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

Marina Santos,Elton P Hudson,Lun Yao,Paula Tamagnini,Catarina C Pacheco

doi:10.3390/life11111198

Abstract

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.

Highlights

It is important to highlight that kpsM has three putative homologues in Synechocystis: slr0977, slr2107, and sll0574 (Figure 1A), and it is necessary to clarify the role of the proteins, encoded by these genes, on extracellular polymeric substances (EPS) production
To pursue the unravelling of cyanobacterial EPS assembly and export pathways, the CRISPRi system was employed as a tool for the multiplex repression of EPS-related genes in Synechocystis, namely for the three kpsM homologues
The kpsM homologues were successfully repressed in the Synechocystis 3-single-guide RNA (sgRNA) kpsM mutant compared to Syn dead Cas9 (dCas9)

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. It is important to highlight that kpsM has three putative homologues in Synechocystis: slr0977, slr2107, and sll0574 (Figure 1A), and it is necessary to clarify the role of the proteins, encoded by these genes, on EPS production. This would be tackled by generating a triple knockout mutant. To pursue the unravelling of cyanobacterial EPS assembly and export pathways, the CRISPRi system was employed as a tool for the multiplex repression of EPS-related genes in Synechocystis, namely for the three kpsM homologues (slr0977, slr2107 and sll0574). The generated mutant was characterized in terms of growth and carbohydrate production, and its phenotype compared to the conventional single knockout mutants generated by double homologous recombination

Experimental Section

Results & Discussion

Conclusions