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Abstract
The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5′-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-5576-z) contains supplementary material, which is available to authorized users.
Highlights
Chiral amines are important building blocks for the synthesis of bioactive molecules, and efficient and environmentally benign methods are required for their preparation in high yield, productivity, product concentration and enantiomeric purity (Höhne and Bornscheuer 2009)
Chiral amines can be prepared using biocatalysis, for example, with the help of enantioselective ω-transaminases (ω-TAs) (Koszelewski et al 2010; Mathew and Yun 2012). ω-TAs catalyze the transfer of an amine group from an amine donor to a ketone moiety with pyridoxal-5′-phosphate (PLP) as a co-factor and can be used to prepare chiral amines either via kinetic resolution or through asymmetric synthesis from a prochiral ketone. (S)-selectivity is more common among ω-TAs; both (R)- and (S)-selective enzymes have previously been used to convert a broad range of substrates with high efficiency, making this enzyme group a versatile tool for the preparation of various chiral amine molecules (Koszelewski et al 2010; Tufvesson et al 2011a)
As endogenous transaminase activity towards-1-PEA might decrease the yield of kinetic resolution, non-engineered E. coli and S. cerevisiae lacking the vanillin aminotransferase (VAMT) enzyme were first investigated for background activity
Summary
Chiral amines are important building blocks for the synthesis of bioactive molecules, and efficient and environmentally benign methods are required for their preparation in high yield, productivity, product concentration and enantiomeric purity (Höhne and Bornscheuer 2009). (S)-selectivity is more common among ω-TAs; both (R)- and (S)-selective enzymes have previously been used to convert a broad range of substrates with high efficiency, making this enzyme group a versatile tool for the preparation of various chiral amine molecules (Koszelewski et al 2010; Tufvesson et al 2011a). Transamination reactions may be limited by unfavourable equilibrium and substrate and/or product inhibition These hurdles have been overcome by addition of excess cosubstrates (Savile et al 2011), in situ product removal (Truppo et al 2010) and/or coupled enzymatic systems (Höhne et al 2008; Koszelewski et al 2008). Most processes with ω-transaminases apply purified enzymes or crude cell extract from recombinant Escherichia coli as a biocatalyst
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