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The development of a novel, CoA-dependent, organic acid modification system as a link to a biosynthetic pathway for the nylon 6 monomer, 6-aminocaproic acid

Hickman, Thomas William Paul; (2020) The development of a novel, CoA-dependent, organic acid modification system as a link to a biosynthetic pathway for the nylon 6 monomer, 6-aminocaproic acid. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The inherently unsustainable production chain of the nylon 6 monomer, 6-aminocaproic acid (6-ACA), has motivated a number of investigations into developing sustainable alternatives. Previous research at UCL developed a biosynthetic pathway from cyclohexanol to 6-ACA. However, the requirement for petroleum-derived cyclohexanol, limited the pathway’s sustainability. Therefore, this investigation aimed to develop a biosynthetic pathway for renewable cyclohexanol production, from an intermediate of central metabolism. The pathway developed here, utilises eight enzymes for the production of cyclohexanol, through the CoA-dependent biotransformation of either shikimate or 3-phosphshikimate. Cyclohexanol production was not fully realised, however, exploring new biosynthetic routes for cyclohexanol production revealed several new enzymes and systems that were characterised and constructed. Four enzymes (MycA1-4), identified in Streptomyces rishiriensis, were recombinantly expressed and assayed. The acyl-CoA reductase, MycA2, showed activity with cyclohex-1-enecarbonyl CoA and 3-hydroxycyclohex-1-enecarbonyl CoA. MycA4, a proposed hydratase/phosphatase, was inactive, attributed to either protein misfolding, or an alternative catalytic mechanism. Combined activity of ChCoADH (Synthrophus aciditrophicus), an acyl-CoA dehydrogenase and BadK (Rhodopseudomonas pseudopalustris), an enoyl-CoA hydratase, facilitated the conversion of cyclohexanecarbonyl CoA into 2-hydroxycyclohexanecarbonyl CoA. Activity analysis of four Hotdog-fold thioesterases, revealed RpaL (Rhodopseudomonas palustris) to have a promiscuous substrate acceptance, and to catalyse the required hydrolysis of 2-hydroxycyclohexanecarbonyl CoA to 2-hydroxycyclohexanocarboxylate. A limited availability of CoA esters prevented the experimental analysis of many enzymatic steps. Resultantly, the CoA ligase, AliA (Rhodopseudomonas pseudopalustris), was screened and found to produce aromatic and alicyclic 4 CoA esters. Finally, a three-module system for the rational production of modified organic acids, via CoA ester intermediates, was tested. By coupling (1) broad AliA activity, (2) CoA modification (ChCoADH/ BadK) and (3) promiscuous RpaL activity, theoretically, a number of modified organic acids could be derived. Initial testing showed this system capable of producing cyclohex-1-enecarboxylate and 2-hydroxycyclohexanecarboxylate from cyclohexanecarboxylate. However, further exploitation of the CoA-utilising enzymes in endogenous metabolism, will undoubtedly expand this systems potential for rationally producing modified organic acids of commercial interest.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The development of a novel, CoA-dependent, organic acid modification system as a link to a biosynthetic pathway for the nylon 6 monomer, 6-aminocaproic acid
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10094703
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