Biosensor‐Enabled Directed Evolution to Improve Muconic Acid Production in <i>Saccharomyces cerevisiae</i>

Leavitt, John M.; Wagner, James M.; Tu, C.C.; Tong, Alice Y.; Liu, Yanyi; Alper, Hal S.

doi:10.1002/biot.201600687

Cited by 108 publications

(90 citation statements)

References 58 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.…”

mentioning

confidence: 99%

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%

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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“…In more and more laboratory evolution campaigns, in vivo (mostly in E. coli ) TF‐based biosensors play a fundamental role as they enable the evaluation of considerable number of genetic designs with reduced experimental effort. Recently, TF‐based biosensors have also been utilized in yeast or phage‐assisted system for strain/enzyme evolution . Still, the current state of affairs in the field indicates that in order for TF biosensors become a routine tool in the hands of the molecular breeder, it would be important that the utilized sensing constructs match strict performance criteria for specificity, sensitivity and detection range, and that the workflow for the rapid development, tuning and application of TF‐based biosensors is also standardized.…”

Section: Conclusion and Prospectsmentioning

confidence: 99%

Transcription Factor‐Based Biosensors in High‐Throughput Screening: Advances and Applications

Cheng

Tang

Kardashliev

2018

Biotechnology Journal

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The molecular mechanisms that cells use to sense changes in the intra- and extracellular environment are increasingly utilized in synthetic biology to build genetic reporter constructs for various applications. Although in nature sensing can be RNA-mediated, most existing genetically-encoded biosensors are based on transcription factors (TF) and cognate DNA sequences. Here, the recent advances in the integration of TF-based biosensors in metabolic and protein engineering screens whereas distinction is made between production-driven and competitive screening systems for enzyme evolution under physiological conditions are discussed. Furthermore, the advantages and disadvantages of existing TF-based biosensors are examined with respects to dynamic range, sensitivity, and robustness, and compared to other screening approaches. The application examples discussed in this review demonstrate the promising potential TF-based biosensors hold as screening tools in laboratory evolution of proteins and metabolic pathways, alike.

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“…Aroma compound production has also benefitted from these methodologies when ample precursor molecules are available [70]. However, the inherent volatile nature of some precursor molecules have excluded them from many directed evolution endeavours, as these are mostly limited to growth-selectable phenotypes, with limited high-throughput possibilities for the rapid screening of mutant libraries [71].…”

Section: Biosensing Aroma Compounds In Yeastmentioning

confidence: 99%

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

2018

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Yeast-especially Saccharomyces cerevisiae-have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast-including S. cerevisiae-to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds.

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Biosensor‐Enabled Directed Evolution to Improve Muconic Acid Production in Saccharomyces cerevisiae

Cited by 108 publications

References 58 publications

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

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

Transcription Factor‐Based Biosensors in High‐Throughput Screening: Advances and Applications

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

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