Lactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria, which are widely distributed in various diverse natural habitats. These are used in a variety of industrial food fermentations and carry numerous traits with utmost relevance to the food industry. Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions. The developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial “pediocin” produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts. The present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.

中文翻译：

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乳酸菌穿梭表达载体的构建

乳酸菌（LAB）是多种革兰氏阳性菌，广泛分布在各种自然栖息地中。这些可用于多种工业食品发酵中，并具有与食品工业最相关的众多特征。基因工程已经成为改善和增强商业上重要的细菌菌株潜力的有效手段。然而，在工业规模实施此类技术期间，重组系统的生物安全性是重要的考虑因素。为了克服这个问题，优选地从编码具有已知功能的基因的充分表征和注释的LAB质粒开发了克隆和表达系统。开发的穿梭载体pPBT-GFP包含两个theta型复制子，拷贝数分别为4.4和2。8个分别在乳酸双球菌MTCC 5101和短乳杆菌MTCC 1750中获得。由嗜酸乳酸杆菌MTCC 5101产生的抗菌“ pediocin”和维多利亚水母的绿色荧光蛋白（GFP）成功表达为选择标记。发酵乳杆菌NCDO 394的异源胆汁盐水解酶（BSH）已在宿主菌株中有效表达，在乳酸乳酸杆菌MTCC 5101中显示高比活性126.12±10.62，在短乳杆菌MTCC 1750中显示95.43±4.26对甘氨酸与牛磺酸缀合的盐相比，优选与胆碱缀合的胆汁盐。本文详细介绍了LAB / LAB穿梭表达载体pPBT-GFP的开发，该载体能够在LAB宿主，乳酸乳酸杆菌MTCC 5101和短乳杆菌MTCC 1750中复制。Pediocin和GFP已被用作选择性标记，可有效生产异源细胞外胆汁盐水解酶。因此，所构建的载体pPBT-GFP具有在多个宿主中复制的能力，低拷贝数和在宿主细胞中的稳定性，可以用作使用遗传工程改良具有商业价值的LAB菌株的理想工具。