Engineering artificial photosynthetic life-forms through endosymbiosis

Abstract

The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined photosynthetic conditions. We anticipate that these genetically tractable endosymbiotic platforms can be used for evolutionary studies, particularly related to organelle evolution, and also for synthetic biology applications.

Fig. 1. Endosymbiotic theory and our platform to recapitulate the evolution of photosynthetic eukaryotic cells.

a The endosymbiotic theory–Mitochondria, M, are proposed to have evolved from a class of α-proteobacteria while the chloroplast, C, is proposed to have originated from cyanobacteria. Golgi apparatus—G, Endoplasmic reticulum—ER, Vacuole—V. b S. cerevisiae (budding yeast) cells produce ATP by glycolysis or mediated oxidative phosphorylation. c Suicide plasmid-based strategy used in this manuscript to engineer cyanobacterial mutants, SynJEC strains. d Our platform: We use suicide plasmid-based strategy to engineer cyanobacterial endosymbionts, SynJEC strains, such that they perform chloroplast-like functions. S. cerevisiae mutants, deficient in ATP synthesis by oxidative phosphorylation under defined photosynthetic selection conditions, are used as host strains. Engineered cyanobacteria strains, SynJEC, are then introduced into the yeast cells by a cell fusion process that is developed and optimized (see Methods). The yeast/cyanobacterial chimera are selected under defined photosynthetic selection conditions where the cyanobacterial endosymbionts provide ATP to the mutant S. cerevisiae host cells, and S. cerevisiae provide essential metabolites to the S. elongatus endosymbionts.

Fig. 2. S. cerevisiae–SynJEC chimeras have a partially rescued respiration-competent phenotype.

a Release of ATP by SynJEC1 cells expressing the UWE25 ADP/ATP translocase in the presence of 80 μM ADP in comparison to SynJEC0 cells. ATP was released when SynJEC1 (expressing the ATP/ADP translocase) and SynJEC0 cells were challenged with extracellular ADP (80 μM), but not with a blank solution lacking ADP (N = 3 biological replicates; data are presented as mean values + /− SEM). Two-sided t-tests were used to compare means without adjustments (95% CI, Cohen’s d = 10.6, DF = 4, P = 0.0002; 95% CI, Cohen’s d = 13.0, DF = 4, P = 0.0001; 95% CI, Cohen’s d = 2.7, DF = 4, P = 0.0002). b Growth of S. cerevisiae cox2-60–SynJEC chimeras on medium containing glycerol as the sole carbon source. No growth was observed in round 4 for yeast lacking intracellular SynJEC. The experiment was repeated independently six times with similar results. c Total DNA isolated from spots grown on selection medium III contain the yeast-encoded MATa gene and SynJEC-encoded chloramphenicol acetyltransferase (CAT) gene. The experiment was repeated independently six times with similar results. d Growth rate of yeast-SynJEC chimeras on Selection Medium III. Cells (3.00 × 10³) were spotted on Selection Medium III and counted after 72 h growth. (N = 3 technical replicates; data are presented as mean values+/− SEM.)P-values were calculated by two-tailed t-test comparing the two means. e Panel 1 describes the growth of S. cerevisiae-cox2-60 and S. cerevisiae-cox2-60-pCOX2n under non-selection conditions. Panel 2 describes the growth of S. cerevisiae-cox2-60 and S. cerevisiae-cox2-60-pCOX2n under selection conditions where the rescue in the growth of S. cerevisiae-cox2-60-pCOX2n is observed but no growth is observed for S. cerevisiae-cox2-60. The experiment was repeated independently three times with similar results. Source data are provided as a Source Data file.

Fig. 3. Rescue of respiration deficient phenotype by yeast-SynJEC chimeras expressing an ATP/ADP translocase and SNARE-like proteins.

a Growth of S. cerevisiae cox2-60–SynJEC2—4 chimeras on medium containing glycerol as the sole carbon source. The experiment was repeated three times independently with similar results. b Total DNA of yeast-SynJEC chimeras contains yeast MATa and SynJEC CAT genes. The experiment was repeated three times independently with similar results. c, d Growth trends of S. cerevisiae cox2-60 (yeast only strain), S. cerevisiae cox2-60- SynJEC2 (yeast-SynJEC2), S. cerevisiae cox2-60- SynJEC3 (yeast-SynJEC3) and S. cerevisiae cox2-60- SynJEC4 (yeast-SynJEC4) chimeras on Selection Medium III. Cells (3.00 × 10³) were spotted on Selection Medium III the final number of cells/spot on plate were determined after 72 h of growth (N = 3 technical replicates; data are presented as mean values+/− SEM). P-values were calculated by two-tailed t-test comparing the two means. Source data are provided as a Source Data file.

Fig. 4. Imaging intracellular endosymbiont Synechococcus by fluorescent microscopy.

a pTIRF microscopic images of Synechococcus cells, control yeast cells, and chimeric cells that were grown under selection conditions (Ex. = 561 nm; Em. = 653/95 nm). Panels are merged images of pTIRF (yellow) and brightfield microscopy (gray). The experiment was repeated three times independently with similar results. b Fluorescence confocal microscopy images of control yeast cells and chimeric cells, which were grown under selection conditions. The yeast cell wall was stained with Con A-FITC (green, Ex.  = 488 nm; Em. = 510/20 nm) and the presence of cyanobacteria was monitored by cyanobacterial fluorescence (red, Ex. = 561 nm; Em. = 650/20 nm). Based on these images, it is possible that multiple cyanobacterial cells could be present in some of the yeast cells. The experiment was repeated three times independently with similar results. c Panel a and b are cyanobacterial samples imaged by Transmission Electron Microscopy (TEM) and Panels c–f are fusions imaged by TEM. Yellow arrows show characteristic cyanobacterial structures within the cytoplasm of the yeast cells. The experiment was repeated twice independently with similar results.

Fig. 5. Rescue of respiration-deficient phenotype by yeast-SynJEC chimeras containing auxotrophic SynJEC5 cells.

a Growth of SynJEC5 cells in BG-11 medium in the presence and absence of L-methionine b Growth of yeast-SynJEC3 and yeast-SynJEC5 chimeras on Selection Medium III. The experiment was repeated three times independently with similar results. c Total DNA analysis of SynJEC5 DNA reveals the presence of genomic SNARE-like proteins Ctr-incA and CT813 (left) and disruption of the metA gene with a kanamycin resistance marker (right). The experiment was repeated twice independently with similar results. d Total DNA of yeast-SynJEC chimeras contains yeast MATa and SynJEC Ctr-incA genes. The experiment was repeated three times independently with similar results. e S. cerevisiae cox2-60-SynJEC3 chimeras are generated under and selected under photosynthetic conditions. After two rounds of growth S. cerevisiae cox2-60-SynJEC3 chimeras are propagated either under standard photosynthetic conditions or under dark; initial number of cells plated per spot is 10⁴ (N = 3 technical replicates; data are presented as mean values+/− SEM). P-values were calculated by two-tailed t-test comparing the two means (95% CI, Cohen’s d = 6.2, P = 0.0016). f SynJEC3 are starved for light for 72 h and the fused to S. cerevisiae cox2-60 and selected under dark conditions (dark fusions). The S. cerevisiae cox2-60-SynJEC3 chimeras generated from dark fusions fail to grow whereas the control S. cerevisiae cox2-60-SynJEC3 chimeras propagated under photosynthetic conditions propagate as previously observed. The experiment was repeated twice independently with similar results. g Plate images to demonstrate the effect of (3-(3,4-dichlorophenyl)−1,1-dimethylurea), DCMU, on S. cerevisiae cox2-60-SynJEC3 chimeras. The experiment was repeated twice independently with similar results. h Cell count analysis to demonstrate the effect of (3-(3,4-dichlorophenyl)−1,1-dimethylurea), DCMU, on S. cerevisiae cox2-60-SynJEC3 chimeras (N = 3 technical replicates; data are presented as mean values+/− SEM). P-values were calculated by two-tailed t-test comparing the two means. Source data are provided as a Source Data file.