Engineering yeast shikimate pathway towards production of aromatics: Rational design of a chassis cell using systems and synthetic biology Nils Jonathan Helmuth Averesch Dipl. Ing. A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Chemical Engineering Abstract Aromatic building blocks are amongst the most important bulk feedstocks in the chemical industry. As these compounds are commonly derived from petrochemistry, obtaining them is becoming more and more a matter of costs and sustainability. Biochemistry gives rise to a wealth of compounds that can potentially replace current petroleum-based chemicals or be used for novel materials. The aromatic compounds para-aminobenzoic acid (pABA) and para-hydroxybenzoic acid (pHBA) and the aromatics derived compound cis,cis- muconic acid (ccMA) can be precursors for, but are not limited to, terephthalic or/and adipic acid. These are essential feedstocks for the production of PET and nylon. The three compounds can be derived from the shikimate pathway, an anabolic pathway leading to the biosynthesis of aromatic amino acids, present in certain prokaryotes and eukaryotes, including fungi. By combination of metabolic modelling with genetic engineering, a microbial production system based on the yeast Saccharomyces cerevisiae can be designed, which effectively channels flux into the target compounds. In order to develop a competitive bio-based process, yields, titers and rates need to be maximized. While productivity or rates in a process can be altered using genetic engineering, carbon yield, and pathway feasibility are stoichiometrically and thermodynamically predetermined. Both limitations need to be considered when designing a microbial production system. For formation of adipic acid and precursors many bio-based routes exists. More rational than just picking one for in vivo studies rather all available biochemical pathways were examined in silico using metabolic modelling. To compare theoretical yields and reaction thermodynamics an interface that allowed network-embedded thermodynamic analysis of elementary flux modes was developed. This allowed distinguishing between thermodynamically feasible and infeasible flux distributions. Feasible maximum theoretical product carbon yields were substantially different in E. coli and S. cerevisiae metabolic models and ranged from 32% to 92%. Further, many pathways appeared to be restricted by a thermodynamic equilibrium lying on the substrate side, some even infeasible. The only routes that deliver significant product yields and were thermodynamically favoured were shikimate pathway based. Being currently of strong scientific interest, recent implementations of these pathways in E. coli and S. cerevisiae were evaluated and strain construction strategies optimized, using the concept of constrained minimal cut-sets. Especially in S. cerevisiae a single non-obvious knock-out target allowed coupling of growth to product formation; in particular, the deletion of the pyruvate kinase reaction resulted in a minimum yield constraint of 28%. Though unique to shikimate pathway, this strategy is transferrable to other products which are derived from chorismate and also involve the formation of pyruvate as a by-product. This applies to pHBA. With further optimizations, the strategy was applied in vivo for the production of this compound in S. cerevisiae. As the pyruvate kinase knock-out entails a growth defect on glucose, a synthetic circuit was used, which allowed conditional knock- down and activation of the determined genetic modifications and by this dynamic control of the phenotype. Thus, production could be separated from growth and it could be proven that the in silico determined genetic intervention strategy holds valid in vivo, resulting in a 1.1 mM final product titer. Further, production of pABA from shikimate pathway in S. cerevisiae was investigated and optimized: Several alleles from different yeast strains of the genes (ABZ1 and ABZ2) for pABA formation from chorismate were screened, using a strain genetically engineered to channel flux to chorismate. ABZ1 of AWRI1631 and ABZ2 of QA23 delivered the highest pABA production. To further increase production, the impact of carbon-source on product yield was investigated in silico using metabolic modelling. It was found that combined glycerol-ethanol was a superior carbon-source than glucose, glycerol or ethanol alone, especially when employed in molar ratios between 1:2 and 2:1. This could be confirmed in vivo with carbon yields reaching up to 2.64%. A fed-batch process on glycerol-ethanol delivered total aromatics titers as high as 1.73 mM. It could be shown that feasibility and viability of adipic acid production greatly depends on the pathway and the organism chosen for engineering. Weaknesses of existing strain construction strategies for ccMA production could be identified and a radical optimization strategy was determined. Inferences from metabolic modelling were proven experimentally for pHBA and pABA production. The obtained concentrations and yields are the highest in S. cerevisiae to date and among the highest in a microbial production system, underlining the potential of yeast as a cell factory for renewable aromatic feedstocks. Declaration by author This thesis is composed of my original work and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Publications during candidature Peer-reviewed papers: Quorum-Sensing Linked RNA interference for Dynamic Metabolic Pathway Control in Saccharomyces cerevisiae. Thomas C. Williams, Nils J. H. Averesch, Gal Winter, Manuel R. Plan, Claudia E. Vickers, Lars K. Nielsen, Jens O. Krömer. Metab. Eng. 2015, 10.1016/j.ymben.2015.03.008 Tailoring strain construction strategies for muconic acid production in S. cerevisiae and E. coli. Nils J. H. Averesch, Jens O. Krömer. Metab. Eng. Commun. 2014, 10.1016/j.meteno.2014.09.001 In vivo instability of chorismate causes substrate loss during fermentative production of aromatics. Gal Winter, Nils J. H. Averesch, Dariela A. Nunez-Bernal, Jens O. Krömer. Yeast. 2014, 10.1002/yea.3025 Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae. Nils J. H. Averesch, Gal Winter, Jens O. Krömer. Submitted to Microb. Cell. Fact., under review Combined elementary flux mode and network-embedded thermodynamic analysis of microbial adipic acid production. Nils J. H. Averesch, Verónica S. Martínez, Lars K. Nielsen, Jens O. Krömer. Previously submitted to Metab. Eng., under revision Conference abstracts: Model-guided strain engineering enables coupling of product formation to growth. Nils J. H. Averesch, Jens O. Krömer, Gal Winter. NZMS, Wellington, 2014 Combining Elementary Mode Analysis with a Network Embedded Thermodynamic Approach for Analysis of Microbial Adipic Acid Production. Nils J. H. Averesch, Verónica S. Martínez, Jens O. Krömer. ME-X, Vancouver, 2014 Publications included in this thesis Combined elementary flux mode and network-embedded thermodynamic analysis of microbial adipic acid production. Nils J. H. Averesch, Verónica S. Martínez, Lars K. Nielsen, Jens O. Krömer. Previously submitted to Metab. Eng., under revision – incorporated as Chapter 2.1. Contributor Statement of contribution Nils J.H. Averesch (Candidate) Designed the experiments (70%) Conducted the experiments (60%) Analysed the data (60%) Drafted the paper (100%) Verónica S. Martínez Designed the experiments (20%) Conducted the experiments (35%) Analysed the data (30%) Reviewed and edited the paper draft (20%) Lars K. Nielsen Reviewed the paper draft (10%) Jens O. Krömer Designed the experiments (10%) Conducted the experiments (5%) Analysed the data (10%) Reviewed and edited the paper draft (70%) Tailoring strain construction strategies for muconic acid production in S. cerevisiae and E. coli. Nils J. H. Averesch, Jens O. Krömer. Metab. Eng. Commun. 2014, 10.1016/j.meteno.2014.09.001 – incorporated as Chapter 2.2. Contributor Statement of contribution Nils J.H. Averesch (Candidate) Jens O. Krömer and Nils J.H.