CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

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Santos, MarinaUniv Porto, I3S Inst Invest & Inovacao Saude, P-4000008 Porto, Portugal.;Univ Porto, IBMC Inst Biol Mol & Celular, P-4000008 Porto, Portugal.;Univ Porto, Inst Ciencias Biomed Abel Salazar ICBAS, Programa Doutoral Biol Mol & Celular MCbiol, P-4000008 Porto, Portugal. (författare)

QC 20220215
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.

Pacheco, Catarina C.Univ Porto, I3S Inst Invest & Inovacao Saude, P-4000008 Porto, Portugal.;Univ Porto, IBMC Inst Biol Mol & Celular, P-4000008 Porto, Portugal. (författare)
Yao, LunKTH,Science for Life Laboratory, SciLifeLab,Proteinvetenskap(Swepub:kth)u1nych9u (författare)
Hudson, Elton P.KTH,Science for Life Laboratory, SciLifeLab,Proteinvetenskap(Swepub:kth)u1ja6ik3 (författare)
Tamagnini, PaulaUniv Porto, I3S Inst Invest & Inovacao Saude, P-4000008 Porto, Portugal.;Univ Porto, IBMC Inst Biol Mol & Celular, P-4000008 Porto, Portugal.;Univ Porto, Fac Ciencias, Dept Biol, P-4000008 Porto, Portugal. (författare)
Univ Porto, I3S Inst Invest & Inovacao Saude, P-4000008 Porto, Portugal.;Univ Porto, IBMC Inst Biol Mol & Celular, P-4000008 Porto, Portugal.;Univ Porto, Inst Ciencias Biomed Abel Salazar ICBAS, Programa Doutoral Biol Mol & Celular MCbiol, P-4000008 Porto, Portugal.Univ Porto, I3S Inst Invest & Inovacao Saude, P-4000008 Porto, Portugal.;Univ Porto, IBMC Inst Biol Mol & Celular, P-4000008 Porto, Portugal. (creator_code:org_t)

Av författaren/redakt...: Santos, Marina; Pacheco, Catarin ...; Yao, Lun; Hudson, Elton P.; Tamagnini, Paula

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