Generation of miniploid cells and improved natural transformation procedure for a model cyanobacterium Synechococcus elongatus PCC 7942

Abstract

The biotechnologically important and naturally transformable cyanobacterium, Synechococcus elongatus PCC 7942, possesses multiple genome copies irrespective of its growth rate or condition. Hence, segregating mutations across all genome copies typically takes several weeks. In this study, Synechococcus 7942 cultivation on a solid growth medium was optimised using different concentrations of agar, the addition of antioxidants, and overexpression of the catalase gene to facilitate the rapid acquisition of colonies and fully segregated lines. Synechococcus 7942 was grown at different temperatures and nutritional conditions. The miniploid cells were identified using flow cytometry and fluorimetry. The natural transformation was carried out using miniploid cells and validated with PCR and high performance liquid chromatography (HPLC). We identified that 0.35% agar concentration and 200 IU of catalase could improve the growth of Synechococcus 7942 on a solid growth medium. Furthermore, overexpression of a catalase gene enhanced the growth rate and supported diluted culture to grow on a solid medium. Our results reveal that high temperature and phosphate-depleted cells contain the lowest genome copies (2.4 ± 0.3 and 1.9 ± 0.2) and showed the potential to rapidly produce fully segregated mutants. In addition, higher antibiotic concentrations improve the selection of homozygous transformants while maintaining similar genome copies at a constant temperature. Based on our observation, we have an improved cultivation and natural transformation protocol for Synechococcus 7942 by optimising solid media culturing, generating low-ploidy cells that ultimately reduced the time required for the complete segregation of engineered lines.

Thermophilic cyanobacteria—exciting, yet challenging biotechnological chassis

“…Even those relatively simple organisms lack reliable genetic modules and advanced genome manipulation methods. In cyanobacteria in general, but especially in Thermosynechococcus, it is more difficult to achieve specific integration due to less effective homologous recombination machinery (unpublished data) and attempts to develop protocols of obtaining miniploid cells are not as successful as those employed for mesophilic model strains (Riaz et al 2021(Riaz et al , 2022. Moreover, in the absence of established genome editing tools based on CRISPR/Cas9 methodology, presumably due to the high toxicity of nucleases (unpublished data) and potential thermal instability of mesophilic proteins, specific genome engineering of thermophilic cyanobacteria remains a tedious task.…”

Current Metabolic Engineering Strategies for Photosynthetic Bioproduction in Cyanobacteria

“…However, this was accompanied by a 100-fold drop in transformation efficiency [ 115 ]. Analogously, Riaz et al found out that the reduction of phosphate and agar concentration in the growth medium and high temperature are key parameters for reducing polyploidy and accelerating segregation process [ 116 ].…”

Construction of an artificial consortium of Escherichia coli and cyanobacteria for clean indirect production of volatile platform hydrocarbons from CO2

“…The cscB containing plasmid (pLJD31) was transformed into E542 through the conjugation via E. coli HB101 harboring pRL443 and pRL623 (Thiel and Peter Wolk, 1987) and E. coli DH5α carrying the pLJD31 plasmids, respectively. Meanwhile, the pLJD32 was transformed into PCC7942 using the recently optimized protocol (Riaz et al, 2022).…”