| Summary: | Abstract Single-cell protein (SCP) production in the methylotrophic yeast Pichia pastoris has the potential to achieve a sustainable protein supply. However, improving the methanol fermentation efficiency and reducing carbon loss has been a long-standing challenge with far-reaching scientific and practical implications. Here, comparative transcriptomics revealed that PAS_0305, a gene directly associated with cell wall thickness under methanol stress, can be used as a target for unlocking cell wall sensors. Intracellular trehalose accumulation confirmed that cell wall sensors were activated after knocking out PAS_0305, which resulted in increased cell wall permeability. Genome-wide signal perturbations were transduced through the HOG module and the CWI pathway, which was confirmed to connected by Pbs2-Mkk. As a consequence of CWI pathway activation, ΔPAS_0305 elicited a rescue response of cell wall remodeling by increasing the β-1,3-glucan content and decreasing the chitin/mannose content. Remarkably, perturbations in global stress signals led to a fine-tuning of the metabolic network of ΔPAS_0305, resulting in a superior phenotype with highest crude protein and methanol conversion rate of 67.21% and 0.46 gDCW/g. Further genome-scale metabolic models were constructed to validate the experimental results, confirming that unlocking cell wall sensors resulted in maximized flux from methanol towards SCP and effectively addressing the issue of carbon loss in methanol fermentation. This work sheds new light on the potential of manipulating cellular signaling pathways to optimize metabolic networks and achieve exceptional phenotypic characteristics, providing new strategies for constructing versatile cell factories in P. pastoris.
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