Cross Kingdom Metabolic Engineering Paradigm Elevating Sustainable Protein Production

ABSTRACT Confronting the dual crisis of escalating global protein demand and unsustainable agriculture necessitates transformative solutions. Here, we pioneer evolutionary insights from maize nitrogen optimization via asparagine synthetase (ASNS) to rewire metabolism in Pichia pastoris . Empirically, the tri‐copy ASNS strain achieved superior protein titers: 62.48% crude protein, 47.86% total amino acids, and 8.05% branched‐chain amino acids, nutritionally surpassing conventional protein sources. Genome‐scale modeling and transcriptomic studies provided convergent evidence that ASNS overexpression drove global metabolic rewiring through predicted synergistic coupling between aspartate metabolism and the tricarboxylic acid cycle. Mechanistically, ASNS overexpression unlocked a previously uncharacterized nitrogen sensor‐regulator circuit by inducing PAS_chr1‐1_0158 , validated in amplifying intracellular nitrogen flux and driving a self‐reinforcing cycle of ammonia assimilation. This work validated evolutionary conservation of nitrogen optimization strategies across kingdoms, established the first scalable blueprint for carbon–nitrogen co‐optimized microbial cell factories, decouples sustainable SCP production from agricultural constraints, and offers a scalable solution to the global protein crisis.