Manuela Avi

Laboratory evolved biocatalysts for stereoselective syntheses of substituted benzaldehyde cyanohydrins.

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).

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

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.