Enhanced Biomass and Protein Synthesis in Engineered Cyberlindnera jadinii Growing on Ethanol/Acetate: Metabolic Engineering and Transcriptomic Mechanism.

Producing single-cell protein (SCP) from syngas-derived ethanol and acetate offers a sustainable solution to global protein shortages, yet microbial utilization mechanisms for these mixtures remain underexplored. This study establishes a systematic bioconversion strategy using Cyberlindnera jadinii TU389. To mitigate acetaldehyde accumulation during ethanol metabolism, we engineered the strain TU546 to overexpress acylating acetaldehyde dehydrogenase (ADA6). TU546 achieved a maximum biomass of 46.7 g/L and a protein yield of 21.69 g/L, representing enhancements of 28.16% and 23.02% over the wild-type, respectively. Transcriptomic analysis revealed extensive metabolic reprogramming. In the C2 assimilation pathway, upregulated aldehyde dehydrogenase and acetyl-CoA Synthetase 1 accelerated acetate conversion to acetyl-CoA, while downregulated pyruvate decarboxylase and alcohol dehydrogenase minimized carbon flux loss. The upregulation of tricarboxylic acid cycle enzymes, the glyoxylate shunt, and acyl-coA oxidase improved carbon skeleton retention. Moreover, the upregulation of transaminases and N-acetylglutamate synthase, synergized with intensified cell proliferation signaling, redirected amino acid metabolism toward a synthesis-enhanced and degradation-controlled paradigm. This synergistic regulatory network drives the high-efficiency bioconversion of ethanol and acetate into SCP, establishing a molecular mechanistic foundation for the valorization of syngas-derived C2 substrates in biological macromolecule production.