Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO2 fixation, or engineer novel CO2-fixing organisms.Author summary: Rising carbon dioxide levels driving climate change prompts us to embrace sustainable resources, such as autotrophic microbes that produce biomass or chemicals by consuming carbon dioxide. As genetic engineering of natural autotrophs is challenging, it is of interest to engineer autotrophy in more pliable microbial species, such as Escherichia coli. We contrasted 1,020 genomes of microbes carrying the most widespread carbon dioxide fixation pathway, the Calvin cycle, to genomes of closest relatives lacking this pathway. This comparison identified and ranked genetic adaptations that may enable Calvin cycle operation. This list of adaptations sheds light on the evolution of autotrophy and represents a recipe for an autotrophic microbe, which can aid genetic engineers in improving autotrophs or creating them from scratch.