Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast.

Williams, TC; Xu, X; Ostrowski, M; Pretorius, IS; Paulsen, IT

Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast.

Williams, TC Xu, X Ostrowski, M

Pretorius, IS Paulsen, IT

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Publisher:: Oxford University Press
Publication Type:: Journal Article
Citation:: Synthetic Biology, 2017, 2, (1), pp. 1-13
Issue Date:: 2017-01

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Field	Value	Language
dc.contributor.author	Williams, TC
dc.contributor.author	Xu, X
dc.contributor.author	Ostrowski, M https://orcid.org/0000-0002-4357-3023
dc.contributor.author	Pretorius, IS
dc.contributor.author	Paulsen, IT
dc.date.accessioned	2022-08-01T03:33:00Z
dc.date.available	2016-11-29
dc.date.available	2022-08-01T03:33:00Z
dc.date.issued	2017-01
dc.identifier.citation	Synthetic Biology, 2017, 2, (1), pp. 1-13
dc.identifier.issn	2397-7000
dc.identifier.issn	2397-7000
dc.identifier.uri	http://hdl.handle.net/10453/159427
dc.description.abstract	Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.
dc.format	Electronic-eCollection
dc.language	eng
dc.publisher	Oxford University Press
dc.relation.ispartof	Synthetic Biology
dc.relation.isbasedon	10.1093/synbio/ysw002
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast.
dc.type	Journal Article
utslib.citation.volume	2
utslib.location.activity	England
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - C3 - Climate Change Cluster
utslib.copyright.status	open_access	*
pubs.consider-herdc	false
dc.date.updated	2022-08-01T03:32:57Z
pubs.issue	1
pubs.publication-status	Published
pubs.volume	2
utslib.citation.issue	1

Abstract:

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.

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http://hdl.handle.net/10453/159427