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<p>Orotic acid is an essential intermediate in the pyrimidine nucleotide biosynthesis pathway and has broad applications in the food, pharmaceutical, and cosmetic industries. In recent years, microbial production of orotic acid has attracted considerable interest. However, its production efficiency remains insufficient for large-scale industrial implementation. Here, rational metabolic engineering strategies were applied to develop an efficient and high-yielding <i>Escherichia coli</i> cell factory for orotic acid production. The entire metabolic pathway is divided into the downstream degradation module, the midstream orotic acid synthesis module, and the upstream precursors supply module. First, the <i>pyrE</i> gene was deleted to block the downstream consumption of orotic acid. Second, the midstream pathway was enhanced by reinforcing the expression of key genes. Third, the supply of the carbamoyl-phosphate and L-aspartic acid was increased to boost the upstream pathway. Finally, an antibiotic-free <i>E. coli</i> cell factory for orotic acid production was established by implementing a novel plasmid stabilization system. The final strain O20 achieved an orotic acid titer of 150.48&#xa0;g/L with a yield of 0.63&#xa0;g/g glucose in fed-batch fermentation. This study developed a competitive microbial platform for orotic acid production and offers a modular engineering framework applicable to the biosynthesis of other pyrimidine nucleotide derivatives.</p>

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Construction of an antibiotic-free orotic acid producer in Escherichia coli via modular molecular engineering combined with a plasmid stabilization system

  • 期刊论文

  • 发表日期：

    2026年3月19日

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  • Zhenhui Pan,
  • Mi Tang,
  • Keren Sun,
  • Yixuan Zhao,
  • Qisheng Sun,
  • Peng Wen,
  • Landuo Sui,
  • Taowei Yang,
  • Xuewei Pan,
  • Zhiming Rao

摘要

Orotic acid is an essential intermediate in the pyrimidine nucleotide biosynthesis pathway and has broad applications in the food, pharmaceutical, and cosmetic industries. In recent years, microbial production of orotic acid has attracted considerable interest. However, its production efficiency remains insufficient for large-scale industrial implementation. Here, rational metabolic engineering strategies were applied to develop an efficient and high-yielding Escherichia coli cell factory for orotic acid production. The entire metabolic pathway is divided into the downstream degradation module, the midstream orotic acid synthesis module, and the upstream precursors supply module. First, the pyrE gene was deleted to block the downstream consumption of orotic acid. Second, the midstream pathway was enhanced by reinforcing the expression of key genes. Third, the supply of the carbamoyl-phosphate and L-aspartic acid was increased to boost the upstream pathway. Finally, an antibiotic-free E. coli cell factory for orotic acid production was established by implementing a novel plasmid stabilization system. The final strain O20 achieved an orotic acid titer of 150.48 g/L with a yield of 0.63 g/g glucose in fed-batch fermentation. This study developed a competitive microbial platform for orotic acid production and offers a modular engineering framework applicable to the biosynthesis of other pyrimidine nucleotide derivatives.