Effects of CO2 and RuBisCO concentration on cyanobacterial growth and carbon isotope fractionation

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope fractionation in cyanobacteria. We found that elevated CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. RuBisCO overexpression in our engineered strain does not significantly affect isotopic discrimination at all tested CO2 concentrations, yielding cellular 13C/12C isotope discrimination ({varepsilon}p) of [~]24{per thousand} for both wild-type and mutant strains at elevated CO2. Understanding the environmental factors that impact gene regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record carbon biosignatures.