Nitrogen Availability Influences Biomass Composition in Yarrowia lipolytica Grown on Acetate

Microbial protein production from acetate represents a promising route for sustainable protein supply, yet its efficiency is constrained by limited understanding of carbon–nitrogen metabolic coordination. In this study, nitrogen availability was systematically varied to investigate its role in regulating biomass composition and protein biosynthesis in Yarrowia lipolytica. Nitrogen limitation markedly reduced cell growth and protein accumulation (19.56% of dry cell weight) while increasing lipid content (up to 34.16%), indicating a altered protein and lipid accumulation under different nitrogen conditions. Transcriptomic analysis revealed a global downregulation of anabolic pathways under nitrogen limitation, accompanied by a shift in nitrogen assimilation from the glutamate dehydrogenase (GDH) pathway to the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway, as well as significant upregulation of genes related to ammonium and amino acid transport. Guided by these findings, metabolic engineering of key nitrogen assimilation pathways was performed. Strains harboring additional copies of GDH and GS expression cassettes showed increased protein content from 48.52% to 55.77% and improved amino acid composition, whereas strains with an additional copy of the GOGAT gene exhibited reduced growth and protein accumulation. These results demonstrate that nitrogen availability regulates biomass composition through coordinated control of nitrogen transport and assimilation, and that balanced upregulation of GDH and GS genes is an effective strategy to improve microbial protein production from acetate, supporting the development of efficient fermentation processes using low-cost carbon sources.