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 Golden Age of Transfer Hydrogenation

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

https://link.springer.com/content/pdf/10.1007%2Fs00253-014-5732-5.pdf

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening–based methods and in vivo selection–based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

Directed evolution drives the next generation of 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

Hydroxynitrile lyases (HNLs) are a family of versatile enzymes that catalyze the reversible cleavage of a-hydroxy nitriles and are utilized for the production of enantiopure cyanohydrins from aldehydes or ketones and HCN. Several HNLs from different sources have been identified, and the HNL-catalyzed synthesis of a large number of cyanohydrins withR and S configuration with excellent stereoselectivity has been demonstrated. In trying to expand the synthetic applicability of the HNL methodology we considered replacing HCN by other nucleophiles to be added to carbonyl compounds catalyzed by these enzymes. Based on the mechanism of this biotransformation crucial parameters for such alternative reagents would be the molecular size and the pKa of the CH-acidic portion, which should be similar to that of HCN (pKa 9). One important substance class that meets these criteria are nitroalkanes. Their reaction with carbonyl compounds—known as the nitroaldol or Henry reaction—constitutes a carboligation process of high synthetic value. The Henry reaction furnishes vicinal nitroalcohols, which can easily be transformed to a series of valuable intermediates such as, for example, 1,2-aminoalcohols and ahydroxycarboxylic acids. First, we examined the addition of nitromethane to aldehydes in the presence of the hydroxynitrile lyase from Hevea brasiliensis (HbHNL). The reaction of benzaldehyde with nitromethane gave 2-nitro-1-phenylethanol in 63% yield with an enantiomeric excess of 92% (Table 1). Besides the expected nitroalcohol product, small amounts (10–15%) of the corresponding elimination product, 1-nitro-2-phenyl-

A Biocatalytic Henry Reaction—The Hydroxynitrile Lyase from Hevea brasiliensis Also Catalyzes Nitroaldol Reactions

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Reliable high‐throughput screening with Pichia pastoris by limiting yeast cell death phenomena

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C–C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry

Comprehensive Step‐by‐Step Engineering of an (R)‐Hydroxynitrile Lyase for Large‐Scale Asymmetric Synthesis

The hydroxynitrile lyase from Hevea brasi- liensis not only catalyzes - according to the natural activity of this enzyme - the formation and cleavage of cyanohydrins but also the reaction of nitroalkanes with aldehydes (Henry reaction). This is the first ex- ample of an enzymatic nitroaldol reaction. With ni- tromethane and nitroethane a broad range of alde- hydes can be transformed into the corresponding nitro alcohols in yields up to 77 % and enantiomeric excess (ee )u p to 99 %.

Wolfgang Skranc

Papers

A Biocatalytic Henry Reaction—The Hydroxynitrile Lyase from Hevea brasiliensis Also Catalyzes Nitroaldol Reactions

Reliable high‐throughput screening with Pichia pastoris by limiting yeast cell death phenomena

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry

Comprehensive Step‐by‐Step Engineering of an (R)‐Hydroxynitrile Lyase for Large‐Scale Asymmetric Synthesis

Hydroxynitrile lyase-catalyzed enzymatic nitroaldol (henry) reaction