Investigation of carbon and energy metabolic mechanism of mixotrophy in Chromochloris zofingiensis

Abstract

Background: Mixotrophy can confer a higher growth rate than the sum of photoautotrophy and heterotrophy in many microalgal species. Thus, it has been applied to biodiesel production and wastewater utilization. However, its carbon and energy metabolic mechanism is currently poorly understood.

Results: To elucidate underlying carbon and energy metabolic mechanism of mixotrophy, Chromochloris zofingiensis was employed in the present study. Photosynthesis and glucose metabolism were found to operate in a dynamic balance during mixotrophic cultivation, the enhancement of one led to the lowering of the other. Furthermore, compared with photoautotrophy, non-photochemical quenching and photorespiration, considered by many as energy dissipation processes, were significantly reduced under mixotrophy. Comparative transcriptome analysis suggested that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO₂ to provide carbon sources for chloroplast organic carbon metabolism under mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped, allowing for more efficient utilization of photoreaction-derived energy. Besides, compared with heterotrophy, photoreaction-derived ATP reduced the need for TCA-derived ATP, so the glucose decomposition was reduced, which led to higher biomass yield on glucose. Based on these results, a mixotrophic metabolic mechanism was identified.

Conclusions: Our results demonstrate that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO₂ to provide carbon for photosynthesis in mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped in mixotrophy, which could reduce energy waste of photosynthesis while promote cell growth. This finding provides a foundation for future studies on mixotrophic biomass production and photosynthetic metabolism.

Keywords: Chromochloris zofingiensis; Mixotrophy; Non-photochemical quenching; Photorespiration; Photosynthesis; RuBisCO.

Fig. 1

Glucose consumption, dry weight, specific growth rate (within 0–30 h), RuBisCO activity, biomass yield on glucose and energy fixation under different culture conditions. Experiments were conducted with three biological replicates. The data points were represented as values ± SD. The statistical significance of the results was tested by t-test (p < 0.05). *M significantly different with P; ^#M + CO₂ significantly different with P + CO₂; ^$M significantly different with H; ^{^}M + CO₂ and M + DCMU significantly different with M

Fig. 2

C13-labeled ratio of eight intermediates under different culture conditions. H + C13Glu: heterotrophy with C13 labeled glucose; M + C13Glu: mixotrophy with C13 labeled glucose; M + C13Glu + CO₂: mixotrophy with C13-labeled glucose and CO₂ aeration; M + C13NaHCO₃ + Glu: mixotrophy with C13 labeled NaHCO₃; P + C13NaHCO₃: photoautotrophy with C13-labeled NaHCO₃; G6P: glucose-6-phosphate; F6P: fructose-6-phosphate; FBP: fructose-1,6-biphosphate; DHAP: dihydroxyacetone phosphate; E4P: erythrose-4-phosphate; S7P: sedoheptulose-7-phosphate; Ru5P: ribulose-5-phosphate; R5P: ribose-5-phosphate. Experiments were conducted with three biological replicates. The data points were represented as values ± SD. The statistical significance of the results was tested by t-test (P < 0.05). *M + C13Glu + CO₂ significantly different with M + C13Glu; ^#P + C13NaHCO₃ significantly differently different with M + C13NaHCO₃ + Glu

Fig. 3

Chlorophyll fluorescence parameters under different culture conditions. Y(II) was represented as the actual quantum yield, Y(NPQ) and Y(NO) were represented as the percentage of the energy consumed by NPQ and chlorophyll fluorescence in the absorbed light energy, respectively, and Y(II) + Y(NPQ) + Y(NO) = 1. Experiments were conducted with three biological replicates. The data points were represented as values ± SD. The statistical significance of the results was tested by t-test (P < 0.05). ^$M significantly different with P; ^{^}M + CO₂ and M + DCMU significantly differently with M

Fig. 4

Compared with photoautotrophy, the expression changes of photorespiration biosynthesis pathway in mixotrophy. Numbers in the blue squares were expression changes that expressed in log₂FC^a. For genes with multiple isoforms, the isoform with the highest FPKM was selected to represent each gene. GK glycerate kinase, PGP phosphoglycolate phosphatase, GO glycolate oxidase, GGAT glutamate-glyoxylate aminotransferase, GlyA glycine hydroxymethyltransferase, AGXT serine-pyruvate transaminase, HPR hydroxypyruvate reductase, G3P glyceraldehyde 3-phosphate, RuBP ribulose-1,5bisphosphate

Fig. 5

The expression changes of central carbon and energy metabolic pathways. Red square: up-regulated; blue square: down-regulated. The numbers in the colored square mean log₂FC^a. For genes with multiple isoforms, the isoform with the highest expression level was selected to represent each gene. Glycolytic pathway: HXT hexose transporter, HK hexokinase, GPI glucosephosphate isomerase, PFK phosphofructokinase, FBA fructose-1,6-bisphosphate aldolase, GAPDH glyceraldehyde phosphate dehydrogenase, PGK phosphoglycerate kinase, PGAM phosphoglyceromutase, ENO enolase, PK pyruvate kinase. Genes in mitochondria: ATPS5 mitochondrial ATP synthase subunit 5, ATPSβ mitochondrial ATP synthase subunit β, PDH pyruvate dehydrogenase, CS citrate synthase, ACO aconitase, IDH isocitrate dehydrogenase, α-KDGH a-ketoglutarate dehydrogenase, SCS succinyl-CoA synthetase, SDH succinate dehydrogenase, FUM fumarase, MDH malate dehydrogenase. Genes in chloroplast: CATPG chloroplast ATP synthase gamma chain, CATPH chloroplast ATP synthase delta chain, FNR ferredoxin-NADP reductase, CPGK chloroplast phosphoglycerate kinase, CGAPDH chloroplast glyceraldehyde phosphate dehydrogenase, TPI triose phosphate isomerase, CFBA chloroplast fructose-1,6-bisphosphate aldolase, FBP fructose-1,6-bisphosphatase, CTKT chloroplast transketolase, SEBP sedoheptulose-1,7-bisphosphatase, CRPI chloroplast ribose 5-phosphate isomerase, PRK phosphoribulokinase. Carbon and energy transporters on chloroplast and mitochondria: CAAC chloroplast ATP/ADP carrier, TPT triose phosphate/phosphate translocator, GPT glucose 6-phosphate/phosphate translocator, NTT nucleotide translocator. Metabolites: G6P glucose-6-phosphate, F6P fructose-6-phosphate, G3P glycerate-3-phosphate, E4P erythrose-4-phosphate, S7P sedoheptulose-7-phosphate, Xu5P xylulose-5-phosphate, Ru5P ribulose-5-phosphate, R5P ribose-5-phosphate, 3PG 3-phosphoglycerate, BPG d-glycerate 1,3-diphosphate, SBP sedoheptulose 1,7-bisphosphate, RuBP ribulose-1,5-bisphosphate

Fig. 6

The differentially expressed genes detected by RT-qPCR. Experiments were conducted with three biological replicates. The data points were represented as values ± SD, *significantly different with P; ^#significantly different with M. CAAC chloroplast ATP/ADP carrier, TPT triose phosphate/phosphate translocator, GPT glucose 6-phosphate/phosphate translocator, NTT nucleotide translocator, activase RuBisCO activase, CPGK chloroplast phosphoglycerate kinase, PRK phosphoribulokinase, HXT hexose transporter, HK hexokinase, GAPDH glyceraldehyde phosphate dehydrogenase, PK pyruvate kinase, PDH pyruvate dehydrogenase, CS citrate synthase, MDH malate dehydrogenase, ATPSβ mitochondrial ATP synthase subunit β

Fig. 7

Proposed mixotrophic metabolic mechanism in C. zofingiensis (red arrow: up-regulated bioprocess; blue arrow: down-regulated bioprocess; black arrow: no significantly different; purple dashed arrow: feedback regulation)