TY - JOUR
T1 - Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria
AU - Jones, Christopher M.
AU - Parrish, Sydney
AU - Nielsen, David R.
N1 - Funding Information:
We thank Zachary Dookeran and Cody Kamoku for technical support. CMJ is supported by DOE EERE (DE-EE0008515). SP is supported by NSF (CBET-1705409) and DOE BES (DE-SC0021645).
Publisher Copyright:
©
PY - 2021/9/17
Y1 - 2021/9/17
N2 - Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.
AB - Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.
KW - CRE-lox
KW - cyanobacteria
KW - genome engineering
KW - polyploidy
KW - synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=85114322559&partnerID=8YFLogxK
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U2 - 10.1021/acssynbio.1c00269
DO - 10.1021/acssynbio.1c00269
M3 - Article
C2 - 34530614
AN - SCOPUS:85114322559
SN - 2161-5063
VL - 10
SP - 2371
EP - 2382
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 9
ER -