Decoupling growth phase dependency and metal ion inhibition: A dual engineering strategy for the high-yield biosynthesis of microcin J25 in Escherichia coli

Abstract

Microcin J25 (MccJ25) has received substantial attention as a potential solution to the global threat of infection caused by antibiotic-resistant bacteria. However, the industrial fermentation of MccJ25 faces production bottlenecks. It is imperative to further explore the production optimization strategies for MccJ25 to formulate comprehensive approaches for its industrial-scale production and other downstream applications. Here, Fe²⁺ in tap water was identified as a critical inhibitor of MccJ25 biosynthesis, selectively repressing mcjA transcription, which was reversible via 2,2′-bipyridine-mediated chelation. To decouple production from growth phase dependency and Fe²⁺ interference, we engineered Escherichia coli BL21 cells by performing two genetic modifications. First, we replaced the native mcjA promoter with a constitutive promoter (P_Q) to allow its mid-log phase expression. Second, we replaced the native mcjBCD promoter with a medium-strength variant (P₂₂₂₃) that delayed production kinetics without affecting final yields. However, the genomic integration of mcjD alleviated plasmid-borne toxicity, increasing the expression timing and doubling the yield to 240 mg/L. Finally, we computationally optimized the mcjA ribosome-binding site (RBS) to enhance translation efficiency. RBS optimization revealed that a moderate translation initiation efficiency (550,584 arbitrary units [au]) maximized production, whereas excessive efficiency (2,019,712 au) impaired growth and output. These interventions synergistically increased the MccJ25 titer 10-fold, reaching 430 mg/L in batch culture. Our findings establish a robust platform for MccJ25 overproduction, highlighting promoter engineering and translational tuning as pivotal strategies for antimicrobial peptide biosynthesis. This study provides insights for overcoming metabolic constraints in microbial fermentation, advancing the development of peptide-based therapeutics against multidrug-resistant pathogens.

Keywords

Antimicrobial peptide / Microcin J25 / Recombinant expression / Biosynthesis

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Haitao Yu reports administrative support was provided by China Agricultural University.

CRediT authorship contribution statement

Guangxin Yang: Writing - original draft, Formal analysis, Data curation. Xinchan Wang: Writing - original draft, Formal analysis, Data curation. Yunting Zhou: Formal analysis, Data curation. Xiuliang Ding: Methodology. Jinxiu Huang: Methodology, Funding acquisition. Shiyan Qiao: Funding acquisition, Conceptualization. Aihua Deng: Writing - review & editing, Supervision, Methodology. Haitao Yu: Writing - review & editing, Supervision, Funding acquisition, Conceptualization.

Acknowledgments

The authors would like to express gratitude to Dr. Xiuliang Ding for providing technical guidance and core facilities at Chongqing Academy of Animal Science.

This research was supported by the Young Scientists Fund of the National Natural Science Foundation of China ( 32402807 ), the National Key Research and Development Program of China (Grant num- ber 32030105 ), the Pioneer Technology Project of the National Center for Technology Innovation for Pigs (NCTIP-XD/B05), the Pinduoduo- China Agricultural University Research Fund (PC2023A01001), and the Chongqing Technology Innovation and Application Development Spe- cial Project (cstc2021jscx dxwtBX0005).

Data Availability Statement

All relevant data supporting the findings of this study are available in this manuscript and the supplementary materials.

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Funding

the Young Scientists Fund of the National Natural Science Foundation of China(32402807)

the National Key Research and Development Program of China(Grant number 32030105)

the Pioneer Technology Project of the National Center for Technology Innovation for Pigs(NCTIP-XD/B05)

the Pinduoduo-China Agricultural University Research Fund(PC2023A01001)

the Chongqing Technology Innovation and Application Development Special Project(cstc2021jscx dxwtBX0005)