Ohdate, K. | bioRxiv Science

bioRxiv2025

Transfer of the synechan biosynthesis and regulatory pathway enables sulfated polysaccharide production in Synechococcus elongatus PCC 7942

Many sulfated polysaccharides (SPSs) possess useful physicochemical properties and biological activities and are widely used in industry. Currently, major SPSs are derived from livestock and marine organisms. To contribute both to addressing global environmental challenges and to the development of diverse SPSs, microbial production systems are therefore required. Nevertheless, bacteria capable of producing SPSs are limited. In contrast, many cyanobacteria synthesize diverse SPSs, making them promising hosts to produce valuable SPSs, sources for novel SPSs, and genetic resources for SPS biosynthesis. However, no study has introduced an entire heterologous SPS biosynthetic system into cyanobacteria to produce a heterologous SPS. We recently identified a novel SPS, synechan, and elucidated the genetic basis for the biosynthesis and regulation of cyanobacterial SPSs. Here, we heterologously expressed the gene set responsible for synechan biosynthesis and its regulation in the non-SPS-producing cyanobacterium Synechococcus elongatus PCC 7942 and successfully induced the production of an SPS. This work provides a foundation for engineering cyanobacteria to produce useful SPSs and, to our knowledge, the first functional heterologous reconstitution in cyanobacteria of a large gene cluster encoding a complex membrane-associated biosynthetic system and thus an important step in synthetic-biology-based engineering of cyanobacteria.

synthetic biology↗

bioRxiv2023

Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid". Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

microbiology↗