex to catalyze the redox isomerization of the allylic alcohol 25 to the saturated ketone 26, followed by the biocatalytic reduction accomplished by a ketoreductase (KRED). A range of commercially available NADPH-dependent KREDs from Codexis were shown to afford high conversions and enantioselectivities on a panel of ketones with isopropanol used for cofactor recycling. First, isomerization was run to completion, before the appropriate KRED and NADPH were added to the reaction. This process was able to achieve very high conversions (92 to 97%), isolated yields (85 to 90%) and ee values (98 to >99%). A concurrent cascade was then developed with both chemo- and biocatalysts present from the start. To achieve this, a compromise in temperature was employed and a higher [Ru]cat loading was required to ensure turnover of the first step. This approach led to good conversion (85 to 94%) and isolated yield of 27 (60 to 86% yield, on 26 – 33 mg of starting material 25). The limiting factors under the concurrent mode were the rate of the ruthenium-catalyzed isomerization and the biocatalyst stability i.e. the enzyme lost activity before complete isomerization had occurred. However, this was overcome in a sequential process as the ketone intermediate produced in the first step could be converted at high rate during the most active period in the lifetime of the biocatalyst.


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Scheme 8. In Vitro Cascades Involving Artificial Transfer Hydrogenases (ATHases) for the Production of Chiral Amines.

The co-location of a transition metal catalyst and a biocatalyst in the same pot often results in the mutual inactivation or attenuation of the activity of both and hence the implementation of such systems is not a trivial undertaking.66–70 One approach to overcome inactivation is to tether the transition metal catalyst within a protein scaffold, creating an artificial metalloenzyme which can then be applied alongside other biocatalysts without inhibitory or deactivating effects.71–73 This concept was demonstrated by Ward, Turner, Hollmann and coworkers, by coupling an artificial transfer hydrogenase (ATHase) with various enzymes to create in vitro cascades that were not possible when employing the free transition metal catalyst.74 The ATHase was constructed by incorporating a biotinylated iridium d6-pianostool complex within a streptavidin isoform and could be regenerated in situ by sodium formate as a source of hydride. The artificial enzyme was then employed in one-pot with a purified monoamine oxidase variant (MAO-N-9) for the deracemization of cyclic amines (Scheme 8a). A variant streptavidin isoform (S112T) was identified that gave some (R)-selectivity for the reduction of cyclic imine 28. The variant was then used in the cascade in which (S)-29 was selectively oxidized by the MAO-N-9, leaving (R)-29, and the imine 28 generated was then reduced by the ATHase to (R)-29. Catalase (CAT) was added to eliminate the build-up of hydrogen peroxide that was speculated to deactivate the iridium catalytic center. A different cascade was also built for the synthesis of L-pipecolic acid L-32 based on the same deracemization principle, however the cyclic imine 31 was generated in an initial step from L-lysine L-30, catalyzed by an L-amino acid


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oxidase (LAAO) (Scheme 8b). In these cascades, the artificial metalloenzyme approach demonstrates how the encapsulating streptavidin protein effectively retains and shields the active iridium core from deactivating effects and provides a local chiral environment for the chemical transformations that occur there. An alternative compartmentalization approach to overcome mutual catalyst inactivation is the use of a polydimethylsiloxane membrane that creates a barrier to separate the biocatalytic and chemocatalytic reactions. This membrane technology was originally employed for chemical cascade reactions75 and has since found application for incompatible chemo- and biocatalysts.69 Recently this strategy was further extended by Micklefield, Greaney and co-workers to enable the operation of a concurrent cascade involving a biocatalytic aryl halogenation followed by palladium-catalyzed Suzuki-Miyaura coupling.76 These innovative solutions to catalyst incompatibility problems have enabled the broader scope and implementation of running concurrent cascades.

IN VIVO CASCADES Construction of an enzymatic cascade within a living host cell offers many advantages over in vitro methods, yet in vivo cascades are currently far less explored. Since whole-cells can be used without further processing (e.g. cell lysis, protein purification) prior to the biotransformation, in vivo biocatalysis is a more cost-effective and easier to implement strategy than in vitro enzyme preparations. Enclosing the entire cascade within a living cell abolishes the need for exogenous cofactor supplementation and regeneration as inherent metabolic pathways within the host can be harnessed for this purpose. However, the expression of multiple enzymes within a host cell towards in vivo biocatalysis is not without its problems. Whilst the cell membranes may provide stabilisation of the expressed proteins, they may also lead to transport issues and can lead to problems with the downstream extraction of reaction products.77 Once the substrate has been transported through membranes it is also then at risk of being metabolized by host cell enzymes, as are any intermediates and products of the cascade reaction. The construction of non-natural enzymatic pathways often involves the application of proteins from a variety of eukaryotic and prokaryotic sources and, as such, a compromise on cell growth and protein expression conditions must be reached when hosting these enzymes within the same cell. In addition to this, the co-expression of multiple recombinant proteins within one cell may result in significant metabolic burden and lead to a decrease in production of the desired enzymes.78 In the strictest terms, ‘in vivo biocatalytic cascade’ describes the compartmentalization of two or more enzymes within a single whole-cell, however recent advances in the field have led to the development of in vivo cascades contained within two or more separate whole-cells.79–81 Containment of the components of an enzymatic cascade within distinct whole-cells (of the same species or of a different species) in a mixed-culture reaction allows for tighter control over the expression conditions for each individual protein and reduces the metabolic burden exerted on each cell.


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Scheme 9. In Vivo Cascade for the Terminal Amino-Functionalization of Fatty Acid Methyl Esters 33 Using a Single Whole-Cell.

In one of the first examples of whole-cell de novo cascade biocatalysis, Schmid, Bühler and coworkers developed a single whole-cell system capable of regiospecific amino-functionalization of the terminal unactivated C-H bonds of fatty acid methyl esters (FAMEs) and alkanes through use of oxygenase and ω-transaminase (ω-TA) enzymes (Scheme 9).82 The alkane monooxygenase AlkBGT and an ω-TA were expressed in a single E. coli BL21 (DE3) cell, with AlkBGT catalyzing the two-step oxidation of alkane 33 to aldehyde 35 (via terminal alcohol 34), and the ω-TA catalyzing the final reduction of 35 to amine 36. Analysis of the reaction mixture indicated that further oxidation to carboxylic acid 37 by the actions of AlkBGT occurred after 15 min reaction time. It was envisaged that employing ω-TA to convert 35 to 36 would intercept the reactive aldehyde intermediate before it could be over-oxidized to the unwanted acid by-product, and indeed addition of ω-TA did result in formation of terminal amine product with lower amounts of acid accumulation seen. The intrinsic host background also raised problems with regards to unwanted side reactions; some hydrolysis of ester intermediate 34 and product 36 was observed, and conversion of aldehyde 35 to alcohol 34 was seen in control experiments, indicating interference from endogenous dehydrogenases in E. coli. The hydrophobic nature of the main substrate investigated in this study, dodecanoic acid methyl ester 33, was seen to be a limiting factor for this cascade system, due to the restricted uptake of FAMEs through the cell membrane. Analysis of the less hydrophobic substrate octane resulted in higher rates of product formation due to the enhanced accessibility of this substrate.


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Scheme 10. In Vivo Cascade for the Production of Chiral Benzylic Amines 41 from Ethylbenzenes 38 Using a Single Whole-Cell.

Recent work by Flitsch and co-workers focussed on the design and construction of a four-enzyme cascade, contained within a single E. coli whole-cell, for the conversion of 4-substituted ethylbenzenes 38 to enantiopure (R)-1-phenylethanamines 41 via a formal C-H amination reaction (Scheme 10).83 An engineered chimeric self-sufficient P450 monooxygenase mutant (P450camY96FRhFRed) catalyzed the oxidation of unactivated C-H to give benzylic alcohol 39, which then underwent further oxidation to the ketone 40 through the actions of two enantiocomplementary alcohol dehydrogenases (LbRADH and ReSADH), and finally an enantioselective ω-TA (ATA-117) converted 40 into the chiral benzylic amine product 41. All genes required for the cascade reaction were originally contained within one plasmid, but development of a two-plasmid system resulted in a more stable whole-cell biocatalyst due to increased gene expression control of the potentially toxic P450 protein. Problems were also encountered when all reagents required for the cascade were added at the start of the reaction, as the sacrificial amine donor used for the ω-TA reaction (isopropylamine, IPA) was seen to inhibit the P450-catalyzed step. To circumvent this issue, a twostep design was implemented with IPA added to the reaction vessel after 24 h. Utilizing these optimized conditions, ethylbenzenes could be converted to their benzylic amine counterparts with ee values of up to 97.5% and conversions of up to 26%. In a very recent study, Reetz et al described another C-H functionalization whereby the substrate cyclohexane could be converted to the corresponding chiral cyclohexane-1,2-diols through simultaneous expression of a P450 and ADH in one whole-cell.84 Directed evolution was used to generate P450-BM3 mutants capable of catalyzing the oxidation of cyclohexane to α-hydroxy cyclohexanone, which was then reduced by the ADH to give final product diol in up to 29% isolated yield. When the cascade was initiated from the intermediary cyclohexanone, isolated yields of final product diol of up to 66% could be accessed.


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Scheme 11. In Vivo Cascade for the Production of Azelaic Acid 46 from Linoleic Acid 42 Using a Single Whole-Cell.

Hauer and coworkers developed a three-enzyme cascade, in a single E. coli host cell, for the conversion of linoleic acid into azelaic acid (Scheme 11).85 The cascade was initiated by the lipoxygenase (LOX)-catalyzed hydroperoxidation of linoleic acid 42, followed by cleavage of the hydroperoxy group by a hydroperoxide lyase (HPL). An endogenous E. coli oxidoreductase (ALDH) completed the conversion to the final product azelaic acid 46 through the oxidation of 9oxononanoic acid 45. Construction of this pathway in vivo, followed by subsequent analysis, indicated that the lipoxygenase requires activation by its product 43 prior to efficient catalysis. For this reason, and because 43 was seen to inhibit the subsequent hydroperoxide lyase reaction, it was necessary to carefully balance the expression levels of both recombinant proteins. The coexpression of a high copy plasmid for LOX and a low copy plasmid for HPL allowed for comparatively lower expression levels of HPL, resulting in sufficient levels of 43 present in the reaction to completely activate LOX. Optimized reaction conditions using a two-phase system with cyclohexane enabled conversion to azelaic acid of up to 26% when using 1 mM linoleic acid as substrate.


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Scheme 12. In Vivo Cascade for the Production of Carvolactone 51 from Limonene 47 Using a MixedCulture Reaction.

Bornscheuer, Mihovilovic, Rudroff and co-workers established a three-enzyme cascade in a single E. coli whole-cell for the transformation of simple allylic alcohol substrates 48 to the corresponding chiral lactone products 51.86 A nonselective alcohol dehydrogenase (ADH) initiated the cascade process by converting the alcohol substrate into the α,β-unsaturated ketone 49. An ene-reductase (ERED) selectively reduced the double bond functionality of the α,β-unsaturated ketone to give saturated ketone 50, which was finally oxidized further by a Baeyer-Villiger monooxygenase (CHMO) to yield lactone product. A potential bottleneck of the cascade, the ADH-catalyzed oxidation of substrate to unsaturated ketone intermediate, was avoided because the ERED successfully shifted the reaction equilibrium towards product formation. However, it was also apparent that a background reaction catalyzed by an endogenous E. coli protein competes with the desired ERED step of the cascade, and an E. coli knockout strain was proposed to overcome this issue. Very recently, the same group expanded the utility of the presented cascade by incorporating a fourth enzyme, cumene dioxygenase (CumDO), into a second host cell (P. putida S12) for the hydroxylation of natural product limonene 47 to provide the alcohol substrate for the previously established cascade (Scheme 12).79 Simultaneous addition of both cultures in a one-pot system achieved around 47% conversion to the chiral carvolactone product 51, whereas an optimized sequential approach to the system was found to result in almost full conversion to the desired product.


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Scheme 13. In Vivo Cascades for the Production of (a) α-Hydroxyacids 56, (b) 1,2-Amino Alcohols 57 and (c) α-Amino Acids 59 from Terminal Alkene 52 Using a Single Whole-Cell.

The Li group recently assembled three distinct whole-cell biocatalysts, harboring four to eight enzymes each, for the production of chiral α-hydroxyacids (Scheme 13a), 1,2-amino alcohols (Scheme 13b) and α-amino acids (Scheme 13c) from terminal alkene substrates.87 The construction of an efficient non-natural enzymatic cascade consisting of four or more reaction steps is very difficult to achieve in a recombinant living system, but was elegantly tackled here through the development of four separate enzyme modules which could be mixed and matched to generate several whole-cell biocatalysts. Module 1 consisted of an epoxidase (EP) and epoxide hydrolase (EH) for the production of 1,2-diol 54 from terminal alkene 52; module 2 consisted of an alcohol


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dehydrogenase (ADH) and an aldehyde dehydrogenase (ALDH) for the production of α-hydroxyacid 56 from 1,2-diol 54; module 3 consisted of ADH, an ω-transaminase (ω-TA) and an alanine dehydrogenase (AlaDH) for the production of 1,2-amino alcohol 57 from 1,2-diol 54; module 4 consisted of a hydroxyacid oxidase (HO), an α-transaminase (α-TA), catalase (CAT) and glutamate dehydrogenase (GluDH) for the production of α-amino acid 59 from α-hydroxyacid 56. Each module was contained on a separate plasmid and coexpressed in E. coli cells, a versatile approach that allowed for the generation of multiple biocatalysts yet may eventually be restricted by the number of compatible plasmids readily available to the researcher. Expression of several genes within the same cell often leads to a mismatch of the expression levels of each cascade protein, which was resolved in this work by testing different combinations of expression constructs until balanced expression of the enzyme profile was reached. During the designing of the cascades, Li et al. selected enzymes based on their relatively high activities in an attempt to avoid any reaction bottlenecks or accumulation of intermediates. By ensuring that each following enzyme had a higher activity than the previous, it was possible for the latter enzymes to pull the reactions towards product formation. Some accumulation of intermediates was seen for module 3, which was attributed to the relatively low activity of the final enzyme ω-TA, and it was proposed that swapping this enzyme for a more efficient homolog could improve this situation. Using these whole-cell biocatalysts, (S)-αhydroxyacids 56, (S)-amino alcohols 57, and (S)-α-amino acids 59 could be accessed on a preparative scale (using 295 – 1042 mg of starting material) with isolated yields of up to 72%, 62%, and 70%, respectively (98% ee). This cascade system was developed further to enable production of (S)phenylglycine from biogenic L-phenylalanine or glucose through employment of two additional enzymes, phenylalanine ammonia lyase (PAL) and phenylacrylic acid decarboxylase (PAD).88

FUTURE PROSPECTS FOR IN VIVO BIOCATALYTIC CASCADES As the cost of custom gene synthesis and gene manipulation continues to fall, the construction of larger de novo biocatalytic cascades in a given host cell is becoming increasingly feasible. Several whole-cell systems expressing four or more recombinant proteins have already been demonstrated successfully, yet it remains to be seen where the edge of failure lies with regards to the number of biocatalysts a single host cell can accommodate. The expression of multiple cascade proteins from a single plasmid system also paves the way for rapid engineering and optimization of cascade processes. The principles of directed evolution, which has found widespread use in the area of biocatalysis as a whole, could potentially be harnessed for the evolution of an entire cascade reaction, rather than the separate evolution of each individual enzyme used in the cascade. For whole-cell systems such as those described above, one can expect instabilities associated with the transformation and expression of large recombinant plasmid DNA.89 However, such issues can be overcome by implanting all of the cascade enzymes into the genome of the host organism.39 Attractive targets for the future could be the funnelling of primary metabolites (instead of synthetic starting materials) into cascades, or the in vivo regeneration of important cofactors. Additionally, strategies such as allosteric regulation and post-translational modifications are prevalent regulatory mechanisms found in nature90 and, although they have been employed in synthetic biology applications,91 these approaches have not yet been fully explored during the construction of de novo enzymatic pathways.

HYBRID IN VIVO/IN VITRO CASCADES


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In the previous sections we have outlined the various advantages and difficulties associated with both in vitro and in vivo cascade biocatalysis. Recent work in the field has led to the development of cascade reactions that use both in vitro and in vivo methodologies within the same system, harnessing the power of each approach whilst minimizing any of the described conflicts/drawbacks.81,92,93 Scheme 14. Hybrid In Vivo/In Vitro Cascade for the Production of Chiral Piperidines and Pyrrolidines 64 from Keto Acids 60.

A three-enzyme cascade using both whole-cell and free enzymes was recently described by Turner and co-workers for the production of chiral mono- and disubstituted piperidines and pyrrolidines 64 from keto acid 60 or keto aldehyde 61 starting materials, via an imine intermediate 63 (Scheme 14).81 Two separate batches of recombinant E. coli cells, one containing a carboxylic acid (CAR) and one containing an imine reductase ((S)- or (R)-IRED), and a crude lysate preparation of an ω-TA (ATA113) were combined for a one-pot reaction alongside a glucose dehydrogenase (GDH)/lactate dehydrogenase (LDH) system to shift the ω-TA equilibrium. The cofactor requirements of CAR (ATP, NADPH) and IRED (NADPH) enzymes encouraged the use of whole-cell preparations of these biocatalysts because of the ability of living cells to provide and regenerate cofactors endogenously. Expressing the enzymes in separate cells allowed for increased control over the levels of each enzyme present in the reaction, and also enabled the use of two different sets of expression conditions tailored to the individual proteins. The ω-TA used was available as a commercial crude lysate and the nucleotide sequence was not in the public domain and, as such, was provided to the reactions in vitro. Applying ATA-113 in vitro also allowed for more efficient use of the GDH/LDH


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system, thus providing a stronger driving force towards product formation for the reversible ω-TA reaction. This hybrid in vivo/in vitro cascade was successfully applied for the production of several chiral piperidines in high isolated yields (up to 83% yield when using 100 mg starting material 60), ee (>98%) and de (98%). Interestingly, it has subsequently been discovered that this work, devised through a de novo design, in fact in part mimics a natural pathway for the production of piperidinecontaining natural products.94 Scheme 15. Hybrid In Vivo/In Vitro Cascade for the Production of α,ω-Dicarboxylic Acids 73 and ωHydroxycarboxylic Acids 70 from Fatty Acids 65.

A cascade established by Park et al involved the utilization of a whole-cell expressing three separate enzymes and an E. coli cell extract, added sequentially, containing a fourth enzyme for the production of α,ω-dicarboxylic acids 73 and ω-hydroxycarboxylic acids 70 from renewable fatty acids 65 (Scheme 15).92 An oleate hydratase, ADH and one of two Baeyer-Villiger monooxygenases (BVMO) were contained within a recombinant E. coli cell, and catalyzed the formation of ester intermediates 68 and 71 via the corresponding alcohol 66 and ketone 67 compounds. Ester hydrolysis to yield α,ω-dicarboxylic acid 73 and ω-hydroxycarboxylic acid 70 products was achieved through the actions of an esterase contained within a separate E. coli cell extract. The products of the esterase reaction were toxic to living cells, and so the cell extract was added once the first three steps were completed to keep the whole-cells viable. Using this optimized sequential approach, ωhydroxynonanoic acid 70 could be produced with 60% conversion.

TOWARDS THE INDUSTRIAL APPLICATION OF BIOCATALYTIC CASCADES


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Biocatalysts have been employed in a wide variety of industrial processes ranging from bulk chemical manufacture to fine chemical synthesis.95–100 Typically these processes involve a single step transformation catalyzed by one enzyme, after which the product is isolated and purified. Industrial examples of de novo multi-step, multi-enzyme reactions occurring in the same reaction vessel are much less common but recent progress in cascade processes, as highlighted in this Perspective, is paving the way for greater industrial scope of these inherently ‘green’ processes in the future. Some cascade reactions have already been successfully implemented and reported in industrial laboratories, for example a recent patent filing by Evonik pertaining to a whole-cell coexpressing a recombinant α-dioxygenase or carboxylic acid reductase (CAR) and transaminase for the production of amines or diamines from carboxylic acids or dicarboxylic acids respectively.101 Additionally, the patent covers the co-expression of other enzymes such as an alanine dehydrogenase for regeneration of the alanine cosubstrate required for the transaminase from pyruvate and ammonia. Lonza have developed an in vivo cascade for the production of 5-hydroxypyrazine-2-carboxylic acid from 2-cyanopyrazine in a whole-cell fermentation process.102 In order for biocatalytic cascades to be more widely implemented for industrial processes, it is first necessary to optimize the selected systems with respect to process cost, product purity, product concentration, catalyst stability, solvent tolerance, catalyst recyclability, amongst other considerations.41,103,104 The choice between using an in vitro or in vivo system (or a hybrid of the two) inherently affects these factors, but process engineering also plays a major role. Enzyme immobilization is a tool currently used during the application of biocatalysts in an industrial setting, yet is still underdeveloped in the context of cascade reactions. Issues such as biocatalyst instabilities and recyclability could potentially be addressed using this technique, yet this introduces another optimizable parameter to the process development. A desirable target for the future would be the development of generally applicable technologies for the successful immobilization of a broad range of enzymes, to avoid the need to screen different immobilization platforms for each biocatalyst individually. The difficulty of successfully generating a biocatalytic cascade reaction increases with the number of different enzymes and reaction steps involved. Each step must be optimized to achieve the highest conversion to product possible, and often each enzyme of the sequence requires varying reaction conditions. For this reason, it is sometimes necessary to operate a sequential, rather than a concurrent, process through the temporal separation of reaction reagents or enzymes, for example.

Scheme 16. In Vitro Cascade for the Production of Chiral Vicinal Diols 76 from Benzaldehyde 9 and Acetaldehyde 74 Using Two Separate Lyophilized Cells.

In the context of process engineering, the Rother group has focussed on developing whole-cell cascade processes suited for industrial application, with particular attention paid to micro-aqueous systems for increased substrate loadings.105 Several reaction parameters such as buffer conditions, substrate concentrations, organic solvents and reaction modes (simultaneous or sequential) were


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investigated for the conversion of benzaldehyde 9 and acetaldehyde 74 to chiral vicinal diols 76 through the actions of a benzaldehyde lyase (BAL) and an ADH contained within two separate lyophilized whole-cells (Scheme 16). A comparison between simultaneous mode and sequential mode indicated that running the cascade concurrently lead to a significantly higher optimal productivity (327 g L-1 d-1) compared to the sequential reaction (222 g L-1 d-1). This space-time yield using the optimized micro-aqueous system is 1600-fold higher than that seen for isolated enzymes in aqueous media, and is of an efficiency relevant to industrial processes.

OUTLOOKS AND CONCLUSIONS Biocatalysis has clearly advanced from single step transformations to de novo cascade reactions that can be used in ‘one-pot’, with either isolated biocatalysts or whole-cell systems, and can also include chemocatalytic steps. With access to an increasing range of different biocatalysts these synthetic cascades are likely to become more highly developed, involving the conversion of simple starting materials, including common metabolic intermediates, to complex target structures. However, the construction of biocatalytic cascades in single whole-cell systems is not always an efficient or feasible approach, and a number of aspects can be noted from the work reviewed here that influence the decision to use an in vitro, in vivo or hybrid system: (a) For an in vivo approach to be adopted, the gene sequence for the enzymes must be freely available to use. Commercially available enzyme preparations, often provided as freeze-dried lysates, as well as chemocatalysts necessitate an in vitro approach. (b) The ease of which the desired cascade enzymes can be successfully expressed to sufficient levels in the chosen chassis enables or restricts the operation of an entirely in vivo cascade. (c) If a substrate, intermediate or product of the reaction is toxic to living cells, then implementation of the cascade lends itself to an in vitro construction. (d) For in vivo cascades, substrates, intermediates and products must be able to traverse the cell membrane to avoid mass transfer limitations. (e) An advantage of employing biocatalytic steps that require cofactor turnover in vivo is that the inherent machinery of the chassis can provide and regenerate most common cofactors.

In conclusion, it is apparent that the development of biocatalytic cascade processes is a rapidly expanding area of research, with many (chemo)enzymatic cascades already reported for the production of a wide range of valuable chemical scaffolds. Cascades of increased complexity can be envisaged through biocatalytic retrosynthetic design,20 which continues to increase in scope and application as more biocatalysts are discovered and developed. The benefits of cascades, whether they be in vitro, in vivo or hybrid systems, have yet to be fully harnessed for industrial manufacture. It is clear that challenges remain that need to addressed in the development of future cascade processes. Often, biocatalyst engineering is undertaken with the aim of broadening the substrate scope of an enzyme. Interestingly, however, for concurrent cascade reactions, it may be necessary to engineer a narrower substrate specificity to reduce unwanted cross reactions with different intermediates of the pathway. Substrate loadings for many of these processes are also still relatively low which limits the scalability of these reactions, although this is a grander challenge of biocatalysis as a whole that is being tackled by engineering of both enzymes and process parameters. Ultimately,


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however, with the significant strides taken in recent years in academic laboratories worldwide, we envisage a wider implementation of cascade processes at large scale in the near future.

AUTHOR INFORMATION Corresponding author(s) [email protected] [email protected]

Funding Sources The research was supported by BBSRC (sLoLa BB/L502005/1 to LJH), (CASE/Pfizer to SPF). NJT and SLF are grateful to the Royal Society for Wolfson Research Merit Awards.

ABBREVIATIONS ADH AHAS-I AlaDH ALDH AmDH ATHase ATP BAL BVMO CAR CAT CumDO DAAO DNA E. coli EH EP ERED FAD FAME g L-1 d-1 Gal GDH GlcNAc GluDH HO HPL

alcohol dehydrogenase acetohydroxyacid synthase I alanine dehydrogenase aldehyde dehydrogenase amine dehydrogenase artificial transfer hydrogenase adenosine triphosphate benzaldehyde lyase Baeyer-Villiger monooxygenase carboxylic acid reductase catalase cumene dioxygenase D-amino acid oxidase deoxyribonucleic acid Escherichia coli epoxide hydrolase epoxidase ene-reductase ferulic acid decarboxylase fatty acid methyl ester grams per liter per day galactose glucose dehydrogenase N-acetyl-glucosamine glutamate dehydrogenase hydroxyacid oxidase hydroperoxide lyase


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IPA IRED KRED LAAD LAAO LDH LOX MAO-N NAD(P)H NCS P. putida P450 PAD PAL POMGnT1 Pyr TAL TcTS ThDP TPL UDP α-TA β-1,4-GalT ω-TA

isopropylamine imine reductase ketoreductase L-amino acid deaminase L-amino acid oxidase lactate dehydrogenase lipoxygenase monoamine oxidase-N nicotinamide adenine dinucleotide (phosphate) norcoclaurine synthase Pseudomonas putida P450 monooxygenase phenylacrylic acid decarboxylase phenylalanine ammonia lyase O-mannose β-1,2-N-acetyl-glucosaminyltransferase 1 pyruvate tyrosine ammonia lyase trans-sialidase thiamine diphosphate tyrosine phenol lyase uridine diphosphate α-transaminase β-1,4-galactosyltransferase ω-transaminase

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Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to

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