Overexpression of hlyB and mdh genes confers halotolerance in Fremyella diplosiphon, a freshwater cyanobacterium

Fremyella diplosiphon is a freshwater cyanobacterium that has great potential as a biofuel agent due to its ability to grow in low light intensity and acclimation to different wavelengths. To enhance its halotolerance for growth in 35 g L−1 sodium chloride (NaCl), plasmids harboring hemolysin B (hlyB) and malate dehydrogenase (mdh) genes were transformed into wild type F. diplosiphon (WT-Fd33). Electroporation-mediated overexpression of the genes resulted in two transformants, HSF33-1 and HSF33-2, with 9- and 20-fold increases in hlyB and mdh transcript levels. In addition, up-regulation of proteins at the expected size ranges of 50-60 kDa for HlyB and 40-50 kDa for MDH was observed. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed a protein spot corresponding to HlyB in HSF33-1 with a significant MOWSE score of 164 and 3% sequence coverage, and a spot corresponding to MDH in HSF33-2 gave a significant MOWSE score of 124 with 10% sequence coverage. Physiological evaluation in BG11/HEPES medium and seawater adjusted to 35 g L−1 NaCl confirmed that the transformants could thrive in high salinity with no loss of photosynthetic pigments. Results of the study indicate that overexpression of hlyB and mdh genes confer halotolerance in F. diplosiphon, thus maximizing its potential as a large-scale biofuel agent.