Method for plasmid-based antibiotic-free fermentation

被引:3
作者
Brechun, Katherine E. [1 ]
Foerschle, Marion [1 ]
Schmidt, Marlen [1 ]
Kranz, Harald [1 ]
机构
[1] Gen H Genet Engn Heidelberg GmbH, Neuenheimer Feld 584, D-69120 Heidelberg, Germany
基金
英国科研创新办公室;
关键词
Escherichia coli; Antibiotic-free; Plasmid maintenance; Genome engineering; Complementation; Fermentation; Antimicrobial resistance; Antibiotic resistance genes; EXPRESSION; SYSTEM; STABILITY; GENES; COMPLEMENTATION; MAINTENANCE; RESISTANCE; VECTORS; PROTEIN;
D O I
10.1186/s12934-023-02291-z
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background Antibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature.Results The developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains.Conclusions This method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.
引用
收藏
页数:11
相关论文
共 50 条
[41]   Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy [J].
Kamensek, Urska ;
Tesic, Natasa ;
Sersa, Gregor ;
Kos, Spela ;
Cemazar, Maja .
PLASMID, 2017, 89 :9-15
[42]   Perspectives on plasmid-based gene therapy: challenges for the product and the process [J].
Kelly, WJ .
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, 2003, 37 :219-223
[43]   A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae [J].
Ishag, H. Z. A. ;
Liu, M. J. ;
Yang, R. S. ;
Xiong, Q. Y. ;
Feng, Z. X. ;
Shao, G. Q. .
GENETICS AND MOLECULAR RESEARCH, 2016, 15 (02)
[44]   Examination of a plasmid-based reverse genetics system for human astrovirus [J].
Chapellier, Benoit ;
Tange, Shoichiro ;
Tasaki, Hidetaka ;
Yoshida, Kazuhiro ;
Zhou, Yan ;
Sakon, Naomi ;
Katayama, Kazuhiko ;
Nakanishi, Akira .
MICROBIOLOGY AND IMMUNOLOGY, 2015, 59 (10) :586-596
[45]   Rapid decline of bacterial drug-resistance in an antibiotic-free environment through phenotypic reversion [J].
Dunai, Anett ;
Spohn, Rake ;
Farkas, Zoltan ;
Lazar, Viktoria ;
Gyorkei, Adam ;
Apjok, Gabor ;
Boross, Gabor ;
Szappanos, Balazs ;
Grezal, Gabor ;
Farago, Aniko ;
Bodai, Laszlo ;
Papp, Balazs ;
Pal, Csaba .
ELIFE, 2019, 8
[46]   A Plasmid-Based Fluorescence Reporter System for Monitoring Oxidative Damage in E. coli [J].
Dandapani, Hariharan ;
Kankaanpaa, Pasi ;
Jones, Patrik R. ;
Kallio, Pauli .
SENSORS, 2022, 22 (17)
[47]   Antibiotic-free responsive biomaterials for specific and targeted Helicobacter pylori eradication [J].
Zheng, Yating ;
Zhang, Tinglin ;
Shao, Juan ;
Du, Yiqi ;
Li, Zhaoshen ;
Zhang, Li ;
Gao, Jie .
JOURNAL OF CONTROLLED RELEASE, 2025, 379 :708-729
[48]   Antibiotic-free Self-assembled Polypeptide Nanomicelles for Bacterial Keratitis [J].
Cheng, Boyuan ;
Cui, Haohao ;
Zhang, Nan ;
Feng, Huayang ;
Chu, Dandan ;
Cui, Bingbing ;
Li, Zhanrong ;
Zhang, Junjie ;
Cao, Shaokui ;
Li, Jingguo .
ACS APPLIED POLYMER MATERIALS, 2022, 4 (10) :7250-7257
[49]   Antibiotic-free production of sucrose isomerase in Bacillus subtilis by genome integration [J].
Li, Mingyu ;
Xu, Ming ;
Bai, Xinrui ;
Wan, Xiang ;
Zhao, Meng ;
Li, Xianzhen ;
Chen, Xiaoyi ;
Wang, Conggang ;
Yang, Fan .
BIOTECHNOLOGY LETTERS, 2024, 46 (05) :781-789
[50]   Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus [J].
Pretorius, Jakobus M. ;
Huismans, Henk ;
Theron, Jacques .
VIROLOGY, 2015, 486 :71-77