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<p>Ectoine, a natural cell protectant produced by a variety of bacteria has commercial value. In this study, <i>Escherichia coli</i> BL21(DE3) was engineered for producing ectoine by using a novel approach. The <i>ectABC</i> gene cluster from <i>Nocardiopsis dassonvillei</i> NCIM 5124 was codon optimized for <i>E. coli</i> and a pET-24a(+) based construct (pEctoineCAB) was designed. Two elements—P2A tag (derived from porcine teschovirus-1 that causes ribosomal skipping) and IRES sequence (internal ribosome entry site from encephalomyocarditis virus) were introduced to ensure sufficient expression levels of all the enzymes relevant to ectoine production. The ability of the transformant harbouring pEctoineCAB was evaluated for its ectoine biosynthetic capability. In the presence of 0.1&#xa0;mM isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer, 31.52&#xa0;mg/g cell dry weight (CDW) of ectoine was obtained. After inclusion of 200&#xa0;mM <span>l</span>-aspartic acid as precursor, this content was further enhanced to 131.41&#xa0;mg/g CDW. With the acquired ability to produce ectoine, the recombinant strain tolerated sodium chloride (NaCl) up to a level of 5%. Compared to the original organism that produced 40.78&#xa0;mg/g CDW of ectoine after 48&#xa0;h in medium containing 5% NaCl, the recombinant produced higher content of ectoine (131.41&#xa0;mg/g CDW) after 24&#xa0;h of incubation in the absence of salt. On account of these features, the process of ectoine production by the recombinant was deemed effectual and economically lucrative for large-scale production. This is the first report on the use of P2A tag in <i>E. coli</i> for effective production of an economically important product.</p>

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Coexpression of the Ectoine Biosynthetic Gene Cluster from Nocardiopsis dassonvillei NCIM 5124 in Escherichia coli by Using a Novel Strategy

  • 期刊论文

  • 发表日期：

    2025年9月18日

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  • Pratik M. Kadam,
  • Vitthal T. Barvkar,
  • Swaranjali S. Patil,
  • Shubham B. Ahirrao,
  • Smita S. Zinjarde

摘要

Ectoine, a natural cell protectant produced by a variety of bacteria has commercial value. In this study, Escherichia coli BL21(DE3) was engineered for producing ectoine by using a novel approach. The ectABC gene cluster from Nocardiopsis dassonvillei NCIM 5124 was codon optimized for E. coli and a pET-24a(+) based construct (pEctoineCAB) was designed. Two elements—P2A tag (derived from porcine teschovirus-1 that causes ribosomal skipping) and IRES sequence (internal ribosome entry site from encephalomyocarditis virus) were introduced to ensure sufficient expression levels of all the enzymes relevant to ectoine production. The ability of the transformant harbouring pEctoineCAB was evaluated for its ectoine biosynthetic capability. In the presence of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer, 31.52 mg/g cell dry weight (CDW) of ectoine was obtained. After inclusion of 200 mM l-aspartic acid as precursor, this content was further enhanced to 131.41 mg/g CDW. With the acquired ability to produce ectoine, the recombinant strain tolerated sodium chloride (NaCl) up to a level of 5%. Compared to the original organism that produced 40.78 mg/g CDW of ectoine after 48 h in medium containing 5% NaCl, the recombinant produced higher content of ectoine (131.41 mg/g CDW) after 24 h of incubation in the absence of salt. On account of these features, the process of ectoine production by the recombinant was deemed effectual and economically lucrative for large-scale production. This is the first report on the use of P2A tag in E. coli for effective production of an economically important product.