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Title: High-CO 2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria

As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO 2-concentrating mechanism (CCM) to impose a high-CO 2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO 2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. In conclusion, we showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 10 8 CFU.
Authors:
 [1] ;  [2] ;  [1] ;  [1] ; ORCiD logo [1] ; ORCiD logo [1]
  1. Univ. of Wisconsin, Madison, WI (United States). Dept. of Chemical and Biological Engineering
  2. Univ. of Wisconsin, Madison, WI (United States). Dept. of Chemical and Biological Engineering; Univ. of Wisconsin, Madison, WI (United States). Microbiology Doctoral Training Program
Publication Date:
Grant/Contract Number:
SC0010329
Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 7; Journal Issue: 2; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Research Org:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org:
USDOE; National Science Foundation (NSF); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biocontainment; carboxysome; CO2-concentrating mechanism; cyanobacteria; horizontal gene transfer; natural competence
OSTI Identifier:
1417031

Clark, Ryan L., Gordon, Gina C., Bennett, Nathaniel R., Lyu, Haoxiang, Root, Thatcher W., and Pfleger, Brian F.. High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria. United States: N. p., Web. doi:10.1021/acssynbio.7b00377.
Clark, Ryan L., Gordon, Gina C., Bennett, Nathaniel R., Lyu, Haoxiang, Root, Thatcher W., & Pfleger, Brian F.. High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria. United States. doi:10.1021/acssynbio.7b00377.
Clark, Ryan L., Gordon, Gina C., Bennett, Nathaniel R., Lyu, Haoxiang, Root, Thatcher W., and Pfleger, Brian F.. 2018. "High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria". United States. doi:10.1021/acssynbio.7b00377.
@article{osti_1417031,
title = {High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria},
author = {Clark, Ryan L. and Gordon, Gina C. and Bennett, Nathaniel R. and Lyu, Haoxiang and Root, Thatcher W. and Pfleger, Brian F.},
abstractNote = {As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. In conclusion, we showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU.},
doi = {10.1021/acssynbio.7b00377},
journal = {ACS Synthetic Biology},
number = 2,
volume = 7,
place = {United States},
year = {2018},
month = {1}
}