University of Groningen
Terpenoid cell factory
Abdallah, Ingy Ibrahim Ahmed Fouad
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2018
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Abdallah, I. I. A. F. (2018). Terpenoid cell factory. Rijksuniversiteit Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Terpenoid Cell Factory
Ingy I. Abdallah
2018
The research described in this thesis was carried out in the Department of Chemical and Pharmaceutical Biology (Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands) and was financially supported by EU FP-7 grant FP7-KBBE-2011-3-6-04-289540 (PROMYSE project) and Erasmus Mundus Action 2, Strand 1, Fatima Al Fihri project ALFI1200161 scholarship.
The research work was carried out according to the requirements of the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands.
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands.
ISBN: 978-94-034-0603-9 (printed version) ISBN: 978-94-034-0602-2 (electronic version) Layout: Ingy Ibrahim Ahmed Fouad Abdallah Cover design: “Remco Wetzels”, Ridderprint BV Printing: Ridderprint BV, www.ridderprint.nl
Copyright © Ingy Ibrahim Ahmed Fouad Abdallah. All rights are reserved. No part of this thesis maybe reproduced or transmitted in any form or by any means without the prior permission in writing of the author.
Terpenoid Cell Factory
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Friday 25 May 2018 at 9.00 hours
by
Ingy Ibrahim Ahmed Fouad Abdallah
born on 12 February 1987
in Alexandria, Egypt
Promoters
Prof. W.J. Quax
Prof. G.J. Poelarends
Assessment Committee
Prof. M.W. Fraaije
Prof. H.J. Haisma
Prof. V.F. Wendisch
“Research is to see what everybody else has seen, and to think what nobody else has thought.”
Dr Albert Szent-Györgi
Paranymphs
Rita Setroikromo
Magdalena Wójcik
Table of Content
Scope and outline of this thesis 9
Chapter 1 A Glimpse into the Biosynthesis of Terpenoids 13
Chapter 2 Metabolic engineering of Bacillus subtilis for terpenoid
production 33
PART ONE Engineering Bacillus subtilis as a cell factory for
terpenoid production 57
Chapter 3 59
Chapter 4 85
Chapter 5
Enhanced C30 carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes
Bacillus subtilis as an optimized cell factory for C30
terpenoid production
Metabolic engineering of Bacillus subtilis towards taxadiene biosynthesis as the first committed step for Taxol production
107
PART TWO Study of terpene synthases with emphasis on
amorphadiene synthase 129
Chapter 6 Insights into the Three-Dimensional Structure of
Amorpha-4,11-diene Synthase and Probing of Plasticity Residues 131
Chapter 7 Catalysis of amorpha-4,11-diene synthase unraveled and
improved by mutability landscape guided engineering
171
Chapter 8 Insights into the promiscuity of amorpha-4,11-diene
synthase obtained from mutability landscape engineering 203
Chapter 9 Insights into the structure-function relations of
amorpha-4,11-diene synthase
223
PART THREE Summary and Future Perspectives 233
Chapter 10 Summary, Concluding Remarks and Future Perspectives 235
Chapter 11 Samenvatting, Conclusies en Toekomstperspectief 247
Appendix Acknowledgments
List of Publications About the author
261 267 269
Scope and outline of this thesis
9 Scope and outline of this thesis
Terpenoids represent a large and diverse class of natural products that offer a trove of prospects to address numerous medical and societal issues. The vast assortment of chemical structures and functions that have been evolved in this class provide a massive pool of molecules for medicinal and industrial use. In spite of the fact that a lot of terpenoids possess therapeutic properties including anticancer, antiparasitic, antimicrobial, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, large scale use of these compounds is limited by their low supply. Most terpenoids are naturally produced in low quantities and require harvesting on a massive scale to obtain amounts sufficient for medicinal use. In addition, their chemical synthesis is difficult and expensive due to the complexity of their structures. Metabolic engineering and synthetic biology offer alternative approaches to boost production in the native organism, and more importantly, transfer the biosynthetic pathways to other hosts. Different microbial hosts were studied and their metabolism was manipulated and optimized for the production of common terpenoid precursors. Hence, the aim of the work described in this thesis is to create a sustainable terpenoid cell factory. To fulfill that objective, the scope of our research focused on two main parts. First is the engineering of the microbial host Bacillus subtilis as a cell factory for terpenoid production. This involves optimizing the biosynthetic pathway of terpenoids in this host and studying the terpene synthase enzymes essential for terpenoid production. The second part explored one of these important terpene synthases, namely amorphadiene synthase which is famous for its role in the biosynthesis of the antimalarial artemisinin. The knowledge obtained from both parts can be considered as two stepping stones onto the road of obtaining a working B. subtilis terpenoid cell factory.
In Chapter 1, we review the different classes of terpenoids and their biosynthetic pathways. We focus on the enzyme family of terpene synthases that represent a prerequisite for formation of terpenoids. An overview of the classes of terpene synthases along with their structures and mechanisms is provided. Finally, current information about metabolic engineering of different hosts for terpenoid production is discussed.
Chapter 2 focuses specifically on reviewing the present knowledge about metabolic
Scope and outline of this thesis
10
pathways of B. subtilis are explained in details showing all the enzymes and intermediates involved. In addition, an outline about the knowledge and challenges of engineering B. subtilis along with different detection and metabolomics methods was provided.
In Chapter 3, we describe the systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway in B. subtilis. The MEP pathway is an inherent terpenoid biosynthesis pathway in B. subtilis. We use the level of production of C30 carotenoids as a readout to illustrate the effect of overexpression of the various
enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids in B. subtilis can be improved significantly by overexpressing the MEP pathway enzymes.
In Chapter 4, we describe a system for engineering synthetic operons to express metabolic pathways in B. subtilis. We clone different genes of the MEP pathway in two different vectors, a rolling circle replication vector (pHB201) and a theta replicating vector (pHCMC04). The structural and segregational stability of the generated constructs was compared along with their level of produced C30 carotenoids. The
construct expressing eight genes of the MEP pathway in pHCMC04 showed the highest amount of carotenoid produced coupled with good stability. In addition, qPCR was used to ensure that all genes in the operon are expressed at similar levels.
In Chapter 5, we report the production of taxadiene, the first precursor for Taxol®, in
B. subtilis. The enzyme taxadiene synthase which is a prerequisite for taxadiene
biosynthesis was successfully expressed for the first time in B. subtilis by inserting the plant gene into the bacterial host genome. It was coupled with the overexpression of nine enzymes in two different vectors (geranylgeranyl pyrophosphate synthase in pBS0E and the eight MEP pathway enzymes in pHCMC04) leading to the formation of the taxadiene precursor (geranylgeranyl pyrophosphate). This strain showed 83 fold increase in taxadiene production compared to the control B. subtilis strain only expressing taxadiene synthase.
In Chapter 6, we concentrate on studying the famous sesquiterpene synthase, amorphadiene synthase, which is responsible for cyclizing farnesyl pyrophosphate to amorphadiene. This is the first and rate limiting step in the production of the antimalarial artemisinin. Since no crystal structure has been reported for this enzyme, a three-dimensional model was generated. Magnesium ions and the substrate were
Scope and outline of this thesis
11
docked into the model. Some active site residues were mutated to examine their function and validate the model. The generated model is the basis for understanding the structure-function relations of the active site residues and gaining insight into the mechanism.
In Chapter 7, we use the amorphadiene synthase model generated from the work in chapter 6 to choose active site residues for mutation. Sixteen active site residues were mutated producing a library of 257 variants. The mutability landscape for catalytic activity showed several variants with improved activity compared to the wild type enzyme especially T399S/H448A double mutant which showed turnover rate 5 times higher than wild type. In addition, the screening of the library allowed for understanding the role of these residues in the mechanism of amorphadiene synthase. In Chapter 8, we examine the impact of mutating a single active site residue on the promiscuity of amorphadiene synthase. This enzyme converts farnesyl pyrophosphate to the major product amorpha-4,11-diene along with several minor products such as β-farnesene, γ-humulene, α-bisabolol, amorpha-4,7-diene and amorpha-4-en-11(7)-ol. We highlight mutants that increase the production of one or more of these minor products at the expense of the major amorpha-4,11-diene and examine the reason for the shift in the major route of the mechanism using the amorphadiene synthase model presented in chapter 6.
Chapter 9 is an editorial comment on “Fang, X., Li, J.X., Huang, J.Q., Xiao,
Y.L., Zhang, P., Chen, X.Y. (2017) Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase. Biochem J, 474, 2191-2202” shedding light on some of the structure-function relations of amorphadiene synthase and reported kinetic properties of the wild type enzyme.
In Chapter 10 and 11, the work presented in this thesis is summarized, final conclusions are drawn, and suggestions for future perspectives are described.
