N-乙酰胞壁质酶缺失对保加利亚乳杆菌LJJ自溶及形态的影响

Effect of N-acetylmuramidase Knockout on Autolysis and Morphology of Lactobacillus bulgaricus LJJ

【目的】乳酸菌自溶在发酵乳制品生产中广泛存在,自溶的速率和程度可以对产品的质量、风味、生产周期产生重要影响,在整个发酵过程中非常重要。N-乙酰胞壁质酶作为肽聚糖水解酶中的重要组成,可以破坏细胞壁完整性,在乳酸菌自溶过程中发挥着重要作用。论文旨在研究N-乙酰胞壁质酶缺失对保加利亚乳杆菌自溶的影响及对其形态的影响。【方法】分别以保加利亚乳杆菌基因组和pMG36e载体为参考序列设计引物,PCR分别扩增保加利亚乳杆菌中N-乙酰胞壁质酶的上下游同源臂基因m-up,m-down和pMG36e中的红霉素抗性基因。上述基因经过测序验证后,将经KpnΙ,XbaΙ双酶切的红霉素抗性基因和pUC19相连接形成重组载体pUC:Emr并进行验证。随后,将经KpnΙ,SacΙ双酶切的m-down和重组载体pUC:Emr相连接形成重组载体pUC:Emr:m-down并进行验证。最后,将经PstΙ,XbaΙ双酶切的m-up与重组载体pUC:Emr:m-down相连接形成重组载体pUC:m-up:Emr:m-down并进行验证。以重组载体pUC:m-up:Emr:m-down作为N-乙酰胞壁质酶的同源重组敲除组件,在电转化条件电压1.5 kV,电阻400Ω和电容25μF下,电转入保加利亚乳杆菌,在含有红霉素的MRS琼脂平板上筛选N-乙酰胞壁质酶基因敲除突变菌株并进行PCR验证。采用核酸溶出法检测基因缺失菌株与野生型菌株间的自溶度变化。扫描电子显微镜观察基因缺失菌株与野生型菌株间的形态变化。【结果】构建得到在N-乙酰胞壁质酶中间插入红霉素抗性基因为筛选标记的敲除组件用于保加利亚乳杆菌中N-乙酰胞壁质酶的基因敲除。获得带有红霉素抗性基因的N-乙酰胞壁质酶缺失的保加利亚乳杆菌突变菌株。突变菌株相比野生型菌株自溶特性及形态均发生显著变化,其中自溶度显著降低,37℃下自溶24 h,野生型菌株的自溶度约为73%,而突变菌株的自溶度约为35%,自溶度降为原来的1/2。野生型菌株的单个菌体细胞长度约10μm,突变菌株的单个菌体细胞约30—40μm,相比野生型菌株约增长了3—4倍。【结论】N-乙酰胞壁质酶在保加利亚乳杆菌自溶与细胞分裂过程中起着重要作用。保加利亚乳杆菌中N-乙酰胞壁质酶的缺失会导致菌体自溶的降低和菌体增殖过程中的细胞分裂受阻,产生菌体长度增长约3—4倍的菌体细胞。

[Objective] Autolysis of lactic acid bacteria exists widely in the production of fermented dairy products. The rate and extent of autolysis not only play an important role in the whole fermentation process, but also have a vital effect on the quality, flavor and production cycle of the product. As main component of peptidoglycan hydrolase, N- acetylmuramidase can destroy the cell wall integrity in lactic acid bacteria autolysis process. It plays a crucial role in the autolysis of lactic acid bacteria. This paper mainly studied the influence of N-acetylmuramidase deletion on autolysis and morphology of Lactobacillus bulgaricus LJJ.[Method] The primers of upstream homologous arm m-up, downstream homologous arm m-down of N-acetylmuramidase and erythromycin resistance genes were designed based on the sequences of L. bulgaricus genome and pMG36e vector. Then m-up, m-down and erythromycin resistance gene were amplified by PCR and verified by gene sequence analysis. Then erythromycin resistance gene and pUC19 were degested with Kpn Ι,Xba Ι and connected together. The recombinant vector pUC：Emr was verified by double-enzyme cleavage method. Next, m-down and recombinant vector pUC： Emr were digested with Kpn Ι, Sac Ι and connected together. The recombinant vector pUC：Emr：m-down was confirmed by double-enzyme cleavage method. Finally, the m-up and recombinant plasmid pUC：Emr：m-down were digested with Pst Ι, Xba Ι and connected together. The recombinant vector pUC：m-up：Emr：m-down was verified by double-enzyme cleavage method and gene sequence analysis. The recombinant vector pUC：m-up：Emr：m-down as the homologous recombinant N-acetylmuramidase knockout component, was transformed into L. bulgaricus under the electroporation conditions of electric voltage, resistence and capacity of 1.5 kV, 400 Ω and 25 μF, respectively. N-acetylmuramidase gene knockout mutant strains were screened on MRS agar containing erythromycin and verified by PCR. Then the autolysis rate of the gene deletion mutant and the wild type strain was detected and the morphological changes of gene deletion mutant and wild-type strain were observed by scanning electron microscope. [Result] N-acetylmuramidase knockout components with erythromycin resistance as selection marker was constructed and used to knock N-acetylmuramidase gene of L. bulgaricus out. N-acetylmuramidase knockout L. bulgaricus mutant strain with erythromycin resistance gene was constructed successfully. Compared with the wild-type strain, the characteristics and morphology of the mutant strain changed significantly. The autolysis rate also decreased significantly. The autolysis rate of the mutant strain was about 35% while the autolysis rate of the wild type strain was about 73% after 24 hours incubation at 37℃. The single cell length of the wild type strain was about 10 μm while that of the mutant strain was approximately 30-40 μm. Compared with the wild-type strain, the single cell length of the mutant strain increased by 3-4 times.[Conclusion]N-acetylmuramidase of L. bulgaricus plays an important role in autolysis and cell division. The deletion of N- acetylmuramidase from L. bulgaricus will result in loss of cell autolysis and the cell length will be increased by about 3-4 times.