Design a reducing CO2 emission system using nonfermentable substrates for carbon-economic biosynthesis of poly-2-hydrobutanedioic acid

Abstract

Reducing carbon dioxide (CO2) emissions in the aerobic biorefinery system is becoming a crucial effort for achieving carbon-economic biosynthesis of biomaterials and chemicals. Poly-2-hydrobutanedioic acid (P2HBD), a long-carbon chain polyester produced by the fungus Aureobasidium pullulans, has garnered significant attention in the biomaterials and chemical industries. In this study, we designed a CO2 emission reduction system using nonfermentable substrates to enable efficient biosynthesis of P2HBD. Based on the genome-scale model iZX637 of A. pullulans, glycerol and ethanol were stimulated to show the potential advantages over glucose in terms of reduced CO2 emission. Additionally, we employed an NADH/NAD+ fluorescent probe called SoNar to dynamically monitor the reductive power ratio during CO2 emission. Subsequently, glycerol metabolism and rTCA carbon fixation pathway were engineered, while implementing a modular assembly strategy to achieve balanced integration between these two modules through precise promoter engineering regulation. The optimal strain ZX-LM03 showed the advantages of less carbon emission with glycerol and ethanol as the co-substrates, and achieved the higher P2HBD titer and yield of 15.07 ± 0.18 g/L and 0.54 ± 0.01 g/g, with the lower carbon emission of 31.08 % compared to glucose in the 5 L fermenter. In conclusion, this study provides novel insights into achieving carbon neutrality using renewable substrates for reducing carbon emission in aerobic fermentation systems.