Aldolase catalyzes the fourth step of glycolysis, cleaving fructose-1,6-bisphosphate into two three-carbon sugars, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Because these triose phosphates feed both the lower half of glycolysis and the glycerol backbone used to build lipids, the enzyme sits at a critical junction of central carbon metabolism. Most of what is known about aldolase comes from medical research, particularly cancer biology, where studies have shown that blocking aldolase can push glucose-6-phosphate into the pentose phosphate pathway, increasing the production of NADPH, the reducing power that cells use for biosynthesis. The Xiamen University team, led by Yiting Zhang and Xueping Ling, asked whether the same logic could be applied to a microbe whose entire commercial value depends on how much reducing power and carbon skeleton it can pour into fatty acid synthesis.

To find out, the researchers constructed a knockout strain by replacing the aldolase gene, designated ALDOA, with a bleomycin resistance cassette through homologous recombination. They amplified roughly 420-base-pair sequences flanking the gene, ligated them into the pBlue-zeo plasmid around the resistance marker, and introduced the construct into wild-type cells by electroporation. Transformants were selected on plates containing 50 milligrams per liter of bleomycin, and PCR sequencing of the homologous arms confirmed that the native gene had been successfully replaced. The resulting strain, called ΔALDOA, was then compared with the wild type over a 168-hour shake-flask fermentation using glucose-based medium.

The immediate consequences of the deletion were negative for bulk productivity. Final biomass in the knockout strain was 9.6 percent lower than the wild-type maximum, and total lipid production fell by 23.2 percent. During active growth the deficits were even larger: at 96 hours the mutant’s cell dry weight was up to 40.61 percent below the wild type, and at 72 hours its lipid yield lagged by as much as 49.34 percent. Glucose consumption told a similar story. The wild-type strain essentially exhausted the sugar supply by 120 hours, whereas the knockout had used only about half of it at that point and needed until 168 hours to finish. Intriguingly, the mutant actually consumed glucose faster than the wild type in the earliest phase of cultivation, apparently ramping up overall metabolism to compensate for the metabolic bottleneck, before its impaired glycolysis caught up with it in the middle and later stages.

The fatty acid composition, however, moved in a very different direction. At 120 hours the proportion of saturated fatty acids in the knockout strain was about 17.4 percent lower than in the wild type, driven mainly by reductions in the C16:0 and C14:0 species. Polyunsaturated fatty acids, including DHA, docosapentaenoic acid, and eicosapentaenoic acid, rose by 28.0 percent overall, with DHA itself up 22.9 percent and EPA up a remarkable 75.0 percent. When the team broke the lipid pool down into triglycerides and phospholipids, they found that polyunsaturated fatty acids are mostly stored in triglycerides, at 35 to 48 percent of that fraction, while phospholipids carry 10 to 15 percent and contain no detectable EPA at all. The knockout boosted the polyunsaturated content of triglycerides specifically, with DHA rising 27.4 percent at 72 hours and 20.8 percent at 120 hours, while the main saturated species, C16:0, dropped by 46.5 percent and 36.0 percent at the same time points.

Phospholipidomics provided a mechanistic explanation rooted in how DHA travels through the cell. Phosphatidylcholine, the most abundant phospholipid in Schizochytrium at roughly half of the total, is known to be the first docking site for newly synthesized DHA before the fatty acid is transferred to triglycerides for storage. In the knockout strain, the proportion of phosphatidylcholine was 11.5 percent higher than the wild type at 72 hours and 7.5 percent higher at 120 hours, and lysophosphatidylcholine, its precursor in the glycerophosphorylcholine acylation pathway, reached 4.8 times the control level at 72 hours. Meanwhile, phosphatidylglycerol, a phospholipid tied to cell growth, collapsed by 92.2 percent at 72 hours, and phosphatidylinositol, phosphatidylserine, and phosphatidic acid all declined as well. Because these species are built through the Kennedy pathway from dihydroxyacetone phosphate-derived precursors, their depletion directly reflects the loss of aldolase activity upstream.

Gene expression analysis reinforced the picture. Transcripts of the fatty acid synthase gene, which produces saturated fatty acids, fell in the mutant, while the chain length factor gene of the anaerobic polyketide synthase cluster, responsible for elongating polyunsaturated chains, rose sharply in the middle and later stages of fermentation. The gene for glucose-6-phosphate dehydrogenase, the gateway enzyme of the pentose phosphate pathway, followed a similar pattern, indicating enhanced flux through that route and a greater supply of NADPH for fatty acid desaturation and elongation. Transcripts of phosphatidic acid phosphatase and diacylglycerol acyltransferase, the enzymes that convert phosphatidic acid into triglycerides via the Kennedy pathway, decreased, whereas the genes for phospholipid diacylglycerol choline phosphate transferase, or PDAT, and lysophosphatidylcholine acyltransferase, or LPCAT, were strongly upregulated. Together these shifts suggest the mutant builds its triglycerides increasingly through the phosphatidylcholine route, shuttling DHA from phospholipid storage into neutral lipid reserves.

Metabolomics by gas chromatography-mass spectrometry identified 35 differential metabolites that mapped the rewiring across glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and amino acid and sterol synthesis. Sugar alcohols associated with the oxidative pentose phosphate route surged, with arabitol up 4.06-fold and ribitol up 7.84-fold at 120 hours. The knockout strain also accumulated protective metabolites: inositol, a signaling molecule against oxidative stress, rose threefold, and mannitol, a hydroxyl radical scavenger, increased 1.23-fold, while glycine rose to supply precursors for glutathione synthesis. Flow cytometry using a fluorescent probe confirmed that reactive oxygen species were lower in the mutant at both measured time points. Since polyunsaturated fatty acids are vulnerable to free-radical oxidation, the reduced oxidative environment plausibly helps preserve the DHA the cells do make, complementing the increased reducing power available for synthesizing it in the first place.

The authors propose a coherent metabolic model: deleting ALDOA weakens glycolysis and the TCA cycle, slowing growth, and starves the Kennedy pathway of dihydroxyacetone phosphate, cutting total lipid and phospholipid output. In compensation, carbon is pushed into the pentose phosphate pathway, raising NADPH for polyunsaturated fatty acid synthesis, while the cell reinforces the glycerophosphorylcholine acylation route and PDAT-mediated transfer so that more DHA is bound to phosphatidylcholine and then moved into triglycerides. The trade-off is clear, with less biomass but a richer omega-3 profile per cell. The team notes that future work using CRISPR or RNA interference to tune, rather than abolish, aldolase expression could improve DHA accumulation without the growth penalty, offering a new metabolic engineering strategy for one of the world’s most important microbial oil producers.

Subject of Research: Aldolase gene disruption and carbon flux redistribution for DHA synthesis in Schizochytrium

Article Title: Functions of aldolase in lipid synthesis of Schizochytrium sp. by gene disruption to switch carbon metabolism

Article References: Zhang, Y., Wu, X., Guo, X., Li, K., Lu, Y., Lin, X., & Ling, X. (2024). Functions of aldolase in lipid synthesis of Schizochytrium sp. by gene disruption to switch carbon metabolism. Blue Biotechnology, 1(1), Article 17. https://doi.org/10.1186/s44315-024-00014-6

Image Credits: AI Generated

DOI: 10.1186/s44315-024-00014-6

Keywords: Schizochytrium, aldolase, DHA, polyunsaturated fatty acids, glycolysis, pentose phosphate pathway, carbon flux, metabolic engineering, phospholipids, triglycerides, NADPH, marine biotechnology

The research team, led by Danhong Ge, YangYang Luo, Ruyu Zhang, Xuechao Hu, and Lujing Ren, set out to answer a deceptively simple question: what happens when you expose a microbe that normally grows in the dark to sustained light stress? Schizochytrium sp. HX-308, the strain used in the study, was isolated from seawater and deposited in the China Center for Type Culture Collection. In industrial settings it is typically fermented in large, dark vessels where it converts glucose into a lipid-rich biomass. Light, in this context, is usually treated as an irrelevance or even a nuisance. The new study suggests that assumption deserves a second look, because light turns out to be a powerful abiotic stress factor that reprograms the cell’s entire metabolic priorities.

The researchers divided the fermentation process into three distinct phases: a cell growth stage from zero to forty-eight hours, a lipid accumulation stage from forty-eight to ninety-six hours, and a lipid turnover stage from ninety-six to one hundred forty-four hours, when the cells begin consuming their own stored fat reserves. Under normal dark conditions, the culture reached a maximum cell dry weight of 50.5 grams per liter at seventy-two hours. When the flasks were illuminated with an array of LED strips delivering a total luminous flux of 18,311.4 lumens, growth slowed noticeably. Glucose consumption was delayed, the fermentation cycle stretched from seventy-two to ninety-six hours, and the peak biomass fell to 41.3 grams per liter, a reduction of 18.21 percent compared with the dark-grown control.

Microscopy revealed that the light-exposed cells were visibly different from their dark-grown counterparts. They grew larger in diameter, became more dispersed and independent in their morphology, and accumulated more prominent lipid droplets as the fermentation progressed. Using propidium iodide staining under a confocal fluorescence microscope, the team showed that cell membrane damage was consistently higher under light stress, peaking at ninety-six hours, precisely when the lipid turnover phase begins. The researchers hypothesize that the enlarged cell diameter is an adaptive response, allowing the organism to meet the elevated energy and material demands imposed by the stressful environment. Similar light-induced changes in cell size have been documented in diatoms such as Ditylum brightwellii, suggesting this may be a widespread strategy among marine microbes.

The real surprise came when the team measured what the cells were actually producing. Although the light-stressed cultures grew more slowly, their final lipid yield reached 8.45 grams per liter, a striking 39.67 percent higher than the 6.05 grams per liter measured in the control after the turnover phase had run its course. The delayed glucose consumption appears to slow the onset of lipid turnover, effectively extending the window during which the cells hold onto their oil. In other words, the stress that cost the microbe some of its growth paid off handsomely in the currency that industry actually cares about: stored lipid that survives to the end of the fermentation.

The composition of that lipid changed too, and in commercially interesting directions. Saturated fatty acids, particularly myristic acid (C14:0) and palmitic acid (C16:0), were 1.49 times more abundant in the light-treated group, reaching 4.97 percent and 15.86 percent of the total fatty acid pool respectively. Saturated fats of this kind are the raw material for neutral lipid formation and have their own market in oleochemical applications. Meanwhile, the polyunsaturated fatty acids docosapentaenoic acid and DHA declined modestly, from 24.74 percent and 59.14 percent in the control to 20.59 percent and 56.28 percent under light. But eicosapentaenoic acid, or EPA, an omega-3 fatty acid with anti-inflammatory applications and a notoriously difficult production process, jumped from 2.14 percent to 8.30 percent of total fatty acids, a 3.88-fold increase that caught the researchers’ attention.

Beyond the fatty acids, light stress proved to be a potent trigger for two of the microbe’s most valuable secondary metabolites. Squalene, a terpenoid used in cosmetics, vaccines, and pharmaceutical formulations, peaked at 67.46 milligrams per liter at seventy-two hours under light stress, compared with 55.67 milligrams per liter at forty-eight hours in the dark, and the light-stressed cultures sustained higher squalene levels through the later stages of fermentation, ending 21.17 percent above the control. Beta-carotene followed a similar pattern, peaking at roughly 7,184 micrograms per liter at ninety-six hours under light versus 5,368 micrograms per liter at seventy-two hours in the dark. By the end of fermentation, the light-stressed cultures contained 5,675 micrograms per liter of beta-carotene, 4.75 times the control level. Astaxanthin, a premium carotenoid synthesized downstream of beta-carotene, rose continuously throughout the light-stressed fermentation, finishing nearly 2,081 micrograms per liter, 11.39 percent above the control, and its accumulation only accelerated after beta-carotene had peaked, consistent with beta-carotene serving as a metabolic precursor.

To understand why light produces these effects, the team measured intracellular reactive oxygen species and performed a full transcriptomic analysis using Illumina RNA sequencing. Light stress elevated oxidative pressure inside the cells, but the light-stressed cultures actually showed lower peak ROS levels than the controls, indicating that the organism mounts a robust antioxidant defense. The transcriptome told the molecular story. A core set of 226 differentially expressed genes was shared across all light-versus-dark comparisons, while the 144-hour comparison alone revealed 788 unique light-responsive genes. At seventy-two hours, light stress predominantly suppressed metabolic genes, an acute stress response; by 144 hours, the pattern flipped to widespread upregulation, signaling a transition to long-term metabolic reprogramming. Gene ontology analysis showed marked enrichment of genes involved in stimulus response, antioxidant activity, and transporter activity under light, while genes for lipid biosynthesis and antioxidant processes became dominant at the later time point.

The pathway-level details are particularly revealing. In the mevalonate pathway, which supplies the building blocks for isoprenoids such as squalene, the enzyme isopentenyl diphosphate isomerase was significantly upregulated under light, especially at 144 hours, along with farnesyl diphosphate synthase and squalene synthase, the enzymes that assemble farnesyl diphosphate into squalene itself. Fatty acid metabolism showed a dual pattern: acetyl-CoA carboxylase and fatty acid synthase were upregulated, driving carbon toward palmitic acid, while a key beta-oxidation gene was downregulated, meaning the cells burned less of the fat they stored. Intriguingly, glycolysis genes were suppressed, suggesting the microbe rerouted carbon flux away from rapid energy production and toward lipid and terpenoid biosynthesis, a strategy that mirrors engineered lipid-boosting interventions in other microalgae. The upregulated polyketide synthase genes hint at the channeling of resources toward EPA production, which supports membrane fluidity under stress.

For the biotechnology industry, the implications are considerable. Light is an inexpensive, non-invasive, and precisely controllable stimulus compared with chemical additives or genetic engineering, and this study demonstrates that it can simultaneously raise lipid yield, enrich EPA, and boost squalene and carotenoid production in a single fermentation run. The trade-off, a longer fermentation cycle and lower biomass, is a real cost, but the researchers argue that the metabolic insights gained here, particularly the coordinated upregulation of the mevalonate pathway and the suppression of fatty acid degradation, could guide further optimization, whether through tuned light regimens, light combined with other stressors, or targeted engineering of the genes identified in the transcriptome. As demand for omega-3 oils and natural antioxidants continues to climb, the idea that a well-placed bank of LED strips could coax more value out of every liter of fermentation broth is an appealingly simple proposition, and one that this marine microbe appears ready to deliver on.

Subject of Research: Effect of light stress on lipid, fatty acid, squalene, and carotenoid biosynthesis in the marine protist Schizochytrium sp.

Article Title: Effect of light stress on the production of lipid compounds by Schizochytrium sp. and its underlying mechanism

Article References: Ge, D., Luo, Y., Zhang, R., Hu, X., & Ren, L. (2025). Effect of light stress on the production of lipid compounds by Schizochytrium sp. and its underlying mechanism. Blue Biotechnology, 2(1), Article 8. https://doi.org/10.1186/s44315-025-00029-7

Image Credits: AI Generated

DOI: 10.1186/s44315-025-00029-7

Keywords: Schizochytrium, light stress, lipid biosynthesis, EPA, DHA, squalene, beta-carotene, astaxanthin, reactive oxygen species, transcriptomics, mevalonate pathway, marine biotechnology