Biochimie et Génétique Moléculaire Bactérienne, Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud 5/L7.07.06, B-1348 Louvain-la-Neuve, Belgium.
Microb Cell Fact. 2011 Aug 30;10 Suppl 1(Suppl 1):S21. doi: 10.1186/1475-2859-10-S1-S21.
In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products.
In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus.
We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.
在工业发酵过程中，嗜热链球菌使牛奶酸化的速度是一个主要的技术指标。先前的研究表明，细胞包膜蛋白酶 PrtS 是该物种牛奶酸化活性的关键决定因素。PrtS 酶通过涉及典型的 sortase A（SrtA）的机制紧密地锚定在细胞壁上，并启动乳清蛋白分解成小寡肽。PrtS 的存在与否将嗜热链球菌菌株分为两个表型群，即缓慢酸化菌株和快速酸化菌株。本研究的目的是提高缓慢酸化的嗜热链球菌菌株的牛奶酸化速度，从而优化乳制品的发酵过程。
在本工作中，我们首次开发了一种基于自然转化的策略，将快速酸化表型赋予缓慢酸化的嗜热链球菌启动子菌株。首先，我们通过基因敲除确定（i）编码细胞包膜蛋白酶的 prtS 是快速酸化菌株快速牛奶酸化的关键因素，（ii）编码 sortase A 的 srtA 不是表达 PrtS 活性所必需的。其次，使用包含 prtS 基因组岛的 15kb PCR 产物，通过自然转化在三个具有或不具有截断 sortase A 基因的不同 prtS 缺陷遗传背景下，用诱导肽转移 prtS。我们表明，在所有情况下，转化体的牛奶酸化速度都显著增加，达到与野生型快速酸化菌株相似的水平。此外，似乎 prtS 编码的活性不依赖于 prtS 拷贝数或其染色体整合位点。
我们成功地使用自然感受态将编码细胞包膜蛋白酶的 prtS 基因座转移到三种缓慢酸化的嗜热链球菌菌株中，使它们转化为快速酸化的衍生物。本文开发的有效方案将为乳制品行业提供新型优化的嗜热链球菌启动子菌株。
