The Effect of Promoter and RBS Combination on the Growth and Glycogen Productivity of Sodium-Dependent Bicarbonate Transporter (SbtA) Overexpressing Synechococcus sp. PCC 7002 Cells

Abstract

Sodium dependent bicarbonate transporter, SbtA is a high-affinity, inducible bicarbonate transporter in cyanobacterial cells. Our previous work has shown that overexpression of this transporter can significantly increase growth and glycogen accumulation in Synechococcus sp. PCC 7002 cells. In this work, we have tested the effect of two different RBS sequences (RBS1: GGAGGA and RBS2: AGGAGA) and three different promoters (PcpcB, PcpcB560, and PrbcL2) on the growth and glycogen production in SbtA-overexpressing Synechococcus sp. PCC 7002 cells. Our results show that PcpcB or PcpcB560 were more effective than PrbcL2 in increasing the growth and glycogen content. The choice of RBS sequence had relatively minor effect, though RBS2 was more effective than RBS1. The transformant E, with PcpcB560 and RBS2, showed the highest growth. The biomass after 5 days of growth on air or 1% CO2 was increased by about 90% in the strain E compared to PCC 7002 cells. All transformants overexpressing SbtA had higher glycogen content. However, growing the cells with bubbling of 1% CO2 did not increase cellular glycogen content any further. The strain E had about 80% higher glycogen content compared to WT PCC 7002 cells. Therefore, the glycogen productivity of the strain E grown with air-bubbling was about 2.5-fold that of the WT PCC 7002 cells grown similarly. Additionally, some of the transformants had higher chlorophyll content while all the transformants had higher carotenoid content compared to the PCC 7002 cells, suggesting interaction between carbon transport and pigment levels. Thus, this work shows that the choice of photosynthetic promoters and RBSs sequences can impact growth and glycogen accumulation in SbtA-overexpressing cells.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses

“…Utilizing the bioreactor, we could obtain similar biomass productivity with air bubbling as seen in shake flasks with bubbling of 1% CO 2 . It is very likely that biomass and glycogen productivity can be increased further by utilizing higher CO 2 concentrations and/or engineered cyanobacteria [ 30 , 34 ]. Alternatively, recently identified marine cyanobacterial strains [ 35 ] that show higher basal biomass productivity or glycogen content [ 36 ] than Synechococcus sp.…”

“…The seeding optical density OD 730 was 0.1. The reactor was illuminated continuously from outside using customized LED lights (Design Innova, New Delhi, India) [ 30 ]. The light intensity was set at 100 µmol m −2 s −1 at the beginning of the experiment and was gradually increased by 100 µmol m −2 s −1 every 8 h until the maximum light intensity (1000 µmol m −2 s −1 ) and kept at that intensity for the rest of the culture.…”

Protein Expression in Synechococcus PCC 7002: A Quantitative Comparison of Promoters and Integration Sites

“…The resulting expression cassette showed superior performance compared to trc constructs and exhibited a 48fold dynamic range. Another comparison of cpc, cpc 560 , and rbcL2revealed that cpc and cpc 560 were more effective than rbcL2 [7].…”