Abstract
Chiral amino alcohols are structural motifs present in sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Their chemical synthesis presents several challenges in establishing at least two chiral centres. Here a de novo metabolic pathway using a transketolase enzyme coupled with a transaminase enzyme has been assembled. To synthesise this motif one of the strategies to obtain high conversions from the transaminase/transketolase cascade is the use of hydroxypyruvate (HPA) as a two-carbon donor for the transketolase reaction; although commercially available it is relatively expensive limiting application of the pathway on an industrial scale. Alternately, HPA can be synthesised but this introduces a further synthetic step. In this study two different biocatalytic strategies were developed for the synthesis of (2S,3R)-2-amino-1,3,4-butanetriol (ABT) without adding HPA into the reaction. Firstly, a sequential cascade of three enzymatic steps (two transaminases and one transketolase) for the synthesis of ABT from serine, pyruvate and glycolaldehyde as substrates. Secondly, a two-step recycling cascade where serine is used as donor to aminate erythrulose (catalysed by a transketolase) for the simultaneous synthesis of ABT and HPA. In order to test the novel pathways, three new transaminases are described, two ω-transaminases able to accept a broad range of amine acceptors with serine as amine donor; and an α-transaminase, which showed high affinity towards serine (KM: 18mM) using pyruvate as amine acceptor. After implementation of the above enzymes in the biocatalytic pathways proposed in this paper, the two-step recycling pathway was found to be the most promising for its integration with E. coli metabolism. It was more efficient (10-fold higher conversion), more sustainable and cost-effective (use of low cost natural substrates and only two enzymes), and the reaction could be performed in a one-pot system.
Highlights
Chiral amino alcohols are key functionalities in several sphingolipids, antibiotics such as chloramphenicol and thiamphenicol, and antiviral glycosidase inhibitors such as the deoxynojirimycin family among others [1]
All the proposed cascades in this work depend on a transaminase able to either aminate pyruvate or erythrulose using serine as an amine donor (Figs. 1 and 2)
It has been reported that transaminases are active with serine at a very low rate [9,16]; but there is not a clear description of an ␣-transaminase that employs serine as amine donor
Summary
Chiral amino alcohols are key functionalities in several sphingolipids, antibiotics such as chloramphenicol and thiamphenicol, and antiviral glycosidase inhibitors such as the deoxynojirimycin family among others [1]. These compounds can be synthesized by either chemical or biocatalytic methods. Directed evolution, an -transaminase was engineered and successfully employed in the synthesis of sitagliptin at higher yields and productivity, and lower waste compared to the chemical method [11] Another example is for the synthesis of valinol, a 1,2 amino alcohol previously synthesised by valine reduction employing agents like LiAlH4. A sustainable biocatalytic process was described using several -transaminases leading to optically pure enantiomers and 99% yield when the R-configuration was produced and 94% conversion when the S-isomer was synthesised [12]
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