Deciphering the ammonia transformation mechanism of a novel marine multi-stress-tolerant yeast, <i>Pichia kudriavzevii</i> HJ2, as revealed by integrated omics analysis

Ammonia nitrogen posed a significant threat to aquatic animals in aquaculture environments, and the substantial potential of microorganisms in removing ammonia nitrogen had garnered considerable attention. This study identified a marine yeast, <i>Pichia kudriavzevii</i> HJ2, which effectively removed ammonia nitrogen. By combining transcriptomics and metabolomics, the ammonia nitrogen transformation mechanism of HJ2 was elucidated. HJ2 achieved 100% ammonia nitrogen removal efficiency within 1 day of fermentation at 35°C with 300 mg/L ammonia nitrogen and 73.56% removal efficiency within 36 h with 600 mg/L ammonia nitrogen. Transcriptomics revealed that exposure to 600 mg/L ammonia nitrogen resulted in 541 up-regulated genes and 567 down-regulated genes in the HJ2 strain. Differentially expressed genes (DEGs) were primarily involved in the tricarboxylic acid (TCA) cycle and amino acid metabolism. Metabolomics revealed that HJ2 facilitated the production of 383 up-regulated metabolites and suppressed 137 down-regulated metabolites when exposed to 600 mg/L ammonia nitrogen. Integrating transcriptomics and metabolomics analyses showed that HJ2 removed ammonia nitrogen by sensing its presence in the extracellular environment, activating the TCA cycle, enhancing amino acid metabolism and nucleotide metabolism, and promoting its robust growth and reproduction. Amino acid metabolism played an important role in the ammonia transformation mechanism of HJ2. The result was confirmed by the increased activity of glutamate dehydrogenase (GDH) and aspartate aminotransferase (GOT). Up-regulated nitrogen metabolites such as L-glutamate, L-aspartic acid, spermidine, and trigonelline were produced. The results of enzyme activity tests, construction of overexpressing strains, and adding exogenous amino acid experiments demonstrated that HJ2 could utilize GDH and GOT ammonia assimilation pathways.<b>IMPORTANCE</b>Ammonia nitrogen removal ability was a universal characteristic among the ammonia-oxidizing bacteria or archaea. Recently, yeast strains from the genus <i>Pichia</i> were found to have ammonia nitrogen removal ability. However, the mechanism of ammonia nitrogen removal in <i>Pichia</i> had not been reported. In the study, the ammonia nitrogen removal efficiency of <i>Pichia kudriavzevii</i> HJ2 was identified, and the mechanisms by which HJ2 transformed ammonia nitrogen into non-toxic organic nitrogen were elucidated, offering potential solutions to pollution challenges in aquaculture and helping minimize resource waste. The study offered new insights into the transformation mechanism of microbial ammonia nitrogen removal and its environmentally friendly application.