Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Carretera de La Coruña Km 7.5, Madrid, 28040, Spain.
Appl Microbiol Biotechnol. 2022 Dec;106(23):7845-7856. doi: 10.1007/s00253-022-12237-x. Epub 2022 Oct 29.
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 P, P P and 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 P and 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, P and 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. • P and P efficiently drove the expression of pedB and nisI in LAB and bifidobacteria.
用于食品应用的细菌基因工程有生物安全要求，包括使用非抗生素选择标记物。这些可以是基因编码细菌素免疫蛋白，如 nisI 和 pedB，它们需要使用启动子来确保最佳表达。我们的目的是寻找 pediocin（pedB）和 nisin（nisI）免疫基因表达的启动子，这可以允许选择各种转化的乳酸菌（LAB）和双歧杆菌菌株。最初使用 evoglow-Pp1 作为报告基因在乳球菌 lactis NZ9000 中研究了来自 LAB 或双歧杆菌的 8 个启动子，结果选择了 P、P P 和 P，它们表现出强组成型表达。这些启动子进一步用于在琼脂扩散测定中用 pediocin 和 nisin 分别表达食品级选择标记物 pedB 和 nisI。获得的结果表明，P 和 P 启动子都允许在测试的 LAB 和双歧杆菌菌株中高水平表达 nisI 和 pedB。可以为携带所选启动子和标记物 nisI 和 pedB 的载体转化的菌株建立合适的 nisin 或 pediocin 浓度，这成功地应用于不同的 LAB 和双歧杆菌菌株。因此，P 和 P 启动子是在 LAB 和双歧杆菌中表达 nisI 和/或 pedB 作为选择标记物的优秀候选者，它们适用于食品级载体，以允许选择基因工程菌株。关键点：• 食品级载体需要非抗生素选择标记物，如 pedB 和 nisI。• 最初在 L. lactis NZ9000 中测试了来自 LAB 或双歧杆菌的 8 个启动子。• P 和 P 有效地驱动了 LAB 和双歧杆菌中 pedB 和 nisI 的表达。
