Biocatalysis and bioprocess engineering for terpenoid production
department or school: Applied Science, Faculty of
department or school: Chemical and Biological Engineering, Department of
data provider: DSpace
degree campus: UBCV
creator: Ayakar, Sonal
issued: 2021-01-31T08:00:00Z
issued: 2019
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: The biomanufacturing of terpenoids is limited by the low yields of heterologously expressed biosynthetic pathway and challenges associated with recovering these products at commercial scales. To enhance the flux through methylerythritol phosphate (MEP) pathway for terpenoid biosynthesis, I screened soil metagenomes for more active and stable orthologs of the rate-limiting enzymes. I successfully identified three entirely novel, natural fusions of IspD and IspF, one of which improved production of lycopene from 235 mg/L to 275 mg/L and production of isoprene from 3.6 mg/L to 6.3 mg/L when compared to the native enzyme overexpression. A comprehensive study of the role of the linking domain revealed the higher activity of each of the catalytic domains and the absence of substrate channeling. Moreover, the non-natural fusions of E. coli enzymes catalyzing consecutive steps were constructed. One such a fusion of IspD and IspE yielded 281 mg/L of lycopene, whereas the best performing fusion of IspE and IspF only yielded 39 mg/L of lycopene. Further investigation of the sequence of this biocatalytic cascade concluded the commencement with the activity of IspE, followed IspD and IspF suggesting the reactive plasticity in MEP pathway. I probed the promiscuous nature of terpene synthases (TSs) through the systematic study of monoterpene synthases from Picea abies in vivo and in vitro. I uncovered the influence of intracellular expression and oxygen supply on the promiscuity of TSs. Computational analysis revealed the putative roles of the amino acid residues within the active sites and their evolutionary trajectory. Finally, the fermentation of engineered E. coli strains for carene and myrcene were scaled up to 1 L and a newer technique was developed for efficient product capture using a fluidized bed capture device (FBCD) using a hydrophobic resin. The device was easy to integrate into the existing bioreactor set up. It yielded 2-fold higher carene titers and 17-fold higher …
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/70141
note: BIOCATALYSIS AND BIOPROCESS ENGINEERING FOR TERPENOID PRODUCTION by Sonal Ayakar B. Pharm., Mumbai University, 2012 M. Tech., Institute of Chemical Technology, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2019 © Sonal Ayakar, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Biocatalysis and bioprocess engineering for terpenoid production submitted by Sonal Ayakar in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical and Biological Engineering Examining Committee: Vikramaditya Yadav, Chemical and Biological Engineering Supervisor Charles Haynes, Chemical and Biological Engineering Supervisory Committee Member Joerg Bohlmann, Botony Supervisory Committee Member Fariborz Taghipour, Chemical and Biological Engineering University Examiner Nobuhiko Tokuriki, Biochemistry and Molecular Biology University Examiner iii Abstract The biomanufacturing of terpenoids is limited by the low yields of heterologously expressed biosynthetic pathway and challenges associated with recovering these products at commercial scales. To enhance the flux through methylerythritol phosphate (MEP) pathway for terpenoid biosynthesis, I screened soil metagenomes for more active and stable orthologs of the rate-limiting enzymes. I successfully identified three entirely novel, natural fusions of IspD and IspF, one of which improved production of lycopene from 235 mg/L to 275 mg/L and production of isoprene from 3.6 mg/L to 6.3 mg/L when compared to the native enzyme overexpression. A comprehensive study of the role of the linking domain revealed the higher activity of each of the catalytic domains and the absence of substrate channeling. More…
has type: Thesis/Dissertation
date issued: 2019-09
is shown at: 10.14288/1.0378690
language: eng
degree discipline: Chemical and Biological Engineering
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/70141