Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

William Rostain; Tom Zaplana; Magali Boutard; Chloé Baum; Sibylle Tabuteau; Mary Sanitha; Mohandass Ramya; Adam Guss; Laurence Ettwiller; Andrew C. Tolonen

doi:10.1021/acssynbio.2c00385

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

William Rostain, Tom Zaplana, Magali Boutard, Chloé Baum, Sibylle Tabuteau, Mary Sanitha, Mohandass Ramya, Adam Guss, Laurence Ettwiller, Andrew C. Tolonen

Synthetic Biology

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

3 Scopus citations

Abstract

Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

Original language	English
Pages (from-to)	4077-4088
Number of pages	12
Journal	ACS Synthetic Biology
Volume	11
Issue number	12
DOIs	https://doi.org/10.1021/acssynbio.2c00385
State	Published - Dec 16 2022

Bibliographical note

Publisher Copyright:
© 2022 American Chemical Society.

Funding

We thank Corinne Cruaud, Shahinaz Gas, and Ioana Popescu for technical expertise. This work was supported by Genoscope-CEA, the Agence Nationale de la Recherche (Grant ANR-16-CE05-0020), Évry Genopole under the “Action Thématique Incitative Genopole” Grant (funding ATIGE 2021 No. 2698), a travel grant from SRM University, and the Oak Ridge National Laboratory at the Center for Bioenergy Innovation, a U.S. Department of Energy (DOE) Bioenergy Research Center. PacBio methylome data were generated by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. DOE under Contract DE-AC02-05CH11231.

Funders	Funder number
Bioenergy Research Center
Oak Ridge National Laboratory
U.S. Department of Energy Joint Genome Institute
U.S. Department of Energy
Office of Science	DE-AC02-05CH11231
SRM Institute of Science and Technology
Agence Nationale de la Recherche	ANR-16-CE05-0020, 2698

Keywords

CRISPRi
Cas12a/Cpf1
Clostridia
electroporation
fermentation
methylome

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10.1021/acssynbio.2c00385

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abstract = "Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.",

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AU - Sanitha, Mary

AU - Ramya, Mohandass

AU - Guss, Adam

AU - Ettwiller, Laurence

AU - Tolonen, Andrew C.

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AB - Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

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Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

Abstract

Bibliographical note

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Keywords

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