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<p>High-yield recombinant protein production in <i>Escherichia coli</i> requires balancing growth with proper folding at low temperatures. Here, we engineered 6&#xa0;S RNA -a reversible σ⁷⁰ antagonist- as a modulator to enhance yields under cold stress (18&#xa0;°C). Transcriptomic reanalysis of GSE74809 revealed growth phase-specific regulation: deletion of 6&#xa0;S RNA upregulated amino acid biosynthesis during logarithmic growth but downregulated ribosomal and translation-related genes in the stationary phase. To test its functional impact, 6&#xa0;S RNA and antisense constructs were cloned into pET‑24a(+), validated by RT‑qPCR, and evaluated at 18&#xa0;°C and 37&#xa0;°C. Both overexpression and knockdown impaired growth yet increased total protein levels (BCA, OD₆₀₀‑normalized). CFU enumeration confirmed reversible attenuation without toxicity, followed by asymmetric 18&#xa0;h growth reduction: controls achieved 5.48‑fold expansion, whereas 6&#xa0;S overexpression reached 2.39‑fold (44% of control) and anti‑6&#xa0;S reached 2.95‑fold (67% of control). Critically, 6&#xa0;S RNA overexpression at 18&#xa0;°C boosted Cas9‑GFP mRNA fivefold and doubled functional GFP fluorescence.</p><p>Overall, 6&#xa0;S RNA enables tunable heterologous protein production by trading proliferation for enhanced folding capacity. Its targeted upregulation significantly improves recombinant protein yield under low‑temperature conditions, highlighting its potential for industrial bioprocessing.</p>

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Modulating 6 S RNA improves recombinant protein yield in Escherichia coli at lower temperatures

  • 期刊论文

  • 发表日期：

    2026年4月28日

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  • Akram Valanik,
  • Yaghoub Safdari,
  • Faezeh Alinia,
  • Mahdi Aalikhani,
  • Zahra Bazi

摘要

High-yield recombinant protein production in Escherichia coli requires balancing growth with proper folding at low temperatures. Here, we engineered 6 S RNA -a reversible σ⁷⁰ antagonist- as a modulator to enhance yields under cold stress (18 °C). Transcriptomic reanalysis of GSE74809 revealed growth phase-specific regulation: deletion of 6 S RNA upregulated amino acid biosynthesis during logarithmic growth but downregulated ribosomal and translation-related genes in the stationary phase. To test its functional impact, 6 S RNA and antisense constructs were cloned into pET‑24a(+), validated by RT‑qPCR, and evaluated at 18 °C and 37 °C. Both overexpression and knockdown impaired growth yet increased total protein levels (BCA, OD₆₀₀‑normalized). CFU enumeration confirmed reversible attenuation without toxicity, followed by asymmetric 18 h growth reduction: controls achieved 5.48‑fold expansion, whereas 6 S overexpression reached 2.39‑fold (44% of control) and anti‑6 S reached 2.95‑fold (67% of control). Critically, 6 S RNA overexpression at 18 °C boosted Cas9‑GFP mRNA fivefold and doubled functional GFP fluorescence.

Overall, 6 S RNA enables tunable heterologous protein production by trading proliferation for enhanced folding capacity. Its targeted upregulation significantly improves recombinant protein yield under low‑temperature conditions, highlighting its potential for industrial bioprocessing.