Immobilised enzyme microreactor for screening of multi-step bioconversions: Characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols

• Published in: Journal of biotechnology Vol. 155; no. 3; pp. 320 - 329
• Main Authors: Matosevic, S., Lye, G.J., Baganz, F.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20.09.2011; Elsevier
• Subjects: Acetaldehyde - analogs & derivatives; Acetaldehyde - metabolism; Acetophenones - metabolism; amino alcohols; Amino Alcohols - metabolism; Asymmetric synthesis; Bioconversions. Hemisynthesis; Biological and medical sciences; Bioreactors; Biotechnology; biotransformation; Enzyme immobilisation; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fundamental and applied biological sciences. Psychology; General aspects; Histidine - chemistry; Histidine - metabolism; Immobilization of enzymes and other molecules; Immobilization techniques; immobilized enzymes; Kinetics; Methods. Procedures. Technologies; Microfluidic Analytical Techniques - instrumentation; Microfluidics; Microreactor; Models, Biological; monitoring; Oligopeptides - chemistry; Oligopeptides - metabolism; Pyruvates - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; screening; Stereoisomerism; Tetroses - metabolism; Transaminase; Transaminases - chemistry; Transaminases - metabolism; Transketolase; Transketolase - chemistry; Transketolase - metabolism; Enzyme immobilisation; Microreactor; Asymmetric synthesis; Transketolase; Transaminase; Microfluidics; Fluid mechanics; Enzyme; Transferases; Immobilization; Alcohol; Chiral compound; Screening; Biotransformation; Transaminases; Miniaturization; Immobilized enzyme
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2011.07.017

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 His 6-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.