Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA

Andrew Hren; William G. Alexander; Joshua P. Abraham; Melissa P. Tumen-Velasquez; Michael Melesse Vergara; Adam M. Guss; Brian F. Pfleger; Jerome M. Fox; Carrie A. Eckert

doi:10.1021/acssynbio.5c00370

Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA

Andrew Hren
, William G. Alexander
, Joshua P. Abraham
, Melissa P. Tumen-Velasquez
, Michael Melesse Vergara
, Adam M. Guss
, Brian F. Pfleger
, Jerome M. Fox
, Carrie A. Eckert

Biological Systems Design Group

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

2 Scopus citations

Abstract

Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.

Original language	English
Pages (from-to)	3258-3264
Number of pages	7
Journal	ACS Synthetic Biology
Volume	14
Issue number	8
DOIs	https://doi.org/10.1021/acssynbio.5c00370
State	Published - Aug 15 2025

Funding

The authors would like to thank Dylan Courtney for his work developing pDC011 from the original, published CRISPR/Cas12a system and Gregory Donovan for his advice and support. This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under Award Numbers DE-SC0018368 and DE-SC0019404. This research used resources of the Compute and Data Environment for Science (CADES) at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05–00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ). The authors would like to thank Dylan Courtney for his work developing pDC011 from the original, published CRISPR/Cas12a system(31)and Gregory Donovan for his advice and support. This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under Award Numbers DE-SC0018368 and DE-SC0019404. This research used resources of the Compute and Data Environment for Science (CADES) at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725.

Keywords

DNA methylation
cyanobacteria
genetic engineering
restriction modification
transformation efficiency

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10.1021/acssynbio.5c00370

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title = "Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA",

abstract = "Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.",

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year = "2025",

month = aug,

day = "15",

doi = "10.1021/acssynbio.5c00370",

language = "English",

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AU - Alexander, William G.

AU - Abraham, Joshua P.

AU - Tumen-Velasquez, Melissa P.

AU - Melesse Vergara, Michael

AU - Guss, Adam M.

AU - Pfleger, Brian F.

AU - Fox, Jerome M.

AU - Eckert, Carrie A.

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N2 - Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.

AB - Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.

KW - DNA methylation

KW - cyanobacteria

KW - genetic engineering

KW - restriction modification

KW - transformation efficiency

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Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA

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Keywords

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