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 plasmid replication proteins (Rep) 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.

Characterization of a cyanobacterial rep protein with broad-host range and its utilization for expression vectors.

Owing to their photosynthetic capabilities, cyanobacteria are regarded as ecologically friendly hosts for production of biomaterials. However, compared to other bacteria, tools for genetic engineering, especially expression vector systems, are limited. In this study, we characterized a Rep protein, exhibiting replication activity in multiple cyanobacteria and established an expression vector using this protein. Our comprehensive screening using a genomic library of Synechocystis sp. PCC 6803 revealed that a certain region encoding a Rep-related protein (here named Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. A reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, GFP expression in the pYS-based system was tightly regulated by IPTG, achieving 10-fold higher levels than in the chromosome-based system. Furthermore, pYS could be used together with the conventional vector pEX, which was constructed from an endogenous plasmid in S. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

Photosynthetic 1,8-cineole production using cyanobacteria.

Terpenoid is an important group of compounds not only as biocomponents but also as useful secondary metabolites. A volatile terpenoid 1,8-cineole, which is used as a food additive, flavoring agent, cosmetic, etc., is also attracting attention from a medical perspective due to its antiinflammation and antioxidation. The 1,8-cineole fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source supplement is necessary for a high-yield 1,8-cineole production. We constructed the 1,8-cineole-producing cyanobacteria toward a carbon-free and sustainable 1,8-cineole production. cnsA, the 1,8-cineole synthase gene in Streptomyces clavuligerus ATCC 27064, was introduced and overexpressed in the cyanobacterium Synechococcus elongatus PCC 7942. We succeeded in producing an average of 105.6 µg g-1 wet cell weight of 1,8-cineole in S. elongatus 7942 without supplementing any carbon source. Using the cyanobacteria expression system is an efficient approach to producing 1,8-cineole by photosynthesis.

Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp. PCC 6803.

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram-negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli, feedback-regulate RNase E expression via the rne 5'-untranslated region (5' UTR) in cis. However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.