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<p>D-allulose is a rare sugar with multiple physiological functions and industrial applications. Efficient microbial production of D-allulose requires appropriate carbon allocation between cell growth and product formation. In this study, an engineered <i>Escherichia coli</i> BL21(DE3) strain was constructed for D-allulose biosynthesis using a glucose–glycerol co-utilization strategy. First, we established a phosphorylation-driven D-allulose pathway, achieving a titer of 1.43&#xa0;g/L of D-allulose. However, knocking out the <i>pfkA</i> gene impaired cell growth, and the biomass OD<sub>600</sub> value was only 1.54. To restore biomass accumulation, the glycerol kinase mutant GlpK<sup>G913S</sup> and the transcriptional regulator Mlc were overexpressed to enhance glycerol utilization efficiency, resulting in a 51.9% increase in biomass, but the glycerol utilization rate was only 45.0%. To further utilize the remaining glycerol, an aldol reaction pathway was constructed by expressing the genes <i>aldO</i> (alditol oxidase), <i>rhaD</i> (L-rhamnulose-1-phosphate aldolase), and <i>yqaB</i> (fructose-1-phosphate), which increased the glycerol utilization rate to 62.5% and the D-allulose yield to 3.46&#xa0;g/L. Under optimized carbon source ratio conditions, the engineered strain produced 3.92&#xa0;g/L of D-allulose with a productivity of 0.054&#xa0;g/L/h, and the cell biomass was successfully increased to an OD<sub>600</sub> of 10.4. These results demonstrate that using glycerol as a co-substrate can promote cell growth and enhance D-allulose synthesis in engineered <i>E. coli</i>, providing a basis for further metabolic optimization of rare sugar production.</p>

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Engineering of glycerol-derived aldolase pathway improves D-allulose synthesis in a glucose–glycerol dual-carbon system

  • 期刊论文

  • 发表日期：

    2026年6月16日

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  • Yifan Tu,
  • Jing Lv,
  • Mulan Wang,
  • Tanxiang Huang,
  • Lei Hu,
  • Chen Liang,
  • Xiyao Cheng,
  • Jidong Liu

摘要

D-allulose is a rare sugar with multiple physiological functions and industrial applications. Efficient microbial production of D-allulose requires appropriate carbon allocation between cell growth and product formation. In this study, an engineered Escherichia coli BL21(DE3) strain was constructed for D-allulose biosynthesis using a glucose–glycerol co-utilization strategy. First, we established a phosphorylation-driven D-allulose pathway, achieving a titer of 1.43 g/L of D-allulose. However, knocking out the pfkA gene impaired cell growth, and the biomass OD600 value was only 1.54. To restore biomass accumulation, the glycerol kinase mutant GlpKG913S and the transcriptional regulator Mlc were overexpressed to enhance glycerol utilization efficiency, resulting in a 51.9% increase in biomass, but the glycerol utilization rate was only 45.0%. To further utilize the remaining glycerol, an aldol reaction pathway was constructed by expressing the genes aldO (alditol oxidase), rhaD (L-rhamnulose-1-phosphate aldolase), and yqaB (fructose-1-phosphate), which increased the glycerol utilization rate to 62.5% and the D-allulose yield to 3.46 g/L. Under optimized carbon source ratio conditions, the engineered strain produced 3.92 g/L of D-allulose with a productivity of 0.054 g/L/h, and the cell biomass was successfully increased to an OD600 of 10.4. These results demonstrate that using glycerol as a co-substrate can promote cell growth and enhance D-allulose synthesis in engineered E. coli, providing a basis for further metabolic optimization of rare sugar production.