An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae

Brückner, Christine; Oreb, Mislav; Kunze, Gotthard; Boles, Eckhard; Tripp, Joanna

doi:10.1093/femsyr/foy017

Cited by 45 publications

(83 citation statements)

References 51 publications

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“…The shikimate pathway has received major attention in recent years by serving as a source of aromatic scaffolds for the production of fine and commodity chemicals, including aromatic amino acids, dopamine, naringenin, p-coumaric acid, resveratrol, vanillin, and MA (3)(4)(5)(6)(7)(8)(9)(10). While Escherichia coli has been engineered to produce up to 36.8 g/liter MA (11), brewer's yeast (Saccharomyces cerevisiae) is often regarded as the superior MA production host because fermentations can be carried out at a low pH to facilitate downstream processing.…”

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confidence: 99%

“…The strain, harboring DHS dehydratase (AroZ), protocatechuic acid (PCA) decarboxylase (AroY), and catechol dioxygenase (CatA), produced only 1.56 mg/liter of MA. Since this initial demonstration, a number of studies have reported iterative improvements in the yield and titer of heterologous DHSderived products (4,8,(13)(14)(15)(16). From these efforts, it is evident that yeast MA production suffers from two major challenges: (i) achieving high performance of the heterologous pathway for efficient conversion of DHS to MA and (ii) balancing carbon flux between the shikimate pathway and the heterologous branch.…”

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confidence: 99%

“…From these efforts, it is evident that yeast MA production suffers from two major challenges: (i) achieving high performance of the heterologous pathway for efficient conversion of DHS to MA and (ii) balancing carbon flux between the shikimate pathway and the heterologous branch. While the MA pathway consists of a three-step conversion of DHS to MA, the AroY-catalyzed decarboxylation of PCA has been identified to be a major bottleneck in yeast (4,(14)(15)(16). For this reason, most studies report MA production in terms of the total heterologous product, as the buildup of pathway intermediates often surpasses that of the target MA product (4,8,16).…”

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confidence: 99%

“…While the MA pathway consists of a three-step conversion of DHS to MA, the AroY-catalyzed decarboxylation of PCA has been identified to be a major bottleneck in yeast (4,(14)(15)(16). For this reason, most studies report MA production in terms of the total heterologous product, as the buildup of pathway intermediates often surpasses that of the target MA product (4,8,16). Indeed, the highest reported yeast MA titer of 2.1 g/liter was accompanied by a 2.6-g/liter buildup of PCA and catechol (8).…”

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confidence: 99%

“…To increase flux through the yeast shikimate pathway, multiple levels of metabolic regulation must be unraveled. The first committed step of the shikimate pathway, synthesis of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), is under allosteric and transcriptional regulation, which can be alleviated by introducing a feedback-resistant version of the Aro4 DAHP synthase (Aro4 FBR ) (19) and, more recently, the Aro3 isozyme (Aro3 FBR ) (4). This flux can then be driven to a heterologous pathway or lost to aromatic amino acid biosynthesis.…”

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confidence: 99%

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An Engineered Aro1 Protein Degradation Approach for Increased cis,cis -Muconic Acid Biosynthesis in Saccharomyces cerevisiae

Pyne

Narcross

Melgar

et al. 2018

Appl Environ Microbiol

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Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in anΔ Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar. Previous efforts to engineer a heterologous MA pathway in have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.

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confidence: 99%

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An Engineered Aro1 Protein Degradation Approach for Increased cis,cis -Muconic Acid Biosynthesis in Saccharomyces cerevisiae

Pyne

Narcross

Melgar

et al. 2018

Appl Environ Microbiol

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An integrated yeast‐based process for cis,cis‐muconic acid production

Wang

Tavares

Schmitz

et al. 2021

Biotech & Bioengineering

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Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply.The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

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