Abstract
The genetic engineering of bacteria for food applications has biosafety requirements, including the use of non-antibiotic selectable markers. These can be gene-encoding bacteriocin immunity proteins, such as nisI and pedB, which require the use of promoters to ensure optimal expression. Our aim was to search for promoters for the expression of pediocin (pedB) and nisin (nisI) immunity genes, which could allow the selection of a wide variety of transformed lactic acid bacteria (LAB) and bifidobacteria strains. Eight promoters from LAB or bifidobacteria were initially studied using evoglow-Pp1 as the reporter gene in Lactococcus lactis NZ9000, resulting in the selection of P32, P3N, PTuR and PEF-P, which exhibited a strong constitutive expression. These promoters were further tested for the expression of the food-grade selectable markers pedB and nisI in agar diffusion assays with pediocin and nisin, respectively. The results obtained demonstrated that both the PTuR and PEF-P promoters allowed a good level of expression of nisI and pedB in the LAB and bifidobacteria strains tested. A suitable concentration of nisin or pediocin could be established for the selection of the strains transformed with vectors harbouring the combination of the selected promoters and markers nisI and pedB, and this was successfully applied to different strains of LAB and bifidobacteria. Therefore, PTuR and PEF-P promoters are excellent candidates for the expression of nisI and/or pedB as selectable markers in LAB and bifidobacteria, and they are suitable for use in food grade vectors to allow the selection of genetically engineered strains.
Key points
• Food-grade vectors require non-antibiotic selectable markers such as pedB and nisI.
• Eight promoters from LAB or bifidobacteria were initially tested in L. lactis NZ9000.
• PTuR and PEF-P efficiently drove the expression of pedB and nisI in LAB and bifidobacteria.
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Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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This study was funded by the project PID2020-11960RB-I00 from the Spanish Ministry of Science, Innovation and Universities.
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JML conceived and designed research, and conducted experiments. AP and SL analyzed the data. JML, SL, AP, JAC, and JLA interpreted the data and wrote the manuscript. All authors read and approved the submitted manuscript.
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Langa, S., Peirotén, Á., Curiel, J.A. et al. Promoters for the expression of food-grade selectable markers in lactic acid bacteria and bifidobacteria. Appl Microbiol Biotechnol 106, 7845–7856 (2022). https://doi.org/10.1007/s00253-022-12237-x
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DOI: https://doi.org/10.1007/s00253-022-12237-x