Development of a Bacterial Biosensor for Rapid Screening of Yeast <i>p</i>-Coumaric Acid Production

Siedler, Solvej; Khatri, Narendar K.; Zsohár, Andrea; Kjærbølling, Inge; Vogt, Michael; Hammar, Petter; Nielsen, Christian Førgaard; Marienhagen, Jan; Sommer, Morten Otto Alexander; Joensson, Haakan N.

doi:10.1021/acssynbio.7b00009

Cited by 114 publications

(94 citation statements)

References 46 publications

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“…As an example, transcription factor-driven biosensors and RNA based aptamer biosensors have been used to detect intracellular and extracellular products, respectively 95,96 and these have been used in combination with droplet-based microfluidics and flow cytometry. Siedler et al 97 used E. coli to express a p-coumaric acid responsive repressor PadR as a biosensor to detect yeast p-coumaric acid production after co-encapsulation. Abatemarco et al 98 used a series of spinach-based aptamers for ultrahigh-throughput screening of various small molecules and proteins produced by Saccharomyces cerevisiae in an approach termed RNA-aptamers-in-droplets (RAPID) high-throughput screening strategy.…”

Section: Ultrahigh-throughput Screening Methodsmentioning

confidence: 99%

“…As an example, transcription factor‐driven biosensors and RNA based aptamer biosensors have been used to detect intracellular and extracellular products, respectively and these have been used in combination with droplet‐based microfluidics and flow cytometry. Siedler et al . used E. coli to express a p ‐coumaric acid responsive repressor PadR as a biosensor to detect yeast p ‐coumaric acid production after co‐encapsulation.…”

Section: Screening and Selection Processesmentioning

confidence: 99%

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Strategies for directed and adapted evolution as part of microbial strain engineering

Zhou

Alper

2018

J of Chemical Tech & Biotech

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The industrial production of chemicals by microorganisms usually requires improvements to the enzymes, pathways, and strain that go beyond the capacity of innate enzymes. To achieve these phenotypes and overcome our limited capacity to de novo design these parts, directed and adaptive evolutionary approaches are used to explore new functions. This review highlights the recent advances in both sequence diversity generation and selection strategies from traditional in vitro mutagenesis to novel in vivo continuous evolution applications. The focus here is on comparison of the different gene diversity methods in an attempt to distinguish the best strategy for protein or strain engineering for a given goal. Furthermore, the important role that screening and selection can play in advancing directed and adapted evolution is discussed. © 2018 Society of Chemical Industry

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Section: Ultrahigh-throughput Screening Methodsmentioning

confidence: 99%

Section: Screening and Selection Processesmentioning

confidence: 99%

Strategies for directed and adapted evolution as part of microbial strain engineering

Zhou

Alper

2018

J of Chemical Tech & Biotech

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“…Droplet sorting, either via microfluidic sorting devices or commercial flow cytometry, is emerging as a valuable tool for screening strain libraries at high-throughputs [40][41][42][43][44][45][46] . With current technology, the highest throughput of sorting is achieved using fluorescence signal.…”

Section: Implementation Of Cross-feeding Co-culture Screening Via Higmentioning

confidence: 99%

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

Saleski

Kerner

Chung

et al. 2019

Preprint

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Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (10 2 -10 4 assays/experiment), spatial separation on agar plates (10 4 -10 5 assays/experiment), and encapsulation in microdroplets (10 5 -10 7 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy.We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules. We expect the cross-feeding system to have two major advantages for high-throughput screening over traditional auxotrophic biosensor implementation: i) amplification of production improvement through the co-culture's exponential growth, and ii) a wider and more tunable dynamic range. The former lies in the co-culture's amplification of differences between library members (Section 3, Calculation). Since strain development generally proceeds through incremental improvements, it is critical to be able to discern modest increases in production.Amplification of differences through the cross-feeding metabolic circuit makes relatively small improvements conspicuous. The second advantage is achieved because, when neither of the cross-fed molecules is exogenously supplied, the production rates of the cross-fed molecules generally limit total co-culture growth, keeping the target molecule within the dynamic range of the sensor strain. This is in contrast to direct application of the sensor to the supernatant of a secretor strain or in co-culture with a non-auxotrophic secretor strain when the level of target molecule will often exceed sensor dynamic range. 6 Materials and MethodsKey elements are described here. Additional details are provided in the Supplementary Information. Strains and plasmidsStrains and plasmids used are lis...

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“…Attempts have been made to translate some of these concepts in an endeavour to sense aroma compounds produced by bioengineered yeast, using encapsulated p-coumaric acid-sensing E. coli to screen for yeast cells producing p-coumaric acid [80]. Table 1.…”

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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Development of a Bacterial Biosensor for Rapid Screening of Yeast p-Coumaric Acid Production

Cited by 114 publications

References 46 publications

Strategies for directed and adapted evolution as part of microbial strain engineering

Strategies for directed and adapted evolution as part of microbial strain engineering

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

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

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