Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

The enormous number of emerging reactions matched with the advances in technology today is pushing the frontiers of biocatalysis. It is becoming a reality that new and even complex reactions are being integrated with ever-increasing speed into industrial processes. This chapter will concentrate on two types of reactions. Those that have very recently emerged on an industrial scale like C=C reduction, some C–X bond forming enzymes and production of chiral amines. It will also describe those new emerging reactions that for various reasons are not yet ready for implementation in industrial processes. Reactions like halogenation, (de)methylation, P450 hydroxylations, dihydroxylation of non-aromatic substrates, new or modified aldolases that accept non-phosphorylated donor substrate aldolases and C=C cleavage fall into this category. A major trend in recent years which will also be discussed in this section is the use of multicomponent reactions for the purpose of deracemization or cascade reactions. The advantage of these reactions is that they decrease the number of unit operations, alleviate the need to isolate intermediates between steps and contribute significantly to decreasing waste production in a process and therefore have the potential to greatly reduce the costs of such processes providing productivity is high enough.

ABSTRACT Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.

This chapter contains sections titled: Introduction Biotrans Outsourcing – AstraZeneca Biotrans Trends – Lonza Biocatalysis in the Pharma Environment Industrial Use of Hydrolases Industrial Biooxidation and Reduction Industrial Application of Transaminases – Cambrex Biocatalyst Discovery and Improvement From Pathway Engineering to Synthetic Biology Prioritization of Future Biocatalysis and Synthetic Biology Needs Concluding Remarks

Lonza began as a small Swiss electricity company and it has successfully adapted to change throughout its history to become a global custom manufacturing company serving the needs of the life-science industry. One of the crucial decisions and changes in its development was the implementation of biotechnology. This article outlines briefly the history of change during Lonza's development, some of the problems that confronted the chemical industry approximately a decade ago and how they affected the area of biotransformation. There are still many chemical reactions that are difficult, inefficient and environmentally unfriendly. Lonza believes that some of these problems can be solved by biotechnology if the biocatalytic platform can be widened and improved so that the biocatalysts can be easily integrated into a chemical process.

A de novo synthesis of pentoses is described starting from (Z)-2-buten-1,4-diol (1). The key step is the enzyme catalysed enantioselective HCN-addition to O-protected 4-hydroxybut-2-enal using the hydroxynitrile lyase from Hevea brasiliensis, followed by an asymmetric dihydroxylation. For the cyanohydrin reaction the influence of the configuration of the double bond and of the protecting group was investigated. The dihydroxylation step was found to be influenced by the protecting group on position 4.

3,4-Dihydro-2H-pyran-2-carbaldehyde (1) and 2-methoxycyclohex-3-encarbaldehyde (2) obtained by thermal or chemocatalytic Diels–Alder reactions were converted into the corresponding cyanohydrins by hydroxynitrile lyase catalysis. Modelling investigations give a clear interpretation for the steric course of the biocatalytic cyanohydrin reaction of these α- and β-oxygenated aldehydes.

A pH-stable hydroxynitrile lyase (HNL) for the conversion of hydroxypivaldehyde (1) and pivaldehyde was developed by screening for stereoselective cyanohydrin synthesis in 96 well plates. Stepwise redesign of PaHNL5 gene and enzyme leads to elevated amounts of a highly efficient biocatalyst for (R)-pantolactone (2) synthesis (er = 98.5:1.5, 100% conversion) .

This chapter contains sections titled: Introduction Biphasic Systems Liquid/Liquid Biphasic Systems Buffer/Organic Solvent Buffer/Ionic Liquids Liquid/Solid Biphasic Systems Crude Enzyme Preparations Inert Carriers Cross-Linking of Oxynitrilases Other Biphasic Systems Encapsulation in Sol-Gel Matrices Comparison of CLEAs and sol-gel HNLs Conclusion

Screening for stereoselective cyanohydrin synthesis in 96-well plates was employed in the development of an efficient, pH-stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site-saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)-cyanohydrins (ee >97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P-AOx1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of today's most efficient biocatalyst for vitamin B-5 synthesis.

Even if biocatalysis is finding increasing application, it still has to gain widespread use in synthetic chemistry. Reasons for this are limitations that enzymes have with regard to substrate range, reaction scope, and insufficient selectivity with unnatural compounds. These shortcomings can be challenged by enzyme and/or substrate engineering, which are employed to alter substrate specificity and enhance the enzyme selectivity toward unnatural substrates. Herein, these two approaches are coupled to improve the hydroxynitrile lyase catalyzed synthesis of 2-hydroxy-(4'-oxocyclohexyl)acetonitrile (4). The ketone functionality is masked as an enol ether, and the oxynitrilase of Hevea brasiliensis is engineered towards this masked substrate to give the product with a high optical purity and to drastically lower the amount of enzyme needed.

The application of (R)-hydroxynitrile lyases ((R)-HNLs) enables a simple chemo-enzymatic approach towards (R)-pantolactone synthesis. For the first time, several new recombinant almond (R)-HNL isoenzymes were compared with native HNLs from different Prunus species with respect to cyanohydrin formation from hydroxypivalaldehyde providing the chiral key precursor in HNL based (R)-pantolactone synthesis. Recombinant PaHNL5 (R-selective hydroxynitrile lyase, isoenzyme 5, from Prunus amygdalus) surpasses all other tested natural and recombinant HNL variants. At low pH even very low amounts of crude enzyme catalysed stereoselective hydroxypivalaldehyde cyanohydrin formation in water based reaction systems.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Hydroxynitrile lyases (HNLs) are biocatalysts employed industrially for the asymmetric addition of HCN to aldehydes or ketones. The resulting optically active cyanohydrins are important building blocks for pharmaceuticals and agrochemicals. Several genes of Rand S-selective HNLs have been cloned and expressed in suitable host systems, and can thus be ACHTUNGTRENNUNGengineered to improve their catalytic properties. The almond (Prunus amygdalus) (R)-HNL isoenzyme 5 (PaHNL5) is particularly suited as a starting point for engineering approaches. PaHNL5 can be heterologously expressed in Pichia pastoris and is highly stable even at pH values lower than 3.0. It is efficiently secreted into the culture supernatant, which can be directly employed for biocatalysis in aqueous or biphasic systems. Reduction of steric hindrance by structure-guided design has resulted in the simple and quick generation of new enzyme variants with significantly improved catalytic rates and enhanced stereoselectivity in cyanohydrin syntheses with employment of non-natural substrates. We wanted to explore whether the so far best, highly active, and stereoselective enzyme variant for the production of (R)-2-chloromandelonitrile (2a, Scheme 1), PaHNL5/L1Q/A111G, already industrially used, can reach an activity similar to that of the WT enzyme with its preferred substrate benzaldehyde. Compound 2a is a key intermediate for the production of a widely administered anticoagulant that reduces the risk of cardiovascular events in patients with acute coronary syndromes. As computational methods did not provide any indications for further improvement, we addressed the challenge of applying directed evolution. Bacteria, especially E. coli, are widely used for laboratory evolution, because of their simple genetic molecular manipulability, high transformation efficiency, and rapid growth rates. However, for eukaryotic proteins their use is often limited because of misfolding and the lack of typical eukaryotic posttranslational modifications. P. pastoris, an efficient system for heterologous protein production, can compensate for such disadvantages. Drawbacks, however, are a high input of linearized plasmid DNA and the concomitant relatively low integration rate into the genome. In addition, we have observed varying copy numbers or incomplete cassette integration, which seemed to impede the utilization of P. pastoris for highthroughput expression, screening, and laboratory evolution of proteins. Although expression of PaHNL in E. coli has been described, large amounts of highly active PaHNL5 could only be obtained with P. pastoris. Consequently, it was necessary to devise a new method for the efficient construction of reliable expression libraries in P. pastoris and the uniform expression of thousands of individual transformants, while circumventing time-consuming ligation and cloning steps in E. coli, which lead to a loss of diversity and library efficiency. Furthermore, there are no stable plasmids for Pichia transformation available. A concise procedure to generate PaHNL5/L1Q/A111G libraries by integration of linear expression cassettes produced by an overlap extension PCR (OE-PCR) strategy was thus designed, and so the randomly mutated Pahnl5 gene, a fragment consisting of a partial GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter, a 5’ secretion signal sequence for the a-mating factor from bakers’ yeast and a 3’ zeocin resistance cassette were linked together (Figure 1). Both flanking arms were generated by proof-reading polymerases. To improve the efficiency of the OE-PCR, the overlapping region between the mutated gene and the selection marker (overlap 2) was redesigned. Then, without any prior ligation step, P. pastoris X33 was directly transformed with the linear PCR-based integration cassettes. In order to test this new strategy, the Pahnl5/L1Q gene was used to reassemble a linear integration cassette by overlap extension PCR. Since an L1Q mutation seemed to enhance exScheme 1. Synthesis of halogen-substituted (R)-mandelonitriles (TBME: tertbutyl methyl ether).