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Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast.

ABSTRACT: Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence-spaces is a labour-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a rate-limiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13-fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

SUBMITTER: Jensen ED

PROVIDER: S-EPMC8601171 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 Saccharomyces cerevisiae 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 an aro3Δ aro4Δ 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 S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 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.IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae 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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Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast.

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