Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

Engineering the third wave of biocatalysis

The [2 + 2] photocycloaddition is undisputedly the most important and most frequently used photochemical reaction. In this review, it is attempted to cover all recent aspects of [2 + 2] photocycloaddition chemistry with an emphasis on synthetically relevant, regio-, and stereoselective reactions. The review aims to comprehensively discuss relevant work, which was done in the field in the last 20 years (i.e., from 1995 to 2015). Organization of the data follows a subdivision according to mechanism and substrate classes. Cu(I) and PET (photoinduced electron transfer) catalysis are treated separately in sections 2 and 4, whereas the vast majority of photocycloaddition reactions which occur by direct excitation or sensitization are divided within section 3 into individual subsections according to the photochemically excited olefin.

http://pubs.acs.org/doi/pdf/10.1021/acs.chemrev.5b00723

Recent Advances in the Synthesis of Cyclobutanes by Olefin [2+2] Photocycloaddition Reactions

https://pure.rug.nl/ws/files/3623614/2006JBiotechnolvBerkel.pdf

Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.

Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.

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Biocatalysis: Enzymatic Synthesis for Industrial Applications

The exploitation of ring strain as a driving force to facilitate chemical reactions is a well-appreciated principle in organic chemistry. The most prominent and most frequently used compound classes in this respect are oxiranes and cyclopropanes. For rather a long time, cyclobutanes lagged behind these three-membered-ring compounds in their development as reactive substrates, but during the past decade an increasing number of useful reactions of four-membered-ring substrates have emerged. This Minireview examines corresponding catalytic reactions ranging from Lewis or Bronsted acid catalyzed processes to enzymatic reactions. The main focus is placed on transition-metal-catalyzed C-C bond-insertion and β-carbon-elimination processes, which enable exciting downstream reactions that deliver versatile building blocks.

Cyclobutanes in Catalysis

The past decade has seen a substantial increase in successful examples of the combination of chemo- and biocatalysis for multistep syntheses. This is driven by obvious advantages such as higher yields, decreased costs, environmental benefits and high selectivity. On the downside, efforts must be undertaken to combine the divergent reaction conditions, reagent tolerance and solvent systems of these ‘different worlds of catalysis’. Owing to progress in enzyme discovery and engineering, as well as in the development of milder and more compatible conditions for operating with various chemocatalysts, many historical limitations can already be overcome. This Review highlights the opportunities available in the chemical space of combined syntheses using prominent examples, but also discusses the current challenges and emerging solutions, keeping in mind the fast progress in transition metal-, organo-, photo-, electro-, hetero- and biocatalysis. Chemical and biological catalysts provide distinct advantages and disadvantages to the synthetic chemist. This Review focuses on efforts to combine chemo- and biocatalysts, outlining the opportunities achievable by this approach and also efforts to overcome any incompatibilities between these different systems.

Opportunities and challenges for combining chemo- and biocatalysis

Over the past few years, industrial interest in biocatalysts that perform selective oxidative reactions has increased significantly. Baeyer–Villiger monooxygenases (BVMOs) have been identified as a highly versatile class of enzymes for the efficient catalysis of chemo-, regio-, and/or enantioselective oxygenation reactions. Although the most prominent transformation catalyzed by these biocatalysts is a chiral variant of the classical Baeyer–Villiger reaction, the oxygenation of heteroatoms and epoxidation reactions have also been reported. Stoichiometric amounts of O2 and NADPH are required for these reactions. A complication for the largescale application of these reactions is the high cost of the reduced nicotinamide coenzyme. To overcome this problem, several electrochemical and photochemical approaches have been explored. However, the efficiency of these approaches is typically poor. Furthermore, it has been shown that BVMOs require NADP for stability and enantioselective catalysis. An efficient and commonly used method for coenzyme regeneration employs whole cells, especially in combination with the recombinant expression of the required biocatalysts. This strategy has been implemented in BVMOmediated biotransformations with wild-type strains and has proved particularly successful with recombinant overexpression systems. The approach avoids laborious enzyme purification steps and exploits the coenzyme regeneration capacity of the host. Although whole cells have been shown to be effective catalysts for Baeyer–Villiger oxidation, they also exhibit limitations, such as cellular toxicity, enzyme inhibition by the substrate/product, degradation of the product, and poor oxygen-transfer rates. Coenzyme regeneration by using isolated enzymes has also been studied extensively in the past few years. Well-known examples of such NADPH-regenerating enzymes are alcohol dehydrogenase and formate dehydrogenase. A phosphite dehydrogenase (PTDH) was also identified as an effective enzyme for coenzyme regeneration. The favorable thermodynamic equilibrium constant makes the oxidation of phosphite a nearly irreversible process. The exquisite selectivity of PTDH for phosphite also precludes any side reactions, such as those that can occur, for example, when an alcohol dehydrogenase is used. These characteristics make PTDH an ideal candidate for use as a coenzyme regenerating enzyme (CRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. Herein, we report a novel approach to the combination of the catalytic activity of a redox biocatalyst with concomitant coenzyme recycling in a single fusion protein (Scheme 1). During the last decade, a number of fusion protein tags have been developed. These tags are used intensely in life-sciencerelated research and commercial activities. Although the fusion of proteins is a widely applied strategy in, for example, enzyme purification (e.g. the use of glutathione S transferase (GST) tags) and the subcellular visualization of target proteins (e.g. with a green fluorescent protein (GFP) tag), this concept is hardly ever encountered in the context of synthetic applications. Only a few isolated examples in the literature provide evidence that the fusion of separate enzymes can result in improved biocatalytic properties. We report herein on the engineering of a number of representative BVMOs that are linked covalently to soluble NADPH-regenerating phosphite dehydrogenase. This construct enables the use of phosphite as a cheap and sacrificial electron donor with whole cells, cell extracts, and purified enzyme. It was our particular goal to design a self-sufficient two-in-one redox biocatalyst that does not require an additional catalytic entity for coenzyme recycling. As model

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Self-sufficient Baeyer-Villiger monooxygenases: effective coenzyme regeneration for biooxygenation by fusion engineering

Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active with a limited number of substrates. By means of a comparison of the PAMO structure and a modeled structure of the sequence-related cyclopentanone monooxygenase, several active-site residues were selected for a mutagenesis study in order to alter the substrate specificity. The M446G PAMO mutant was found to be active with a number of aromatic ketones, amines and sulfides for which wild-type PAMO shows no activity. An interesting finding was that the mutant is able to convert indole into indigo blue: a reaction that has never been reported before for a Baeyer-Villiger monooxygenase. In addition to an altered substrate specificity, the enantioselectivity towards several sulfides was dramatically improved. This newly designed Baeyer-Villiger monooxygenase extends the scope of oxidation reactions feasible with these atypical monooxygenases.

Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ange.202006648

Radka Snajdrova

Papers

Biocatalysis: Enzymatic Synthesis for Industrial Applications

Opportunities and challenges for combining chemo- and biocatalysis

Self-sufficient Baeyer-Villiger monooxygenases: effective coenzyme regeneration for biooxygenation by fusion engineering

Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

Biokatalyse: Enzymatische Synthese für industrielle Anwendungen