Single-Cell Protein Production from Corn Straw by Kluyveromyces marxianus Evolved through Integrated Adaptive Evolution and Physical Mutagenesis

The development of sustainable bioprocesses for utilizing lignocellulosic biomass to produce single-cell protein (SCP) remains a significant challenge in industrial biotechnology. Here, we developed a novel integrated strategy combining adaptive laboratory evolution (ALE) with atmospheric room-temperature plasma (ARTP) mutagenesis to simultaneously enhance multiple performance traits of Kluyveromyces marxianus, including substrate utilization, tolerance to toxic compounds, biomass accumulation, and SCP content. A superior mutant strain, J368, was obtained through directed evolution and high-throughput screening. Strain J368 exhibited markedly enhanced xylose consumption, a shortened lag phase during glucose-xylose cofermentation, and robust growth in undetoxified hydrolysate, reducing chemical detoxification demand by 80% and wastewater discharge by 60%. Genomic and transcriptomic analyses revealed mutations and regulatory rewiring in pathways related to ribosomal activity and stress resistance, providing mechanistic insights into the enhanced phenotypes. Strain J368 produced SCP with a crude protein content of 57.3 g per 100 g with a protein yield of 0.14 g·g –1, exhibiting significantly improved nutritional quality. The essential amino acid (EAA) content reached 19.43 g per 100 g, with an essential amino acid index (EAAI) of 0.91, surpassing both soybean meal and the parental strain GQ7. Notably, lysine and threonine contents were significantly increased, underscoring the potential of J368 as a high-quality microbial protein source. This study establishes a general and environmentally friendly strategy for enhancing SCP production from lignocellulosic feedstocks and elucidates the intrinsic regulatory mechanisms underlying enhanced SCP production through multiomics analyses.