title: Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.
abstract: Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E7JE, UK.
authors.forename: S
authors.initials: S
authors.lastname: Matosevic
authors.forename: G J
authors.initials: GJ
authors.lastname: Lye
authors.forename: F
authors.initials: F
authors.lastname: Baganz
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000098:Acetophenones / Q000378:metabolism; D000605:Amino Alcohols / Q000378:metabolism*; D019149:Bioreactors; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008954:Models, Biological; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013780:Tetroses / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*; D014174:Transketolase / Q000737:chemistry / Q000378:metabolism*
chemical_list: C445732:2-amino-1,3,4-butanetriol; D000098:Acetophenones; D000605:Amino Alcohols; D004800:Enzymes, Immobilized; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011773:Pyruvates; D011993:Recombinant Fusion Proteins; D013780:Tetroses; C100073:erythrulose; D006639:Histidine; C012375:hydroxypyruvic acid; D014174:Transketolase; D000637:Transaminases; D000079:Acetaldehyde; C038699:acetophenone; C010972:glycolaldehyde