扣囊复膜酵母A11菌株酸性蛋白酶基因和MIG1基因敲除对酶的生产和海藻积累的影响
|
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。
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.
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。
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.
引文
陈其军,肖玉梅,王学臣,周海梦.植物功能基因组研究中的基因敲除技术.植物生理学通讯[J], 2004, 40(1):121-126.
黄留玉. PCR最新技术原理、方法及应用.化学工业出版社[M], 2005.
史兆兴,王恒梁,苏国富,黄留玉.简并PCR及其应用.生物技术通讯[J], 2004, 15(2): 172-175.
王德培,孙伟,李明春,魏东盛,张颖慧,邢来君.利用长引物嵌套式反向PCR方法克隆雅致枝霉Δ6-脂肪酸脱氢酶基因上游序列.生物工程学报[J], 2006, 22(4) :581-586.
王洪振,周晓馥.简并PCR技术及其在基因克隆中的应用.遗传[J], 2003, 25(2): 201-204.
朱玉贤,李毅.现代分子生物学.高等教育出版社[M], 2002.
Abouzied MM, Reddy CA. Fermentation of starch to ethanol by a complementary mixture of an amylolytic yeast and Saccharomyces cerevisiae. Biotechnol Lett, 1987, 9: 59–62.
Adams A, Gottschling DE, Kaiser CA, Stearns T. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1998, pp 84–88.
Adams DJ. Fungal cell wall chitinases and glucanases. Microbiol, 2004, 150: 2029–2035.
Alexandar I, Segundo PS, Venkov P, del Rey F, Va′zquez de Aldana CR. Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene. Intern J Biochem Cell Biol, 2004, 36:2196–2213.
Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G. Insights on the evolution of trehalose biosynthesis. BMC Evol Biol, 2006, 6:109–124.
Benaroudj N, Lee DH, Goldberg AL. Trehalose accumulation during cellular stressprotects cells and cellular proteins from damage by oxygen radicals. J Biol Chem, 2001, 276:24261–7.
Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA. Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awunzori. Gene (Amst), 1990, 86: 153-162.
Bernstein E, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001, 409 (6818):363-366.
Bodie AE, Bower B, Berka RM, Dunn-Coleman NS. Economically important organic acid and enzyme products. Prog Indu Microbiol, 1994, 29: 561-602.
Cabib E, Duran A. Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures. J Bacteriol, 1975, 124:1604–1606.
Capecchi MR. Altering the genome by homologous recombination. Science, 1989, 244(4910):1288-1292.
Carlson M, Glucose repression in yeast. Curr Opin Microbiol, 1999, 2: 202–207.
Carlson M. Regulation of sugar utilization in Saccharomyces species. J Bacteriol, 1987, 169: 4873–4877.
Cassart JP, Georis I, Ostling J, Ronne H, Vandenhaute J. The MIG1 repressor from Kluyveromyces lactis, cloning, sequencing and functional analysis in Saccharomyces cerevisiae. FEBS Lett, 1995, 371: 191-194.
Chen L, Chi ZM, Chi Z, Li M. Amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation for hydrolysis of cassava starch. Applied Biochemistry and Biotechnology, 2010, 62(1):252-263.
Cheng, C., N. Kacherovsky, K. M. Dombek, S. Camier, S. K. Thukral, E. Rhim, and E. T. Young. Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1. Mol Cell Biol, 1994, 14: 3842–3852.
Chi Z, Gao J. Recent advances in the mechanisms of ethanol tolerance in yeasts. Microbiology, 1999, 26(5): 373–376.
Chi Z, Wang JM, Chi ZM, Ye F. Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalosepurification. J Ind Microbiol Biotechnol, 2010, 37:19–25.
Chi ZM, Arneborg N. Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol. J Indu Microbiol Biotechnol, 2000, 24:75–8.
Chi ZM, Chi Z, Liu GL, Wang F, Ju L, Zhang T . Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv, 2009, 27:423–431.
Chi ZM, Chi Z, Zhang T, Yue LX, Li J,Wang XH. Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications. Biotechnol Adv, 2009b, 27:236–55.
Chi ZM, Li JF, Wang XH, Yao SM. Inositol and phosphatidylinositol mediated glucose derepression, gene expression and invertase secretion in yeasts. Acta Biochim Biophys Sin, 2004, 36: 443–449.
Chi ZM, Liang LK, Zhu KL, Zhang FL. Advanced in metabolism and regulation of trehalose in yeast. J Ocean Univer China, 2006, 36(2):209–214.
Chi ZM, Liu J, Ji JR, Meng ZL. Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu. J Biotechnol, 2003, 102:135–141.
Chi ZM, Liu J, Zhang W. Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzyme Microb Technol, 2001, 28:240–246.
Chi ZM, Liu ZR. High-concentration alcoholic production from hydrolyzate of raw ground corn by a tetraploid yeast strain. Biotechnol Lett, 1993, 15:877–82.
Chi ZM, Su CD, Lu WD. A new exopolysaccharide produced by marine Cyanothece sp. 113. Biores Technol, 2007, 98:1329–1332.
Chi ZM, Wang F, Chi Z, Yue LX, Liu GL, Zhang T. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Appl Microbiol Biotechnol, 2009a, 2009(82):793–804.
Cho YJ, Park OJ, Shin HJ. Immobilization of thermostable trehalose synthase for the production of trehalose. Enzym Microb Technol, 2006, 39:108–113.
Choi SH, Sung C, Oh MJ, Kim CJ. Intergeneric protoplast fusion in Saccharomycopsis fibuligera and Saccharomyces cerevisiae. J Ferment Bioeng, 1997, 84:158–61.
Dariush N, Azim A, Jeno MS, Murray MY. Fungal glucoamylases. Biotechnol Adv, 2006, 24:80–5.
Davies DR. The structure and function of the aspartic proteinases. Annu Rev Biophys Chem, 1990, 19:189–215.
Ejiofor AO, Chisti Y, Moo-Young M. Culture of Saccharomyces cerevisiae on hydrolyzed waste cassava starch for production of baking-quality yeast. Enzyme Microb Technol, 1996, 18:519–25.
Eksteen JM, Rensburg PV, Otero RRC, Pretorius IS. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the a-Amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84:639-46.
Elbashir SM, Harborth J, Lendeckel WSM, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411:494-498.
Elbein AD, Pan YT, Pastuszak I, Carroll D. New insights on trehalose: a multifunctional molecule. Glycobiology, 2003, 13:17–27.
Francois JM, Parrou JL. Reserve carbohydrates metabolism in the yeast S. cerevisiae. FEMS Microbiol Rev, 2001, 25: 125–45.
Futatsugi M, Ogawa T, Fukuda H. Purification and properties of two forms of glucoamylase from Saccharomycopsis fibuligera. J Ferment Bioeng, 1993, 76:521–3.
Gancedo C, Flores CL. The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res, 2004, 4:351–9.
Gancedo JM. Yeast carbon catabolite repression. Microbiol Mol Biol Rev, 1998, 62: 334–361.
Gao J, Chi ZM. The relationship between trehalose content and ethanol tolerance in high ethanol tolerant yeast and ethanol sensitive yeast. Food Ferment Indu, 2001, 27:4–7.
Gasperik J, Hostinova E. Glucoamylases encoded by variant Saccharomycopsis fibuligera genes: structure and properties. Curr Microbiol, 1993, 27:11–4.
Georis I, Cassart JP, Breuning KD, Vandenhaute J. Glucose repression of the Kluyveromyces lactis invertase gene KIINV1 does not require Mig1p. Mol Gen Genet, 1999, 261: 862–870.
González CF, Fari?a JI, de Figueroa LIC. Optimized amylolytic enzymes production in Saccharomycopsis fibuligera DSM-70554 An approach to efficient cassava starch utilization. Enzyme Microb Technol, 2008, 42:272–7.
Gottesman S. Minimizing proteolysis in Escherichia coli: genetic solutions. Meth Enzymol, 1990, 185: 119-129.
Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell, 2001, 106 (1):23-34.
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial a-amylases: a biotechnological perspective. Proc Biochem, 2003, 38:1599–616.
Hasan K, Ismaya WT, Kardi I, Andiyana Y, Kusumawidjaya S, Ishmayana S, et al. Proteolysis ofα-amylase from Saccharomycopsis fibuligera: characterization of digestion products. Biol, 2008, 63:1044–50.
He XS, Shyu YT, Nathoo S, Wong SL, Doi RH. Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. Ann NY Acud Sci, 1991, 646: 69-77.
Herdeiro RS, Pereira MD, Panek AD, Eleutherio ECA. Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress. Biochim Biophys Acta, 2006, 1760: 340–6.
Hinnen A, Buxton F, Chaudhuri B, Heim J, Hottinger T, Meyhack B, Pohlig G. Gene expression in recombinant yeast, in gene expression in recombinant microorganisms (Smith, A,, ed.). Marcel Dekker, New York. 1994, pp. 121-193.
Hostinova E, Solovicov A, Dvorsky R, Gasperik J. Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain. Arch Biochem Biophys, 2003, 411:189–95.
Hu Z, Nehlin JO, Ronne H, Michels CA. MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae. CurrGenet, 1995, 28: 258-266.
Hua G, Jamey DM, Paul CO, et al. Deletion of a DNA polymeraseβgene segment in T cells using cell type-specific gene targeting. Science, 1993, 265:103-106.
Itoh T, Ohtsuki I, Yamashita I, Fukui S. Nucleotide sequence of the glucoamylase gene GLUl in the Yeast Saccharomycopsis fibuligera. J Bacteriol. 1987b, 169:4171–6.
Itoh T, Yamashita I, Fukui S. Nucleotide sequence of theα-amylase gene (ALP1) in the yeast Saccharomycopsis fibuligera. FEBS Lett, 1987a, 219:339–42.
Jan ON and Hans R. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms` tumour finger proteins. The EMBO Jourmal, 1990, 9(9): 2891-2898.
JeffWu CF, Hamada M. Experiments Planning, Analysis and Parameter Design Optimization. John Wiley and Sons, New York, 2000, pp 23-34.
Johannes PTW, Hombergh VD, Gelpke MDS, Peter JI, Vondervoort VD, Buxton FP, Visser J. Disruption of three acid proteases in Aspergillus niger effects on protease spectrum, intracellular proteolysis, and degradation of target proteins. Euro J Biochem, 1997, 247: 605-613.
Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C. Expression of laccase lllb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol, 2005, 66: 450–456.
Kamada M, Oda K, Murao S. The purification of the extracellular acid protease of Rhodotorula glutinis K-24 and its general properties. Agric Biol Chem, 1972, 36: 1095–101.
Kitano H, Kataoka K, Furukawa K, Hara S. Specific expression and temperature-dependent expression of the acid protease-encoding gene (PepA) in Aspergillus oryzae in solid-state culture (rice-koji). J Biosci Bioengineer, 2002, 93:563–7.
Knox AM, du Preez JC, Kilian SG. Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes fromLipomyces kononenkoae and Saccharomycopsis fibuligera. Enz Microb Technol, 2004, 34: 453–60.
Kobayashi K, Komeda K, Miura Y, Kettoku M, Kato M. Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1. J Ferment Eng, 1997, 83:296–8.
Kobayashi N, McEntee K. Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae. Mol Cell Biol, 1993, 13: 248–56.
Kocabiyik S, Ozel H. An extracellular-pepstatin in sensitive acid protease produced by Thermoplasma volcanium. Biores Technol, 2007, 98:112–7.
Kurtzman CP, Fell JW. The yeasts: a taxonomic study. 4th revised and enlarged edition. Amsterdam: Elsevier, 2000, pp. 1-525.
Larson MK, Whltaker JR. Endothia parasitica protease. Parameters affecting activity of the rennin-like enzyme. J Dair Sci, 1970, 53: 253–261.
Li HF, Chi ZM, Duan XH, Wang L, Sheng J, Wu LF. Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme. Proc Biochem, 2007a, 42:462–5.
Li HF, Chi ZM, Wang XH, Duan XH, Ma LY, Gao LM. Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion. Enzyme Microb Technol, 2007b, 40:1006–12.
Li J, Chi ZM, Chi Z,Wang XH. Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli. Microbiol Res, 2008b, doi:10.1016/j.micres, 2008.08.003.
Li J, Chi ZM, Liu ZQ, Yue LX, Peng Y,Wang L. Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli. Appl Biochem Biotechnol, 2008a, doi:10.1007/s12010-008-8400-3.
Li M, Liu GL, Chi Z, Chi ZM. Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a. BiomaBioenerg, 2010, 34: 101-107.
Liang LK, Chi ZM, Gao LM, Ma LY. Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11. Ind J Biochem Biophys, 2006b, 43:289–94.
Liang LK, Wang XK, Zhu KL, Chi ZM. Trehalose accumulation in a high-trehalose accumulating mutant of Saccharomycopsis fibuligera sdu does not respond to stress treatments. Biochem, 2006, 71: 1291–1297.
Liu ZR, Zhang G, Long ZF, Liu SG. Heterologous expression of amylase gene from Saccharomycopsis fibuligera in an industrial strain of Saccharomyces cerevisiae. Wuhan University Journal of Natural Sciences, 2005, 10(6): 1041–6.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCt method. Meth, 2001, 25: 402–408.
Lloyd AM, Davis RW. Functional expression of the yeast FLP/FRT site-specific recombination system in nicotiana tabacum. Molecular and General Genetics, 1994, 242: 653-657.
Lutfiyya LL, Iyer VR, de Risi J, de Vit MJ, Brown PO, Johnston M. Characterization of three related glucose repressors and genes they regulate in Saccharomyces cerevisiae. Genetics, 1998, 150:1377–1391.
Lutfiyya LL, Johnston M. Two zinc-finger-containing repressors are responsible for glucose repression of SUC2 expression. Mol Cell Biol, 1996, 16: 4790–4797.
Machida M, Ohtsuki I, Fukui S, Yamsshida I. Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular b-glucosidases as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol, 1988, 54:3147–55.
Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol, 2004, 109: 63–68.
Matsui I, Ishikawa K, Miyairi S, Fukui S, Honda K. An increase in the transglycosylation activity of Saccharomycopsisα-amylase altered by site-directed mutagenesis. Biochem Biophys Acta, 1991, 1077(3): 416–9.
Mehrabi R, Ding S, Xu JR. MADS-Box transcription factor Mig1 is required forinfectious growth in Magnaporthe grisea. Eukaryotic Cell, 2008, 7: 791–799.
Miyazaki JI, Miyagawa KI, Sugiyama Y. Trehalose accumulation by a basidiomycotious yeast, Filobasidium Xoriforme. J Ferment Bioeng, 1996, 4:315–319.
Mukai K, Tabuchi A, Nakada T, Shibuya T, Chaen H, Fukuda S, Kurimoto M, Tsujisaka Y. Production of trehalose from starch by thermostable enzymes from Sulfolobus acidocaldarius. Starch, 1997, 49:26–30.
Nehlin JO, Carlberg M, Ronne H. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J, 1991, 10: 3373–3377.
Nga BH, Chiu LL, Koh SI, Yip CW, Harashima S, Oshima Y. Occurrence of genetic segregation in a putative haploid strain of Endomyces fibuliger met by spontaneous sectoring of protoplast fusants. World J Microbiol Biotechnol, 1994, 10:465–471.
Nga WY, Nga BH, Pridmore D, Mollet B, Tan HM. Transformation of Endomyces fibuliger based on its gene for orotidine-5-phosphate decarboxylase. Gene, 2000, 254: 97-103.
Ochman H, Ajioka JW, Garza D, Hartl, DL. Inverse polymerase chain reaction. Biotechnology (NY), 1990, 8(8): 759-760.
Pandey A. Solid state fermentation. Biochem Eng, 2003, 13:81–4.
Papamichos-Chronakis M, Gligoris T, Tzamarias D. The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8–Tup1 co-repressor. EMBO Rep, 2004, 5: 368-372.
Reddy OVSR, Basappa SC. Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: synergism and limitations. Biotechnol Lett, 1996, 18: 1315–1318.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Beijing, 1989, pp. 367–370 (Chinese translating ed.).
Sanchez OJ, Cardona CA. Trends in biotechnological production of fuel ethanol from different feedstocks. Biores Technol, 2008, 99:5270–95.
Sandhu DK, Vilkhu KS, Soni SK. Production ofα-amylase by Saccharomycopsis fibuligera(Syn. Endomycopsis fibuligera). J Ferment Bioeng, 1987, 65:387–94.
Satto K, Kase T, Takahashi E, Takahashi E, Horinouchi S. Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl Environ Microbiol, 1998, 64:4340–5.
Schick I, Haltrich D, Kulbe KD. Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose. Appl Microbiol Biotechnol, 1995, 43:1088–95.
Shen Y, Zhang Y, Ma T, Bao XM, Du FG, Zhuang GQ, et al. Simultaneous saccharification and fermentation of acid-pretreated corncobs with a recombinant Saccharomyces cerevisiae expressingβ-glucosidase. Biores Technol, 2008, 99:5099–103.
Shimada A, Koda T, Nakamura I. Concanavalin A-agarose gel system capable of accumulating extracellular glucoamylase produced by immobilized Saccharomycopsis fibuligera. J Ferment Bioeng, 1998, 85:542–5.
Slightom JL, Metzger BT, Luu HT, Elhammer AP. Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938. Gene, 2009, 431: 67–79.
Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chrosomoal betaglobin locus by homologous recombination. Nature, 1985, 317(6034):230-234.
Solovicova A, Gasperik J, Hostinova E. High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms. Biochem Biophys Res Commun, 1996, 224:790–5.
Spiro RG. Analysis of sugars found in glycoproteins. Meth Enzymol, 1966, 8: 3-26.
Stewart PR. Trehalose extraction and determination. In: Prescott DM (ed) Methods in cell biology, Academic Press, London and New York, 1982, 12:111–147.
Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA. Degerate oligonucleotide primed PCR: general amplification of target DNA by a singledegenrate primer[J]. Genomic, 1992, 13:718-725.
Ueda M, Tanaka A. Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv, 2000, 18:121–140.
Valenzuela-Soto EM, Marquez-Escalante JA, Iturriaga G, Figueroa-Soto CG. Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties. Biochem Biophys Res Commun, 2004, 313:314–9.
Vivier MA, Lambrechts MG, Pretorius S. Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol, 1997,32:405–35.
Westholm JO, Nordberg N. Murén E, Ameur A, Komorowski J, Ronne H. Combinatorial control of gene expression by the three yeast repressors Mig1, Mig2 and Mig3. BMC Genom, 2008, 9: 601-607.
Wickerham LJ, Lockwood LB, Pettijohn OG, Ward GE. Starch hydrolysis and fermentation by the yeast Endomycopsis fibuliger. J Bacteriol,1944,48:413–27.
Yamamoto T, Omoto S, Mizuguchi M, et al. Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol Immunol, 2002, 46 (11) : 809-817.
Yamashita I, Hirata D, Machida M, Fukui S. Cloning and expression in Saccharomyces cerevisiae of the secretable acid protease gene from Saccharomycopsis fibuligera. Agric Biol Chem, 1986, 50:109–13.
Yoshida M, Nakamura N, Horikoshi K. Production of trehalose from starch by maltose phosphorylase and trehalose phosphorylase from a strain of Plesiomonas. Starch, 1997, 49: 21–26.
Yoshikawa Y, Matsumoto K, Nagata K, Sato T. Extraction of trehalose from thermally-treated bakers yeast. Biosci Biotech Biochem, 1994, 58:1226–1230.
Yu XJ, Li HJ, Li J, Chi, ZM. Overexpression of acid protease of Saccharomycopsis fibuligera in Yarrowia lipolytica and characterization of the recombinant acid protease for skimmed-milk-clotting. Biotechnol Bioproc Engineer, 2010, 87: 669–677.
Yue LX, Chi ZM, Wang L, Liu J, Madzak C, Li J, et al. Construction of a newplasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods, 2008, 72:116–23.
Zaragoza O, Rodriguez C, Gancedo C. Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression. J Bacteriol, 2000, 182: 320–326.
Zhang H, Liu J, Chi ZM. Studies on enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera. Food Ferment Indus, 2002, 28:1–4.
Zhang T, Chi ZM, Sheng J. A highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a potentially useful for single-cell protein production and its nutritive components. Mar Biotechnol, 2009, 11:280–286.
Zhang Y, Zhang T, Chi Z, Wang JM, Liu GL, Chi ZM. Conversion of cassava starch to trehalose by Saccharomycopsis fibuligera A11 and purification of trehalose. Carbohyd Polym, 2010, 80: 13–18.
Zhao C, Zhang T, Chi ZM, Chi Z, Li J, Wang X. Single cell protein production from yacon extract using a highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a and its nutritive analysis. Bioproc Biosyst Eng, 2010, 33: 549–556.
黄留玉. PCR最新技术原理、方法及应用.化学工业出版社[M], 2005.
史兆兴,王恒梁,苏国富,黄留玉.简并PCR及其应用.生物技术通讯[J], 2004, 15(2): 172-175.
王德培,孙伟,李明春,魏东盛,张颖慧,邢来君.利用长引物嵌套式反向PCR方法克隆雅致枝霉Δ6-脂肪酸脱氢酶基因上游序列.生物工程学报[J], 2006, 22(4) :581-586.
王洪振,周晓馥.简并PCR技术及其在基因克隆中的应用.遗传[J], 2003, 25(2): 201-204.
朱玉贤,李毅.现代分子生物学.高等教育出版社[M], 2002.
Abouzied MM, Reddy CA. Fermentation of starch to ethanol by a complementary mixture of an amylolytic yeast and Saccharomyces cerevisiae. Biotechnol Lett, 1987, 9: 59–62.
Adams A, Gottschling DE, Kaiser CA, Stearns T. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1998, pp 84–88.
Adams DJ. Fungal cell wall chitinases and glucanases. Microbiol, 2004, 150: 2029–2035.
Alexandar I, Segundo PS, Venkov P, del Rey F, Va′zquez de Aldana CR. Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene. Intern J Biochem Cell Biol, 2004, 36:2196–2213.
Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G. Insights on the evolution of trehalose biosynthesis. BMC Evol Biol, 2006, 6:109–124.
Benaroudj N, Lee DH, Goldberg AL. Trehalose accumulation during cellular stressprotects cells and cellular proteins from damage by oxygen radicals. J Biol Chem, 2001, 276:24261–7.
Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA. Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awunzori. Gene (Amst), 1990, 86: 153-162.
Bernstein E, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001, 409 (6818):363-366.
Bodie AE, Bower B, Berka RM, Dunn-Coleman NS. Economically important organic acid and enzyme products. Prog Indu Microbiol, 1994, 29: 561-602.
Cabib E, Duran A. Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures. J Bacteriol, 1975, 124:1604–1606.
Capecchi MR. Altering the genome by homologous recombination. Science, 1989, 244(4910):1288-1292.
Carlson M, Glucose repression in yeast. Curr Opin Microbiol, 1999, 2: 202–207.
Carlson M. Regulation of sugar utilization in Saccharomyces species. J Bacteriol, 1987, 169: 4873–4877.
Cassart JP, Georis I, Ostling J, Ronne H, Vandenhaute J. The MIG1 repressor from Kluyveromyces lactis, cloning, sequencing and functional analysis in Saccharomyces cerevisiae. FEBS Lett, 1995, 371: 191-194.
Chen L, Chi ZM, Chi Z, Li M. Amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation for hydrolysis of cassava starch. Applied Biochemistry and Biotechnology, 2010, 62(1):252-263.
Cheng, C., N. Kacherovsky, K. M. Dombek, S. Camier, S. K. Thukral, E. Rhim, and E. T. Young. Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1. Mol Cell Biol, 1994, 14: 3842–3852.
Chi Z, Gao J. Recent advances in the mechanisms of ethanol tolerance in yeasts. Microbiology, 1999, 26(5): 373–376.
Chi Z, Wang JM, Chi ZM, Ye F. Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalosepurification. J Ind Microbiol Biotechnol, 2010, 37:19–25.
Chi ZM, Arneborg N. Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol. J Indu Microbiol Biotechnol, 2000, 24:75–8.
Chi ZM, Chi Z, Liu GL, Wang F, Ju L, Zhang T . Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv, 2009, 27:423–431.
Chi ZM, Chi Z, Zhang T, Yue LX, Li J,Wang XH. Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications. Biotechnol Adv, 2009b, 27:236–55.
Chi ZM, Li JF, Wang XH, Yao SM. Inositol and phosphatidylinositol mediated glucose derepression, gene expression and invertase secretion in yeasts. Acta Biochim Biophys Sin, 2004, 36: 443–449.
Chi ZM, Liang LK, Zhu KL, Zhang FL. Advanced in metabolism and regulation of trehalose in yeast. J Ocean Univer China, 2006, 36(2):209–214.
Chi ZM, Liu J, Ji JR, Meng ZL. Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu. J Biotechnol, 2003, 102:135–141.
Chi ZM, Liu J, Zhang W. Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzyme Microb Technol, 2001, 28:240–246.
Chi ZM, Liu ZR. High-concentration alcoholic production from hydrolyzate of raw ground corn by a tetraploid yeast strain. Biotechnol Lett, 1993, 15:877–82.
Chi ZM, Su CD, Lu WD. A new exopolysaccharide produced by marine Cyanothece sp. 113. Biores Technol, 2007, 98:1329–1332.
Chi ZM, Wang F, Chi Z, Yue LX, Liu GL, Zhang T. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Appl Microbiol Biotechnol, 2009a, 2009(82):793–804.
Cho YJ, Park OJ, Shin HJ. Immobilization of thermostable trehalose synthase for the production of trehalose. Enzym Microb Technol, 2006, 39:108–113.
Choi SH, Sung C, Oh MJ, Kim CJ. Intergeneric protoplast fusion in Saccharomycopsis fibuligera and Saccharomyces cerevisiae. J Ferment Bioeng, 1997, 84:158–61.
Dariush N, Azim A, Jeno MS, Murray MY. Fungal glucoamylases. Biotechnol Adv, 2006, 24:80–5.
Davies DR. The structure and function of the aspartic proteinases. Annu Rev Biophys Chem, 1990, 19:189–215.
Ejiofor AO, Chisti Y, Moo-Young M. Culture of Saccharomyces cerevisiae on hydrolyzed waste cassava starch for production of baking-quality yeast. Enzyme Microb Technol, 1996, 18:519–25.
Eksteen JM, Rensburg PV, Otero RRC, Pretorius IS. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the a-Amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84:639-46.
Elbashir SM, Harborth J, Lendeckel WSM, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411:494-498.
Elbein AD, Pan YT, Pastuszak I, Carroll D. New insights on trehalose: a multifunctional molecule. Glycobiology, 2003, 13:17–27.
Francois JM, Parrou JL. Reserve carbohydrates metabolism in the yeast S. cerevisiae. FEMS Microbiol Rev, 2001, 25: 125–45.
Futatsugi M, Ogawa T, Fukuda H. Purification and properties of two forms of glucoamylase from Saccharomycopsis fibuligera. J Ferment Bioeng, 1993, 76:521–3.
Gancedo C, Flores CL. The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res, 2004, 4:351–9.
Gancedo JM. Yeast carbon catabolite repression. Microbiol Mol Biol Rev, 1998, 62: 334–361.
Gao J, Chi ZM. The relationship between trehalose content and ethanol tolerance in high ethanol tolerant yeast and ethanol sensitive yeast. Food Ferment Indu, 2001, 27:4–7.
Gasperik J, Hostinova E. Glucoamylases encoded by variant Saccharomycopsis fibuligera genes: structure and properties. Curr Microbiol, 1993, 27:11–4.
Georis I, Cassart JP, Breuning KD, Vandenhaute J. Glucose repression of the Kluyveromyces lactis invertase gene KIINV1 does not require Mig1p. Mol Gen Genet, 1999, 261: 862–870.
González CF, Fari?a JI, de Figueroa LIC. Optimized amylolytic enzymes production in Saccharomycopsis fibuligera DSM-70554 An approach to efficient cassava starch utilization. Enzyme Microb Technol, 2008, 42:272–7.
Gottesman S. Minimizing proteolysis in Escherichia coli: genetic solutions. Meth Enzymol, 1990, 185: 119-129.
Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell, 2001, 106 (1):23-34.
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial a-amylases: a biotechnological perspective. Proc Biochem, 2003, 38:1599–616.
Hasan K, Ismaya WT, Kardi I, Andiyana Y, Kusumawidjaya S, Ishmayana S, et al. Proteolysis ofα-amylase from Saccharomycopsis fibuligera: characterization of digestion products. Biol, 2008, 63:1044–50.
He XS, Shyu YT, Nathoo S, Wong SL, Doi RH. Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. Ann NY Acud Sci, 1991, 646: 69-77.
Herdeiro RS, Pereira MD, Panek AD, Eleutherio ECA. Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress. Biochim Biophys Acta, 2006, 1760: 340–6.
Hinnen A, Buxton F, Chaudhuri B, Heim J, Hottinger T, Meyhack B, Pohlig G. Gene expression in recombinant yeast, in gene expression in recombinant microorganisms (Smith, A,, ed.). Marcel Dekker, New York. 1994, pp. 121-193.
Hostinova E, Solovicov A, Dvorsky R, Gasperik J. Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain. Arch Biochem Biophys, 2003, 411:189–95.
Hu Z, Nehlin JO, Ronne H, Michels CA. MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae. CurrGenet, 1995, 28: 258-266.
Hua G, Jamey DM, Paul CO, et al. Deletion of a DNA polymeraseβgene segment in T cells using cell type-specific gene targeting. Science, 1993, 265:103-106.
Itoh T, Ohtsuki I, Yamashita I, Fukui S. Nucleotide sequence of the glucoamylase gene GLUl in the Yeast Saccharomycopsis fibuligera. J Bacteriol. 1987b, 169:4171–6.
Itoh T, Yamashita I, Fukui S. Nucleotide sequence of theα-amylase gene (ALP1) in the yeast Saccharomycopsis fibuligera. FEBS Lett, 1987a, 219:339–42.
Jan ON and Hans R. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms` tumour finger proteins. The EMBO Jourmal, 1990, 9(9): 2891-2898.
JeffWu CF, Hamada M. Experiments Planning, Analysis and Parameter Design Optimization. John Wiley and Sons, New York, 2000, pp 23-34.
Johannes PTW, Hombergh VD, Gelpke MDS, Peter JI, Vondervoort VD, Buxton FP, Visser J. Disruption of three acid proteases in Aspergillus niger effects on protease spectrum, intracellular proteolysis, and degradation of target proteins. Euro J Biochem, 1997, 247: 605-613.
Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C. Expression of laccase lllb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol, 2005, 66: 450–456.
Kamada M, Oda K, Murao S. The purification of the extracellular acid protease of Rhodotorula glutinis K-24 and its general properties. Agric Biol Chem, 1972, 36: 1095–101.
Kitano H, Kataoka K, Furukawa K, Hara S. Specific expression and temperature-dependent expression of the acid protease-encoding gene (PepA) in Aspergillus oryzae in solid-state culture (rice-koji). J Biosci Bioengineer, 2002, 93:563–7.
Knox AM, du Preez JC, Kilian SG. Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes fromLipomyces kononenkoae and Saccharomycopsis fibuligera. Enz Microb Technol, 2004, 34: 453–60.
Kobayashi K, Komeda K, Miura Y, Kettoku M, Kato M. Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1. J Ferment Eng, 1997, 83:296–8.
Kobayashi N, McEntee K. Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae. Mol Cell Biol, 1993, 13: 248–56.
Kocabiyik S, Ozel H. An extracellular-pepstatin in sensitive acid protease produced by Thermoplasma volcanium. Biores Technol, 2007, 98:112–7.
Kurtzman CP, Fell JW. The yeasts: a taxonomic study. 4th revised and enlarged edition. Amsterdam: Elsevier, 2000, pp. 1-525.
Larson MK, Whltaker JR. Endothia parasitica protease. Parameters affecting activity of the rennin-like enzyme. J Dair Sci, 1970, 53: 253–261.
Li HF, Chi ZM, Duan XH, Wang L, Sheng J, Wu LF. Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme. Proc Biochem, 2007a, 42:462–5.
Li HF, Chi ZM, Wang XH, Duan XH, Ma LY, Gao LM. Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion. Enzyme Microb Technol, 2007b, 40:1006–12.
Li J, Chi ZM, Chi Z,Wang XH. Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli. Microbiol Res, 2008b, doi:10.1016/j.micres, 2008.08.003.
Li J, Chi ZM, Liu ZQ, Yue LX, Peng Y,Wang L. Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli. Appl Biochem Biotechnol, 2008a, doi:10.1007/s12010-008-8400-3.
Li M, Liu GL, Chi Z, Chi ZM. Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a. BiomaBioenerg, 2010, 34: 101-107.
Liang LK, Chi ZM, Gao LM, Ma LY. Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11. Ind J Biochem Biophys, 2006b, 43:289–94.
Liang LK, Wang XK, Zhu KL, Chi ZM. Trehalose accumulation in a high-trehalose accumulating mutant of Saccharomycopsis fibuligera sdu does not respond to stress treatments. Biochem, 2006, 71: 1291–1297.
Liu ZR, Zhang G, Long ZF, Liu SG. Heterologous expression of amylase gene from Saccharomycopsis fibuligera in an industrial strain of Saccharomyces cerevisiae. Wuhan University Journal of Natural Sciences, 2005, 10(6): 1041–6.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCt method. Meth, 2001, 25: 402–408.
Lloyd AM, Davis RW. Functional expression of the yeast FLP/FRT site-specific recombination system in nicotiana tabacum. Molecular and General Genetics, 1994, 242: 653-657.
Lutfiyya LL, Iyer VR, de Risi J, de Vit MJ, Brown PO, Johnston M. Characterization of three related glucose repressors and genes they regulate in Saccharomyces cerevisiae. Genetics, 1998, 150:1377–1391.
Lutfiyya LL, Johnston M. Two zinc-finger-containing repressors are responsible for glucose repression of SUC2 expression. Mol Cell Biol, 1996, 16: 4790–4797.
Machida M, Ohtsuki I, Fukui S, Yamsshida I. Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular b-glucosidases as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol, 1988, 54:3147–55.
Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol, 2004, 109: 63–68.
Matsui I, Ishikawa K, Miyairi S, Fukui S, Honda K. An increase in the transglycosylation activity of Saccharomycopsisα-amylase altered by site-directed mutagenesis. Biochem Biophys Acta, 1991, 1077(3): 416–9.
Mehrabi R, Ding S, Xu JR. MADS-Box transcription factor Mig1 is required forinfectious growth in Magnaporthe grisea. Eukaryotic Cell, 2008, 7: 791–799.
Miyazaki JI, Miyagawa KI, Sugiyama Y. Trehalose accumulation by a basidiomycotious yeast, Filobasidium Xoriforme. J Ferment Bioeng, 1996, 4:315–319.
Mukai K, Tabuchi A, Nakada T, Shibuya T, Chaen H, Fukuda S, Kurimoto M, Tsujisaka Y. Production of trehalose from starch by thermostable enzymes from Sulfolobus acidocaldarius. Starch, 1997, 49:26–30.
Nehlin JO, Carlberg M, Ronne H. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J, 1991, 10: 3373–3377.
Nga BH, Chiu LL, Koh SI, Yip CW, Harashima S, Oshima Y. Occurrence of genetic segregation in a putative haploid strain of Endomyces fibuliger met by spontaneous sectoring of protoplast fusants. World J Microbiol Biotechnol, 1994, 10:465–471.
Nga WY, Nga BH, Pridmore D, Mollet B, Tan HM. Transformation of Endomyces fibuliger based on its gene for orotidine-5-phosphate decarboxylase. Gene, 2000, 254: 97-103.
Ochman H, Ajioka JW, Garza D, Hartl, DL. Inverse polymerase chain reaction. Biotechnology (NY), 1990, 8(8): 759-760.
Pandey A. Solid state fermentation. Biochem Eng, 2003, 13:81–4.
Papamichos-Chronakis M, Gligoris T, Tzamarias D. The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8–Tup1 co-repressor. EMBO Rep, 2004, 5: 368-372.
Reddy OVSR, Basappa SC. Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: synergism and limitations. Biotechnol Lett, 1996, 18: 1315–1318.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Beijing, 1989, pp. 367–370 (Chinese translating ed.).
Sanchez OJ, Cardona CA. Trends in biotechnological production of fuel ethanol from different feedstocks. Biores Technol, 2008, 99:5270–95.
Sandhu DK, Vilkhu KS, Soni SK. Production ofα-amylase by Saccharomycopsis fibuligera(Syn. Endomycopsis fibuligera). J Ferment Bioeng, 1987, 65:387–94.
Satto K, Kase T, Takahashi E, Takahashi E, Horinouchi S. Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl Environ Microbiol, 1998, 64:4340–5.
Schick I, Haltrich D, Kulbe KD. Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose. Appl Microbiol Biotechnol, 1995, 43:1088–95.
Shen Y, Zhang Y, Ma T, Bao XM, Du FG, Zhuang GQ, et al. Simultaneous saccharification and fermentation of acid-pretreated corncobs with a recombinant Saccharomyces cerevisiae expressingβ-glucosidase. Biores Technol, 2008, 99:5099–103.
Shimada A, Koda T, Nakamura I. Concanavalin A-agarose gel system capable of accumulating extracellular glucoamylase produced by immobilized Saccharomycopsis fibuligera. J Ferment Bioeng, 1998, 85:542–5.
Slightom JL, Metzger BT, Luu HT, Elhammer AP. Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938. Gene, 2009, 431: 67–79.
Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chrosomoal betaglobin locus by homologous recombination. Nature, 1985, 317(6034):230-234.
Solovicova A, Gasperik J, Hostinova E. High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms. Biochem Biophys Res Commun, 1996, 224:790–5.
Spiro RG. Analysis of sugars found in glycoproteins. Meth Enzymol, 1966, 8: 3-26.
Stewart PR. Trehalose extraction and determination. In: Prescott DM (ed) Methods in cell biology, Academic Press, London and New York, 1982, 12:111–147.
Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA. Degerate oligonucleotide primed PCR: general amplification of target DNA by a singledegenrate primer[J]. Genomic, 1992, 13:718-725.
Ueda M, Tanaka A. Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv, 2000, 18:121–140.
Valenzuela-Soto EM, Marquez-Escalante JA, Iturriaga G, Figueroa-Soto CG. Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties. Biochem Biophys Res Commun, 2004, 313:314–9.
Vivier MA, Lambrechts MG, Pretorius S. Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol, 1997,32:405–35.
Westholm JO, Nordberg N. Murén E, Ameur A, Komorowski J, Ronne H. Combinatorial control of gene expression by the three yeast repressors Mig1, Mig2 and Mig3. BMC Genom, 2008, 9: 601-607.
Wickerham LJ, Lockwood LB, Pettijohn OG, Ward GE. Starch hydrolysis and fermentation by the yeast Endomycopsis fibuliger. J Bacteriol,1944,48:413–27.
Yamamoto T, Omoto S, Mizuguchi M, et al. Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol Immunol, 2002, 46 (11) : 809-817.
Yamashita I, Hirata D, Machida M, Fukui S. Cloning and expression in Saccharomyces cerevisiae of the secretable acid protease gene from Saccharomycopsis fibuligera. Agric Biol Chem, 1986, 50:109–13.
Yoshida M, Nakamura N, Horikoshi K. Production of trehalose from starch by maltose phosphorylase and trehalose phosphorylase from a strain of Plesiomonas. Starch, 1997, 49: 21–26.
Yoshikawa Y, Matsumoto K, Nagata K, Sato T. Extraction of trehalose from thermally-treated bakers yeast. Biosci Biotech Biochem, 1994, 58:1226–1230.
Yu XJ, Li HJ, Li J, Chi, ZM. Overexpression of acid protease of Saccharomycopsis fibuligera in Yarrowia lipolytica and characterization of the recombinant acid protease for skimmed-milk-clotting. Biotechnol Bioproc Engineer, 2010, 87: 669–677.
Yue LX, Chi ZM, Wang L, Liu J, Madzak C, Li J, et al. Construction of a newplasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods, 2008, 72:116–23.
Zaragoza O, Rodriguez C, Gancedo C. Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression. J Bacteriol, 2000, 182: 320–326.
Zhang H, Liu J, Chi ZM. Studies on enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera. Food Ferment Indus, 2002, 28:1–4.
Zhang T, Chi ZM, Sheng J. A highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a potentially useful for single-cell protein production and its nutritive components. Mar Biotechnol, 2009, 11:280–286.
Zhang Y, Zhang T, Chi Z, Wang JM, Liu GL, Chi ZM. Conversion of cassava starch to trehalose by Saccharomycopsis fibuligera A11 and purification of trehalose. Carbohyd Polym, 2010, 80: 13–18.
Zhao C, Zhang T, Chi ZM, Chi Z, Li J, Wang X. Single cell protein production from yacon extract using a highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a and its nutritive analysis. Bioproc Biosyst Eng, 2010, 33: 549–556.