CN101175848A - 抗噬菌体的乳酸菌 - Google Patents
抗噬菌体的乳酸菌 Download PDFInfo
- Publication number
- CN101175848A CN101175848A CNA2006800017877A CN200680001787A CN101175848A CN 101175848 A CN101175848 A CN 101175848A CN A2006800017877 A CNA2006800017877 A CN A2006800017877A CN 200680001787 A CN200680001787 A CN 200680001787A CN 101175848 A CN101175848 A CN 101175848A
- Authority
- CN
- China
- Prior art keywords
- yjae
- leu
- milk
- acid bacteria
- phage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 241000894006 Bacteria Species 0.000 title claims abstract description 69
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 54
- 241001515965 unidentified phage Species 0.000 title claims abstract description 32
- 239000004310 lactic acid Substances 0.000 title abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 136
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 76
- 239000000203 mixture Substances 0.000 claims abstract description 18
- 239000007858 starting material Substances 0.000 claims abstract description 14
- 235000013305 food Nutrition 0.000 claims abstract description 12
- 230000001580 bacterial effect Effects 0.000 claims description 59
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 26
- 230000002779 inactivation Effects 0.000 claims description 23
- 230000035772 mutation Effects 0.000 claims description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 22
- 241000194035 Lactococcus lactis Species 0.000 claims description 19
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 19
- 230000008859 change Effects 0.000 claims description 16
- 239000000463 material Substances 0.000 claims description 16
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- BSNZTJXVDOINSR-JXUBOQSCSA-N Thr-Ala-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BSNZTJXVDOINSR-JXUBOQSCSA-N 0.000 description 4
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- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 2
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 2
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Abstract
一种乳酸菌(LAB),其中的YjaE蛋白质是基本上失活性,从而该LAB得到改善的抗噬菌体抗性,以及一种起子培养物组合物包含该乳酸菌和使用该起子培养物制备食品或饲料制品。
Description
技术领域
本发明涉及乳酸菌(LAB),其中的YjaE蛋白是基本上失活性,从而该LAB具有改善的抗噬菌体性,此外,还涉及含有乳酸菌的起子培养物组合物,以及使用该起子培养物制备食品和饲料制品。
背景技术
噬菌体感染所引起的细菌培养物的产品失效被认为是细菌培养物工业中的主要问题之一。该工业中使用的许多菌株中均已发现存在噬菌体,例如乳酸菌种中的如乳球菌属、乳酸杆菌属、明串珠菌属、小球菌属或链球菌属。
在食品工业中,乳酸菌起子培养物被广泛用于食物发酵。在所有的乳酸菌种类中,乳球菌属是最容易被噬菌体感染所破坏的。导致乳酸菌起子培养物中频繁的噬菌体感染的一个因素就是在食品工业中,包括乳品工业中的发酵条件通常不是无菌条件。因此,在这样的工业条件下还不太可能消除噬菌体污染。
噬菌体促使细胞溶解的过程包括噬菌体被吸附在宿主细胞表面、噬菌体DNA被注入到细胞内、噬菌体蛋白质的合成、噬菌体DNA的复制、后代噬菌体的装配以及后代从宿主中释放的过程。这些过程中的任何一步受到细胞介导的机理的干涉都能防止噬菌体感染。在工业应用中的细菌培养物对噬菌体感染的抵抗能力在很大程度上取决于宿主菌株的特征,该特征能够影响上述机理中的一个或多个。
乳酸乳球菌中含有用于细胞膜蛋白质的染色体基因(pip),其可充当扁长噬菌体c2以及其它c2种的噬菌体的受体。当前,制造抗噬菌体乳球菌株的工业优选方法是制备pip基因失活的菌株。
在Kraus等的文章(Kraus J.et al,1998J.Dairy Science 81:2339-2335)中描述了构建一系列与商用相关的乳酸乳球菌株,其pip基因未被激活(pip-菌株)。pip-菌株对c2种的扁长噬菌体具有完全的抗性,但对936种的小的等轴噬菌体sk1、小的等轴噬菌体mm210b和31(p335种),以及大的等轴噬菌体949(949种)非常敏感。
乳酸乳球菌IL1403的完整的基因组已经测序并发表在基因库数据库中。
yjaE是乳酸乳球菌IL1403的基因。
在本发明的申请日,基因库进入号AE006322显示出乳酸乳球菌乳酸亚种IL1403的完整基因组的218个区中的84个。yjaE基因的编码DNA序列的CDS序列为从5892到8291。就其功能而言,可被简单地称为“假定的蛋白质”。另外,yjaE基因也具有与pip基因的低的同源性。具体而言,“与噬菌体感染蛋白质pip具有22%的等同性”。
基因库进入号NC_002662显示出乳酸乳球菌乳酸亚种IL1403的完整序列。这里,yjaE基因的编码DNA序列的CDS序列为从904024到906423。
WO01/77334公开了乳酸乳球菌乳酸亚种IL1403的完整基因组。在该文件中,yjaE基因对应于ORF 900。该ORF 900没有相关功能。在第27-29页,描述了涉及抗噬菌体的一些基因。在第29页,第2-3行总结了这些噬菌体相关的基因,例如ORF 38、41、448、452、518、1461和1472。
总之,从技术的角度考虑,yjaE基因的功能在本申请的申请日时是不为人所知的。
发明内容
本发明所解决的问题是提供新的抗噬菌体的乳酸菌(LAB)。
解决方案的提出是基于本发明者们对yjaE基因的参与的确认。本发明者们对不同乳酸乳球菌菌株的yjaE基因进行失活,并发现这些yjaE菌株对噬菌体具有抗性。具体参见实施例。
按如上所述,乳酸乳球菌乳酸亚种IL1403的yjaE基因的DNA序列在基因库中已有公开。其编码DNA序列示于这里的SEQ ID NO 1中,相应的yjaE蛋白质的氨基酸序列示于这里的SEQ ID NO 2中。
因此,本发明的第一个方面涉及乳酸菌,其中由yjaE基因表达的YjaE蛋白质基本上没有活性,且yjaE基因表达的YjaE蛋白质包含了选自由以下序列组成的组中的DNA序列:
(a)在SEQ ID NO 1(IL1403 yjaE DNA编码序列)中位于1-2400处的DNA序列;
(b)编码多肽的DNA序列,可选择性地具有YjaE蛋白质活性,即与位于SEQ ID NO 2(IL1403 YjaE蛋白质序列)1-799处的多肽序列的至少70%相同,例如,至少与80%,优选为至少90%,例如至少95%或至少99%的相同。
术语“基本上失活性”应该根据本发明的目的而理解。本发明的目的是制备一种菌株,其中YjaE蛋白质所起作用远不及其对应的母体野生型菌株。如下面将说明的,制备这样的菌株属于本领域技术人员的日常工作。例如,通过向yjaE基因中引入终止密码子或进行移码插入,从而产生非功能性基因,该基因例如不表达YjaE蛋白质,或者表达部分长度的失活性YjaE蛋白质。另外,也可在基因中引发突变,例如,给YjaE蛋白质突变变异体,其虽具有一些活性,但对于与在此相关的实际目的而言基本上是失活性的。检测YjaE蛋白质的失活性的方法是分析细菌对适当的有代表性的不同噬菌体组的抗性的增加。如以下将要解释的,本领域技术人员可以理解,当这里描述的细菌对代表性的不同噬菌体的抗性的有所增加时,表明YjaE蛋白质基本上是失活性的。如上所解释,已知乳酸菌可对某些噬菌体敏感,而对其他的不敏感。因此,在本领域范围内应该可以理解,如果说这里所述的细菌具有对适当的有代表性的不同噬菌体组的增加的抗性,则意味着该细菌对噬菌体组中的至少一种噬菌体具有增加的抗性。当然,通常优选的是细菌具有对噬菌体组中的两种或多种噬菌体的增加的抗性。
基本使YjaE蛋白质失去活性的优点是得到的细菌不但可对c2型噬菌体具有抗性,而且可对其他类型的噬菌体也具有抗性。这里的实施例表明了这里描述的LAB不但对c2种的噬菌体具有抗性,而且对936种的小的等轴噬菌体也具有抗性。
这是对pip-菌株的改进。如上所述,pip-菌株通常只对c2种的噬菌体具有抗性。
使YjaE蛋白质基本上失去活性通常不会对LAB的存活性、生长速率或酸的产生带来负面影响。例如,这里给出的一些实施例中展示了两种不同的菌株。
本发明的第二个方面涉及起子培养物组合物,其包含第一个方面的乳酸菌。
本发明的第三个方面涉及制备食品或饲料制品的方法,包含向食品或饲料制品起始材料中加入根据第二方面的起子培养物组合物,并在具有正常代谢活性的乳酸菌的条件下将该接种的起始材料保持。
本发明的第四个方面涉及制备乳酸菌的方法,其中,由yjaE基因表达的YjaE蛋白质是基本上失活性的,包含对yjaE基因进行适当改性,从而不产生活性YjaE蛋白质的表达,其中,yjaE基因包含了选自由以下序列组成的组中的DNA序列:
(a)在SEQ ID NO 1(IL1403 yjaE DNA编码序列)中位于1-2400处的DNA序列;
(b)编码多肽的DNA序列,可选择性具有YjaE蛋白质活性,即与位于SEQ ID NO 2(IL1403 YjaE蛋白质序列)1-799处的多肽序列的至少80%相同,例如,至少与85%,优选为至少90%,例如至少95%或至少99%的相同。
以下描述本发明的实施方式,但仅以实施例的形式描述。
发明的详细描述
根据本发明,对问题的解决方法是提供新的乳酸菌(LAB),其对噬菌体具有抗性,其中,YjaE蛋白质是基本上失活性,从而YjaE蛋白质对于噬菌体感染的功能而言失活性。
这里的表达“YjaE蛋白质对于噬菌体感染的功能而言失活性”是指一种YjaE蛋白质与所述的YjaE蛋白质序列SE Q ID No.2中的不同,其特征是携带YjaE基因而编码所述功能上失活性的YjaE蛋白质的细菌对至少一种噬菌体有改善的抗性,其中,噬菌体是选自适当的有代表性的不同的噬菌体组。适当的有代表性的不同的噬菌体组优选含有不同的相关的噬菌体,并代表c2种的扁长噬菌体,936种的小的等轴噬菌体,p335种的小的等轴噬菌体,949种的大的等轴噬菌体。一个具体的有代表性的不同的噬菌体组是包含了c2种的扁长噬菌体:bIL67、CHL92、MPC100、c2、3、24、116、122、134、180、199、227、364、670;936种的小的等轴噬菌体:234、649;以及P335种的小的等轴噬菌体:228。
术语“对噬菌体改善的抗性”是指当在空斑测定,例如下面描述的“通过琼脂覆盖法确定噬菌体抗性”对细菌菌株进行测试时,细菌菌株对至少一种噬菌体具有改善的噬菌体抗性,表现为对给定菌株,由所述至少一种噬菌体获得的pfu/ml(每ml的空斑形成单位)与在亲菌株上由相同的噬菌体获得的pfu/ml相比较的差别。对噬菌体具有改善的抗性的菌株优选地显示出pfu/ml的降低的因子至少为50,例如,至少100,如500,优选至少为1000,更优选至少为10000或更多。
乳酸菌
这里用的术语“乳酸菌”是指革兰阳性、微嗜气或厌氧菌,它们对糖进行发酵,并产生酸,包括其主要为乳酸,以及乙酸、甲酸和丙酸等。
工业上最为有用的乳酸菌被发现存在于乳球菌属、链球菌属、乳酸杆菌属、明串珠菌属、小球菌属或肠球菌属中。并且,属于双歧杆菌属中的严格厌氧菌也通常包含在乳酸菌组中。
如上所述,乳酸菌中的成员乳球菌亚种多被噬菌体感染所破坏。
优选的实施方式中的乳酸菌是乳球菌属,优选为乳酸乳球菌种。
优选的乳球菌亚种的例子有乳酸乳球菌乳脂亚种、乳酸乳球菌乳酸亚种或乳酸乳球菌乳酸亚种双乙酰乳酸生物变种(Lactococcus lactissubsp.lactis biovar.Diacetylactis)。
如上所述,乳球菌亚种含有膜蛋白质的染色体基因(pip),其可充当扁长噬菌体c2以及其它c2种的噬菌体的受体。在一个优选的实施方式中,这里描述的LAB也具有失活的pip基因。
YjaE蛋白质和yjaE基因
基于已知技术中的与yjaE相关的序列信息,以及在此所揭露的内容,本领域技术人员应当能够相对容易地确认在另一种类型的LAB中的基因是否为yjaE基因。
例如,本发明者们拥有3种不同菌株的乳球菌亚种中的yjaE基因。在氨基酸水平上,这3种序列基本上与IL1430菌株(SEQ ID NO 2)的序列相同。例如,菌株CAa120的序列在IL1430的200位处具有Lys(K)而不是Glu(E)。序列的其余部分与IL1430的相同。
因此,目前认为,特别是当LAB是乳球菌亚种时,不同菌株中的不同yjaE序列颇为相同。然而,在没有任何理论限制下,在今后的测序从其他属中找出具有类似yjaE序列的LAB。
如上所述,YjaE蛋白质可以是由yjaE基因表达的YjaE蛋白质,该基因包含选自由以下序列组成的组的DNA序列:
(b)编码多肽的DNA序列,可选择性具有YjaE蛋白质活性,即与位于SEQ ID NO 2(IL1403 YjaE蛋白质序列)1-799处的多肽序列的至少70%相同。
(b)中编码多肽的DNA序列,优选为与位于SEQ ID NO 2的1-799处的多肽序列的至少80%相同,例如至少85%相同,更优选为有至少90%相同,进一步优选为有至少93%相同的编码多肽的DNA序列和最优选的是(b)中编码多肽的DNA序列优选为与位于SEQ ID NO 2的1-799处的多肽序列的至少96%相同,更优选为有至少99%相同的编码多肽的DNA序列。
(b)中的DNA序列可以是如(a)中的DNA序列的非天然的变异体,以编码多肽的非天然的变异体。本领域的技术人员知道如何制备这样的变异体,例如,通过定点突变或随机突变或变换相似基因。
使YjaE蛋白质基本失活的方法
如上所讨论的,本领域技术人员可按常规操作制备出这里所描述的菌株,其中的YjaE蛋白质是基本上失活性的。可参见这里给出的实施例以及背景技术部分Kraus J.等的描述。
通常而言,一条常规的方法可以在yjaE基因组的基因序列中通过同源重组引入或替代适当的片段(例如,通过使用公众可获得的pGhost载体)。如果引入的片段包含无义(停止)密码子,则基因会失活,从而该LAB会成为具有失活性的YjaE蛋白质的LAB。其他适合的改性可以是移码突变,缺失、突变或插入。此外,适当的改性还可以被引入到相关区域,例如,启动区。
如上所解释的,yjaE基因的适当的改性可以是指许多情况,例如,终止密码子,可导致如移码、缺失、突变等的插入。
对于本领域技术人员而言,可按常规选择适当的方法向yjaE基因中引进一适当的改性从而不产生活性YjaE蛋白质的表达。
另外,还可以进行随机突变(例如,通过UV辐射)和对突变进行选择,其中,YjaE蛋白质基本上失活性。此外,可以对相关的自发的突变进行选择,其中,YjaE蛋白质基本上失活性。
在一优选的实施方式中,YjaE蛋白质是失活性的。
在一优选的实施方式中,YjaE蛋白质是失活性的。
例如,可通过在YjaE蛋白质中如引入移码或终止密码子进行突变,而使YjaE蛋白质失去活性,特别是当突变发生在蛋白质的大约一半处,见实施例1和3。然而,包括84核苷酸在2007-2090位上的框内缺失在内的任何突变,将导致蛋白质缺少至少一个对应于推导蛋白质序列中的氨基酸14-33,625-642,664-683,691-710,719-738或774-792的可预测的跨膜区,都将导致YjaE蛋白质对噬菌体感染在功能上失活性的一种失活的蛋白质。因此,本发明的一种实施方式涉及的乳酸菌中编码YjaE蛋白质的yjaE基因缺少至少一个对应于由推导蛋白质序列中的氨基酸14-33,625-642,664-683,691-710,719-738或774-792的可预测的跨膜区。
如在实施例4中所讨论的,可以对YjaE蛋白质的个体区域的细胞内和细胞外定位进行预测。YjaE蛋白质的个体区域的细胞内和细胞外定位被认为在噬菌体感染中对其功能有重要影响。对此,84核苷酸在2007-2090位(氨基酸670-697)上的框内缺失显得尤为有趣。这是由于该缺失导致的一种蛋白质,其中的其余部分的可预测的细胞内和细胞外定位发生了交换。因此,本发明的另一实施方式中的乳酸菌,其中,由yjaE基因编码的YjaE蛋白质,其中,YjaE蛋白质的可预测的细胞内和细胞外定位区的分布随着在菌株IL1403中的可预测的情况而发生了改变。
使YjaE蛋白质失去活性的测定方法
如上所述,检测YjaE蛋白质的失活性的方法是分析细菌对适当的有代表性的不同噬菌体组的抗性的增加。
通常可使用标准的空斑测定法来完成。可参见实施例1中根据琼脂覆盖法对合适的空斑测定的描述。空斑测定通过将特定的噬菌体作用于目标菌株得到的pfu/ml(每毫升中含有的空斑单位),相比于相同的噬菌体作用于亲菌株(YjaE蛋白质具有天然的野生型活性)所得到的pfu/ml,以它们之间的差别来测定目标菌株的抗噬菌体性(YjaE失活的蛋白质)。
相应地,这里所描述的乳酸菌对至少一种噬菌体具有增强的抵抗性的特点,其中该噬菌体选自于适当的有代表性的不同噬菌体组。可参见这里的实施例中的优选方法来分析抗噬菌体性。
适当的有代表性的不同噬菌体组应优选含有不同且相关的噬菌体,包括c2种的扁长噬菌体,936种的小的等轴噬菌体,p335种的小的等轴噬菌体,949种的大的等轴噬菌体。
c2种的扁长噬菌体的适当的例子为bIL67,CHL92,MPC100,c2,3,24,364,P001。
936种小的等轴噬菌体的适当的例子为sk1,p2,jj50,234,649。
p335种的小的等轴噬菌体的适当的例子为mm210b,31,p335。
949种的大的等轴噬菌体的适当的例子为949。
上述所列的所有噬菌体均可在文献中查到或通过向Chr.Hansen A/S,Denmark请求而得到。
优选地,这里所述的乳酸菌对c2种的扁长噬菌体和/或936种小的等轴噬菌体已具有改善的抵抗性。
另一种测量YjaE蛋白质的失活性的方法就是分析yjaE的基因序列,看其是否含有适当的能够引起诸如基因失活的改性。如上面所叙述的,适当的改性可通过多种手段,例如终止密码子、可导致如移码、缺失、突变等的插入。这是本领域的技术人员例行确定(如通过排序基因)是否基因含有这样的适当的改性。
相应地,在优选的实施方式中,这里所描述的乳酸菌含有对yjaE基因的适当的改性,其中的改性导致基本上不表达活性的YjaE蛋白质。
更优选地,改性导致活性YjaE蛋白质不被表达。
测量YjaE蛋白质失活性的进一步方法就是分析活性YjaE蛋白质是否会存在于细菌的细胞膜中。这可以通过此处实施例中描述的标准分离法测量。
相应地,在优选的实施方式中此处所描述的乳酸菌并不合有细胞膜中活性YjaE蛋白质的可测量的量。
含有这里描述的LAB的起子培养物
这里描述的乳酸菌被用作食品和饲料制品生产中的起子培养物。通常,这样的起子培养物组合物包含浓缩形式的细菌,包括冷冻的,干燥的或冻干的浓缩物,在每克的组合物中,通常活细胞的浓度范围在104到1012cfu(菌落形成单位),每克组合物至少包括104cfu,例如,至少为105cfu/g,如至少为106cfu/g,如至少为107cfu/g,如至少为108cfu/g,如至少为109cfu/g,如至少为1010cfu/g,如至少为1011cfu/g。
组合物还可进一步含有防冻剂和/或传统的添加剂,例如营养物,如酵母提取物、糖和维生素。
在乳酸菌发酵过程中,一般会采用混合的乳酸菌的培养物。因此,在一些实施方式中,组合物包含了多种菌株,它们可以是属于相同的种或可属于不同的种。在起子培养物组合物中的这种有用的乳酸菌组合的典型例子是以下菌株的混合物:明串珠菌属,一种或多种乳球菌亚种,例如,乳酸乳球菌乳酸亚种,乳酸乳球菌乳脂亚种,或乳酸乳球菌乳酸亚种双乙酰乳酸生物变种。
制备食品和饲料制品的方法
如上所述,本发明的一方面涉及制备食品或饲料制品的方法,包含向食品或饲料制品起始材料中加入这里描述的起子培养物组合物,并在具有正常代谢活性的乳酸菌的条件下将该接种的起始材料保持。
有用的食品起始材料包括任何可用于传统的乳酸菌发酵步骤的材料,例如,奶、蔬菜材料、肉制品、水果汁、果汁、生面团和牛奶鸡蛋面糊。由该方法得到的发酵产品,包括典型的乳制品,例如奶酪,包括新鲜的奶酪制品和酪乳。
在进一步的实施方式中,底物材料为用于动物饲养的起始材料,例如,青贮饲料,如草、谷类材料、豆、紫花苜蓿或甜根的叶,其中,细菌培养物在被青贮的饲料农作物中进行接种,以保存这些材料。并且,在富含蛋白质的动物废弃物中,例如屠宰后的碎屑和鱼的废弃物中,发酵的目的也是保存这些废弃物,并为动物饲养之用。
本发明的乳酸菌的另一重要应用是将细菌培养物用作所谓的益生菌。本文中的术语“益生菌”是指微生物培养物,当它们被人或动物以活细胞的形式摄食后,能够改善健康条件,例如,通过抑制胃肠道内的有害微生物,通过增强免疫系统,或通过有助于营养物的消化。
鉴定DNA序列
这里所述的DNA序列鉴定二个序列之间的相似程度,其表示第一个序列偏离第二个序列的程度。
在本发明的申请日,National Center for Biotechnology Information(NCBI)在Intemet站点(http://www.ncbi.nlm.nih.gov/)中提供了对标准的BLAST计算机序列同源性检索的可能性。
在[Altschul et al(1997),″Gapped BLAST and PSI-BLAST:a newgeneration of protein database search programs″,Nucleic Acids Res.25:3389-3402]中对BLAST程序进行了描述。
在本文中,优选的计算机同源性检索程序为“标准的核苷酸-核苷酸BLAST[blastn]”检索,该定义与在本发明的申请日,在NCBI的Intemet站点上设置检索条件:Low complexity;Expect:10,Word Size:11相一致。
在程序中还引进了参考序列,并且该程序能够鉴定出发布序列的片段以及其与对应的参考序列片段的相同百分比。
鉴定氨基酸序列
类似于核苷酸同源性分析,在本文中,优选的计算机同源性检索程序为“标准的蛋白质-蛋白质BLAST[blastn]”检索,该定义与在本申请的申请日,在NCBI的Internet站点上设置Composition-based statistics:yes,检索条件Low complexity;Expect:10,Word Size:3,Matrix:BLOSUM 62,Gap Costs:Existence 11 Extension 1相一致。
图例
图1显示了细菌细胞膜中可预测的跨膜区域以及从CAa120中推断出的YjaE蛋白质位置。箭头处的数字表示在氨基酸序列中的位置(SEQID No.2)以及表示围绕跨膜区域的氨基酸。
实施例
材料和方法
空斑测定-通过琼脂覆盖法确定对噬菌体的抗性。
该方法用于评价给定的乳酸乳球菌突变菌株对噬菌体的抗性。将该给定菌株在给定噬菌体下获得的pfu/ml(每ml的空斑形成单位)与亲菌株在相同噬菌体作用下获得的pfu/ml的差别进行比较。各个空斑均起源于一个噬菌体,且在生长着细菌的菌苔上呈现出清晰的无细菌生长的圆形区域。
期望的菌株在盘上(M17+所需的添加剂)呈现清晰的条纹。5-10个单独的菌落被接种在培养液体中,且检测呈指数生长的培养物的OD600。当培养物的OD600在0.5和0.8之间时,将100μl的培养物与100μl的噬菌体混合。对噬菌体-溶液进行这样的重复操作,滴定度范围在1011pfu/ml到10pfu/ml,如在亲菌株中所测量的。细胞和噬菌体在总量为3ml的上层琼脂中混合(M17,10mMCa CI2,0.75%琼脂),再倒到盘(M17,10mM CaCl2,1.5%琼脂)上,并在30℃培育过夜。
通过计算形成的空斑数目来评价盘。确定用于给定菌株的噬菌体的pfu/ml。给定菌株的噬菌体抗性由在该菌株中发现的pfu/ml与在亲菌株中发现的来决定。
检验在细菌膜中是否存在活性YjaE蛋白质
用溶解酵素消化细胞,使细胞壁与膜分离,并通过标准步骤,如差速离心收集膜。使用用于膜蛋白质的标准的蛋白质增溶试剂盒,例如Pierce Biotechnology(Rockford,IL,USA)的用于膜蛋白质的2-D样品制剂(2-D Sample Preparation for Membrane Proteins)或BioRad(Hercules,CA,USA)ReadyPrep Protein Extraction Kit(Membrane I or II),通过增溶作用将蛋白质从膜中分离。然后,按照标准程式将溶解的蛋白质在pH7.5-9.5(YjaE蛋白质的pI是8.6-8.9)的范围内进行二维凝胶电泳。YjaE蛋白质的分子量(85kDa)和pl将决定其在二维凝胶中的位置,而YjaE蛋白质的缺席或改变(例如,截短的)将与亲菌株形成对比。亲菌株的YjaE蛋白质的确认可通过凝胶内消化和质谱来验证。
文献:
Valyasevi,R.,Sandine,W.E.,Geller,B.L.,1991.A membrane protein isrequired for bacteriophage c2 infection of Lactococcus lactis subsp.lactis C2.J.Bact.173(19),6095-6100.
Silveira,M.G.,M.,Rombouts,F.M.,Abee,T.,2004.Effect ofadaptation to ethanol on cytoplasmic and membrane protein profiles ofOenococcus oeni.Appl.Environ.Microbiol.70(5),2748-2755.
Nouwens,A.S.,Cordwell,S.J.,Larsen,M.R.,Molloy,M.P.,Gillings,M.,Willcox,M.D.P.,Walsh,B.J.,2000.Complementing genomics withproteomics:the membrane subproteome of Pseudomonas aeruginosa PAO1.Electrophoresis 21,3797-3809.
Molloy,M.P.,Herbert,B.R.,Slade,M.B.,Rabilloud,T.,Nouwens,A.S.,Williams,K.L.,Gooley,A.A.,2000.Proteomic analysis of the Escherichia coliouter membrane.Eur.J.Biochem.267,2871-2881.
实施例1:使乳球菌菌株中的yjaE基因失去活性
菌株:
IL1403:参见WO01/77334
CAa120:
Stuer-Lauridsen,B.,Janzen,T.,Schnabl,J.,Johansen,E.,2003.Identificationof the host determinant of two prolate-headed phages infecting Lactococcuslactis.Virology 309,10-17.
载体:
pGhost载体:详细说明参见下列文章
Maguin,E.,Prévost,H.,Gruss,A.,1996.Construction of food-grade mutantsof lactic acid bacteria.Lait 76,139-146.
Biswas,I.,Gruss,A.,Ehrlich,S.D.,Maguin,E.,1993.High-efficiency geneinactivation and replacement system for Gram-positive bacteria.Journal ofBacteriology 175(11),3628-3635.
失活步骤的描述:
生成650bp的PCR片段,覆盖在yjaE基因从nt703-1344的中间部分。使用的模板是来自乳酸乳球菌CAa120的染色体DNA。该片段被克隆于载体pGhost9中。该PCR片段含有独特的BsrGI位点,当其被克列诺片段填充并再连接时,将变为SnaBI位点并导致一个碱基的移码。通过同源重组,将此构建并入到CAa120的yiaE的染色体版本中,则载体pGhost9随后成功杂交,留下移码突变。产生的突变通过限制性分析和对PCR片段测序进行验证,突变株被命名为CAa120ΔyjaE。在乳酸乳球菌IL1403上进行类似的构建,其突变株命名为IL1403ΔyjaE。通过空斑测定测试这些菌株对感染亲菌株的噬菌体的抗性,发现这些构建的菌株对一些噬菌体(即用YjaE蛋白质感染的噬菌体)产生了抗性。该失活性的yjaE基因是基于与构建的片段的同源重组,当代替正常片段时,该构建的片段经改性产生功能障碍的基因。代替是通过使用pGhost9作为载体进行,但也可用任何其他没有在乳酸乳球菌中进行重组功能的载体。
结果:
以下的噬菌体组被用于进行如上描述的空斑测定。
c2种的扁长噬菌体:bIL67、CHL92、MPC100、c2、3、24、116、122、134、180、199、227、364、670。
936种的小的等轴噬菌体:234、649。
p335种的小的等轴噬菌体:228。
结果示于以下表1中。
表1示出了一些噬菌体对两种乳酸乳球菌菌株和他们的yjaE突变株产生感染(+)或不产生感染(-)
IL1403 | IL1403Δyja | CAa120 | CAa120Δyja | |
3 | + | - | + | - |
24 | + | - | + | - |
116 | + | + | - | - |
122 | + | + | - | - |
134 | + | + | - | - |
180 | + | + | - | - |
199 | + | + | - | - |
227 | + | + | - | - |
228 | + | + | - | - |
234 | - | - | + | - |
364 | - | - | + | - |
649 | - | - | + | - |
670 | - | - | + | + |
CHL92 | + | - | + | - |
bIL67 | + | - | - | - |
MPC100 | + | + | - | - |
c2 | + | + | - | - |
结果表明,具有失活性的yjaE基因的菌株对c2种的扁长噬菌体和936种的小的等轴噬菌体均具有改善的抗噬菌体性。
进一步测试菌株的存活性、生长速率和产酸性。
对比相应的野生型菌株,没有可测量的负面影响被发现。
实施例2:用于产生自发性抗噬菌体突变体的一般方法
该方法可通过在yjaE基因中由DNA测序确定的突变而被用于获得自发性抗噬菌体突变体。
从保存的冷冻储存中刮下期望的菌株在10ml的培养液体M17(Oxoid CM0817,Oxoid Ltd.,Basingstoke,Hampshire,England)+所需的添加剂中进行接种。在本例中,所述的添加剂是0.5%的乳糖用于菌株CAa120和34,0.5%的葡萄糖用于菌株IL1403和Bu2-60。检测呈指数生长的培养物的OD600。当培养物的OD600达到0.5和0.8之间时,将100μl的培养物与感染复数(MOI)为每个细胞1-10个噬菌体的噬菌体混合。细胞和噬菌体在总量为3ml的上层琼脂中混合(M17,10mMCa Cl2,0.75%琼脂),再倒到盘(M17,10mM Ca Cl2,1.5%琼脂)上,并在30℃培育一天或两天。大多数的细胞都被感染性的噬菌体杀灭,但自发性抗噬菌体的突变体菌落最终将出现。自发性抗噬菌体的突变体的出现的频率在不同菌株中可有所不同。
取出抗噬菌体的突变体,在盘上整齐划线,通过在本文中它处所描述的空斑测定,测试对产生这些突变体的步骤中所用的噬菌体以及对感染亲菌株的其他噬菌体的噬菌体抗性。
随后,将突变体在培养液体中接种,并取出染色体DNA,用于产生包括了期望基因的PCR片段,本例中,该期望基因是yjaE基因。然后对这些PCR片段进行测序,以确定各突变体中的DNA序列与亲株中的是怎样不同。
实施例3:自发性抗噬菌体yjaE突变体的产生
菌株:
IL1403:乳酸乳球菌。参见WO01/77334
CAa120:乳酸乳球菌。参见Chr.Hansen Culture Collection.Stuer-Lauridsen,B.,Janzen,T.,Schnabl,J.,Johansen,E.,2003.Identification of thehost determinant of two prolate-headed phages infecting Lactococcus lactis.Virology 309:10-17。
Bu2-60:乳酸乳球菌。Wetzel,A.,Neve,H.,Geis,A.,Teuber,M.,1986.Transfer of plasmid-mediated phage resistance in lactic acid Streptococci.Chem.Mikrobiol.Technol.Lebensm.10:86-89。
菌株34:乳酸乳球菌。参见Chr.Hansen Culture Collection.
菌株CAa120和34可由Chr.Hansen购得。
步骤的描述:
按照在“材料和方法”中所描述的,菌株经过该步骤而产生自发性抗噬菌体的突变体。在不同组合下的一个菌株和一个噬菌体分别为:菌株34和Φ24,Bu2-60和Φ3,Bu2-60和Φ364,IL1403和ΦbIL67,CAa120和Φ24。以染色体DNA为基础,从每个自发性突变体产生包括了全部yjaE基因的PCR片段以及产生从该基因的起始和终止密码子开始的大约150nt上游和下游的基因的PCR片段。对这些PCR片段进行测序,并将每个突变体的序列与亲菌株的比较。
结果:
用PCR和DNA测序对总共21个自发性突变体进行了研究。所有的突变体菌株均具有yjaE基因的突变(详细情况见表2)。
表2.自发性突变。所有的位置均指DNA SEQ no.1
突变 | 描述 |
192位的a | 在8个自发性突变体中缺失a(3个来自34,1个来自CAa 120,1个来自IL1403,以及3个来自Bu2-60),导致在224-226位出现一个终止密码子。 |
192位的a | 1个自发性突变体(来自34)具有一个额外的a,导致在204-206位出现一个终止密码子。 |
291位的a | 在2个自发性突变体中缺失a(来自IL1403),导致在350-352位出现一个终止密码子。 |
419位的c | 在1个自发性突变体(来自CAa120)中c被a取代,导致在418-420位出现一个终止密码子。 |
766位的c | 在2个自发性突变体(来自CAa120)中c被t取代,导致在766-768位出现一个终止密码子。 |
865位的c | 在1个自发性突变体(来自Bu2-60)中c被t取代,导致在865-867位出现一个终止密码子。 |
998位的a | 在2个自发性突变体中缺少a(一个来自IL1403,一个来自CAa120),导致在1040-1042位出现一个终止密码子。 |
1869位的c | 在1个自发性突变体(来自Bu2-60)中c被a取代,导致在1867-1869位出现一个终止密码子。 |
-111到-16位 | 在1个自发性突变体(来自Bu2-60)中发现96nt的缺失,导致从yjaE基因的上游发生启动子的缺失。 |
601到1987位 | 在1个自发性突变体(来自CAa120)中发现1386nt的框内缺失,其跨越了推导YjaE蛋白质的预期的大外环的主要部分和在预期的膜锚定区的第一个跨膜区(对应于推导蛋白质序列中的氨基酸201-663) |
2007到2090位 | 在1个自发性突变体(来自Bu2-60)中的推导YjaE蛋白质的预期的膜锚定区中发现84nt的框内缺失(对应于推导蛋白质序列中的氨基酸670-697) |
还测试了这些突变体对以下表3中所示的其他噬菌体的噬菌体抗性。
所有的自发性抗噬菌体突变体均显示出对c2种的扁长噬菌体和936种的小的等轴噬菌体的改善的抗噬菌体性。每个菌株的所有自发性抗噬菌体突变体显示出相同的噬菌体感染分布谱,因此表中示出自发性抗噬菌体突变体的栏代表了本例中所有被研究的自发性突变体。
表3.示出了不同乳酸乳球菌菌株和它们的构建的(ΔyjaE)或自发性的(spont)yjaE突变体被一些噬菌体感染(+)或不被感染(-)。自发性突变体的详细情况参见表2。
c2种的扁长噬菌体:bIL67、CHL92、MPC100、c2、3、24、116、122、134、180、199、227、364、670。
936种的小的等轴噬菌体:234、649。
p335种的小的等轴噬菌体:228。
实施例4:预测推导YjaE蛋白质中的跨膜区
一些公共网站提供从氨基酸序列预测蛋白质的跨膜区的服务。例如Swiss Embnet的www.ch.embnet.org,和DTU的Prediction Servers ofCenter for Biological Sequence Analysis的www.genome.cbs.dtu.dk/services/。将由CAa120的yjaE基因序列推导出的氨基酸序列输入Swiss Embnet的TMPRED,则给出了如图1所示的预测的跨膜区和在YjaE蛋白质的细菌膜中的位置。由DTU的CBS Server的预测给出了类似的结果。
序列表
<110>科·汉森有限公司
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Met Leu Lys Lys Glu Trp Gln Ala Ile Leu Lys His Lys Phe Phe Ile
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att gtt att atc gct ttg gca ctt gta cca gca att tat aac tat att 96
Ile Val Ile Ile Ala Leu Ala Leu Val Pro Ala Ile Tyr Asn Tyr Ile
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Phe Leu Gly Ser Met Trp Asp Pro Tyr Gly Lys Leu Asn Asp Leu Pro
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Val Ala Val Val Asn Leu Asp Lys Thr Ser Glu Leu Asn Gly Lys Lys
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Phe Lys Leu Gly Asp Asp Val Ile Ala Glu Met Lys Lys Ser Lys Asp
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tta gat tat cat ttt gtt tcg gag tat aaa gct tct aaa ggg ata aaa 288
Leu Asp Tyr His Phe Val Ser Glu Tyr Lys Ala Ser Lys Gly Ile Lys
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aaa ggt gat tat tac atg gtt atc act ttt cca gaa aac ttt tca gaa 336
Lys Gly Asp Tyr Tyr Met Val Ile Thr Phe Pro Glu Asn Phe Ser Glu
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aat gca aca acc tta atg aat aag aaa cca aaa acg gtt cag tta gat 384
Asn Ala Thr Thr Leu Met Asn Lys Lys Pro Lys Thr Val Gln Leu Asp
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tat caa acg aca cgt ggt cat aac tat att tca tca aaa atg agc gaa 432
Tyr Gln Thr Thr Arg Gly His Asn Tyr Ile Ser Ser Lys Met Ser Glu
130 135 140
agt gcg atg aat cag ctg aaa tca gag gtt tct aaa aat atc acg gaa 480
Ser Ala Met Asn Gln Leu Lys Ser Glu Val Ser Lys Asn Ile Thr Glu
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act tac acc aaa gaa att ttc gct aaa ctt ggt gat atg aag tca gga 528
Thr Tyr Thr Lys Glu Ile Phe Ala Lys Leu Gly Asp Met Lys Ser Gly
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atg aaa gaa gct tct gac ggt tca aat aaa ttg gct gat gga act tcg 576
Met Lys Glu Ala Ser Asp Gly Ser Asn Lys Leu Ala Asp Gly Thr Ser
180 185 190
tca gca tta aat ggt tca aaa gaa tta agt agt aat ttg aat act tta 624
Ser Ala Leu Asn Gly Ser Lys Glu Leu Ser Ser Asn Leu Asn Thr Leu
195 200 205
tct tca tca agt tta act ttt act gac ggc gca aat aat ttg aat tca 672
Ser Ser Ser Ser Leu Thr Phe Thr Asp Gly Ala Asn Asn Leu Asn Ser
210 215 220
ggc tta aat aaa tat gta tca gga gtg aat caa gct gcc aat ggg gga 720
Gly Leu Asn Lys Tyr Val Ser Gly Val Asn Gln Ala Ala Asn Gly Gly
225 230 235 240
caa caa ctt tct tct ggt gca gac caa ttt gtc agt gga acg caa caa 768
Gln Gln Leu Ser Ser Gly Ala Asp Gln Phe Val Ser Gly Thr Gln Gln
245 250 255
tta gca gca ggt aca caa aca ctg gct gat aaa tca aaa gag ctg tca 816
Leu Ala Ala Gly Thr Gln Thr Leu Ala Asp Lys Ser Lys Glu Leu Ser
260 265 270
gca gga att tca caa att tct caa ggg tca gaa gcg gtg agt caa ttg 864
Ala Gly Ile Ser Gln Ile Ser Gln Gly Ser Glu Ala Val Ser Gln Leu
275 280 285
caa tcg ggt gtg caa caa tta tct gaa ggc tta agt caa atg gct caa 912
Gln Ser Gly Val Gln Gln Leu Ser Glu Gly Leu Ser Gln Met Ala Gln
290 295 300
aaa aca act tta tcc gaa act caa cag caa aat att agt gct gta caa 960
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Asn Gly Leu Thr Glu Leu Asn Gln Glu Leu Gln Lys Asn Thr Ile Asp
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Thr Val Thr Asn Leu Ala Asn Asp Gln Val Ala Ala Ser Lys Thr Ala
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gtt gaa agt act gag act ttt aaa act tta agc gaa agt cag aaa aca 1152
Val Glu Ser Thr Glu Thr Phe Lys Thr Leu Ser Glu Ser Gln Lys Thr
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Asp Ile Arg Asn Ala Leu Asp Asn Ser Val Gly Ala Gly Ala Val Gln
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Thr Asp Leu Leu Ser Leu Ash Lys Asn Ile Lys Gly Val Gln Asp Ser
405 410 415
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Leu Thr Ser Leu Leu Pro Met Phe Gly Lys Ile Ala Asp Leu Lys Thr
420 425 430
tta gtt cca gga gca agt caa ata att gct gac ctt tca ggt ggt ctg 1344
Leu Val Pro Gly Ala Ser Gln Ile lle Ala Asp Leu Ser Gly Gly Leu
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Gln Gln Ile Ser Ser Val Ser Gln Gln Leu Ala Asn Gly Gly Ser Ser
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tta aca agt ggt ttg gga acg cta aat aca ggg gct aat aac ttg tca 1728
Leu Thr Ser Gly Leu Gly Thr Leu Asn Thr Gly Ala Asn Asn Leu Ser
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aca gcg tta acc aaa gca gat gat act ctc tca gca act aat aat tct 1776
Thr Ala Leu Thr Lys Ala Asp Asp Thr Leu Ser Ala Thr Asn Asn Ser
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aat gaa aat gct aaa aaa gta gct gca ccg ttg aaa ctt aaa cat act 1824
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gac cat gac aat gta cca gaa aat ggt gca gga atg aca cca tac atg 1872
Asp His Asp Asn Val Pro Glu Asn Gly Ala Gly Met Thr Pro Tyr Met
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Ile Asn Val Ala Leu Phe Ile Gly Ala Leu Ala Thr Asn Val Val Ile
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atc gtc tgg gga gca gtt gct ctt ctt gga ttg aga cca aat cat ttg 2064
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tgg gaa atg ttg ttg tca gta ctt tta atc agc ttc gct tac atg gca 2112
Trp Glu Met Leu Leu Ser Val Leu Leu Ile Ser Phe Ala Tyr Met Ala
690 695 700
atc aat act ttc ttc tta acg gct cta ggt aag att ggt gaa ttc ctt 2160
Ile Asn Thr Phe Phe Leu Thr Ala Leu Gly Lys Ile Gly Glu Phe Leu
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Pro Met Thr Tyr Gly Leu Lys Met Leu Arg Glu Thr Ile Gly Leu Asn
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Gly Ala Ile Leu Pro Glu Ala Ile Leu Phe Val Val Ile Ile Ala Leu
770 775 780
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Phe Thr Phe Met Leu Ser Phe Phe Lys Lys Phe Ser Arg Phe Ala
785 790 795
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Ile Val Ile Ile Ala Leu Ala Leu Val Pro Ala Ile Tyr Asn Tyr Ile
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Phe Leu Gly Ser Met Trp Asp Pro Tyr Gly Lys Leu Asn Asp Leu Pro
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Val Ala Val Val Asn Leu Asp Lys Thr Ser Glu Leu Asn Gly Lys Lys
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Phe Lys Leu Gly Asp Asp Val Ile Ala Glu Met Lys Lys Ser Lys Asp
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Asn Ala Thr Thr Leu Met Asn Lys Lys Pro Lys Thr Val Gln Leu Asp
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Tyr Gln Thr Thr Arg Gly His Asn Tyr Ile Ser Ser Lys Met Ser Glu
130 135 140
Ser Ala Met Asn Gln Leu Lys Ser Glu Val Ser Lys Asn Ile Thr Glu
145 150 155 160
Thr Tyr Thr Lys Glu Ile Phe Ala Lys Leu Gly Asp Met Lys Ser Gly
165 170 175
Met Lys Glu Ala Ser Asp Gly Ser Asn Lys Leu Ala Asp Gly Thr Ser
180 185 190
Ser Ala Leu Asn Gly Ser Lys Glu Leu Ser Ser Asn Leu Asn Thr Leu
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Ser Ser Ser Ser Leu Thr Phe Thr Asp Gly Ala Asn Asn Leu Asn Ser
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Gly Leu Asn Lys Tyr Val Ser Gly Val Asn Gln Ala Ala Asn Gly Gly
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Gln Ser Gly Val Gln Gln Leu Ser Glu Gly Leu Ser Gln Met Ala Gln
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305 310 315 320
Asn Gly Leu Thr Glu Leu Asn Gln Glu Leu Gln Lys Asn Thr Ile Asp
325 330 335
Pro Asn Leu Ala Ala Asn Ile Gln Asn Asn Leu Lys Gly Leu Gly Ala
340 345 350
Thr Val Thr Asn Leu Ala Asn Asp Gln Val Ala Ala Ser Lys Thr Ala
355 360 365
Val Glu Ser Thr Glu Thr Phe Lys Thr Leu Ser Glu Ser Gln Lys Thr
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Asp Ile Arg Asn Ala Leu Asp Asn Ser Val Gly Ala Gly Ala Val Gln
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Thr Asp Leu Leu Ser Leu Asn Lys Asn Ile Lys Gly Val Gln Asp Ser
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Leu Thr Ser Leu Leu Pro Met Phe Gly Lys Ile Ala Asp Leu Lys Thr
420 425 430
Leu Val Pro Gly Ala Ser Gln Ile Ile Ala Asp Leu Ser Gly Gly Leu
435 440 445
Asn Glu Val Asn Thr Asn Val Asn Thr Lys Phe Ile Pro Gly Ile Asn
450 455 460
Gln Leu Asn Ala Gly Leu Thr Asn Phe Asn Asp Gln Leu Thr Ser Gly
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Ser Gln Lys Leu Thr Ser Gly Phe Ala Gln Tyr Ala Asp Gly Val Asn
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Gln Ala Asn Ala Gly Ala Gln Gln Leu Ala Ser Lys Ser Lys Asp Leu
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Gln Ser Gly Thr Met Gln Leu Val Ser Gly Leu Ser Gln Leu Gln Ala
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Asn Gly Ser Thr Leu Thr Ser Gly Ser Asn Gln Leu Ala Gly Gly Ala
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Gln Gln Ile Ser Ser Val Ser Gln Gln Leu Ala Asn Gly Gly Ser Ser
545 550 555 560
Leu Thr Ser Gly Leu Gly Thr Leu Asn Thr Gly Ala Asn Asn Leu Ser
565 570 575
Thr Ala Leu Thr Lys Ala Asp Asp Thr Leu Ser Ala Thr Asn Asn Ser
580 585 590
Asn Glu Asn Ala Lys Lys Val Ala Ala Pro Leu Lys Leu Lys His Thr
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Asp His Asp Asn Val Pro Glu Asn Gly Ala Gly Met Thr Pro Tyr Met
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Ile Asn Val Ala Leu Phe Ile Gly Ala Leu Ala Thr Asn Val Val Ile
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Gly Ile Gly Phe Ser Gly Glu Lys Trp Lys Ser Gly Arg Glu Phe Met
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Ile Val Trp Gly Ala Val Ala Leu Leu Gly Leu Arg Pro Asn His Leu
675 680 685
Trp Glu Met Leu Leu Ser Val Leu Leu Ile Ser Phe Ala Tyr Met Ala
690 695 700
Ile Asn Thr Phe Phe Leu Thr Ala Leu Gly Lys Ile Gly Glu Phe Leu
705 710 715 720
Met Ile Val Val Leu Val Leu Gln Leu Ala Thr Ser Ala Gly Thr Tyr
725 730 735
Pro Leu Gln Leu Ala Pro Lys Ile Tyr Gln Val Ile Ser Pro Trp Leu
740 745 750
Pro Met Thr Tyr Gly Leu Lys Met Leu Arg Glu Thr Ile Gly Leu Asn
755 760 765
Gly Ala Ile Leu Pro Glu Ala Ile Leu Phe Val Val Ile Ile Ala Leu
770 775 780
Phe Thr Phe Met Leu Ser Phe Phe Lys Lys Phe Ser Arg Phe Ala
785 790 795
Claims (15)
1.乳酸菌,其中,由yjaE基因表达的YiaE蛋白质是基本上失活性,其中,yjaE基因表达的YjaE蛋白质包含了选自由以下序列组成的组中的DNA序列:
(a)在SEQ ID NO 1(IL1403 yjaE DNA编码序列)中位于1-2400处的DNA序列;
(b)编码多肽的DNA序列,选择性地具有YjaE蛋白质活性,即与位于SEQ ID NO 2(IL1403 Yjae蛋白质序列)1-799处的多肽序列的至少70%相同。
2.权利要求1的乳酸菌,其中,所述的基本上失活性的YjaE蛋白质对于噬菌体感染而言是功能上失活性。
3.权利要求2的乳酸菌,其中,所述细菌具有对噬菌体改善的抗性,优选地显示出在pfu/ml上降低的因子至少为50,例如,至少100,如500,优选至少为1000,更优选至少为10000或更多。
4.前述任何一项权利要求中的乳酸菌,其中,乳酸菌是乳球菌属,优选是选自由以下乳球菌属的菌种所组成的组:乳酸乳球菌乳脂亚种,乳酸乳球菌乳酸亚种和乳酸乳球菌乳酸亚种双乙酰乳酸生物变种。
5.前述任何一项权利要求中的乳酸菌,其中,编码多肽(b)的DNA序列是编码与位于SEQ ID NO 2中的1-799处的多肽序列至少90%相同的多肽的DNA序列,更优选的是编码与位于SEQ ID NO 2中的1-799处的多肽序列至少96%相同的多肽的DNA序列。
6.前述任何一项权利要求中的乳酸菌,其中,yjaE基因编码的YjaE蛋白质缺少至少一个可预测的跨膜区。
7.前述任何一项权利要求中的乳酸菌,其中,yjaE基因编码的YjaE蛋白质,其中,YjaE蛋白质的细胞内和细胞外定位区的可预期分布相对于在菌株IL1403中位置发生了变化。
8.前述任何一项权利要求中的乳酸菌,其中,YjaE蛋白质是失活性。
9.前述任何一项权利要求中的乳酸菌,其中,yjaE基因是失活性。
10.前述任何一项权利要求中的乳酸菌,其中,yjaE蛋白质是失活性,其是由于向yjaE基因引入了适当的改性,优选的适当的改性选自由以下情况组成的组:终止密码子,可导致如移码、缺失和突变的插入。
11.前述任何一项权利要求中的乳酸菌,其中,乳酸菌的特征是具有对至少一种噬菌体有改善的抗性,其中,噬菌体是选自适当的有代表性的不同的噬菌体组,其中,适当的有代表性的不同的噬菌体组优选含有不同的相关的噬菌体,并代表c2种的扁长噬菌体、936种的小的等轴噬菌体、p335种的小的等轴噬菌体和949种的大的等轴噬菌体。
12.前述任何一项权利要求中的乳酸菌,其中,乳酸菌在细胞膜中不含可测量的活性YjaE蛋白质的量。
13.一种起子培养物组合物,包含权利要求1-12中任何一项的乳酸菌,其中,优选的起子培养物组合物具有活细胞的浓度范围是每克组合物中为104-1012cfu。
14.一种制备食品或饲料制品的方法,包含向食品或饲料制品起始材料中加入权利要求13的起子培养物组合物,并在具有正常代谢活性的乳酸菌的条件下将该接种的起始材料保持。
15.一种制备权利要求1-12中任何一项的乳酸菌的方法,其中,由yjaE基因表达的YjaE蛋白质是基本上失活性,包含对yjaE基因进行适当改性,从而不产生活性YjaE蛋白质的表达,其中,yjaE基因包含了选自由以下序列组成的组中的DNA序列:
(a)在SEQ ID NO 1(IL1403 yjaE DNA编码序列)中位于1-2400处的DNA序列;
(b)编码多肽的DNA序列,可选择性具有YjaE蛋白质活性,即与位于SEQ ID NO 2(IL1403 YjaE蛋白质序列)1-799处的多肽序列的至少80%相同。
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US64359105P | 2005-01-14 | 2005-01-14 | |
US60/643,591 | 2005-01-14 | ||
PCT/EP2006/050078 WO2006072631A1 (en) | 2005-01-06 | 2006-01-06 | Bacteriophage resistant lactic acid bacteria |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102770527A (zh) * | 2010-01-28 | 2012-11-07 | 科·汉森有限公司 | 基于噬菌体抗性选择的用于对食品进行质构化的乳酸细菌 |
US9453231B2 (en) | 2010-10-22 | 2016-09-27 | Chr. Hansen A/S | Texturizing lactic acid bacteria strains |
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ATE500742T1 (de) | 2006-07-14 | 2011-03-15 | Legarth Lone | Homofermentierte produkte |
EP2173763A1 (en) | 2007-07-03 | 2010-04-14 | Danmarks Tekniske Universitet | Phage resistance |
US9462817B2 (en) | 2011-02-28 | 2016-10-11 | Franklin Foods Holdings Inc. | Processes for making cheese products utilizing denatured acid whey proteins |
US9635870B2 (en) | 2011-02-28 | 2017-05-02 | Franklin Foods Holdings Inc. | Direct-set cheese |
WO2017178518A1 (en) * | 2016-04-15 | 2017-10-19 | Chr. Hansen A/S | New bacteria |
JP2022548735A (ja) * | 2019-09-18 | 2022-11-21 | アンシリア, インコーポレイテッド | マイクロバイオーム調節のための組成物及び方法 |
WO2021250183A1 (en) * | 2020-06-11 | 2021-12-16 | Chr. Hansen A/S | Method of increasing nisin production in lactococcus lactis |
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FR2807446B1 (fr) | 2000-04-11 | 2005-05-06 | Agronomique Inst Nat Rech | Genomes de lactococcus lactis, polypeptides et utilisations |
FR2807764B1 (fr) * | 2000-04-18 | 2004-09-10 | Agronomique Inst Nat Rech | Mutants de bacteries lactiques surproducteurs d'exopolysaccharides |
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- 2006-01-06 WO PCT/EP2006/050078 patent/WO2006072631A1/en active Application Filing
- 2006-01-06 DK DK06701244.3T patent/DK1838839T4/da active
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102770527A (zh) * | 2010-01-28 | 2012-11-07 | 科·汉森有限公司 | 基于噬菌体抗性选择的用于对食品进行质构化的乳酸细菌 |
US8865238B2 (en) | 2010-01-28 | 2014-10-21 | Chr. Hansen A/S | Bacterium |
US9060524B2 (en) | 2010-01-28 | 2015-06-23 | Chr. Hansen A/S | Bacterium |
CN102770527B (zh) * | 2010-01-28 | 2016-01-20 | 科·汉森有限公司 | 基于噬菌体抗性选择的用于对食品进行质构化的乳酸细菌 |
CN105713853A (zh) * | 2010-01-28 | 2016-06-29 | 科·汉森有限公司 | 基于噬菌体抗性选择的用于对食品进行质构化的乳酸细菌 |
US9416351B2 (en) | 2010-01-28 | 2016-08-16 | Chr. Hansen A/S | Lactic bacterium for texturizing food products selected on basis of phage resistance |
US9562221B2 (en) | 2010-01-28 | 2017-02-07 | Chr. Hansen A/S | Lactic bacterium for texturizing food products selected on the basis of phage resistance |
CN105713853B (zh) * | 2010-01-28 | 2020-07-17 | 科·汉森有限公司 | 基于噬菌体抗性选择的用于对食品进行质构化的乳酸细菌 |
US9453231B2 (en) | 2010-10-22 | 2016-09-27 | Chr. Hansen A/S | Texturizing lactic acid bacteria strains |
US10392597B2 (en) | 2010-10-22 | 2019-08-27 | Chr. Hanse A/S | Texturizing lactic acid bacteria strains |
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DK1838839T3 (da) | 2011-11-07 |
DK1838839T4 (da) | 2020-07-13 |
RU2007129848A (ru) | 2009-02-20 |
EP1838839B2 (en) | 2020-04-15 |
WO2006072631A1 (en) | 2006-07-13 |
PL1838839T3 (pl) | 2012-01-31 |
EP1838839B1 (en) | 2011-07-27 |
ATE517981T1 (de) | 2011-08-15 |
EP1838839A1 (en) | 2007-10-03 |
US8137950B2 (en) | 2012-03-20 |
CN101175848B (zh) | 2014-07-02 |
AU2006204470A1 (en) | 2006-07-13 |
BRPI0606433A2 (pt) | 2009-06-30 |
US20080317903A1 (en) | 2008-12-25 |
PL1838839T5 (pl) | 2020-11-16 |
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