Carbon-economic biosynthesis of poly-2-hydrobutanedioic acid driven by nonfermentable substrate ethanol
2022
Wang, Pan | Li, Bingqin | Li, Baiyue | Yang, Jing | Xu, Xingran | Yang, Shang-Tian | Zou, Xiang
Poly-2-hydrobutanedioic acid (P2HBD), produced by the yeast-like fungus Aureobasidium pullulans, is a new type of water-soluble polyhydroxy acid with potential applications in the biomaterial and biomedical fields. Generally, aerobic P2HBD fermentation with glucose as a carbon source causes carbon loss (releasing CO₂) via the decarboxylation of pyruvate to form acetyl-CoA. Compared with sugars, the nonfermentable substrate ethanol exhibits a higher degree of reduction per carbon atom and a shorter route to generate acetyl-CoA. In this study, the carbon-economic biosynthesis of P2HBD driven by ethanol as the sole carbon source was investigated. Ethanol was first found to be efficiently converted into P2HBD via a biosynthetic mechanism, and specially activated the transcription factor Cat8 to regulate the glyoxylic acid shunt in A. pullulans. Based on transcriptomic analysis under ethanol stress, a modular assembly strategy was designed to balance three modules of ethanol oxidation, glyoxylic acid shunt, and the gluconeogenesis pathway, followed by precise regulation with promoter engineering. The congruent strain showed comparable yields with ethanol as the sole substrate. Moreover, an adaptive evolution strategy was performed to enhance ethanol tolerance. As a result, we obtained the mutant strain EGG 47, and resting cell fermentation achieved a comparable P2HBD titer and yield of 66.7 ± 0.77 g L⁻¹ and 0.87 g g⁻¹ ethanol in a 5 L fermenter, respectively. Our findings provide new insights into carbon-economic transformation from ethanol substrates into biopolymers and chemicals in third-generation biorefineries.
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