PDF(Pubmed)
Abstract：
Inducible gene expression systems are invaluable tools for the functional characterization of genes and in the construction of protein overexpression hosts. Controllable expression is especially important for the study of essential and toxic genes or genes where the level of expression tightly influences their cellular effect. Here, we implemented the well-characterized tetracycline-inducible expression system in two industrially important lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus. Using a fluorescent reporter gene, we show that optimization of the repression level is necessary for efficient induction using anhydrotetracycline in both organisms. Random mutagenesis in the ribosome binding site of the tetracycline repressor TetR in Lactococcus lactis indicated that altering the expression levels of TetR was necessary for efficient inducible expression of the reporter gene. Through this approach, we achieved plasmid-based, inducer-responsive, and tight gene expression in Lactococcus lactis. We then verified the functionality of the optimized inducible expression system in Streptococcus thermophilus following its chromosomal integration using a markerless mutagenesis approach and a novel DNA fragment assembly tool presented herein. This inducible expression system holds several advantages over other described systems in lactic acid bacteria, although more efficient techniques for genetic engineering are still needed to realize these advantages in industrially relevant species, such as S. thermophilus. Our work expands the molecular toolbox of these bacteria, which can accelerate future physiological studies. IMPORTANCE Lactococcus lactis and Streptococcus thermophilus are two industrially important lactic acid bacteria globally used in dairy fermentations and, therefore, are of considerable commercial interest to the food industry. Moreover, due to their general history of safe usage, these microorganisms are increasingly being explored as hosts for the production of heterologous proteins and various chemicals. Development of molecular tools in the form of inducible expression systems and mutagenesis techniques facilitates their in-depth physiological characterization as well as their exploitation in biotechnological applications.
摘要：
诱导型基因表达系统是用于基因功能表征和蛋白质过表达宿主构建的宝贵工具。可控表达对于研究必需和毒性基因或表达水平紧密影响其细胞效应的基因尤其重要。这里,我们在两种工业上重要的乳酸菌中实施了特征明确的四环素诱导表达系统，乳酸乳球菌和嗜热链球菌。使用荧光报告基因，我们表明，在两种生物中使用脱水四环素有效诱导抑制水平的优化是必要的。乳酸乳球菌中四环素阻遏物TetR的核糖体结合位点的随机诱变表明，改变TetR的表达水平对于报告基因的有效诱导型表达是必要的。通过这种方法，我们实现了基于质粒的，感应响应，和乳酸乳球菌中紧密的基因表达。然后，我们使用无标记诱变方法和本文提出的新型DNA片段组装工具验证了嗜热链球菌染色体整合后优化的诱导型表达系统的功能。这种诱导型表达系统在乳酸菌中与其他描述的系统相比具有几个优点。尽管仍需要更有效的基因工程技术来实现工业相关物种的这些优势，比如嗜热链球菌。我们的工作扩展了这些细菌的分子工具箱，这可以加速未来的生理研究。重要乳酸乳球菌和嗜热链球菌是两种工业上重要的乳酸菌,因此,对食品工业具有相当大的商业利益。此外，由于其安全使用的一般历史，这些微生物越来越多地被探索作为生产异源蛋白质和各种化学物质的宿主。以诱导型表达系统和诱变技术的形式开发分子工具有助于其深入的生理表征以及其在生物技术应用中的开发。
