Methanol is a promising renewable C1 feedstock for sustainable single‐cell protein (SCP) production. However, its inherent cytotoxicity and metabolic trade‐offs between cell growth and protein synthesis remain significant bottlenecks. Here, we established an “evolutionary‐rational” dual‐driven paradigm to construct a high‐yield Pichia pastoris chassis. Through UV mutagenesis and adaptive laboratory evolution, we developed a highly tolerant strain A40, capable of growing in 70 g/L methanol. Notably, at 30 g/L methanol, A40 achieved a 3.4‐fold higher maximum biomass than the wild‐type. Whole‐genome resequencing and reverse genetics revealed that this superior performance stems from a multi‐gene synergistic network rather than a single dominant mutation. To further optimize SCP production, we rationally co‐overexpressed nitrogen assimilation genes (GLN1, GDH1) and a translation elongation factor (PpeEF3) in the A40 background. This targeted metabolic engineering effectively redirected carbon flux toward protein biosynthesis. The engineered strain A40‐2Ge3 achieved a peak intracellular crude protein content of 67.9% and a 51.3 g/L total titer in a 5‐L bioreactor, representing a 23.1% increase over the wild‐type strain. Collectively, this study provides deep insights into the synergistic mechanisms of methanol adaptation and establishes an efficient, scalable strategy for sustainable SCP production from C1 feedstocks.
Chong Xie, Cheng-Chao Zhu, Jun-Ze Liu et al.· Biotechnology Journal· 0 citations
Developing sustainable single-cell protein (SCP) from non-food feedstocks offers a promising strategy to address the escalating global demand for sustainable nutrition. However, the lack of industrially robust platforms capable of cost-efficient multi-substrate assimilation remains a key bottleneck. Here, we discover a Cyberlindnera jadinii strain, CGMCC34730, which can efficiently utilize diverse non-food carbon sources (e.g., acetate, ethanol, and xylose) for SCP production. Metabolic analysis reveals the assimilation mechanisms for these substrates, notably identifying the reductive glycine pathway as central to formic acid (FA) utilization. Scaled-up production in a 5-L bioreactor demonstrates excellent performance, with ethanol- and acetate-driven cultures reaching protein contents of 68.60 and 67.34% and volumetric productivities of 8.91 and 7.90 t m–3 y–1, respectively. The resulting SCP surpasses soybean meal and approaches the quality of fish meal, exhibiting superior essential amino acid indices alongside elevated carbohydrate and B-vitamin contents. Furthermore, we validate a circular bioeconomy model by converting electrochemically synthesized 13C-labeled acetate into SCP. Techno-economic analysis confirms that acetate-based fermentation offers optimal cost-effectiveness for industrial deployment. In summary, this GRAS-certified platform establishes a highly efficient and economically viable route for the sustainable production of high-value SCP from non-food substrates.
This study systematically elucidates the synergistic lignin conversion mechanism of CDGs by a fungal consortium from structural, enzymatic, and metabolic perspectives, uncovering a temporal division of labor together with new lignin catabolic pathways that provide a mechanistic framework for the biological valorization of lignocellulosic biomass.
