酸性蛋白酶基因在毕赤酵母及黑曲霉中的表达
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摘要
酸性蛋白酶是一种能在酸性环境下水解蛋白质的酶类,其最适作用pH值为2.5~5.0。由于酸性蛋白酶具有较好的耐酸性,因此被广泛地应用于食品、医药、轻工、皮革工艺以及饲料加工工业中。宇佐美曲霉是工业化生产酸性蛋白酶的主要菌种,但是酸性蛋白酶的表达受碳源和氮源阻遏的调控和蛋白质的诱导,并受严格的pH控制,导致其发酵条件不易控制,发酵酶活不稳定。利用宇佐美曲霉工业化生产酸性蛋白酶的酶活还有待进一步提高。
真核生物表达系统是高效表达蛋白的有效手段。酵母作为表达外源蛋白的工程菌日益受到重视。它具有基因表达调控和蛋白修饰功能,克服了原核表达系统中产物活性低以及包涵体的变性、复性等的问题。而且,酵母可以通过高密度发酵,且分泌产物与天然蛋白相似,临床应用较安全。同样黑曲霉卓越的表达和分泌能力也一直是吸引人们的最大特点。并具有真核分泌机制,很可能还具有与哺乳动物系统相似的蛋白修饰性能,如高甘露糖型和N-糖基化等,也是表达真核基因的理想系统。通过基因工程手段在酵母和黑曲霉中高效表达酸性蛋白酶,对于简化发酵条件、提高酶制剂产量都具有重要意义。
本研究以酸性蛋白酶基因生产菌种宇佐美曲霉为基因供体,克隆酸性蛋白酶基因(pepA),构建其毕赤酵母表达载体,并通过电击法转化毕赤酵母,以获得高效表达酸性蛋白酶的工程菌。同时,试图通过农杆菌介导法敲除糖化酶生产菌黑曲霉的pyrA基因和ku70基因,获得黑曲霉受体菌,并构建农杆菌介导的pepA基因同源重组载体,为黑曲霉表达系统的建立和实现pepA基因在黑曲霉中的高效表达奠定基础。
主要研究成果如下:
1.基因的克隆
采用RT-PCR方法克隆得到了宇佐美曲霉pepA基因的cDNA序列,采用PCR方法克隆得到了黑曲霉pyrA基因,黑曲霉ku70基因5′和3′同源臂基因,黑曲霉glaA基因启动子和终止子,瑞氏木霉pyrG基因,以及宇佐美曲霉pepA基因。测序结果正确。
2.酸性蛋白酶基因在毕赤酵母中的表达
构建了pepA基因的毕赤酵母表达载体pGAPHαm-pepAc。将载体线性化并通过电击转化毕赤酵母。经转化子PCR鉴定得到重组子GS115-pGAPHαm-pepAc。在最适发酵条件下,GS115-pGAPHαm-pepAc分泌表达的酸性蛋白酶最高酶活为1289.68u/mL。
3.黑曲霉表达载体构建
构建了pyrA、ku70、glaA、pepA基因的相关载体pEMT-pyrA,pEMT-△pyrA,pEMT-△kusA,pEMT-△kusA-pyrG,pEMT-△glaA-pepA,并将这些载体通过冻融法转入到根癌农杆菌GV3101中,以用于下一步转化黑曲霉。
4.黑曲霉pyrA基因缺失突变菌株的构建
以黑曲霉UV48为受体材料,利用农杆菌介导法将表达载体pEMT-△pyrA对黑曲霉进行遗传转化。筛选培养基中加入5-氟乳清酸,获得了一些抗性菌株,但经过PCR鉴定后,发现这些抗性菌株并非pyrA基因缺失突变菌株。此实验在进一步进行中。
Acidic protease is an enzyme which can hydrolyze proteins in the acidic environment, and the optimum pH is from 2.5 to 5.0.As has good acid resistance, Acidic protease is widely used in food, medicine, light industry, leather technology and forage processing industry. Aspergillus usamii is the main species in the industrial production of acid protease, but the expression is regulated by the deter at cabon and nitrogen, induced by proteins, and controlled by the pH. The result is that it is difficult to control the ferment condition and the activity is unstable.In addition, the activity of acid protease produced in Aspergillus usamii needs to be further improved.
Eukaryotic expression system is an effective method in the efficient expression of proteins. Yeast as an engineering strain has received more and mor attention. It has the functions that regulates gene expression and modifies proteins. Meanwhile it overcomes the questions of low activity, denaturation and renaturation of Inclusion body, and so on. Moreover, it could be high-density fermented and the product is similar to natural proteins. The clinical application is safe. Aspergillus niger has been more and more attractive because of the excellent expression and secretion ability. It has the eukaryotic secretion mechanism and may also has the protein modification performance, such as high mannose-type and N-glycosylation. It is an ideal expression system for eukaryotic gene. Efficient expression of acidic protease in yeast and Aspergillus niger by means of genetic engineering has important significance in simplifing the fermentation conditions and increasing the production.
In this study, cloned pepA gene with gene donor Aspergillus usamii, then constructed yeast expression vector, transformated into Pichia pastoris by electroporation, and got engineering strain that express acidic protease gene effectively. Meanwhile, tried to obtain Aspergillus niger as the acceptor with the pyrA gene and kusA gene disrupted mediated by Agrobacterium, and constructed homologous recombination vector of pepA. Laied the basis for establishing the Aspergillus niger expression system and achieving the efficient expression of pepA gene in Aspergillus niger. The major results were as follows:
1. Cloning of genes
The cDNA sequence of pepA gene was cloned from Aspergillus usamii by RT-PCR. PyrA gene, 5' and 3' homologous arm of ku70 gene, promoter and terminator of glaA gene were cloned from Aspergillus niger by PCR. PyrG and pepA genes were cloned from Trichoderma reesei and Aspergillus usamii respectively using PCR. The sequences of these genes were right.
2. The expression of pepA gene in Pichia pastoris
Pichia pastoris expression vector pGAPHαm-pepAc was constructed. The linear expression vectors were transformed into Pichia pastoris by electroporation. Transformants were identified by PCR: GS115-pGAPHαm-pepAc. Under optimum fermentative condition, the activity of the acid protease expression was 1289.68u/mL.
3. Construction of Aspergillus niger expression vectors
Five Aspergillus niger expression vectors were constructed, incuding pEMT-pyrA、pEMT-△pyrA、pEMT-△kusA、pEMT-△kusA-pyrG、pEMT-△glaA–pepA,and then were translated into Agrobacterium GV3101 for Aspergillus niger transformation.
4. Construction of the pyrA gene disruption mutant in Aspergillus niger
The expression vector pEMT-△pyrA was transferred into Aspergillus niger UV48 mediated by Agrobacterium Tumefaciens. At the stage of screening, 5-FOA was added in screening medium.Some resistant mutants were obtained via screening. However, these were not pyrA gene disruption mutants by PCR identified. The study is still in research.
真核生物表达系统是高效表达蛋白的有效手段。酵母作为表达外源蛋白的工程菌日益受到重视。它具有基因表达调控和蛋白修饰功能,克服了原核表达系统中产物活性低以及包涵体的变性、复性等的问题。而且,酵母可以通过高密度发酵,且分泌产物与天然蛋白相似,临床应用较安全。同样黑曲霉卓越的表达和分泌能力也一直是吸引人们的最大特点。并具有真核分泌机制,很可能还具有与哺乳动物系统相似的蛋白修饰性能,如高甘露糖型和N-糖基化等,也是表达真核基因的理想系统。通过基因工程手段在酵母和黑曲霉中高效表达酸性蛋白酶,对于简化发酵条件、提高酶制剂产量都具有重要意义。
本研究以酸性蛋白酶基因生产菌种宇佐美曲霉为基因供体,克隆酸性蛋白酶基因(pepA),构建其毕赤酵母表达载体,并通过电击法转化毕赤酵母,以获得高效表达酸性蛋白酶的工程菌。同时,试图通过农杆菌介导法敲除糖化酶生产菌黑曲霉的pyrA基因和ku70基因,获得黑曲霉受体菌,并构建农杆菌介导的pepA基因同源重组载体,为黑曲霉表达系统的建立和实现pepA基因在黑曲霉中的高效表达奠定基础。
主要研究成果如下:
1.基因的克隆
采用RT-PCR方法克隆得到了宇佐美曲霉pepA基因的cDNA序列,采用PCR方法克隆得到了黑曲霉pyrA基因,黑曲霉ku70基因5′和3′同源臂基因,黑曲霉glaA基因启动子和终止子,瑞氏木霉pyrG基因,以及宇佐美曲霉pepA基因。测序结果正确。
2.酸性蛋白酶基因在毕赤酵母中的表达
构建了pepA基因的毕赤酵母表达载体pGAPHαm-pepAc。将载体线性化并通过电击转化毕赤酵母。经转化子PCR鉴定得到重组子GS115-pGAPHαm-pepAc。在最适发酵条件下,GS115-pGAPHαm-pepAc分泌表达的酸性蛋白酶最高酶活为1289.68u/mL。
3.黑曲霉表达载体构建
构建了pyrA、ku70、glaA、pepA基因的相关载体pEMT-pyrA,pEMT-△pyrA,pEMT-△kusA,pEMT-△kusA-pyrG,pEMT-△glaA-pepA,并将这些载体通过冻融法转入到根癌农杆菌GV3101中,以用于下一步转化黑曲霉。
4.黑曲霉pyrA基因缺失突变菌株的构建
以黑曲霉UV48为受体材料,利用农杆菌介导法将表达载体pEMT-△pyrA对黑曲霉进行遗传转化。筛选培养基中加入5-氟乳清酸,获得了一些抗性菌株,但经过PCR鉴定后,发现这些抗性菌株并非pyrA基因缺失突变菌株。此实验在进一步进行中。
Acidic protease is an enzyme which can hydrolyze proteins in the acidic environment, and the optimum pH is from 2.5 to 5.0.As has good acid resistance, Acidic protease is widely used in food, medicine, light industry, leather technology and forage processing industry. Aspergillus usamii is the main species in the industrial production of acid protease, but the expression is regulated by the deter at cabon and nitrogen, induced by proteins, and controlled by the pH. The result is that it is difficult to control the ferment condition and the activity is unstable.In addition, the activity of acid protease produced in Aspergillus usamii needs to be further improved.
Eukaryotic expression system is an effective method in the efficient expression of proteins. Yeast as an engineering strain has received more and mor attention. It has the functions that regulates gene expression and modifies proteins. Meanwhile it overcomes the questions of low activity, denaturation and renaturation of Inclusion body, and so on. Moreover, it could be high-density fermented and the product is similar to natural proteins. The clinical application is safe. Aspergillus niger has been more and more attractive because of the excellent expression and secretion ability. It has the eukaryotic secretion mechanism and may also has the protein modification performance, such as high mannose-type and N-glycosylation. It is an ideal expression system for eukaryotic gene. Efficient expression of acidic protease in yeast and Aspergillus niger by means of genetic engineering has important significance in simplifing the fermentation conditions and increasing the production.
In this study, cloned pepA gene with gene donor Aspergillus usamii, then constructed yeast expression vector, transformated into Pichia pastoris by electroporation, and got engineering strain that express acidic protease gene effectively. Meanwhile, tried to obtain Aspergillus niger as the acceptor with the pyrA gene and kusA gene disrupted mediated by Agrobacterium, and constructed homologous recombination vector of pepA. Laied the basis for establishing the Aspergillus niger expression system and achieving the efficient expression of pepA gene in Aspergillus niger. The major results were as follows:
1. Cloning of genes
The cDNA sequence of pepA gene was cloned from Aspergillus usamii by RT-PCR. PyrA gene, 5' and 3' homologous arm of ku70 gene, promoter and terminator of glaA gene were cloned from Aspergillus niger by PCR. PyrG and pepA genes were cloned from Trichoderma reesei and Aspergillus usamii respectively using PCR. The sequences of these genes were right.
2. The expression of pepA gene in Pichia pastoris
Pichia pastoris expression vector pGAPHαm-pepAc was constructed. The linear expression vectors were transformed into Pichia pastoris by electroporation. Transformants were identified by PCR: GS115-pGAPHαm-pepAc. Under optimum fermentative condition, the activity of the acid protease expression was 1289.68u/mL.
3. Construction of Aspergillus niger expression vectors
Five Aspergillus niger expression vectors were constructed, incuding pEMT-pyrA、pEMT-△pyrA、pEMT-△kusA、pEMT-△kusA-pyrG、pEMT-△glaA–pepA,and then were translated into Agrobacterium GV3101 for Aspergillus niger transformation.
4. Construction of the pyrA gene disruption mutant in Aspergillus niger
The expression vector pEMT-△pyrA was transferred into Aspergillus niger UV48 mediated by Agrobacterium Tumefaciens. At the stage of screening, 5-FOA was added in screening medium.Some resistant mutants were obtained via screening. However, these were not pyrA gene disruption mutants by PCR identified. The study is still in research.
引文
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