An Organic Acid Based Counter Selection System for Cyanobacteria

Begemann, Matthew B.; Zess, Erin K.; Walters, Eric M.; Schmitt, Emily F.; Markley, Andrew L.; Pfleger, Brian F.

doi:10.1371/journal.pone.0076594

An Organic Acid Based Counter Selection System for Cyanobacteria

Journal Article · Tue Oct 01 00:00:00 EDT 2013 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0076594· OSTI ID:1904672

Begemann, Matthew B. ^[1]; Zess, Erin K. ^[2]; Walters, Eric M. ^[2]; Schmitt, Emily F. ^[2]; Markley, Andrew L. ^[2]; Pfleger, Brian F. ^[2]

University of Wisconsin, Madison, WI (United States); Univ. of Wisconsin, Madison, WI (United States)
University of Wisconsin, Madison, WI (United States)

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).

View Accepted Manuscript (DOE)

Research Organization:: University of Wisconsin, Madison, WI (United States)

Sponsoring Organization:: National Science Foundation (NSF); US Air Force Office of Scientific Research (AFOSR); USDOE Office of Science (SC)

Grant/Contract Number:: FG02-04ER25627

OSTI ID:: 1904672

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 10 Vol. 8; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002 Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E. ACS Synthetic Biology, Vol. 4, Issue 5 https://doi.org/10.1021/sb500260k	journal	September 2014
Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002 Vogel, Anne Ilse Maria; Lale, Rahmi; Hohmann-Marriott, Martin Frank Journal of Biological Engineering, Vol. 11, Issue 1 https://doi.org/10.1186/s13036-017-0061-8	journal	June 2017
Synechocystis: Not Just a Plug-Bug for CO2, but a Green E. coli Branco dos Santos, Filipe; Du, Wei; Hellingwerf, Klaas J. Frontiers in Bioengineering and Biotechnology, Vol. 2 https://doi.org/10.3389/fbioe.2014.00036	journal	September 2014
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Enhanced Production of D-Lactate in Cyanobacteria by Re-Routing Photosynthetic Cyclic and Pseudo-Cyclic Electron Flow Selão, Tiago Toscano; Jebarani, Jasmin; Ismail, Nurul Aina Frontiers in Plant Science, Vol. 10 https://doi.org/10.3389/fpls.2019.01700	journal	January 2020
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Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology Gale, Grant A. R.; Schiavon Osorio, Alejandra A.; Mills, Lauren A. Microorganisms, Vol. 7, Issue 10 https://doi.org/10.3390/microorganisms7100409	journal	September 2019

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59 BASIC BIOLOGICAL SCIENCES
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An Organic Acid Based Counter Selection System for Cyanobacteria

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