Overexpression of bicarbonate transporters in the marine cyanobacterium Synechococcus sp. PCC 7002 increases growth rate and glycogen accumulation

Abstract

Background: Synechococcus sp. PCC 7002 is an attractive organism as a feedstock and for photoautotrophic production of biofuels and biochemicals due to its fast growth and ability to grow in marine/brackish medium. Previous studies suggest that the growth of this organism is limited by the HCO 3 − transport across the cytoplasmic membrane. Tools for genetic engineering are well established for this cyanobacterium, which makes it possible to overexpress genes of interest. Results: In this work, we overexpressed two different native Na +-dependent carbon transporters viz., SbtA and BicA in Synechococcus sp. PCC 7002 cells under the influence of a strong light-inducible promoter and a strong RBS sequence. The overexpression of these transporters enhanced biomass by about 50%, increased intracellular glycogen about 50%, and increased extracellular carbohydrate up to threefold. Importantly, the biomass and glycogen productivity of the transformants with air bubbling was even higher than that of WT cells with 1% CO 2 bubbling. The overexpression of these transporters was associated with an increased carotenoid content without altering the chl a content. Conclusions: Our work shows the utility of increased carbon transport in improving the growth as well as product formation in a marine cyanobacterium and will serve to increase the utility of this organism as a potential cell factory.

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

“…SbtA is a high-affinity, low flux, inducible sodium (Na +) dependent HCO 3 − transporter, functioning as an Na + /HCO 3 − symporter, that has been shown to be upregulated in carbon limiting conditions in earlier studies (Shibata et al, 2002;Price, 2011). Our work had shown that overexpression of SbtA can promote the growth of PCC 7002 cells to the same levels as that observed with the overexpression of BicA (Gupta et al, 2020).…”

“…RNA and proteins were extracted as in our earlier study (Gupta et al, 2020). For RNA isolation, 50 mL culture in exponential phase was centrifuged for 5 min at 4000 × g. The pellet was crushed in presence of liquid nitrogen using a pestle wiped with RNaseZAP and washed with RNase-free water.…”

“…An earlier published protocol was used (Gupta et al, 2020) for western blotting. 20 mL culture was centrifuged and the pellet collected.…”

“…However, the growth rate and cell titers are still low to be commercially viable (Markley et al, 2015). We have previously engineered PCC 7002 with the bicarbonate transporters SbtA and BicA and shown improvement in their growth and intracellular product (glycogen) content (Gupta et al, 2020). While BicA is a relatively well-studied bicarbonate transporter, there are only a few studies on SbtA.…”

Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120

“…PCC 7120 in response to CO 2 levels ( Figure 2 ). High CO 2 in light promotes a high rate of photosynthesis and the storage of photosynthate in the form of glycogen [ 13 , 109 , 110 , 111 ]. Strong expression of PEP synthase and PFOR under these conditions indicate glycolysis/gluconeogenesis through the metabolism of pyruvate, which is consumed in the TCA cycle to facilitate respiratory electron transport and to provide 2OG for amino acid synthesis (reviewed in [ 112 , 113 ]).…”