【作者】 王东升；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2011， 博士

【摘要】 扣囊复膜酵母菌(Saccharomycopsis fibuligera)是子囊菌属的一个种,它可以利用淀粉积累海藻糖,并能分泌大量的淀粉酶、酸性蛋白酶、β-葡糖苷酶和其它酶。所以扣囊复膜酵母在发酵行业、医药行业和生物能源工业有着巨大的潜在应用价值,但是人们对它的生理遗传系统了解得还很少。本实验室最近几年对高产海藻糖的扣囊复膜酵母A11菌株进行了大量研究,获得许多重要结果。由于酸性蛋白酶在酸性条件下可以分解其它酶,对其它酶的产生会造成不利的影响。所以本研究试图通过基因敲除法敲掉扣囊复膜酵母A11菌株酸性蛋白酶基因,然后研究该基因的敲除对其它酶的产生和稳定会产生什么样的影响。首先利用编码潮霉素B磷酸转移酶的基因HPT (hygromycin B phosphotransferase)的ORF (Open Reading Frame)替换A11菌株的酸性蛋白酶基因的ORF。获得的敲除菌株A11-a能在含有潮霉素的培养基中生长,并且在牛奶平板上不能形成水解透明圈,而原始菌株A11在牛奶平板上能形成大而清晰的水解透明圈。在培养3 d的情况下,敲除菌株A11-a的酸性蛋白酶和淀粉酶的活性分别为0.89 U/mL和424.7 U/mL,而原始菌株A11分别为13.5 U/mL和259.9 U/mL,说明了酸性蛋白酶的缺失会大大提高敲除菌株淀粉酶的合成能力。敲除菌株A11-a和原始菌株A11产生的粗淀粉酶放置在37℃下2 d,它们残余的活性分别为原来的88.8%和45.5%,说明了酸性蛋白酶的缺失会大大提高敲除菌株淀粉酶的稳定性。敲除菌株A11-a细胞的海藻糖积累量达到了细胞干重的28.3% (w/w),而原始菌株A11只有23.6 (w/w) ,说明了酸性蛋白酶的缺失还会大大提高敲除菌株转化淀粉合成海藻糖的能力。目前如何有效地从酵母细胞中提取海藻糖是一个问题,一般是用0.5M三氯乙酸提取,但是三氯乙酸是蛋白质变性剂,并且具有很强的腐蚀作用。有研究表明当高热敏渗透突变株(highly thermosensitive and permeable mutant)酵母细胞在37℃的低渗透压水中孵育时,细胞内含物能被释放出来。所以本研究利用上述获得的高含海藻糖扣囊复膜酵母A11-a菌株经亚硝基胍诱变获得了这样的高热敏渗透突变菌株A11-b,在37℃的蒸馏水中过夜处理能释放自身海藻糖含量的73.8%,而扣囊复膜酵母菌A11-a菌株在同样条件下仅能释放10%的海藻糖。突变菌株A11-b细胞悬浮在蒸馏水中经37℃过夜处理后,细胞体积比经过相同条件处理的A11-a菌株要大,说明由于细胞通透性发生变化,导致细胞在蒸馏水中发生膨胀。在摇瓶和5-l发酵罐中培养,突变菌株A11-b的生长量和海藻糖积累量与菌株A11-a几乎相同。突变菌株A11-b和菌株A11-a都能利用木薯淀粉在细胞中累积大约28.0% (w/w)的海藻糖。用蒸馏水从突变株A11-b中提取和纯化了海藻糖,最后获得了高纯度的海藻糖晶体。从而建立了一种从酵母菌细胞中提取海藻糖的简单、经济、安全和有效的新工艺。在微生物中葡萄糖阻遏是一种非常普遍的现象,在葡萄糖存在情况,许多酶的合成和基因表达都会受到抑制。参与葡萄糖阻遏作用的主要两种蛋白是Snf1和Mig1,Snf1是一种蛋白激酶,在培养基中有葡萄糖存在情况下可以使Mig1发生磷酸化反应,磷酸化的Mig1可以使有关的基因表达受到阻遏,所以如果把编码Mig1的基因敲除,酵母细胞不管是否生长在葡萄糖培养基中有关基因表达都不会受到阻遏。本研究首先用简并引物PCR法和反向PCR法从扣囊复膜酵母A11菌株的基因组DNA中克隆出了MIG1(Multicopy Inhibitor of Glucose)基因。克隆的MIG1基因(NCBI的登录号:HM450676)全长1152 bp,编码384个氨基酸的蛋白质,该蛋白质的氨基酸序列与其它真菌的Mig1s非常类似,有高度保守的两个锌指结构,Mig1蛋白还有一个短的保守模序,可能在葡萄糖阻遏中有重要作用。然后敲除扣囊复膜酵母A11菌株的MIG1基因,利用上述的HPT基因ORF替换了MIG1基因的ORF,结果在含有潮霉素的YPD平板上获得了敲除菌株A11-c,它能在含有2-脱氧-D葡萄糖的YPS平板上生长,而原始菌株A11则都不能,说明敲除菌株的葡萄糖阻遏得到了解除。与菌株A11相比,敲除菌株A11-c淀粉酶、酸性蛋白酶和β-葡糖苷酶的产量有很大提高,同时编码α-淀粉酶、葡糖淀粉酶、酸性蛋白酶和β-葡糖苷酶基因的表达量也有极大提高。这就证明,扣囊复膜酵母中的Mig1确实能阻遏某些基因的转录,它对一些基因的表达和一些胞外酶的生产起调控作用。综上所述,本论文研究了扣囊复膜酵母A11酸性蛋白酶基因和MIG1基因被敲除后对多种酶生产及海藻糖积累的影响,这对于了解扣囊复膜酵母菌生理和遗传,开发和利用扣囊复膜酵母具有积极意义。更多还原

【Abstract】 Saccharomycopsis fibuligera is one species of the teleomorphic ascomycetous genera. It is found to actively accumulate trehalose from starch. This yeast is also found to secrete a large amount of amylases, acid protease andβ-glucosidase. Although S. fibuligera has great potential for application in both fermentation industry and medicine, very little has been known of the genetic system of S. fibuligera.The acid protease gene in S. fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the acid protease gene. The mutant A11-a obtained could grow in the medium containing hygromycin. No clear zone formed by the mutant grown on the plate containing milk protein was observed whereas a big clear zone formed by the strain A11 was detected. The acid protease and amylases activities produced by the mutant within 3 days were 0.89 U/mL and 424.7 U/mL, respectively while those produced by the strain A11 were 13.5 U/mL and 259.9 U/mL, respectively. The amylases preparations produced by the mutant A11-a and the strain A11 kept 88.8% and 45.5% of amylase activity, respectively after they were incubated at 37℃for two days. Trehalose amount accumulated in the mutant cells was 28.3% (w/w) while that accumulated in the cells of S. fibuligera A11 was 23.6 (w/w).Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37℃). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from S. fibuligera A11-a, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37℃overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-a treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37℃overnight was much bigger than that of S. fibuligera A11-a treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-a during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.The MIG1(Multicopy Inhibitor of Glucose)gene of S. fibuligera A11 was cloned from the genomic DNA using the degenerated primers and inverse PCR. The MIG1 gene (1152 bp accession number: HM450676) encodes a 384-amino acid protein very similar to Mig1s from other fungi. Besides their highly conserved zinc fingers, the Mig1 proteins display short conserved motifs of possible significance in glucose repression. The MIG1 gene in S. fibuligera A11 was disrupted by integrating the HPT gene into ORF of the MIG1 gene. The disruptant A11-c obtained could grow in the media containing hygromycin and 2-deoxy-D-glucose, respectively. Amylases, acid protease andβ-glucosidase production by the disruptant and expression of the genes encodingα-amylase, glucoamylase, acid protease andβ-glucosidase in the disruptant were enhanced. This confirms that Mig1, the transcriptional repressor indeed also regulate expression of many genes and production of many extracellular enzymes in S. fibuligera A11.更多还原