Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

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,cismuconic 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...

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

“…In addition to what has been described in Box 2 , two commonly applied targets beyond the shikimate pathway are overexpression of the transketolase and deletion of the glucose-6-phosphate dehydrogenase. The latter, once a popular knockout target in yeast ( ZWF1 ) is rarely applied anymore, a trend that is in accordance with our findings from in silico metabolic modeling (Averesch, 2016 ): we found that the knockout rarely benefits the yield (opposing to its original intention); especially in case of reducing pathways it negatively impacts redox cofactor availability (NADPH production from PPP). This is supported by our previous study on production of pHBA (described above) (Williams et al, 2015 ), where knock-down of ZWF1 had an adverse effect on the product titer.…”

“…In combination with the usual feedback-resistant DHAP synthase (and chorismate mutase) respective titers in the range of 1.3 and 0.37 g/L could be reached (Kim et al, 2014 ; Romagnoli et al, 2015 ). It is noteworthy that in a study on production of pABA in S. cerevisiae [(Averesch et al, 2016 ), see above], in glucose batch cultures phenylethanol titers of almost 0.1 g/L were obtained, which exceeded the target product, even though the chorismate mutase ( ARO7 ) was knocked out (Averesch, 2016 ). This was attributed to a flux overflow that degraded chorismate into prephenate and phenylpyruvate (Winter et al, 2014 ).…”

“…Considering that in general the reactions of the shikimate pathway are thermodynamically favored (Averesch, 2016 ), it can be concluded that limitations are mostly kinetic and/or regulatory. This is supported by a study showing the performance of the shikimiate pathway in S. cerevisiae to be greatly dependent on the type strain (Suástegui et al, 2016 ).…”

“…Biotechnology is anticipated to be a key to fulfilling these objectives, as biochemical pathways are extremely versatile, giving rise to a wealth of diverse organic compounds (Chen and Nielsen, 2013 ; Becker and Wittmann, 2015 ; Becker et al, 2015 ). It has been predicted that bio-feedstocks could increase to 17% (Webster, 2012 ) of the global chemical business by 2025, equivalent to 425 billion USD (de Jong et al, 2013 ; Averesch, 2016 ), with global demand for biomass derived chemicals exceeding 8.5 Mt by 2023 (Insights, 2016 ). Unfortunately, to date, the majority of the bio-replacements is hardly cost competitive.…”

“…Parts of this review are based on the Ph.D. thesis of NA (Averesch, 2016 ), in particular the introductory chapters and some descriptive parts of the strain construction strategies contain sections that have been adapted.…”