Engineered Saccharomyces cerevisiae for the Enhancement of Glucosamine Accumulation by the Consumption of Glucose and Ammonium Based on Synthetic Biological Pathways

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, <i>Saccharomyces cerevisiae</i> genes <i>PFK1</i>, <i>PDB1</i>, <i>GNA1</i>, <i>ISR1</i>, and <i>PCM1</i> 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 <i>GlmD</i>, glucosamine-6-phosphate phosphatase <i>GlmP</i>, and ammonium transporter <i>AMT1</i>, were introduced to construct engineered strains for GlcN synthesis in the presence of high-concentration inorganic ammonium ions. The results indicated that <i>S. cerevisiae</i> HPG5 with <i>GlmD</i>, <i>GlmP</i>, and <i>AMT1</i> integration and simultaneous deletion of <i>PFK1</i>, <i>PDB1</i>, <i>GNA1</i>, <i>PCM1</i>, and <i>ISR1</i> achieved the highest GlcN yield (1.95 ± 0.02 g/L) during fermentation with 10 g/L (NH₄)₂SO₄, 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 (NH₄)₂SO₄. Thus, the results indicated that <i>S. cerevisiae</i> HPG5 could effectively produce GlcN in the presence of high-concentration ammonium sulphate. This study provides a promising alternative, <i>S. cerevisiae</i> HPG5, for GlcN production.