Development of a Tightly Controlled Off Switch for <i>Saccharomyces cerevisiae</i> Regulated by Camphor, a Low-Cost Natural Product

Ikushima, Shigehito; Zhao, Yu; Boeke, Jef D.

doi:10.1534/g3.114.012765

Cited by 30 publications

(44 citation statements)

References 16 publications

(14 reference statements)

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“…Toward assessing metabolic heterogeneity, several novel experimental tools have recently been developed to measure metabolite levels in single cells (Qiu et al, 2019), e.g., by exploiting the autofluorescence of specific metabolites (Papagiannakis et al, 2016), Förster resonance energy transfer (FRET) (Hou et al, 2011), or metabolite-binding transcription factors (Mahr & Frunzke, 2016). For instance, transcription factor (TF)-based biosensors now exist to detect amino acids (Mustafi et al, 2012), sugars (Raman et al, 2014), succinate and 1-butanol (Dietrich et al, 2013), triacetic acid lactone (Tang et al, 2013), and malonyl CoA (Xu et al, 2014), partly enabled by the transplantation of prokaryotic metabolite-responsive TFs to eukaryotes (Ikushima et al, 2015;Li et al, 2015;Skjoedt et al, 2016;Wang et al, 2016;Ikushima & Boeke, 2017).…”

Section: Introductionmentioning

confidence: 99%

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Monteiro

Hubmann

Takhaveev

et al. 2019

Molecular Systems Biology

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Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

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Section: Introductionmentioning

confidence: 99%

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Monteiro

Hubmann

Takhaveev

et al. 2019

Molecular Systems Biology

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“…We wished to construct a TET-based SG strain. Unfortunately, the current TET system available to us (31,32) was too leaky and showed no significant reduction in growth without doxycycline for all of our 44 essential gene SG strains. Therefore, based on success with the SPAL and SPAZ promoters, we assembled an SPAL containing Tet repeats ("SPET") promoter.…”

Section: Resultsmentioning

confidence: 90%

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Agmon

Tang

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial-and yeastbased safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional-and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiaebased transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.genome safety | escape mutants | Rpd3L | histone deacetylase | yeast W ith the increasing use of genetically modified organisms, there is a growing need for biocontainment to secure biosystems from outside malice as well as to prevent propagation in an open system in the event of advertent or inadvertent release to the environment. Although recombinant DNA technology was established more than four decades ago (1), the fact that genetically modified organisms have not caused any substantial deleterious incident is due, in part, to precautionary measures taken by professional genetic engineers and the high cost of genetic engineering, largely restricting its use to academic and industrial laboratories. However, in the current era of "do-ityourself" synthetic biology, with rapid technological advances in DNA synthesis and assembly (2), genome editing (3, 4) and computational tools for design (5-9), and with the decreasing cost of DNA synthesis, the need for genomic safeguards (SGs) is clear.Early biocontainment efforts focused on the use of metabolic auxotrophy dependence (10, 11), toxin/antitoxin-dependent suicide (12-15), or both (16,17). Although top performers using these strategies do comply with the NIH standard for SGs (10

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“…Several groups have engineered native yeast promoters for the design of biosensors. A common strategy has been to insert aTF operator sites (see section ‘Operator sequence’) into a native yeast promoter chassis in which upstream activation sequences or upstream repression sequences have been deleted whenever present, in order to minimise the complexity of native regulation (Ikushima, Zhao and Boeke 2015 ; Li et al. 2015 ; Skjoedt et al.…”

Section: Biosensor Design Engineeringmentioning

confidence: 99%

Lighting up yeast cell factories by transcription factor-based biosensors

D’ambrosio

Jensen

2017

FEMS Yeast Research

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Our ability to rewire cellular metabolism for the sustainable production of chemicals, fuels and therapeutics based on microbial cell factories has advanced rapidly during the last two decades. Especially the speed and precision by which microbial genomes can be engineered now allow for more advanced designs to be implemented and tested. However, compared to the methods developed for engineering cell factories, the methods developed for testing the performance of newly engineered cell factories in high throughput are lagging far behind, which consequently impacts the overall biomanufacturing process. For this purpose, there is a need to develop new techniques for screening and selection of best-performing cell factory designs in multiplex. Here we review the current status of the sourcing, design and engineering of biosensors derived from allosterically regulated transcription factors applied to the biotechnology work-horse budding yeast Saccharomyces cerevisiae. We conclude by providing a perspective on the most important challenges and opportunities lying ahead in order to harness the full potential of biosensor development for increasing both the throughput of cell factory development and robustness of overall bioprocesses.

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Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product

Cited by 30 publications

References 16 publications

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Lighting up yeast cell factories by transcription factor-based biosensors

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