A Bioprocess Engineering Approach to Boost Selection of Fully Segregated Transformants in Cyanobacteria

Cyanobacteria are photoautotrophic microorganisms with significant applications in biotechnology. Although many cyanobacteria, including Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 11901 (Picosynechococcus) and Synechocystis sp. PCC 6803 (Synechocystis), are readily and naturally transformable, their polyploidy poses a major challenge. To obtain a stable phenotype, transgenic strains must be fully segregated, i.e. mutations must appear in all chromosome copies. Traditional protocols rely on re-streaking of colonies on increasingly selective plates, a time-intensive laboratory procedure that requires continuous intervention from the operator. This study proposes an alternative protocol that combines transformation in a batch system in liquid culture with transformant selection in a continuous-flow stirred-tank reactor system. This protocol led to the successful selection of homoplasmic transformants of Picosynechococcus containing, alternatively, an antibiotic resistance alone (construct "SmR") or a more complex construct ("bKT") that leads to the accumulation of a ketocarotenoid. The stability of SmR transformants under semi-continuous cultivation in the absence of antibioticsf was tested for 42 days, proving their potential fitness to industrial cultivation conditions. The selection process was also validated on the model species Synechocystis, demonstrating its applicability to other cyanobacterial strains.