Deciphering cyanobacterial phenotypes for fast photoautotrophic growth via isotopically nonstationary metabolic flux analysis

Abstract

Background: Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium characterized to date. Its genome was found to be 99.8% identical to S. elongatus 7942 yet it grows twice as fast. Current genome-to-phenome mapping is still poorly performed for non-model organisms. Even for species with identical genomes, cell phenotypes can be strikingly different. To understand Synechococcus 2973's fast-growth phenotype and its metabolic features advantageous to photo-biorefineries, ¹³C isotopically nonstationary metabolic flux analysis (INST-MFA), biomass compositional analysis, gene knockouts, and metabolite profiling were performed on both strains under various growth conditions.

Results: The Synechococcus 2973 flux maps show substantial carbon flow through the Calvin cycle, glycolysis, photorespiration and pyruvate kinase, but minimal flux through the malic enzyme and oxidative pentose phosphate pathways under high light/CO₂ conditions. During fast growth, its pool sizes of key metabolites in central pathways were lower than suboptimal growth. Synechococcus 2973 demonstrated similar flux ratios to Synechococcus 7942 (under fast growth conditions), but exhibited greater carbon assimilation, higher NADPH concentrations, higher energy charge (relative ATP ratio over ADP and AMP), less accumulation of glycogen, and potentially metabolite channeling. Furthermore, Synechococcus 2973 has very limited flux through the TCA pathway with small pool sizes of acetyl-CoA/TCA intermediates under all growth conditions.

Conclusions: This study employed flux analysis to investigate phenotypic heterogeneity among two cyanobacterial strains with near-identical genome background. The flux/metabolite profiling, biomass composition analysis, and genetic modification results elucidate a highly effective metabolic topology for CO₂ assimilatory and biosynthesis in Synechococcus 2973. Comparisons across multiple Synechococcus strains indicate faster metabolism is also driven by proportional increases in both photosynthesis and key central pathway fluxes. Moreover, the flux distribution in Synechococcus 2973 supports the use of its strong sugar phosphate pathways for optimal bio-productions. The integrated methodologies in this study can be applied for characterizing non-model microbial metabolism.

Keywords: 13C labeling experiments; Channeling; Energy charge; Glycogen; Metabolites; Photobioreactor.

Fig. 1

Growth performances of cyanobacterial species. a Growth rate of Synechococcus 2973, Synechococcus 7942 and the Synechococcus 2973 Δzwf mutant in PBR and SF conditions under continuous light conditions. Doubling times in hours are reported below for each strain. ^† Synechococcus 7942 was grown at 300 μmol photons/m²s. Standard deviations are a result of 3–5 biological replicates. *p value < 0.02 between Syn. 2973 and 7942 PBR conditions using two-tailed equal variance Student’s t test. b Diurnal growth curve and rates of Synechococcus strains under SF conditions. 12-h diurnal growth curve of Synechococcus 2973 WT, Synechococcus 7942 WT, Synechococcus 2973 ∆zwf and Synechococcus 2973 ∆pgl. Standard deviations are based on three biological replicates

Fig. 2

Synechococcus 2973 flux map determined in PBRs and SF and Synechococcus 7942 from the PBR. Syn. 2973 PBR values are listed first, followed by SF in italicized values and the third values are Syn. 7942 PBR. Net fluxes are normalized to net uptake of 100 mol CO₂. Mean flux values and 95% confidence intervals are given in Additional file 1: Tables S3–S5. Estimated net CO₂ uptake rates (mmol/gDCW/h) were 12.2 (PBR), 6.7 (SF), and 5.1 (PBR- 7942). Dotted lines represented pathways with enzymes that are not annotated for Synechococcus elongatus on the KEGG Genome database. ˫ indicates the inhibition of pyruvate kinase by ATP

Fig. 3

Relative pool sizes of Synechococcus 2973, Synechococcus 7942, and Synechocystis 6803 to E.coli K-12 under continuous light PBR conditions. A ratio of 1 indicates the same metabolite concentrations (normalized to gram biomass) between cyanobacteria and Escherichia coli. A ratio greater than 1 indicates a larger pool size in cyanobacteria strain than Escherichia coli. Standard deviations are based on three cyanobacteria biological replicates. a Average relative pool size of energy molecules compared to E. coli in PBR conditions. b Average relative pool size of UDP glucose to E. coli in PBR conditions. ADP glucose ratio is relative to Synechococcus 6803 (normalized to 10) due to the lack of ADP glucose in the E. coli control. c Relative metabolite pool size of Synechococcus 2973, Synechococcus 7942 and Synechocystis 6803 in PBR conditions to E. coli K-12. d Relative metabolite pool size normalized to free glutamate between the shaking flask and PBR conditions for Synechococcus 2973

Fig. 4

Shaking flask biomass growth of Synechococcus 2973 in the presence of carbon sources. a Growth rate (h⁻¹) of Synechococcus 2973 in two conditions when fed with 4 g L⁻¹ sodium bicarbonate and 6 mM of malate (MAL), acetate (AC), citrate (CIT), OAA, alpha-ketoglutarate (aKG), and a mix. Standard deviation is calculated from biological duplicates. There was no significant difference between growth rates. b Percent ¹²C-enrichment in amino acids through provision of 4 g L⁻¹ of ¹³C-bicarbonate and 6 mM of unlabeled organic acids in SF cultures (labeling for 48 h) compared against the control of just 4 g L⁻¹ of ¹³C-bicarbonate. The amino acids were chosen because of their direct relation to key metabolic precursors (Ala->PYR, Ser->3PGA, Phe->PEP/E4P, Glu->α-KG, Asp->OAA). [M-57] represents the mass signals of unlabeled amino acid (without fragmentation by GC–MS). Note: Synechococcus 2973 used unlabeled organic carbon sources to synthesize proteinogenic amino acids, the [M-57] % of amino acids increased compared to the control sample that was only fed with ¹³C-bicarbonate. Standard deviations are a result of biological duplicates *represents a p value of < 0.02 using two-tailed equal variance Student’s t Test

Fig. 5

Labeling dynamics for Synechococcus 2973 and 7942 in photobioreactor conditions for Citrate, 3PGA, and Malate. a The mass isotopomer distribution for citrate (M+0) and (M+4) fractions as a function of time. Experimentally measured MIDs are the data points, while the line represents the fitted data from INCA. b The mass isotopomer distribution for malate (M+0) and (M+4) fractions as a function of time. Experimentally measured MIDs are the data points, while the line represents the fitted data from INCA. c The total ¹³C-enrichment of 3PGA compared to citrate as a function of time from experimentally measured MIDs. d Substrate channel scheme proposed based on labeling data from Synechococcus 2973. Experimentally measured MIDs with error bars represent standard deviations from biological duplicates