Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to de Novo Multi-Enzyme Pathways

Hepworth, Lorna J.; Turner, Nicholas J.; Flitsch, Sabine L.

doi:10.1021/acscatal.6b02979

Cited by 354 publications

(296 citation statements)

References 103 publications

(132 reference statements)

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“…The basic principle of cascade reactions is also frequently used in biocatalysis, using enzymes in isolation, as well as in combination with chemocatalysts (see Fig. 1 and Box 1 for definition of terms) [1][2][3][4][5][6][7] . As there is no need for purification and isolation of intermediates, operating time, production costs and waste are reduced, and concomitantly, overall yields are improved.…”

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Opportunities and challenges for combining chemo- and biocatalysis

et al. 2018

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N ature has evolved highly efficient systems in the form of cascade reactions, which assemble the metabolic networks that support life (growth and survival). The basic principle of cascade reactions is also frequently used in biocatalysis, using enzymes in isolation, as well as in combination with chemocatalysts (see Fig. 1 and Box 1 for definition of terms) [1][2][3][4][5][6][7] . As there is no need for purification and isolation of intermediates, operating time, production costs and waste are reduced, and concomitantly, overall yields are improved. In addition, the problem of unstable or difficult to handle intermediates can be overcome, and reactivity as well as selectivity can be enhanced by avoiding unfavourable reaction equilibria through the cooperative effects of multiple catalysts 8 . Starting in the 1980s, the early examples of the combination of chemo-and biocatalysts were reported by the van Bekkum group, who pioneered the development of a process to make the sugar substitute d-mannitol from readily available d-glucose through the combination of a heterogeneous metal-catalysed hydrogenation and an enzyme-catalysed isomerization 9 . The first broadly applied technology for the combination of enzyme and metalcatalysts, which was the research subject of many academic groups as well as industry, emerged in the 1990s from the Williams group 10,11 and aimed to achieve higher yields than classical kinetic resolution of racemates, thus overcoming the limitation of a maximum yield of 50% in the latter case. A prominent example of work that developed this theme is the combination of lipase-catalysed kinetic resolution via acylation of secondary alcohols with Pd-or Rh-catalysed racemization via reversible transfer hydrogenation to achieve a dynamic kinetic resolution (DKR) [12][13][14] . This example was facilitated in part because lipases are active and stable in organic solvents. Later, for instance, Turner's group combined a monoamine oxidase-catalysed imine formation with a chemical reduction 15 to achieve the 100% theoretical yield through a deracemization process. In addition to the combination of metalcatalysis with enzymes, organocatalysis, electrochemistry and light-induced reaction couples have since then been studied extensively, going far beyond the scope of a DKR.The challenges to combining chemo-and biocatalysis in cascades (see Box 1 for definitions and Table 1) can be daunting, not least the requirement for the chemical step to occur in the presence of water, the preferred solvent for enzymes 3 . This Review therefore highlights recent examples of the combination of chemo-and biocatalysts in aqueous multistep syntheses, and looks at how to overcome limitations by, for example, design of appropriate reaction conditions, protein engineering and advanced reactor concepts. Furthermore, trends such as the integration of transition metalcatalysis into microorganisms and the introduction of novel chemistry into engineered enzymes are discussed, and a critical assessment of the impact of this research ...

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Opportunities and challenges for combining chemo- and biocatalysis

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“…Thea denylation domain activates carboxylic acids by catalyzing the formation of an acyl adenylate intermediate,a tt he expense of adenosine triphosphate (ATP). [11][12][13] nicotinamide adenine dinucleotide phosphate (NADPH) is then expended to reduce the intermediate to an aldehyde ( Figure 1).…”

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Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

et al. 2017

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Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

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“…It is evident from natural examples that compartmentalized reaction systems offer advantages when intermediate species are prone to escape into competing reaction channels [3][4][5][6][7][8] . The GDH-ST/SC-LbADH hydrogels can be used to investigate this phenomenon because the NADPH consuming LbADH reduction is spatially and kinetically coupled to the NADPH regeneration by GDH.…”

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“…Towards the goal of the efficient use of renewable biomass as an alternative to petrochemical synthesis for sustainable production processes and energy supply in the future, multistep enzymatic cascade reactions are currently attracting much attention [3][4][5][6][7][8] . Such cascades are abundant in living systems, for instance, to maintain the regulation of metabolic activity or signal transduction [9][10][11] .…”

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Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Peschke

Gallus

Bitterwolf

et al. 2017

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______________________________________________________________We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported. ______________________________________________________________

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Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to de Novo Multi-Enzyme Pathways

Cited by 354 publications

References 103 publications

Opportunities and challenges for combining chemo- and biocatalysis

Opportunities and challenges for combining chemo- and biocatalysis

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

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