title: Multi-step biocatalytic strategies for chiral amino alcohol synthesis.
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.
authors.affiliation: The Advanced Centre for Biochemical Engineering, University College London, Department of Biochemical Engineering, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address: maria.torres.09@ucl.ac.uk.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Villegas-Torres
authors.affiliation: Research Department of Structural and Molecular Biology, ISMB, The Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: R Julio
authors.initials: RJ
authors.lastname: Martinez-Torres
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom.
authors.forename: Armando
authors.initials: A
authors.lastname: Cázares-Körner
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom. Electronic address: h.c.hailes@ucl.ac.uk.
authors.forename: Helen
authors.initials: H
authors.lastname: Hailes
authors.affiliation: The Advanced Centre for Biochemical Engineering, University College London, Department of Biochemical Engineering, Gordon Street, London WC1H 0AH, United Kingdom.
authors.forename: Frank
authors.initials: F
authors.lastname: Baganz
authors.affiliation: The Advanced Centre for Biochemical Engineering, University College London, Department of Biochemical Engineering, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address: j.ward@ucl.ac.uk.
authors.forename: John
authors.initials: J
authors.lastname: Ward
mesh_terms: D000605:Amino Alcohols / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D057005:Bioengineering; D053898:Biosynthetic Pathways; D007700:Kinetics; D011773:Pyruvates / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D000637:Transaminases / Q000235:genetics / Q000378:metabolism; D014174:Transketolase / Q000235:genetics / Q000378:metabolism
chemical_list: C445732:2-amino-1,3,4-butanetriol; D000605:Amino Alcohols; D001426:Bacterial Proteins; D011773:Pyruvates; D011994:Recombinant Proteins; C012375:hydroxypyruvic acid; D014174:Transketolase; D000637:Transaminases; C022638:serine-pyruvate aminotransferase
keywords: Cascades; Chiral amino alcohols; Recycling system; Transaminase; Transketolase