LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

Zeyu Lu; Qianyi Shen; Naga Chandra Bandari; Samuel Evans; Liam McDonnell; Lian Liu; Wanli Jin; Carlos Horacio Luna-Flores; Thomas Collier; Gert Talbo; Tim McCubbin; Lygie Esquirol; Chris Myers; Matt Trau; Geoff Dumsday; Robert Speight; Christopher B. Howard; Claudia E. Vickers; Bingyin Peng

doi:10.1093/nar/gkae460

LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

Zeyu Lu^*, Qianyi Shen, Naga Chandra Bandari, Samuel Evans, Liam McDonnell, Lian Liu, Wanli Jin, Carlos Horacio Luna-Flores, Thomas Collier, Gert Talbo, Tim McCubbin, Lygie Esquirol, Chris Myers, Matt Trau, Geoff Dumsday, Robert Speight, Christopher B. Howard^*, Claudia E. Vickers^*, Bingyin Peng^*

^*Corresponding author for this work

School of Natural Sciences

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

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Abstract

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30^◦C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. ‘Turbo’ mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80psupressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37–33^◦C) to a low temperature (≤30^◦C). Overall, we present a synthetic biology procedure that leverages ‘leaky’ biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Original language	English
Pages (from-to)	7367-7383
Number of pages	17
Journal	Nucleic Acids Research
Volume	52
Issue number	12
Early online date	29 May 2024
DOIs	https://doi.org/10.1093/nar/gkae460
Publication status	Published - 8 Jul 2024

Bibliographical note

Copyright the Author(s) 2024. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Access to Document

10.1093/nar/gkae460

Publisher version (open access)Final published version, 2.64 MBLicence: CC BY

Cite this

Lu, Z., Shen, Q., Bandari, N. C., Evans, S., McDonnell, L., Liu, L., Jin, W., Luna-Flores, C. H., Collier, T., Talbo, G., McCubbin, T., Esquirol, L., Myers, C., Trau, M., Dumsday, G., Speight, R., Howard, C. B., Vickers, C. E., & Peng, B. (2024). LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae. Nucleic Acids Research, 52(12), 7367-7383. https://doi.org/10.1093/nar/gkae460

@article{b470829fd9f54dfc9553b99be4cfd405,

title = "LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae",

abstract = "Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30◦C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. {\textquoteleft}Turbo{\textquoteright} mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80psupressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37–33◦C) to a low temperature (≤30◦C). Overall, we present a synthetic biology procedure that leverages {\textquoteleft}leaky{\textquoteright} biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.",

author = "Zeyu Lu and Qianyi Shen and Bandari, \{Naga Chandra\} and Samuel Evans and Liam McDonnell and Lian Liu and Wanli Jin and Luna-Flores, \{Carlos Horacio\} and Thomas Collier and Gert Talbo and Tim McCubbin and Lygie Esquirol and Chris Myers and Matt Trau and Geoff Dumsday and Robert Speight and Howard, \{Christopher B.\} and Vickers, \{Claudia E.\} and Bingyin Peng",

note = "Copyright the Author(s) 2024. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.",

year = "2024",

month = jul,

day = "8",

doi = "10.1093/nar/gkae460",

language = "English",

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Lu, Z, Shen, Q, Bandari, NC, Evans, S, McDonnell, L, Liu, L, Jin, W, Luna-Flores, CH, Collier, T, Talbo, G, McCubbin, T, Esquirol, L, Myers, C, Trau, M, Dumsday, G, Speight, R, Howard, CB, Vickers, CE & Peng, B 2024, 'LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae', Nucleic Acids Research, vol. 52, no. 12, pp. 7367-7383. https://doi.org/10.1093/nar/gkae460

TY - JOUR

T1 - LowTempGAL

T2 - a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

AU - Lu, Zeyu

AU - Shen, Qianyi

AU - Bandari, Naga Chandra

AU - Evans, Samuel

AU - McDonnell, Liam

AU - Liu, Lian

AU - Jin, Wanli

AU - Luna-Flores, Carlos Horacio

AU - Collier, Thomas

AU - Talbo, Gert

AU - McCubbin, Tim

AU - Esquirol, Lygie

AU - Myers, Chris

AU - Trau, Matt

AU - Dumsday, Geoff

AU - Speight, Robert

AU - Howard, Christopher B.

AU - Vickers, Claudia E.

AU - Peng, Bingyin

N1 - Copyright the Author(s) 2024. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

PY - 2024/7/8

Y1 - 2024/7/8

N2 - Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30◦C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. ‘Turbo’ mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80psupressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37–33◦C) to a low temperature (≤30◦C). Overall, we present a synthetic biology procedure that leverages ‘leaky’ biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

AB - Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30◦C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. ‘Turbo’ mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80psupressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37–33◦C) to a low temperature (≤30◦C). Overall, we present a synthetic biology procedure that leverages ‘leaky’ biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

UR - https://www.scopus.com/pages/publications/85197379591

UR - https://purl.org/au-research/grants/arc/CE200100029

U2 - 10.1093/nar/gkae460

DO - 10.1093/nar/gkae460

M3 - Article

C2 - 38808673

AN - SCOPUS:85197379591

SN - 0305-1048

VL - 52

SP - 7367

EP - 7383

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 12

ER -

LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

Abstract

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COESB: ARC Centre of Excellence in Synthetic Biology

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LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

Abstract

Bibliographical note

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Projects

COESB: ARC Centre of Excellence in Synthetic Biology

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