Natural biosynthesis assembles a vast array of complex natural products starting from a limited set of building blocks, under physiological conditions, and in the presence of numerous other biomolecules. Organisms rely on the extraordinary selectivity of enzymes and their ability to operate under similar reaction conditions, meaning that these catalysts are perfectly adapted to mediate cascade reactions. In these multistep processes, the product of one biocatalytic step becomes the substrate for the next transformation (Display footnote number:1-3). On page 1255 of this issue, Huffman et al. (Display footnote number:4) report the development of an impressive nine-enzyme biocatalytic cascade for the synthesis of the investigational drug islatravir for the treatment of human HIV.

This study represents a partnership between scientists from Merck and Codexis. These two companies have a history of successfully collaborating to develop biocatalysts for the synthesis of important pharmaceuticals. Almost a decade ago, they developed a chemoenzymatic route for the synthesis of the type 2 diabetes drug sitagliptin (Januvia), relying on a key enzyme-catalyzed transamination with a highly engineered (R)-selective transaminase (Display footnote number:5). The work was considered a landmark example of directed evolution and functioned to highlight the potential application of biocatalysis to revolutionize industrial chemical processes.

The cascade for synthesizing islatravir was inspired by the bacterial nucleoside salvage pathway, which recycles precious nucleosides by using three key enzymes: a purine nucleoside phosphorylase (PNP), a phosphopentomutase (PPM), and a deoxyribose-5-phosphate aldolase (DERA) (see the figure). However, to achieve the synthesis of the target molecule, Huffman et al. required the natural nucleoside degradative cascade to run in reverse. The reversible nature of enzymes is central to the design of this cascade and is one of the important features that sets biocatalysts apart from the majority of traditional chemical catalysts.

The success of the cascade developed by the team also relied on all three enzymes accepting non-natural substrates bearing a fully substituted carbon at the C-4 position of the 2-deoxyribose ring. The authors reconstructed the reverse nucleoside salvage pathway from a PNP and PPM found in Escherichia coli and a DERA from Shewanella halifaxensis. The native E. coli enzymes required engineering to improve their activity. The DERA displayed existing high activity and stereoselectivity for the formation of the desired sugar phosphate enantiomer, but it required engineering to improve its ability to operate at high substrate concentration.

One of the many advantages of performing biocatalytic cascade reactions is the effective displacement of unfavorable reaction equilibria that can be achieved through product removal. However, despite performing the PNP and PPM steps in tandem, the reaction proceeded with poor conversion, and the inorganic phosphate by-product inhibits the enzymes. An elegant solution to these issues was the inclusion of an auxiliary sucrose phosphorylase, along with its sugar substrate, which removed free phosphate and effectively displaced the reaction equilibrium toward product formation.

Having assembled enzymes for the three key steps in the cascade, Huffman et al. sought to develop a biocatalytic route for the synthesis of the DERA substrate 2-ethynylglyceraldehyde 3-phosphate. Extensive screening of a broad range of kinases resulted in the identification of pantothenate kinase (PanK) from E. coli, which displayed low levels of activity (∼1% conversion) toward the (R)-enantiomer of the target aldehyde. Despite the modest initial activity, directed evolution was successfully used to substantially improve the productivity and stability of this enzyme. Finally, after 12 rounds of evolution, the authors reversed the enantioselectivity and improved the activity, stability, and expression of a galactose oxidase variant for the desymmetrization of the starting substrate, 2-ethynylglycerol.

engineering a biocatalytic cascade

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Advancements in protein engineering, rapid gene sequencing, and the availability of low-cost DNA synthesis have made it possible to alter the properties of enzymes and fine-tune them for biocatalytic applications (Display footnote number:6-8). The work by Huffman et al. is a milestone in cascade design, largely because of the number of biocatalysts operating in tandem and the engineering feat required to optimize five of the nine enzymes involved in the synthesis. It also highlights how biosynthetic or degradative pathways can be a source of inspiration for the design of efficient biocatalytic cascades and how sequences can be reconstituted using enzymes recruited from multiple sources—in this case, of bacterial, fungal, plant, and mammalian origin. The diverse role that biocatalysts can play is also exemplified in this work, where five engineered enzymes are directly involved in the synthesis of the target molecule, and four additional enzymes function to recycle coenzyme, remove inhibitory by-products, and maintain the correct oxidation state of the copper cofactor.

Previous approaches reported for the synthesis of islatravir relied on multistep syntheses and require protecting group manipulations and intermediate purification steps (Display footnote number:9, 10). The incorporation of a key biocatalytic step or steps has the potential to revolutionize synthetic design strategies by making possible transformations that are not accessible using solely chemical approaches (Display footnote number:11, 12). The application of enzymes in industry and the development of chemoenzymatic routes to complex molecules is now well established. However, multistep syntheses exclusively comprising biocatalytic transformations are rare (Display footnote number:13), and this contribution sets a new standard for the synthesis of complex molecules with enzymatic cascades.

School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland. Email:


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J.R. acknowledges the School of Chemistry, University College Dublin, for support.