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<p>L-<i>threo</i>-3-hydroxyaspartic acid (L-THA) is a non-proteinogenic amino acid that has garnered significant attention due to its diverse biological activities. However, the synthesis of L-THA through enzymatic and whole-cell catalysis requires the expensive substrate L-aspartic acid or L-asparagine, and co-substrate α-ketoglutarate, which limits their large-scale application. Here, this is the first report of engineering <i>E. coli</i> as a cell factory for de novo production of L-THA from glucose by fermentation. Firstly, the <i>asnO</i> gene encoding asparagine hydroxylases from <i>Streptomyces coelicolor</i> was heterologously expressed in <i>E. coli</i> to yield the L-THA producing strain. The formation and configuration of L-THA were characterized by LC–MS and HPLC after FDAA derivatization. Secondly, the pathway genes <i>aspC</i> and <i>asnB</i>, which encode aspartate aminotransferase and asparagine synthase, respectively, were overexpressed to enhance L-THA titer from 49.9 to 90.84&#xa0;mg/L. Thirdly, the efforts were made to improve the key precursor L-aspartic acid pool by overexpressing the aspartase encoding gene <i>aspA</i> and knocking out aspartate kinase (AK) III encoding gene <i>lysC</i>. The best strain CC03 was obtained and L-THA titer reached 278.3&#xa0;mg/L in a shake flask, representing an approximately 5.6-fold increase compared to the original strain. Ultimately, 2.87&#xa0;g/L L-THA was obtained after 32&#xa0;h fed-batch fermentation. This research underscores the potential use of <i>E. coli</i>&#xa0;fermentation as a feasible platform for de novo biosynthesis of L-THA from glucose, which is amenable to industrial application.</p>

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Constructing Genetically Engineered Escherichia coli for De Novo Production of L-Threo-3-Hydroxyaspartic Acid

  • 期刊论文

  • 发表日期：

    2025年3月22日

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  • Jing Guo,
  • Jiayi Cui,
  • Mingyue Xun,
  • Wencheng Zhang,
  • Mo Xian,
  • Rubing Zhang

摘要

L-threo-3-hydroxyaspartic acid (L-THA) is a non-proteinogenic amino acid that has garnered significant attention due to its diverse biological activities. However, the synthesis of L-THA through enzymatic and whole-cell catalysis requires the expensive substrate L-aspartic acid or L-asparagine, and co-substrate α-ketoglutarate, which limits their large-scale application. Here, this is the first report of engineering E. coli as a cell factory for de novo production of L-THA from glucose by fermentation. Firstly, the asnO gene encoding asparagine hydroxylases from Streptomyces coelicolor was heterologously expressed in E. coli to yield the L-THA producing strain. The formation and configuration of L-THA were characterized by LC–MS and HPLC after FDAA derivatization. Secondly, the pathway genes aspC and asnB, which encode aspartate aminotransferase and asparagine synthase, respectively, were overexpressed to enhance L-THA titer from 49.9 to 90.84 mg/L. Thirdly, the efforts were made to improve the key precursor L-aspartic acid pool by overexpressing the aspartase encoding gene aspA and knocking out aspartate kinase (AK) III encoding gene lysC. The best strain CC03 was obtained and L-THA titer reached 278.3 mg/L in a shake flask, representing an approximately 5.6-fold increase compared to the original strain. Ultimately, 2.87 g/L L-THA was obtained after 32 h fed-batch fermentation. This research underscores the potential use of E. coli fermentation as a feasible platform for de novo biosynthesis of L-THA from glucose, which is amenable to industrial application.