Alanine aminotransferase-acetyl-CoA carboxylase switching produces starch-rich bioethanol feedstock in Desmodesmus.

Effective control of intracellular carbon allocation is essential for the production of targeted microalgal feedstocks. This study evaluated the sequential manipulation of alanine aminotransferase (ALT)-mediated alanine-pyruvate metabolism and acetyl CoA carboxylase (ACC)-dependent lipid synthesis in nitrogen-limited Desmodesmus sp. CNW-N. An integrated approach combined physiological analysis, transcriptomics, enzyme assays, inducible CrAAT1 overexpression, pharmacological interventions, [U 13C6]glucose labelling, and simultaneous saccharification and fermentation. Nitrogen depletion initially induced a starch-rich state, followed by lipid accumulation, accompanied by transient increases in alanine and aspartate levels and elevated ALT and ACC activities. Inhibition of aminotransferase activity increased alanine retention and decreased fatty acid accumulation, while clethodim treatment reduced ACC activity and lipid accumulation but enhanced carbohydrate retention. [U-13C6]glucose labelling demonstrated reciprocal 13C enrichment in carbohydrate- and lipid-associated pools, although direct tracing of alanine carbon was not observed. The combination of ALT overexpression and ACC inhibition produced 4.78 g/L biomass containing 75.32% glucose plus xylose (approximately 3.60 g/L fermentable sugar), a 37% increase over clethodim-treated wild type. Fermentation produced 378 g/kg dry biomass with 98-99% conversion efficiency. These findings link improved ethanol output to feedstock enrichment and indicate two metabolic control points for directing carbon toward fermentable carbohydrates in Desmodesmus sp. CNW-N.