Journal
LIFE-BASEL
Volume 11, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/life11111198
Keywords
CRISPRi; cyanobacteria; Synechocystis; extracellular polymeric substances (EPS); KpsM
Categories
Funding
- FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020
- Portuguese funds through the Fundacao para a Ciencia e a Tecnologia (FCT)/Ministerio da Ciencia, Tecnologia e Ensino Superior [POCI-01-0145-FEDER-028779 (PTDC/BIA-MIC/28779/2017)]
- FCT, I.P [UIDB/04293/2020, UIDP/04293/2020]
- FCT [SFRH/BD/119920/2016, CEECIND/00259/2017]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/119920/2016, UIDP/04293/2020] Funding Source: FCT
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By repressing three putative kpsM homologues using the CRISPRi multiplex system, this study confirmed the effectiveness of CRISPRi in unraveling complex EPS biosynthetic pathways in cyanobacteria.
The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.
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