Hydroxynitrile lyases expression

Hasslacher, M., Schall, M., Hayn, M. et al. (1997) High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expression and Purification, 11, 61-71. 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

M. Hasslacher, M. Schall, M. Hayn, H. Griengl, S. D. Kohlwein, H. Schwab, Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea hrasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue. J. Biol. Chem. 1996, 271, 5884-5891. 

H. Breithaupt, Klonierung und Expression der (R)-Hydroxynitrile Lyase aus Linum usitatissimum (Lein) [Doctoral Thesis], Heinrich-Heine University Dtisseldorf 1997. 

Cloning and expression of (R)-hydroxynitrile lyase from Linum usitatissimum (flax)./. Mol. Catal. B -Enzymaticl999, 6, 315-332. 

The highest reported heterologous production of a protein in yeast comes from the work by Hasslacher et al. (1997). The enzyme hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis was reported to produce levels of 22g/L intracellularly in P. pastoris. In the same study, S. cerevisiae and E. coli were tested in parallel experiments but were not competitive. Levels of proteins produced by yeasts have more typically been in the range of 1-15 g/L (Schmidt, 2004). A list of reported yields for expression of proteins in P. pastoris is provided by Cregg at http / faculty.kgi.edu/cregg/index.htm... 

Siritunga, D., Arias-Garzon, D., White, W. and Sayre R.T. (2004) Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification. Plant Biotechnol. /., 2, 37-43. 

A. (2012) Application of a recombinant Escherichia coli whole-cell catalyst expressing hydroxynitrile lyase and nitrilase activities in ionic liquids for the production of (S)-mandelic acid and (S)-mandeloamide. Adv. Synth. Catal., 354, 113-122. 

Historically, enzyme catalysis has played a highly prominent role, with the first enzyme-catalyzed asymmetric addition of HCN to aldehydes dating back to 1908 [167]. A wide range of both aromatic and aliphatic ketones are suitable substrates and produce cyanohydrins of high optical purity. The most readily available and hence most commonly employed enzyme for asymmetric cyanohydrin formation is (R)-hydroxynitrile lyase isolated from almonds. Recent cloning and over-expression techniques have also made a number of (S)-hydroxynitrile lyases available for organic synthesis [164, 165]. This was utilized in Griengl s synthesis of coriolic acid (255), a natural product that displays calcium ionophoric activity and acts as a prostacyclin mimic (Scheme 2.32) [168]. Thus, an (S)-hydroxynitrile lyase was cloned from rubber trees (Hevea brasiliensis), overexpressed in Pichia pastoris, and used to provide cyanohydrin 254 in 99 % ee. 

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