The global demand for sustainable biofuels, oleochemicals, and nutraceuticals necessitates the development of efficient, non-food-competing bioproduction platforms. Yeast, particularly *Saccharomyces cerevisiae*, stands out as a robust and industrially scalable microorganism for these applications. However, achieving high-titer accumulation of neutral lipids, primarily triacylglycerols (TAGs), presents significant metabolic challenges. Native lipid accumulation in *S. cerevisiae* is often limited by metabolic flux bottlenecks and the inherent trade-off between rapid biomass growth and efficient lipid synthesis. Therefore, significant metabolic redirection and optimization of the carbon flux are required to prioritize fatty acid synthesis over competing pathways like cell wall component production or primary metabolism.
Lipid biosynthesis in yeast is fundamentally rooted in the Acetyl-CoA pool. The primary pathway involves the conversion of acetyl-CoA into malonyl-CoA, which serves as the essential building block for fatty acids. The process begins with Fatty Acid Synthesis (FAS), where two acetyl-CoA molecules condense to form acetoacetyl-CoA. Subsequent steps involve the action of malonyl-CoA:ACP transacylase and methylmalonyl-CoA reductase to extend the carbon chain, ultimately yielding a saturated fatty acyl-ACP, which is then released as a free fatty acid (FFA).
The accumulated FFAs are then activated into acyl-CoA derivatives. The final, critical step in lipid assembly is the esterification of these acyl-CoA units onto a glycerol backbone to form TAGs. This reaction is catalyzed by key enzymes, notably Diacylglycerol Acyltransferase (DGAT) and Phosphatidic Acid Phosphatase (PAP). DGAT activity is a major target for metabolic engineering because it directly dictates the rate-limiting step of TAG formation, making its enhancement crucial for maximizing lipid yield.
To enhance lipid titer, metabolic engineering efforts are strategically focused on three main areas: increasing carbon flux, enhancing key enzymatic activities, and managing redox balance. One key strategy is the overexpression of genes involved in the pyruvate dehydrogenase complex (PDC) or pyruvate carboxylase to boost the availability of acetyl-CoA, the primary precursor. Furthermore, enhancing the activity of acetyl-CoA carboxylase (ACC) boosts the conversion of acetyl-CoA to malonyl-CoA, thereby increasing the pool of building blocks for fatty acids. Crucially, constitutive overexpression of the rate-limiting enzyme, DGAT, significantly enhances the final assembly of TAGs, effectively diverting carbon away from competing storage pathways and towards the desired end product.
Beyond boosting synthesis, metabolic flux must be carefully managed by redirecting carbon away from competing pathways, such as those involved in cell wall synthesis or ethanol production. Genetic knockout or down-regulation of these competing pathways forces the excess carbon flux toward the lipid synthesis pathway. For successful industrial scale-up, operational control is paramount. The most effective approach involves a two-stage fermentation process: the first stage optimizes rapid biomass accumulation under nutrient-rich conditions, followed by a second stage involving nutrient limitation (e.g., nitrogen or phosphate starvation). This stress condition triggers the metabolic shift, forcing the yeast to accumulate energy reserves in the form of TAGs, thereby maximizing lipid yield per cell.
Economically viable bioproduction also requires utilizing low-cost, non-food feedstocks, such as lignocellulosic hydrolysates or industrial waste streams. Pre-treatment of these substrates is essential to ensure a consistent availability of key carbon sources (e.g., glucose, xylose), which maintains stable metabolic flux during large-scale operation. By integrating targeted metabolic pathway engineering with optimized industrial fermentation protocols, *S. cerevisiae* can be transformed into a highly efficient, robust, and sustainable platform for the bioproduction of high-titer lipids.