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<p>Androsta-1,4-diene-3,17-dione (ADD) is a key intermediate in the synthesis of various steroidal pharmaceuticals, but the biocatalytic conversion of androst-4-ene-3,17-dione (AD) to ADD is limited by hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), a byproduct that inhibits the enzyme 3-ketosteroid-∆1-dehydrogenase (KstD). This study aimed to improve the bioconversion efficiency by introducing catalase (katA) to decompose H<sub>2</sub>O<sub>2</sub>, thereby alleviating its toxic effects on the enzyme. The <i>kstD</i><sub><i>2</i></sub> gene from <i>Mycobacterium neoaurum</i> DSM 1381 was mutated using error-prone PCR to generate the KstD<sub>2</sub><sup>ep</sup> variant, which was then coupled with the <i>katA</i> gene in <i>E. coli</i> strains. The engineered strain <i>E. coli</i> BL21-pET28a-KstD<sub>2</sub><sup>ep</sup>-L-katA exhibited the highest catalytic efficiency under optimized conditions, achieving a 98.6% conversion of 40&#xa0;g/L AD to ADD in 14&#xa0;h (optimized conditions: 40&#xa0;°C, pH 8.0, 40&#xa0;g/L wet cell concentration, and 1:1 cosolvent HP-β-CD with AD). Fermentation in a 5L fermenter further increased the conversion to 98.2%, using 80&#xa0;g/L AD in repeated batch feeding, significantly improving the conversion efficiency compared to shake flask conditions. These results suggest that the coupled KstD<sub>2</sub><sup>ep</sup> and catalase system, along with optimized fermentation parameters, could provide an efficient and scalable biocatalytic process for the industrial production of ADD and related steroidal compounds.</p>

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High-Efficiency Biocatalytic Production of Androsta-1,4-diene-3,17-dione via a Dual-Enzyme Coupling System in Escherichia coli

  • 期刊论文

  • 发表日期：

    2025年6月20日

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  • Zijuan Tao,
  • Yusong Zhang,
  • Yanmei Dai,
  • Liangli Luo,
  • Changshun Huang,
  • Lan Tang,
  • Hanbing Shi,
  • Zhimin Ou

摘要

Androsta-1,4-diene-3,17-dione (ADD) is a key intermediate in the synthesis of various steroidal pharmaceuticals, but the biocatalytic conversion of androst-4-ene-3,17-dione (AD) to ADD is limited by hydrogen peroxide (H2O2), a byproduct that inhibits the enzyme 3-ketosteroid-∆1-dehydrogenase (KstD). This study aimed to improve the bioconversion efficiency by introducing catalase (katA) to decompose H2O2, thereby alleviating its toxic effects on the enzyme. The kstD2 gene from Mycobacterium neoaurum DSM 1381 was mutated using error-prone PCR to generate the KstD2ep variant, which was then coupled with the katA gene in E. coli strains. The engineered strain E. coli BL21-pET28a-KstD2ep-L-katA exhibited the highest catalytic efficiency under optimized conditions, achieving a 98.6% conversion of 40 g/L AD to ADD in 14 h (optimized conditions: 40 °C, pH 8.0, 40 g/L wet cell concentration, and 1:1 cosolvent HP-β-CD with AD). Fermentation in a 5L fermenter further increased the conversion to 98.2%, using 80 g/L AD in repeated batch feeding, significantly improving the conversion efficiency compared to shake flask conditions. These results suggest that the coupled KstD2ep and catalase system, along with optimized fermentation parameters, could provide an efficient and scalable biocatalytic process for the industrial production of ADD and related steroidal compounds.