Abstract
Glucosamine (GlcN) is a high-value compound with significant health applications. GlcN is widely used in the food and health industry as a food additive or functional food. The development of a green, efficient, and safe method for GlcN production is of great significance due to the complexity of traditional production methods, environmental pollution, and sensitization of raw materials. In this study, Saccharomyces cerevisiae genes PFK1, PDB1, GNA1, ISR1, and PCM1 were knocked out using the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) method. In addition, three key enzyme genes, glucosamine-6-phosphate deaminase GlmD, glucosamine-6-phosphate phosphatase GlmP, and ammonium transporter AMT1, were introduced to construct engineered strains for GlcN synthesis in the presence of high-concentration inorganic ammonium ions. The results indicated that S. cerevisiae HPG5 with GlmD, GlmP, and AMT1 integration and simultaneous deletion of PFK1, PDB1, GNA1, PCM1, and ISR1 achieved the highest GlcN yield (1.95 ± 0.02 g/L) during fermentation with 10 g/L (NH(4))(2)SO(4), which was 2.47-fold higher than the control. The conversion rate of glucose to GlcN in HPG5 was 9.75% in liquid YPD medium containing 20 g/L of glucose and 10 g/L of (NH(4))(2)SO(4). Thus, the results indicated that S. cerevisiae HPG5 could effectively produce GlcN in the presence of high-concentration ammonium sulphate. This study provides a promising alternative, S. cerevisiae HPG5, for GlcN production.