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<p><i>Corynebacterium glutamicum</i> S9114, a non-model strain with enhanced glutamate biosynthesis, is a promising microbial chassis for the production of glutamine-derived compounds. However, existing gene editing platforms show limited efficiency and adaptability in this strain. Therefore, establishing a more efficient and adaptable gene editing platform is essential to accelerate the construction of microbial cell factories. Herein, endogenous recombinases were integrated with the CRISPR-Cpf1 system to develop a gene editing platform for <i>C. glutamicum</i> S9114. Based on BLAST and recombination efficiency evaluation, the recombinase CauR from <i>Corynebacterium aurimucosum</i> was identified with best efficiency in <i>C. glutamicum</i> S9114. Recombinase CauR was combined with the CRISPR-Cpf1 system to construct the editing platform. By systematically optimizing Cpf1 expression, homology arm length, inducer concentration, and cultivation conditions, a gene knockout efficiency of 77% was achieved. This platform was then employed to construct a microbial cell factory capable of efficiently synthesizing <i>N</i>-acetylglucosamine (GlcNAc) by reducing flux through central carbon metabolism and enhancing precursor supply. A GlcNAc titer of 141.2&#xa0;g·L<sup>− 1</sup> and a productivity of 1.88&#xa0;g·L<sup>− 1</sup>·h<sup>− 1</sup> were achieved in a 50&#xa0;L bioreactor. This study establishes an effective gene editing platform for <i>C. glutamicum</i> S9114, facilitating the development of microbial cell factories for GlcNAc biosynthesis and providing a valuable reference for gene editing in other non-model strains.</p>

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Development of a CRISPR-Cpf1 and endogenous recombinase synergy platform for N-acetylglucosamine overproduction in Corynebacterium glutamicum S9114

  • 期刊论文

  • 发表日期：

    2025年11月25日

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  • Siqi Liu,
  • Jiangong Lu,
  • Wenwen Yu,
  • Yanfeng Liu,
  • Jianghua Li,
  • Guocheng Du,
  • Xueqin Lv,
  • Long Liu

摘要

Corynebacterium glutamicum S9114, a non-model strain with enhanced glutamate biosynthesis, is a promising microbial chassis for the production of glutamine-derived compounds. However, existing gene editing platforms show limited efficiency and adaptability in this strain. Therefore, establishing a more efficient and adaptable gene editing platform is essential to accelerate the construction of microbial cell factories. Herein, endogenous recombinases were integrated with the CRISPR-Cpf1 system to develop a gene editing platform for C. glutamicum S9114. Based on BLAST and recombination efficiency evaluation, the recombinase CauR from Corynebacterium aurimucosum was identified with best efficiency in C. glutamicum S9114. Recombinase CauR was combined with the CRISPR-Cpf1 system to construct the editing platform. By systematically optimizing Cpf1 expression, homology arm length, inducer concentration, and cultivation conditions, a gene knockout efficiency of 77% was achieved. This platform was then employed to construct a microbial cell factory capable of efficiently synthesizing N-acetylglucosamine (GlcNAc) by reducing flux through central carbon metabolism and enhancing precursor supply. A GlcNAc titer of 141.2 g·L− 1 and a productivity of 1.88 g·L− 1·h− 1 were achieved in a 50 L bioreactor. This study establishes an effective gene editing platform for C. glutamicum S9114, facilitating the development of microbial cell factories for GlcNAc biosynthesis and providing a valuable reference for gene editing in other non-model strains.