Development of genetic manipulation tools for marine synechococcus

Abstract

As one of the most abundant photosynthetic picoplankton in the oceans, the cyanobacterium Synechococcus is an ideal organism to produce a wide range of biofuels, industrially relevant chemicals, and commodity bioproducts. However, progress in engineering marine Synechococcus to synthesize bioproducts and understanding them at the molecular level has been hampered by a lack of genetic manipulation tools for these organisms.

In this study, we sought to develop efficient conjugation-based genetic manipulation tools for a model strain, Synechococcus sp. WH 7803. These include a pour plating method to obtain isolated colonies with high efficiencies and a novel E. coli-mediated conjugation method for the introduction of foreign DNA. Using these tools, we integrated a suicide vector into the chromosome of Synechococcus WH 7803 by the homologous recombination events between the sequences cloned into the plasmid and _ their counterparts on the chromosome. The mutagenesis tool developed in this study can be employed for the insertional inactivation of target genes in marine Synechococcus strains.

Simultaneously, we attempted to develop an efficient and precise genome editing tool using CRISPR technology with a novel nuclease, Cpfl. The markerless nature of Cpfl would allow rapid and multiplex genome modifications in model strains. Through the conjugation, we demonstrated the replicative plasmid pSL2680 that previously generated markerless modifications in freshwater cyanobacterial strains could not be stably maintained in Synechococcus WH 7803. An alternative method to integrate CRISPR/Cas systems to the chromosome of model strains may overcome this limitation and enable CRISPR-based genome editing in these organisms.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Qinglu ZENG (Supervisor)