Overproduction and easy recovery of target gene products from cyanobacteria, photosynthesizing microorganisms

Abstract

New cyanobacterial expression vectors, possessing an origin of replication that functions in a broad range of Gram-negative bacteria, were constructed. To inspect the shuttle vectors, the gene gfp was cloned downstream from the expression control element (ECE) originating from the regulatory region of the Microcystis aeruginosa gene psbA2 (for photosystem II D1 protein), and the vectors were introduced into three kinds of cyanobacteria ( Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, and Limnothrix / Pseudanabaena sp. ABRG5-3) by conjugation. Multiple copy numbers of the expression vectors (in the range of 14–25 copies per cell) and a high expression of green fluorescent protein (GFP) at the RNA/protein level were observed in the cyanobacterial transconjugants. Importantly, GFP was observed in a supernatant from the autolysed transconjugants of ABRG5-3 and easily collected from the supernatant without centrifugation and/or further cell lysis. These results indicate the vectors together with the recombinant cells to be useful for overproducing and recovering target gene products from cyanobacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-3989-0) contains supplementary material, which is available to authorized users.

Characterization of Lysis of the Multicellular CyanobacteriumLimnothrix/Pseudanabaenasp. Strain ABRG5-3

“…12,13) Under these conditions, lysis was also observed with TEM. Just before lysis, we observed large numbers of black particles stained with uranyl acetate around the tip and focal adhesion areas in these cells (Fig.…”

“…13) After we partially clarified the mechanism of lysis (Figs. 2 to 6), we investigated the factors that effectively induced lysis.…”

“…Bottom, Autolysed cells were prepared under static cultivation under bench-work conditions following the shaking condition described in previous reports. 12,13) The cells were fixed by the procedure described above, and then subjected to TEM observation (Â18,400) at the same acceleration voltage. The bar indicates 1 mm.…”

“…Middle, Early stationary cells under continuous shaking conditions were prepared and subjected to static cultivation under bench-work conditions, for 12 h to achieve a stage just before autolysis. 12,13) The resulting cells were fixed with the reagents named above, and then subjected to TEM observation (Â9,690) by the ultrathin sectioning method. The acceleration voltage was 80 kV.…”

“…The cyanobacterium semi-filamentous multicellular strain ABRG5-3 was isolated from a freshwater area in Japan, and its unique nature has been characterized. 12,13) This axenic strain formed colonies and was motile on an agarose plate. The 16S rRNA gene of ABRG5-3 exhibited similarities to those of the Limnothrix and Pseudanabaena strains, which are filamentous and nonheterocystous cyanobacteria.…”