Knocking Out One Glycolysis Gene Rewires a Marine Microbe to Make More DHA

Scientists in China have shown that deleting a single glycolytic enzyme in an industrially important marine microorganism can dramatically change where its carbon goes, trading overall growth for a striking boost in the production of docosahexaenoic acid, the omega-3 fatty acid better known as DHA. The study, published in the journal Blue Biotechnology, focused on Schizochytrium limacinum SR21, an oleaginous thraustochytrid fungus that can accumulate 50 to 70 percent of its dry weight as oil and has become the dominant industrial source of DHA for nutritional supplements and fortified foods. By disrupting the gene encoding fructose-1,6-bisphosphate aldolase, the research team demonstrated that the enzyme acts as a control point for carbon flux distribution, and that redirecting that flux can shift the fatty acid profile of the cells toward more valuable polyunsaturated products.

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