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

Area of Science:

• * Biotechnology
• * Microbiology
• * Genetic Engineering

Background:

• * Cyanobacteria are vital photoautotrophs with broad biotechnological uses.
• * Polyploidy in cyanobacteria like Picosynechococcus and Synechocystis complicates achieving stable transgenic strains.
• * Traditional methods for segregating mutations across all chromosome copies are laborious and time-consuming.

Purpose of the Study:

• * To develop an efficient and rapid protocol for selecting homoplasmic cyanobacterial transformants.
• * To overcome the challenge of polyploidy in genetic modification of cyanobacteria.
• * To demonstrate the applicability of the new protocol for industrial cultivation and other strains.

Main Methods:

• * Combined batch transformation in liquid culture with continuous-flow stirred-tank reactor selection.
• * Utilized antibiotic resistance (SmR) and ketocarotenoid accumulation (bKT) constructs for selection.
• * Validated the protocol on Picosynechococcus sp. PCC 11901 and Synechocystis sp. PCC 6803.

Main Results:

• * Successfully selected homoplasmic transformants in Picosynechococcus with SmR and bKT constructs.
• * Demonstrated the stability of SmR transformants over 42 days of semi-continuous cultivation without antibiotics.
• * Confirmed the protocol's effectiveness and applicability in the model cyanobacterium Synechocystis.

Conclusions:

• * The novel protocol significantly accelerates the selection of stable, homoplasmic cyanobacterial transformants.
• * This method is suitable for industrial cultivation conditions and applicable to diverse cyanobacterial species.
• * Offers a more efficient alternative to traditional, labor-intensive segregation techniques.