Use of the alr gene as a food-grade selection marker in lactic acid bacteria
Use of the alr gene as a food-grade selection marker in lactic acid bacteria
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DOI：
10.1128/aem.68.11.5663-5670.2002
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发表时间：
2002-11-01
影响因子：
4.4
通讯作者：
Hols, P
中科院分区：
文献类型：
--
作者：
Bron, PA;Benchimol, MG;Hols, P
乳酸乳球菌和植物乳杆菌均含有单个alr基因，编码丙氨酸消旋酶(EC 5.1.1.1)，其催化D-丙氨酸和L-丙氨酸的相互转化。研究了这些乳酸菌的 alr 基因在异源互补方法中作为食品级选择标记的应用。由于携带alr缺失(Deltaalr)的两个物种的同基因突变体显示D-丙氨酸营养缺陷型，因此构建并可以选择携带异源alr的质粒，因为它们补充了植物乳杆菌Deltaalr和乳酸乳球菌Deltaalr菌株中的D-丙氨酸营养缺陷型。发现选择是高度严格的，并且质粒稳定地维持了200代以上的培养。此外，通过选择对Alr竞争性抑制剂D-环丝氨酸（分别为600和200μg/ml）的抗性，携带异源alr基因的质粒可以在植物乳杆菌和乳酸乳球菌的野生型菌株中稳定维持。此外，构建了携带受调控nisA启动子控制的植物乳杆菌alr基因的质粒，以证明乳酸乳球菌的D-环丝氨酸抗性与alr表达水平线性相关。最后，由nisA启动子控制的乳酸乳球菌alr基因与乳链菌肽调节基因nisRK一起整合到植物乳杆菌Deltaalr的染色体中。仅当用乳链菌肽诱导alr基因表达时，所得菌株才能在缺乏D-丙氨酸的情况下生长。
Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-Cycloserine, a competitive inhibitor of Alr (600 and 200 mug/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.