Integrated 13C Metabolic Flux and Multi-Omics Analyses Reveal Central Carbon and Redox Constraints in a Salt-Sensitive Chromochloris zofingiensis Mutant

Saline cultivation can reduce freshwater demand and reshape the production physiology of high-value microalgae, yet the metabolic basis of strain-specific salinity tolerance remains unclear. Here, the astaxanthin-producing green microalga Chromochloris zofingiensis was examined by comparing the wild type (Cz-WT) with a cGMP-dependent protein kinase (PKG)-deficient mutant (Cz-pkg) under 100 mM NaCl. Physiological measurements were integrated with parallel 13C-glucose tracing, 13C metabolic flux analysis, LC–MS metabolomics, and time-series RNA sequencing. Relative to Cz-pkg, Cz-WT showed higher normalized fluxes from phosphoenolpyruvate to pyruvate and oxaloacetate, greater pyruvate-to-acetyl-CoA conversion, and enhanced tricarboxylic acid (TCA)-cycle activity, suggesting a greater potential to support energy and reducing-equivalent metabolism. Cz-pkg exhibited lower glucose-6-phosphate and 6-phosphogluconate levels, early NADH depletion, and reduced pools of several amino-acid- and nucleotide-related metabolites. At 24 h, 141 genes were co-upregulated and 228 co-downregulated in both strains, involving photosynthetic electron transport, antioxidant metabolism, glycolysis/gluconeogenesis, amino-acid metabolism, and terpenoid biosynthesis. Phosphatidylglycerophosphate synthase was upregulated in both strains, supporting membrane-lipid remodeling. Overall, the data provide flux-level evidence that the salt-sensitive mutant is constrained in glycolysis–TCA cycle coupling and energy–redox homeostasis during salinity acclimation. Direct PKG substrates and causal phosphorylation events were not examined in this study.