甘油去阻遏表型巴斯德毕赤酵母（Pichia Pastoris）的构建及其初步研究
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摘要
1983年美国Wegner等人最先发展了以甲基营养型酵母(methylotrophic yeast)为代表的第二代酵母表达系统。其中以Pichia pastoris(巴斯德毕赤酵母)为宿主的外源基因表达系统近年来发展最为迅速,应用也最为广泛。毕赤酵母系统的广泛应用,原因在于该系统除了具有一般酵母所具有的特点外,还有以下几个优点:1)具有醇氧化酶1(AOX1)基因启动子,这是目前最强,调控机理最严格的启动子之一;2)表达质粒是整合型载体,能在基因组的特定位点以单拷贝或多拷贝的形式稳定整合,克服了酿酒酵母中附加体形式质粒的遗传不稳定性缺陷;3)生产繁殖速度快,营养要求低,培养基经济廉价,具强烈的好氧生长偏爱性,能够耐受较高的流体净压,故可高密度发酵培养,在发酵罐中细胞干重可达120g/L以上,便于大规模工业化生产;4)毕赤酵母中存在过氧化物酶体,表达的蛋白贮存其中,可免受蛋白酶的降解,而且减少对细胞的毒害作用;5)超糖基化发生的概率较小,分泌蛋白的核心糖链结构是Man8GlcNAc2或Man9GlcNAc2,糖链平均为8-14个甘露糖基,可避免酿酒酵母菌中高甘露糖型寡糖链的超糖基化,而且Pichia pastoris可形成分支糖链,但糖链末端不存在α-1,3甘露聚糖(α-1,3甘露聚糖是人体内不存在的一种糖链结构,所以这种结构能引起人体不良有害的免疫反应),故N-乙酰糖基化修饰较酿酒酵酵母更接近于高等真核生物;6)毕赤酵母分泌到胞外的内源性蛋白的量很少,分泌到胞外的重组蛋白的纯度相对较高,易于纯化,非常适用于基础研究领域;7)另外,表达的外源蛋白既可存在于胞内,又可分泌到胞外。
到目前为止,巴斯德毕赤酵母基因表达系统已高效表达了HBsAg、HSA、TNF、EGF、破伤风毒素C片段和基因工程抗体等700多种外源基因,表达量最高可达到每升克级水平,证实该系统为高效、实用、简便,以提高表达量并保持产物生物学活性为突出特征的外源基因表达系统,而且非常适宜扩大为工业规模。
虽然毕赤酵母表达系统由于其一些显著的优点,不仅应用于工业生产而且被应用到了基础研究领域,但是它还不是一个非常完善的表达系统,目前还不可能完全替代其他的表达系统,具有一定的相对局限性:1)发酵周期较长,易污染,且长时间发酵不利于外源蛋白的表达;2)一些外源蛋白在毕赤酵母分泌过程中,会受到某些蛋白酶的作用而发生降解,而且降解的蛋白随成熟的蛋白一起分泌至胞外,不仅降低了目的蛋白的的产量,而且由于在表达上清中降解蛋白的存在,给后续分离纯化带来了不利的影响;3)虽然毕赤酵母表达系统具有一个较完善的蛋白质翻译后修饰加工和正确折叠的机制,但是与高等真核生物的对应机制还是有所差别,尤其是在糖基化修饰方面:毕赤酵母中N-连接糖链的结构可表示为MannGlcNAc2,但高等真核生物表达的糖蛋白(尤其是复杂型寡糖链结构)还包括唾液酸、半乳糖和果糖等糖基,由于寡糖连的结构常常涉及蛋白的免疫原性,所以在表达高等真核来源的糖蛋白时,毕赤酵母表达系统所得的产物很可能就不是其天然结构,故而影响蛋白的一些生物学性质;4)到目前为止,毕赤酵母表达的重组蛋白的表达水平不均一,有的已达到每升克级的水平,但有些蛋白有可能只有每升毫克级甚至是微克级水平,尤其是在表达一些低分子量的蛋白时,表达水平往往比较低,因此,毕赤酵母表达系统还不是个很广谱的表达宿主;5)由于AOX1启动子受分解代谢物阻遏作用的调控,Mut+表型的毕赤酵母菌如果进行大规模发酵罐培养,一般分成三相发酵,即甘油批次相、甘油补料相和甲醇诱导相。然而,由于甘油批次相和甘油补料相积累了大量的菌体,在诱导相需提供大量的甲醇才能维持所有菌体正常的碳源需要以及满足AOX1启动子的高效诱导,但是高浓度甲醇易挥发具易燃性,以甲醇为原料培养具一定的安全隐患,对发酵罐安全系数要求高,增加发酵罐设计成本,从而增加了生产成本;同时,由于采用三相发酵法,人为延长了发酵周期,导致过程消耗增加,从而也增加了生产成本。
毕赤酵母的这些局限,尤其是三相发酵法引起的毕赤酵母一些工业发酵急需解决的问题,是本课题关注的核心。导致这些问题的根本原因,还是由于AOX1启动子受分解代谢物阻遏作用的调控,这里主要表现为甲醇对AOX1启动子的诱导活化作用受到甘油等抑制性碳源的抑制。因此,要解决三相发酵法带来的这些问题,首先需要对AOX1启动子顺式作用元件及其反式作用因子进行探索,弄清楚AOX1启动子分解代谢物阻遏机制。
在酿酒酵母、假丝酵母和汉逊多形酵母中,通过各种突变技术,分离得到与分解代谢物阻遏相关的基因,如GCR1、MIG1等。在巴斯德毕赤酵母分解代谢物阻遏方面,日本已有研究者通过化学诱变技术筛选得到一株葡萄糖去阻遏的酵母菌株,并申请了专利。因此,本研究拟使用LacZ蛋白作为报告蛋白,在GS115 yps1?菌株中通过两个突变方案构建甘油去阻遏的巴斯德毕赤酵母菌株:第一,通过REMI技术在GS115 yps1?菌株基因组引入随机突变,致使与甘油阻遏相关基因失活,然后筛选去阻遏菌株;第二,通过Error-prone PCR技术,扩增AOX1启动子,得到含有多个突变的启动子,再将此突变启动子克隆至表达载体LacZ基因上游,以控制LacZ基因表达,然后,将此载体转化GS115 yps1?酵母细胞并筛选甘油去阻遏的菌株。通过REMI技术,本研究获得在BMGMY培养基中表达LacZ的GS115-pPIC9-LacZ yps1? GR?突变株,并通过质粒拯救和TAIL-PCR技术从该菌株中分离得到一个与甘油阻遏相关的基因GR1,结合Cre-loxP重组敲除技术将其敲除失活,得到GS115 yps1? gr1?菌株,该菌株表现为甘油去阻遏,即能够在甘油存在下由甲醇诱导表达外源蛋白,如LacZ和HSA-AX15(R13K)蛋白。通过Error-prone PCR技术,本研究构建了AOX1启动子随机突变库,并将其转化GS115 yps1?酵母细胞,筛选得到三株在BMGMY培养基中表达LacZ的GS115-pPIC9ZαA-LacZ yps1? ep1,2,3菌株,在此基础上初步确定了AOX1启动子上与甘油阻遏相关的区域,而且将该相关区域突变的启动子克隆至pPIC9b载体,构建得到pPIC9AM载体,然后,将该质粒转化GS115 yps1?酵母细胞酵母细胞,所得菌株能够在甘油存在下由甲醇诱导表达外源蛋白,如LacZ和HSA-AX15(R13K)蛋白。
In 1983, Wegner, et al. in America had firstly developed the methylotrophic yeasts representative of the second generation of yeast expression system. Wherein, the expression system using Pichia pastoris as a host was developed rapidly and used extensively. Extensive application of Pichia pastoris expression system is due to the fact that, with the exception of all characteristics belonging to common yeasts, it has also the followings advantages: 1) it uses alcohol oxidase 1 (AOX1) promoter which is one of the strongest and most strict promoters at present; 2) the expression plasmids used are integrating vectors and can integrate in the form of single or multiple copies, overcoming genetic instability of episomal plasmid in Saccharomyces cerevisiae; 3) it grows rapidly, has a low requirement for nutrition with lost cost in medium, has a strong aerobic preference, and can tolerate a higher hydrodynamic pressure, which thus is able to be fermented in high density, be up to 120g/L for the dry weight of cells in fermentor, and suitable for large-scale industrialized production; 4) there is peroxisome in Pichia pastoris cell, in which the expressed proteins are stored, can avoid being degraded by protease, and reduces the toxicity to cell; 5) the core carbohydrate in secreted proteins is Man8GlcNAc2 or Man9GlcNAc2 with low probability of superglycosylation and average 8-14 mannose residues in sugar chain, which can avoid the superglycosylation of high mannose oligosaccharide chain in Saccharomyces cerevisiae, and there is branched sugar chain in Pichia pastoris but noα-1,3 mannan (it is not present naturally in human, so this structure may cause adverse immune reaction in human body), so its N-acetyl glycosylation modification is closer to that in high eukaryotes than that in Saccharomyces cerevisiae; 6) there is seldom endogenous proteins secreted to outside of Pichia pastoris cell and extracellular recombinant proteins have relative high purity and are easy to be purified, so it is believable that it is very suitable for fundamental study; 7) in addition, the expressed foreign proteins can be not only present intracellularly but also secreted extracellularly.
So far, high performance expression using Pichia pastoris has been achieved for more than 700 heterologous proteins, e.g., HBsAg, HSA, TNF, EGF, the C fragment of tetanus toxin, genetic engineering antibodies, among which the expression level can be up to gram grade per liter, confirming that this system is an expression system for heterologous gene characterized by high performance, utility, convenience, increased expression, and retention of biological activity in product, and suitable extremely for industrial scale.
Although the Pichia pastoris expression system is used, due to its some outstanding advantages, to both industrial production and fundamental study, it is still not a very complete expression system and, at present, cannot substitute for other expression system, which thus has some limitations: 1) there is longer fermentation period, tendency of being easy to be polluted, and detrimental effect on the expression of heterologous proteins during long-time fermentation; 2) some foreign proteins are degraded by some proteases during secretion from Pichia pastoris, and the degraded proteins are secreted along with intact proteins to cell outside, which not only reduces the yield of protein of interest but also cause adverse effect on subsequent purification because of the degraded proteins in fermentation supernatant; 3) there are more complete mechanisms of post-translational modification, processing and correct folding, although they are different somewhat from the counterparts in high eukaryotes, especially, for the glycosylation modification: the N-linked sugar chain in Pichia pastoris can be expressed to be Mann GlcNAc2, but the glycoproteins in high eukaryotes also contain some saccharide residues, e.g., sialic acid, galactose, fructose; therefore, due to the structure of oligosaccharide chain is often involved in protein immunogenicity, those glycoproteins derived from high eukaryotes may not have their own natural structure when expressed in Pichia pastoris expression system, thus influencing their biological activities; 4) so far, the expression level in Pichia pastoris are different for different recombinant proteins, e.g., gram grade per liter for some proteins, only milligram even microgram grade per liter for others, especially the proteins with low molecular weight which are often lower in expression level; therefore, Pichia pastoris is still not a very extensive host for expression; 5) the Pichia pastoris strains with Mut+ phenotype are generally cultured by a three-step fermentation process during large-scale fermentation, i.e., glycerol batch phase, glycerol fed-batch phase, and methanol fed-batch induction phase, because of carbon catabolite repression to AOX1 promoter, but a large bulk of methanol, due to high-density cells achieved at glycerol batch phase and glycerol fed-batch phase, is required at methanol fed-batch induction phase to supply the carbon for maintaining cell growth and the inducer for high-performance induction of AOX1 promoter; however, high-concentration methanol volatilizes easily and is inflammable, and culture using methanol at high level as sole carbon has a certain risk which increases the design parameters of fermentor required for safety, thus increasing design cost of fermentor and finally the production cost; meanwhile, using the three-step fermentation process prolongs fermentation period, resulting in increased consumption during fermentation and thus increasing production cost.
In these limitations, the problems caused due to the three-step fermentation process in the large-scale fermentation of Pichia pastoris are focuses in this study. The root cause leading to these problems are due to the fact that AOX1 promoter is regulated by carbon catabolite repression, here mainly showing that the induction and activation of AOX1 promoter by methanol are inhibited by some inhibitory carbon sources, e.g. glycerol. Therefore, to overcome these problem caused by the three-step fermentation process, first effort should be done for exploring cis-acting elements in AOX1 promoter and their trans-acting factors to make clear the mechanism of carbon catabolite repression occurring in AOX1 promoter.
For Saccharomyces cerevisiae, Candida mycoderma, and Hansenula polymorpha, many genes relevant to carbon catabolite repression, e.g., GCR1, MIG1, are isolated using various kinds of mutation techniques. In addition, in the aspect of carbon catabolite repression in Pichia pastoris, some scientists in Japan had contructed a strain deficient in glucose catabolite repression using a chemical mutagenesis technique and obtained a relevant patent. Therefore, this study is to construct the Pichia pastoris strain deficient in glycerol catabolite repression in GS115 yps1? strain using LacZ as reporter by two mutation protocols: in the first one, random mutation is introduced into the genome of GS115 yps1? strain using REMI technique to inactivate the genes related to glycerol catabolite repression, and screening is then done for the strain deficient in glycerol catabolite repression; in the second one, error-prone PCR is used to amplify the AOX1 promoter, the product of which will contain multiple mutations and cloned into upstream of LacZ gene in pPIC9-LacZ plasmid to control its expression, and the resulting plasmid is used to transform GS115 yps1? cell followed by screening for the strain deficient in glycerol catabolite repression. A GS115-pPIC9-LacZ yps1? GR? able to expression LacZ in BMGMY medium was obtained by REMI technique and a gene related to glycerol catabolite repression, called GR1 gene, was also isolated with plasmid rescue and TAIL-PCR techniques from this strain. GS115 yps1? gr1? strain was obtained after disruption of GR1 gene using Cre-loxP system, and showed the deficiency in glycerol catabolite repression, that is, expression of heterologous proteins can be induced in GS115 yps1? gr1? strain by methanol in the presence of glycerol, e.g., LacZ, HSA-AX15(R13K). A random mutant library was constructed using Error-prone PCR and then introduced into GS115 yps1? cell by transformation. Finally, three strains that can express LacZ in BMGMY were obtained and refered to as GS115-pPIC9ZαA-LacZ yps1? ep1, 2 and 3, respectively. Based on the three strains, we initially determined those regions related to glycerol catabolite repression in AOX1 promoter, and cloned the relevant regions with mutations into pPIC9b vector. As a result, pPIC9AM vector was obtained and then introduced into GS115 yps1? cell by transformation. The resulting strain can express the heterologous proteins, e.g., LacZ, HSA-AX15(R13K), under induction by methanol in the presence of glycerol.
到目前为止,巴斯德毕赤酵母基因表达系统已高效表达了HBsAg、HSA、TNF、EGF、破伤风毒素C片段和基因工程抗体等700多种外源基因,表达量最高可达到每升克级水平,证实该系统为高效、实用、简便,以提高表达量并保持产物生物学活性为突出特征的外源基因表达系统,而且非常适宜扩大为工业规模。
虽然毕赤酵母表达系统由于其一些显著的优点,不仅应用于工业生产而且被应用到了基础研究领域,但是它还不是一个非常完善的表达系统,目前还不可能完全替代其他的表达系统,具有一定的相对局限性:1)发酵周期较长,易污染,且长时间发酵不利于外源蛋白的表达;2)一些外源蛋白在毕赤酵母分泌过程中,会受到某些蛋白酶的作用而发生降解,而且降解的蛋白随成熟的蛋白一起分泌至胞外,不仅降低了目的蛋白的的产量,而且由于在表达上清中降解蛋白的存在,给后续分离纯化带来了不利的影响;3)虽然毕赤酵母表达系统具有一个较完善的蛋白质翻译后修饰加工和正确折叠的机制,但是与高等真核生物的对应机制还是有所差别,尤其是在糖基化修饰方面:毕赤酵母中N-连接糖链的结构可表示为MannGlcNAc2,但高等真核生物表达的糖蛋白(尤其是复杂型寡糖链结构)还包括唾液酸、半乳糖和果糖等糖基,由于寡糖连的结构常常涉及蛋白的免疫原性,所以在表达高等真核来源的糖蛋白时,毕赤酵母表达系统所得的产物很可能就不是其天然结构,故而影响蛋白的一些生物学性质;4)到目前为止,毕赤酵母表达的重组蛋白的表达水平不均一,有的已达到每升克级的水平,但有些蛋白有可能只有每升毫克级甚至是微克级水平,尤其是在表达一些低分子量的蛋白时,表达水平往往比较低,因此,毕赤酵母表达系统还不是个很广谱的表达宿主;5)由于AOX1启动子受分解代谢物阻遏作用的调控,Mut+表型的毕赤酵母菌如果进行大规模发酵罐培养,一般分成三相发酵,即甘油批次相、甘油补料相和甲醇诱导相。然而,由于甘油批次相和甘油补料相积累了大量的菌体,在诱导相需提供大量的甲醇才能维持所有菌体正常的碳源需要以及满足AOX1启动子的高效诱导,但是高浓度甲醇易挥发具易燃性,以甲醇为原料培养具一定的安全隐患,对发酵罐安全系数要求高,增加发酵罐设计成本,从而增加了生产成本;同时,由于采用三相发酵法,人为延长了发酵周期,导致过程消耗增加,从而也增加了生产成本。
毕赤酵母的这些局限,尤其是三相发酵法引起的毕赤酵母一些工业发酵急需解决的问题,是本课题关注的核心。导致这些问题的根本原因,还是由于AOX1启动子受分解代谢物阻遏作用的调控,这里主要表现为甲醇对AOX1启动子的诱导活化作用受到甘油等抑制性碳源的抑制。因此,要解决三相发酵法带来的这些问题,首先需要对AOX1启动子顺式作用元件及其反式作用因子进行探索,弄清楚AOX1启动子分解代谢物阻遏机制。
在酿酒酵母、假丝酵母和汉逊多形酵母中,通过各种突变技术,分离得到与分解代谢物阻遏相关的基因,如GCR1、MIG1等。在巴斯德毕赤酵母分解代谢物阻遏方面,日本已有研究者通过化学诱变技术筛选得到一株葡萄糖去阻遏的酵母菌株,并申请了专利。因此,本研究拟使用LacZ蛋白作为报告蛋白,在GS115 yps1?菌株中通过两个突变方案构建甘油去阻遏的巴斯德毕赤酵母菌株:第一,通过REMI技术在GS115 yps1?菌株基因组引入随机突变,致使与甘油阻遏相关基因失活,然后筛选去阻遏菌株;第二,通过Error-prone PCR技术,扩增AOX1启动子,得到含有多个突变的启动子,再将此突变启动子克隆至表达载体LacZ基因上游,以控制LacZ基因表达,然后,将此载体转化GS115 yps1?酵母细胞并筛选甘油去阻遏的菌株。通过REMI技术,本研究获得在BMGMY培养基中表达LacZ的GS115-pPIC9-LacZ yps1? GR?突变株,并通过质粒拯救和TAIL-PCR技术从该菌株中分离得到一个与甘油阻遏相关的基因GR1,结合Cre-loxP重组敲除技术将其敲除失活,得到GS115 yps1? gr1?菌株,该菌株表现为甘油去阻遏,即能够在甘油存在下由甲醇诱导表达外源蛋白,如LacZ和HSA-AX15(R13K)蛋白。通过Error-prone PCR技术,本研究构建了AOX1启动子随机突变库,并将其转化GS115 yps1?酵母细胞,筛选得到三株在BMGMY培养基中表达LacZ的GS115-pPIC9ZαA-LacZ yps1? ep1,2,3菌株,在此基础上初步确定了AOX1启动子上与甘油阻遏相关的区域,而且将该相关区域突变的启动子克隆至pPIC9b载体,构建得到pPIC9AM载体,然后,将该质粒转化GS115 yps1?酵母细胞酵母细胞,所得菌株能够在甘油存在下由甲醇诱导表达外源蛋白,如LacZ和HSA-AX15(R13K)蛋白。
In 1983, Wegner, et al. in America had firstly developed the methylotrophic yeasts representative of the second generation of yeast expression system. Wherein, the expression system using Pichia pastoris as a host was developed rapidly and used extensively. Extensive application of Pichia pastoris expression system is due to the fact that, with the exception of all characteristics belonging to common yeasts, it has also the followings advantages: 1) it uses alcohol oxidase 1 (AOX1) promoter which is one of the strongest and most strict promoters at present; 2) the expression plasmids used are integrating vectors and can integrate in the form of single or multiple copies, overcoming genetic instability of episomal plasmid in Saccharomyces cerevisiae; 3) it grows rapidly, has a low requirement for nutrition with lost cost in medium, has a strong aerobic preference, and can tolerate a higher hydrodynamic pressure, which thus is able to be fermented in high density, be up to 120g/L for the dry weight of cells in fermentor, and suitable for large-scale industrialized production; 4) there is peroxisome in Pichia pastoris cell, in which the expressed proteins are stored, can avoid being degraded by protease, and reduces the toxicity to cell; 5) the core carbohydrate in secreted proteins is Man8GlcNAc2 or Man9GlcNAc2 with low probability of superglycosylation and average 8-14 mannose residues in sugar chain, which can avoid the superglycosylation of high mannose oligosaccharide chain in Saccharomyces cerevisiae, and there is branched sugar chain in Pichia pastoris but noα-1,3 mannan (it is not present naturally in human, so this structure may cause adverse immune reaction in human body), so its N-acetyl glycosylation modification is closer to that in high eukaryotes than that in Saccharomyces cerevisiae; 6) there is seldom endogenous proteins secreted to outside of Pichia pastoris cell and extracellular recombinant proteins have relative high purity and are easy to be purified, so it is believable that it is very suitable for fundamental study; 7) in addition, the expressed foreign proteins can be not only present intracellularly but also secreted extracellularly.
So far, high performance expression using Pichia pastoris has been achieved for more than 700 heterologous proteins, e.g., HBsAg, HSA, TNF, EGF, the C fragment of tetanus toxin, genetic engineering antibodies, among which the expression level can be up to gram grade per liter, confirming that this system is an expression system for heterologous gene characterized by high performance, utility, convenience, increased expression, and retention of biological activity in product, and suitable extremely for industrial scale.
Although the Pichia pastoris expression system is used, due to its some outstanding advantages, to both industrial production and fundamental study, it is still not a very complete expression system and, at present, cannot substitute for other expression system, which thus has some limitations: 1) there is longer fermentation period, tendency of being easy to be polluted, and detrimental effect on the expression of heterologous proteins during long-time fermentation; 2) some foreign proteins are degraded by some proteases during secretion from Pichia pastoris, and the degraded proteins are secreted along with intact proteins to cell outside, which not only reduces the yield of protein of interest but also cause adverse effect on subsequent purification because of the degraded proteins in fermentation supernatant; 3) there are more complete mechanisms of post-translational modification, processing and correct folding, although they are different somewhat from the counterparts in high eukaryotes, especially, for the glycosylation modification: the N-linked sugar chain in Pichia pastoris can be expressed to be Mann GlcNAc2, but the glycoproteins in high eukaryotes also contain some saccharide residues, e.g., sialic acid, galactose, fructose; therefore, due to the structure of oligosaccharide chain is often involved in protein immunogenicity, those glycoproteins derived from high eukaryotes may not have their own natural structure when expressed in Pichia pastoris expression system, thus influencing their biological activities; 4) so far, the expression level in Pichia pastoris are different for different recombinant proteins, e.g., gram grade per liter for some proteins, only milligram even microgram grade per liter for others, especially the proteins with low molecular weight which are often lower in expression level; therefore, Pichia pastoris is still not a very extensive host for expression; 5) the Pichia pastoris strains with Mut+ phenotype are generally cultured by a three-step fermentation process during large-scale fermentation, i.e., glycerol batch phase, glycerol fed-batch phase, and methanol fed-batch induction phase, because of carbon catabolite repression to AOX1 promoter, but a large bulk of methanol, due to high-density cells achieved at glycerol batch phase and glycerol fed-batch phase, is required at methanol fed-batch induction phase to supply the carbon for maintaining cell growth and the inducer for high-performance induction of AOX1 promoter; however, high-concentration methanol volatilizes easily and is inflammable, and culture using methanol at high level as sole carbon has a certain risk which increases the design parameters of fermentor required for safety, thus increasing design cost of fermentor and finally the production cost; meanwhile, using the three-step fermentation process prolongs fermentation period, resulting in increased consumption during fermentation and thus increasing production cost.
In these limitations, the problems caused due to the three-step fermentation process in the large-scale fermentation of Pichia pastoris are focuses in this study. The root cause leading to these problems are due to the fact that AOX1 promoter is regulated by carbon catabolite repression, here mainly showing that the induction and activation of AOX1 promoter by methanol are inhibited by some inhibitory carbon sources, e.g. glycerol. Therefore, to overcome these problem caused by the three-step fermentation process, first effort should be done for exploring cis-acting elements in AOX1 promoter and their trans-acting factors to make clear the mechanism of carbon catabolite repression occurring in AOX1 promoter.
For Saccharomyces cerevisiae, Candida mycoderma, and Hansenula polymorpha, many genes relevant to carbon catabolite repression, e.g., GCR1, MIG1, are isolated using various kinds of mutation techniques. In addition, in the aspect of carbon catabolite repression in Pichia pastoris, some scientists in Japan had contructed a strain deficient in glucose catabolite repression using a chemical mutagenesis technique and obtained a relevant patent. Therefore, this study is to construct the Pichia pastoris strain deficient in glycerol catabolite repression in GS115 yps1? strain using LacZ as reporter by two mutation protocols: in the first one, random mutation is introduced into the genome of GS115 yps1? strain using REMI technique to inactivate the genes related to glycerol catabolite repression, and screening is then done for the strain deficient in glycerol catabolite repression; in the second one, error-prone PCR is used to amplify the AOX1 promoter, the product of which will contain multiple mutations and cloned into upstream of LacZ gene in pPIC9-LacZ plasmid to control its expression, and the resulting plasmid is used to transform GS115 yps1? cell followed by screening for the strain deficient in glycerol catabolite repression. A GS115-pPIC9-LacZ yps1? GR? able to expression LacZ in BMGMY medium was obtained by REMI technique and a gene related to glycerol catabolite repression, called GR1 gene, was also isolated with plasmid rescue and TAIL-PCR techniques from this strain. GS115 yps1? gr1? strain was obtained after disruption of GR1 gene using Cre-loxP system, and showed the deficiency in glycerol catabolite repression, that is, expression of heterologous proteins can be induced in GS115 yps1? gr1? strain by methanol in the presence of glycerol, e.g., LacZ, HSA-AX15(R13K). A random mutant library was constructed using Error-prone PCR and then introduced into GS115 yps1? cell by transformation. Finally, three strains that can express LacZ in BMGMY were obtained and refered to as GS115-pPIC9ZαA-LacZ yps1? ep1, 2 and 3, respectively. Based on the three strains, we initially determined those regions related to glycerol catabolite repression in AOX1 promoter, and cloned the relevant regions with mutations into pPIC9b vector. As a result, pPIC9AM vector was obtained and then introduced into GS115 yps1? cell by transformation. The resulting strain can express the heterologous proteins, e.g., LacZ, HSA-AX15(R13K), under induction by methanol in the presence of glycerol.
引文
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