Poly-2-hdyrobutanedioic acid (P2HBD), produced by the yeast-like fungus Aureobasidium pullulans, is a brand new kind of water-soluble polyhydroxy acid with potential functions within the biomaterial and biomedical fields. Typically, cardio P2HBD fermentation with glucose as a carbon supply causes carbon loss (releasing CO2) by way of the decarboxylation of pyruvate to kind acetyl-CoA. In contrast with sugars, the nonfermentable substrate ethanol displays the next diploma of discount per carbon atom and a shorter path to generate acetyl-CoA. On this research, the carbon-economic biosynthesis of P2HBD pushed by ethanol as the only carbon supply was investigated. Ethanol was first discovered to effectively convert into P2HBD by way of a biosynthetic mechanism, and specifically actuate the transcription issue Cat8 to manage the glyoxylic acid shunt in A. pullulans. Primarily based on transcriptomic evaluation beneath ethanol stress, a modular meeting technique was designed to steadiness three modules of ethanol oxidation, glyoxylic acid shunt, and the gluconeogenesis pathway, adopted by exact regulation with promoter engineering. The congruent pressure confirmed comparable yields with ethanol as the only substrate. Furthermore, an adaptive evolution technique was carried out to boost ethanol tolerance. In consequence, we obtained the mutant pressure EGG 47, and resting cell fermentation achieved a comparable P2HBD titer and yield of 66.7± 0.77 g/L and 0.87 g/g ethanol in a 5-L fermenter, respectively. Our findings present new insights into carbon-economic transformation from ethanol substrates to biopolymers and chemical substances in third-generation biorefineries.