UAS重复对脆壁克鲁维酵母乳糖酶基因表达的影响
摘要
乳糖酶(lactase)又称为β-D-半乳糖苷半乳糖水解酶(β-D-galactohydrolase.E.C.3.2.1.23),简称β-半乳糖苷酶(β-galactosidase)。生产食品级乳糖酶菌株主要有乳酸克鲁维酵母、脆壁克鲁维酵母等克鲁维酵母。克鲁维酵母是天然新鲜牛乳中含有的微生物,来源于克鲁维酵母菌的乳糖酶是一种中性乳糖酶,其最适pH值与天然牛乳的pH值(6.6-6.8)最为接近,同时,来源于克鲁维酵母菌的乳糖酶的活性较高,适合工业化处理牛乳和乳清。此外,来源于克鲁维酵母菌的乳糖酶的另一优点是在低温时仍具有较好的活力,在低温环境下,容易引起牛奶酸败变质的腐败菌生长速度基本停止,可利用此性质在低温下分解牛奶和乳清的乳糖,避免了乳品加工过程中的杂菌污染。
但是,来源于克鲁维酵母的乳糖酶产量并不理想,限制了乳糖酶的快速发展。采用基因工程手段大量提高乳糖酶表达量是促进乳糖酶的发展方法之一。因此,本研究以脆壁克鲁维酵母乳糖酶基因为研究对象,并从启动子的改造入手,设计和构建了新型脆壁克鲁维表达载体,为乳糖酶在脆壁克鲁维酵母中安全高效表达探讨了条件和方法。研究内容及结果如下:
1.脆壁克鲁维酵母Kan抗性标记载体的构建
根据Genbank上已发表的序列,设计引物,应用PCR法克隆得到了含有Lac启动子上游激活序列和部分乳糖酶基因的目的片段PLac。根据Genbank上已发表的酵母乳糖酶基因序列和质粒pPic9k序列设计双酶切位点,构建质粒pPkan。此质粒在含有Kan抗性基因的基础上,在Kan抗性基因的两端各连接1 kb左右的同源臂,用以转入酵母。将线性化的质粒pPkan,利用电击法转入脆壁克鲁维酵母,构建具有Kan抗性的脆壁克鲁维酵母K.f.-kan,同时,Kan基因整合在启动子的位置上,利用同源重组敲除启动子区域以及部分乳糖酶基因,使乳糖酶基因失活,相当于在增加Kan抗性基因的同时增加了一个标记。脆壁克鲁维酵母K.f.-kan可作为构建改造启动子,尝试不同信号肽的中间载体。
2.脆壁克鲁维酵母UAS重复载体的构建
根据Genbank上已发表的酵母乳糖酶基因序列,设计两对引物,应用重叠延伸PCR法,构建了含有一个重复的上游激活序列的质粒pTUASR,此质粒在UAS重复区两端含有和pPkan相同的同源臂,用以进行转化。用电击转化法将线性化质粒质粒pTUASR转化脆壁克鲁维酵母K.f.-kan,获得脆壁克鲁维酵母重组转化子K.f.-UASR。脆壁克鲁维酵母K.f.-UASR在原启动子的基础上增加了一个UAS重复区域,并恢复敲除掉的乳糖酶基因,恢复了乳糖酶活性。
3.转化子的筛选及乳糖酶活性检测
PCR筛选脆壁克鲁维酵母转化子,ONPG法测定阳性转化子发酵液乳糖酶活性。结果表明,具有UAS重复的K.f.-UASR转化子较出发菌株Kluyveromyces fragilis乳糖酶活性从15.646 U/ml提高至22.823 U/ml,提高了乳糖酶的表达量。
Lactose is also known asβ-D-galactohydrolase . Product food-grade lactose enzyme strains are mainly Pichia, Kluyveromyces Fragilis, etc. Kluyveromyces Fragilis is from the natural fresh milk,and its lactose enzyme is a kind of neutral lactose enzymes,its optimal pH is 6.6-6.8 which is close to milk ,and at the same time ,Kluyveromyces Fragilis has a higher production and enzyme activity of lactose.In addition, the lactose enzyme from Kluyveromyces Fragilis still has good enzyme activity in low temperature,and the low temperature environment will avoid the contamination from process of miscellaneous bacteria which is apt to cause the milk contaminated.But the traditional industrial methods of enzyme production such as has a low volume, not easy to separate purpose protein, not accord with food-grade disadvantages express requirement have limited the fast development of lactose. Using genetic engineering means to improve the expression of lactose will promote the development of lactose .
In this paper,we study on lactose gene of Kluyveromyces Fragilis, and from the transformation of promoters, design and construct the new Kluyveromyces Fragilis expression vector for lactose, and it is to efficiently express discusses the safety in conditions and methods, the content and the results are as follows:
1. Construction of Kluyveromyces Fragilis resistant vector K.f.-kan
According to the Genbank published lactose gene sequence of Kluyveromyces Fragilis and the sequence of plasmid pPic9k,we design double enzyme cutting sites to construct the plasmid pPkan. The plasmid contains the resistance gene Kan, and based on the gene of Kan, each side of the resistance gene has a homologous arm around 1kb to turn to Kluyveromyces Fragilis . Using the linearization of plasmids pPkan transformate into Kluyveromyces Fragilis to construct Kluyveromyces Fragilis-kan which with resistance of kan ,and meanwhile, we make it instead of Lac promoter integration in its position.Using the homologous recombination to knockout promoter region and part of lactose enzyme gene, to make the lactose gene inactivation.It is meant that the resistance gene kan is adding a mark. Kluyveromyces Fragilis-kan can be used as constructing promoter, try different transformation among carrier signal peptide.
2. Construction of contains a repeat of upstream activation sequence vector K.f.-UASR
According to the Genbank published lactose gene sequence of Kluyveromyces Fragilis , design a pair of primers, using the method of SOE-PCR, constructing the plasmid pTUASR of which contains a repeat of the upstream activation sequence, the plasmid contains the same homologous arm with pPkan.With Kluyveromyces Fragilis for receptor bacteria, using the method of electric shocks reforming process we transformate the linearization plasmid of pTUASR into Kluyveromyces Fragilis-kan, and get Kluyveromyces Fragilis-UASR. The Kluyveromyces Fragilis -UASR has a repeat of UAS instead of the original promoters, and restore the lactose enzyme gene knock put away, restored lactose .
3.Screening of transformation, and detection of enzyme activity
Using PCR to determate the positive K.f.-UASR ,and using the method of ONPG to measure the activity of lactose in fermentation. The results show that the activity of lactose of K.f.-UASR is 22.823 U/ml instead of 15.646 U/ml of Kluyveromyces fragilis.
但是,来源于克鲁维酵母的乳糖酶产量并不理想,限制了乳糖酶的快速发展。采用基因工程手段大量提高乳糖酶表达量是促进乳糖酶的发展方法之一。因此,本研究以脆壁克鲁维酵母乳糖酶基因为研究对象,并从启动子的改造入手,设计和构建了新型脆壁克鲁维表达载体,为乳糖酶在脆壁克鲁维酵母中安全高效表达探讨了条件和方法。研究内容及结果如下:
1.脆壁克鲁维酵母Kan抗性标记载体的构建
根据Genbank上已发表的序列,设计引物,应用PCR法克隆得到了含有Lac启动子上游激活序列和部分乳糖酶基因的目的片段PLac。根据Genbank上已发表的酵母乳糖酶基因序列和质粒pPic9k序列设计双酶切位点,构建质粒pPkan。此质粒在含有Kan抗性基因的基础上,在Kan抗性基因的两端各连接1 kb左右的同源臂,用以转入酵母。将线性化的质粒pPkan,利用电击法转入脆壁克鲁维酵母,构建具有Kan抗性的脆壁克鲁维酵母K.f.-kan,同时,Kan基因整合在启动子的位置上,利用同源重组敲除启动子区域以及部分乳糖酶基因,使乳糖酶基因失活,相当于在增加Kan抗性基因的同时增加了一个标记。脆壁克鲁维酵母K.f.-kan可作为构建改造启动子,尝试不同信号肽的中间载体。
2.脆壁克鲁维酵母UAS重复载体的构建
根据Genbank上已发表的酵母乳糖酶基因序列,设计两对引物,应用重叠延伸PCR法,构建了含有一个重复的上游激活序列的质粒pTUASR,此质粒在UAS重复区两端含有和pPkan相同的同源臂,用以进行转化。用电击转化法将线性化质粒质粒pTUASR转化脆壁克鲁维酵母K.f.-kan,获得脆壁克鲁维酵母重组转化子K.f.-UASR。脆壁克鲁维酵母K.f.-UASR在原启动子的基础上增加了一个UAS重复区域,并恢复敲除掉的乳糖酶基因,恢复了乳糖酶活性。
3.转化子的筛选及乳糖酶活性检测
PCR筛选脆壁克鲁维酵母转化子,ONPG法测定阳性转化子发酵液乳糖酶活性。结果表明,具有UAS重复的K.f.-UASR转化子较出发菌株Kluyveromyces fragilis乳糖酶活性从15.646 U/ml提高至22.823 U/ml,提高了乳糖酶的表达量。
Lactose is also known asβ-D-galactohydrolase . Product food-grade lactose enzyme strains are mainly Pichia, Kluyveromyces Fragilis, etc. Kluyveromyces Fragilis is from the natural fresh milk,and its lactose enzyme is a kind of neutral lactose enzymes,its optimal pH is 6.6-6.8 which is close to milk ,and at the same time ,Kluyveromyces Fragilis has a higher production and enzyme activity of lactose.In addition, the lactose enzyme from Kluyveromyces Fragilis still has good enzyme activity in low temperature,and the low temperature environment will avoid the contamination from process of miscellaneous bacteria which is apt to cause the milk contaminated.But the traditional industrial methods of enzyme production such as has a low volume, not easy to separate purpose protein, not accord with food-grade disadvantages express requirement have limited the fast development of lactose. Using genetic engineering means to improve the expression of lactose will promote the development of lactose .
In this paper,we study on lactose gene of Kluyveromyces Fragilis, and from the transformation of promoters, design and construct the new Kluyveromyces Fragilis expression vector for lactose, and it is to efficiently express discusses the safety in conditions and methods, the content and the results are as follows:
1. Construction of Kluyveromyces Fragilis resistant vector K.f.-kan
According to the Genbank published lactose gene sequence of Kluyveromyces Fragilis and the sequence of plasmid pPic9k,we design double enzyme cutting sites to construct the plasmid pPkan. The plasmid contains the resistance gene Kan, and based on the gene of Kan, each side of the resistance gene has a homologous arm around 1kb to turn to Kluyveromyces Fragilis . Using the linearization of plasmids pPkan transformate into Kluyveromyces Fragilis to construct Kluyveromyces Fragilis-kan which with resistance of kan ,and meanwhile, we make it instead of Lac promoter integration in its position.Using the homologous recombination to knockout promoter region and part of lactose enzyme gene, to make the lactose gene inactivation.It is meant that the resistance gene kan is adding a mark. Kluyveromyces Fragilis-kan can be used as constructing promoter, try different transformation among carrier signal peptide.
2. Construction of contains a repeat of upstream activation sequence vector K.f.-UASR
According to the Genbank published lactose gene sequence of Kluyveromyces Fragilis , design a pair of primers, using the method of SOE-PCR, constructing the plasmid pTUASR of which contains a repeat of the upstream activation sequence, the plasmid contains the same homologous arm with pPkan.With Kluyveromyces Fragilis for receptor bacteria, using the method of electric shocks reforming process we transformate the linearization plasmid of pTUASR into Kluyveromyces Fragilis-kan, and get Kluyveromyces Fragilis-UASR. The Kluyveromyces Fragilis -UASR has a repeat of UAS instead of the original promoters, and restore the lactose enzyme gene knock put away, restored lactose .
3.Screening of transformation, and detection of enzyme activity
Using PCR to determate the positive K.f.-UASR ,and using the method of ONPG to measure the activity of lactose in fermentation. The results show that the activity of lactose of K.f.-UASR is 22.823 U/ml instead of 15.646 U/ml of Kluyveromyces fragilis.
引文
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何梅,杨月欣.1999乳糖酶缺乏和乳糖不耐受[J].国外医学卫生学分册,16(6):339-342
李宁,贾英民,韩军.2005.高温乳糖酶产生菌株的诱变选育[J].河北农业大学学报,28(2):64-66
刘文玉,史应武,王杏芹.2007.B-半乳糖苷酶的研究进展[J].新疆农业科学,(5):647-651.
刘媛美. 1996.酵母细胞的电击高频转化[J].暨南大学学报.,16(3):6-9
马孟根,王红宁. 2001巴斯德毕赤酵母表达外源蛋白研究进展.四川农业大学学报.19 (3):277-280
秦燕,宁正祥,胡新宇.2000β-半乳糖苷酶的应用研究进展[J].沈阳农业大学学报, 31(6): 595-599
沈为群,郭杰炎,李水福.1993乳酸克鲁维酵母B-半乳糖苷酶的分离纯化及性质研究[J].生物工程学报,9(4):348-354
王逢,袁勤生,徐康森.2004产乳糖酶价目Kluyveromyces marxianus发酵条件的研究[J].中过医药工业杂志,25(10):587-589
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