2 孢子的组装过程。
在孢子的形成过程中,孢子质膜和孢子壁都是在母体细胞的细胞质以从头合成的方式进行的[8]
.孢子的形成需要 2 个重要的步骤: 第一是前孢子膜的形成并包裹减数分裂Ⅱ期的子细胞核形成前孢子; 第二是孢子壁的组装。
2. 1 前孢子膜及前孢子的形成。
如图 2 所示,在减数分裂Ⅱ期,在核被膜的外侧对 称 形 成 了 4 个 纺 锤 极 体 SPB ( spindle polebodies)[23 -24].囊泡开始形成并定位在 SPB 的外侧,在可溶性 N-乙基马来酰亚胺敏感蛋白受体 SNARE( soluble N-ethylmaleimide- sensitive protein recep-tor)[25]帮助下逐渐融合,形成扁平的双层膜结构,这个结构便是前孢子膜形成的开端。随着减数分裂Ⅱ期核分裂的进行,囊泡不断地融入双层膜结构,最终扩大并包裹各自的子细胞核。核分裂完成后,双层膜最终将各自的细胞核和部分母细胞中的细胞质封闭在内部。这样在原来的母细胞中,最终形成含有四个前孢子的子囊。在每个核外部形成的双层膜结构称之为前孢子膜[23 -24,26].
2. 2 孢子壁的组装。
孢子壁的组装即在前孢子膜双层膜之间的内腔填充孢子壁材料,该过程的起始发生在双成膜结构的内腔[27].在填充孢子壁的过程中,前孢子膜包裹的部分细胞质进行蛋白质的合成和分泌,可溶性分泌蛋白经过前孢子膜分泌到孢子壁外侧。在反馈机制的协调作用下,孢子壁从内而外依层组装,只有靠内的一层完成组装后,靠外的一层才开始组装( 图 3) : 首先是最内层甘露糖层,其次为 β-葡聚糖层,再次为壳聚糖层,最后为二酪氨酸层[20].一旦组装完成,孢子壁便赋予孢子一系列抗逆性能[28].
2. 2. 1 孢子壁合成的起始。
孢子壁合成起始所必需的基因一般被认为是GIP1 基因,同时有学者发现 AMA1 基因也是孢子壁合成起始所必需的[29 -31].和 GIP1 的突变相似,AMA1 突变后将导致孢子壁无法合成[32 -33].研究表明,AMA1 可能在孢子壁形成的起始和减数第二次分裂完成的衔接中起协调作用。
2. 2. 2 甘露糖层和 β-葡聚糖层的形成。
前孢子膜闭合之后,分泌性囊泡将甘露糖蛋白运送至前孢子膜双层膜内腔并大量积累以形成甘露糖层。
β-1,3-葡聚糖层的形成需要 β-葡聚糖合成酶的参与,该酶在孢子质膜上合成 β-葡聚糖链后将其释放入前孢子膜的内腔,然后这些糖链进一步连接组装形成 β-葡聚糖层。在酿酒酵母中,该 β-葡聚糖合成酶由 3 个亚基组成,其中,FKS1 基因编码的亚基主要在营养生长时起作用,GSC2/FKS2 基因编码的亚基主要在孢子壁组装中起作用,Fks2 是负责该层合成的主要蛋白[34].FKS3 基因编码的亚基对孢子组装起一定作用[35],而 fks3Δ 缺陷菌株展现出孢子壁缺陷的性状[19].还有研究表明,多个基因对 β-葡聚糖层的正 常 组 装 都 起 到 重 要 作 用,如 SPO77,SPS2,SPS22,CRR1,SSP2[36 -39].其中许多基因是序列簇产孢特有成员,例如,SPS2,SPS22 分别与 ECM33,PST1相关,CRR1 与葡糖水解酶基因 CRH1 和 CRH2 相关,GAS 与营养细胞壁蛋白合成基因 PIR 相关[40].这些成孢特有蛋白在组装甘露糖层和 β-葡聚糖层时发挥的作用等同于相应基因在营养细胞壁构建时发挥的作用。在 β-葡聚糖层组装完毕后,前孢子膜外膜消失; 虽然其具体机制尚不清楚; 但是该膜的消失可以使某些孢子壁组装因子如 Osw1 进入,从而形成孢子外壁[36,39]; 同时孢子装配好甘露糖层和 β-葡聚糖层后可捕获折射光,使得孢子在光学显微镜下可见。
在研究酵母孢子固定化酶的同时,我们发现: 由于壳聚糖具有吸附性的特点,作为壳聚糖球的 dit1Δ孢子可以吸附金属离子和负电性化合物。例如,本研究室张海妮[11]将二酪氨酸层缺失的 dit1Δ 孢子用作壳聚糖球,用来吸附 Cu2 +,并与孢子壁完整的野生型和同时缺失二酪氨酸层和壳聚糖层的 chs3Δ 孢子进行比较。结果显示,作为壳聚糖球的 dit1Δ 孢子,吸附 Cu2 +能力最强; 其次是孢子壁完整的野生型; 而缺失了二酪氨酸层和壳聚糖层的 chs3Δ 孢子吸附 Cu2 +能力最弱。之后,又对孢子吸附 Cr3 +、Ni2 +、Zn2 +、Cd2 +、Pb2 +等离子的能力进行了研究。同样,作为壳聚糖球的 dit1Δ 孢子,对各种离子的吸附量是所有细胞类型中最高的,而营养细胞是最低的; 即便是缺失了最外 2 层的 chs3Δ 孢子,其吸附量也是营养细胞的2 倍多。由此说明,缺失了二酪氨酸层的 dit1Δ 孢子作为壳聚糖球用于重金属离子的吸附是最佳选择。同时,由于壳聚糖带正电,又对作为壳聚糖球的 dit1Δ孢子吸附带有负电荷的牛磺胆酸能力进行研究。与吸附金属离子结果相似,dit1Δ 孢子吸附牛黄胆酸能力优于野生型孢子和 chs3Δ 孢子。上述两个实验说明 dit1Δ 孢子作为壳聚糖球对重金属及某些负电荷分子的吸附是可行的。该研究将酿酒酵母孢子的应用从固定化酶领域进一步拓展到了环保领域,为酿酒酵母孢子的应用开拓了道路。
综上所述,除了对孢子吸附能力的研究外,本实验室对于酵母孢子固定化酶的研究是酵母孢子应用研究的重点。随着微胶囊固定化酶技术的发展,酿酒酵母孢子固定化酶作为微胶囊固定化酶技术的新成员,以其独特的优点首次展现在大家面前: 固定化酶的组装“自然”完成,具有天然抗逆性,大小均一,可重复使用,绿色环保,“一孢多酶体系”已经初步建立,等等。基于以上优点及研究结果,相信,酵母孢子固定化酶将会为酶的微胶囊固定化增光添彩!
参 考 文 献:
[1] BENITA S. Microencapsulation: methods and industrialapplications [M]. CRC Press,2005.
[2] PEYRATOUT C S,DAEHNE L. Tailor-made polyelectro-lyte microcapsules: from multilayers to smart containers[J]. Angewandte Chemie International Edition,2004,43( 29) : 3 762 -3 783.
[3] MEIER W. Polymer nanocapsules [J]. Chemical SocietyReviews,2000,29( 5) : 295 - 303.
[4] 姚善泾,梅乐和。 一种新型生物微胶囊体系及其在生物质固定化过程中的应用 [J]. 膜科学与技术,1999,19( 1) : 19 - 23.
[5] HANEFELD U,GARDOSSI L,MAGNER E. Understand-ing enzyme immobilisation [J]. Chemical Society Re-views,2009,38( 2) : 453 - 468.
[6] 陈诗一。 能源消耗,二氧化碳排放与中国工业的可持续发展[J]. 经济研究,2009( 4) : 41 -55.
[7] 付允,马永欢,刘怡君,等。 低碳经济的发展模式研究[J]. 中国人口 资源与环境,2008,18( 3) : 14 -19.
[8] NEIMAN A M. Ascospore formation in the yeast Saccharo-myces cerevisiae [J]. Microbiology and Molecular BiologyReviews,2005,69( 4) : 565 - 584.
[9] NEIMAN A M. Sporulation in the budding yeast Saccharo-myces cerevisiae [J]. Genetics,2011,189 ( 3 ) : 737 -765.
[10] SHI L,LI Z,TACHIKAWA H,et al. Use of yeast sporesfor microencapsulation of enzymes [J]. Applied and en-vironmental microbiology,2014,80 ( 15 ) : 4 502 -4 510.
[11] ZHANG H,TACHIKAWA H,GAO X-D,et al. Appliedusage of yeast spores as chitosan beads [J]. Applied andenvironmental microbiology,2014,80 ( 16 ) : 5 098 -5 105.
[12] LI Z,LI Y,DUAN S,et al. Bioconversion of d-glucoseto d-psicose with immobilized d-xylose isomerase andd-psicose 3-epimerase on Saccharomyces cerevisiae spores[J]. Journal of industrial microbiology & biotechnology,2015,42( 8) : 1 117 - 1 128.
[13] CHEN S-C,DUAN K-J. Production of Galactooligosac-charides Using β-Galactosidase Immobilized on Chitosan-Coated Magnetic Nanoparticles with Tris ( hydroxymethyl)phosphine as an Optional Coupling Agent [J]. Interna-tional journal of molecular sciences,2015,16 ( 6 ) :12 499 - 12 512.
[14] HONIGBERG S M. Ime2p and Cdc28p: Co-pilots drivingmeiotic development [J]. Journal of cellular biochemis-try,2004,92( 5) : 1 025 - 1 033.
[15] LEE B,AMON A. Meiosis: how to create a specializedcell cycle [J]. Current opinion in cell biology,2001,13( 6) : 770 -777.
[16] MITCHELL A P. Control of meiotic gene expression inSaccharomyces cerevisiae [J]. Microbiological reviews,1994,58( 1) : 56 - 70.
[17] ROEDER G S. Sex and the single cell: meiosis in yeast[J]. Proceedings of the National Academy of Sciences,1995,92( 23) : 10 450 - 10 456.
[18] STERN B M. FEARless in meiosis [J]. Molecular cell,2003,11( 5) : 1 123 - 1 125.
[19] SUDA Y,RODRIGUEZ R K,COLUCCIO A E,et al. Ascreen for spore wall permeability mutants identifies a se-creted protease required for proper spore wall assembly[J]. PLoS One,2009,4( 9) : e7184.
[20] TACHIKAWA H,BLOECHER A,TATCHELL K,et al.A Gip1p-Glc7p phosphatase complex regulates septin or-ganization and spore wall formation [J]. The Journal ofcell biology,2001,155( 5) : 797 - 808.
[21] EGEL R. Selective spore survival during replica-plating offission yeast [J]. Archives of microbiology,1977,112( 1) : 109 -110.
[22] KUPIEC M,BYERS B,ESPOSITO R E,et al. 11 Meio-sis and Sporulation in Saccharomyces cerevisiae [J]. ColdSpring Harbor Monograph Archive,1997,21: 889 -1 036.
[23] BYERS B. Cytology of the yeast life cycle [J]. ColdSpring Harbor Monograph Archive,1981,11: 59 - 96.
[24] STRATHERN J N,JONES E W,BROACH J R. Molecu-lar biology of the yeast saccharomyces,life cycle and in-heritance [M]. Cold Spring Harbor Laboratory,1981.
[25] NAKANISHI H,MORISHITA M,SCHWARTZ C L,etal. Phospholipase D and the SNARE Sso1p are necessaryfor vesicle fusion during sporulation in yeast [J]. J CellSci,2006,119( 7) : 1 406 - 1 415.
[26] MOENS P B. Fine structure of ascospore development inthe yeast Saccharomyces cerevisiae [J]. Canadian Journalof Microbiology,1971,17( 4) : 507 - 510.
[27] LYNN R R,MAGEE P T. Development of the spore wallduring ascospore formation in Saccharomyces cerevisiae[J]. The Journal of cell biology,1970,44 ( 3) : 688- 692.
[28] SMITS G J,VAN DEN ENDE H,KLIS F M. Differentialregulation of cell wall biogenesis during growth and devel-opment in yeast [J]. Microbiology,2001,147( 4) : 781- 794.
[29] COOPER K F,MALLORY M J,EGELAND D B,et al.Ama1p is a meiosis-specific regulator of the anaphase pro-moting complex / cyclosome in yeast [J]. Proceedings ofthe National Academy of Sciences, 2000, 97 ( 26 ) :14 548 - 14 553.
[30] OELSCHLAEGEL T,SCHWICKART M,MATOS J,etal. The yeast APC / C subunit Mnd2 prevents prematuresister chromatid separation triggered by the meiosis-specif-ic APC / C-Ama1 [J]. Cell,2005,120( 6) : 773 - 788.
[31] PENKNER A M,PRINZ S,FERSCHA S,et al. Mnd2,an essential antagonist of the anaphase-promoting complexduring meiotic prophase [J]. Cell,2005,120( 6) : 789- 801.
[32] COLUCCIO A,NEIMAN A M. Interspore bridges: a newfeature of the Saccharomyces cerevisiae spore wall [J].Microbiology,2004,150( 10) : 3 189 - 3 196.meiotic exit to cytokinesis during sporulation in Saccharo-myces cerevisiae [J]. Molecular biology of the cell,2009,20( 1) : 134 - 145.
[33] DIAMOND A E,PARK J-S,INOUE I,et al. The ana-phase promoting complex targeting subunit Ama1 links
[34] HIMURO Y,TAKAI M,ISHIHARA K. Poly ( vinylferro-cene-co-2-hydroxyethyl methacrylate) mediator as immo-bilized enzyme membrane for the fabrication of ampero-metric glucose sensor [J]. Sensors and Actuators B:Chemical,2009,136( 1) : 122 - 127.
[35] DEUTSCHBAUER A M,WILLIAMS R M,CHU A M,etal. Parallel phenotypic analysis of sporulation and post-germination growth in Saccharomyces cerevisiae [J]. Pro-ceedings of the National Academy of Sciences,2002,99( 24) : 15 530 -15 535.
[36] COLUCCIO A,BOGENGRUBER E,CONRAD M N,etal. Morphogenetic pathway of spore wall assembly in Sac-charomyces cerevisiae [J]. Eukaryotic Cell,2004,3( 6) : 1 464 -1 475.
[37] GOMEZ-ESQUER F,RODRGUEZ-PEA J M,DAZG,et al. CRR1,a gene encoding a putative transglycosi-dase,is required for proper spore wall assembly in Sac-charomyces cerevisiae [J]. Microbiology, 2004, 150( 10) : 3 269 -3 280.
[38] SARKAR P,FLORCZYK M,MCDONOUGH K,et al.SSP2, a sporulation-specific gene necessary for outerspore wall assembly in the yeast Saccharomyces cerevisiae[J]. Molecular Genetics and Genomics,2002,267( 3) :348 - 358.
[39] LI J,AGARWAL S,ROEDER G S. SSP2 and OSW1,two sporulation-specific genes involved in spore morpho-genesis in Saccharomyces cerevisiae [J]. Genetics,2007,175( 1) : 143 - 154.
[40] CHU S,DERISI J,EISEN M,et al. The transcriptionalprogram of sporulation in budding yeast [J]. Science,1998,282( 5389) : 699 - 705.
[41] CHRISTODOULIDOU A,BOURIOTIS V,THIREOS G.Two sporulation-specific chitin deacetylase- encodinggenes are required for the ascospore wall rigidity of Sac-charomyces cerevisiae [J]. Journal of Biological Chemis-try,1996,271( 49) : 31 420 - 31 425.
[42] MISHRA C,SEMINO C E,MCCREATH K J,et al. Clo-ning and expression of two chitin deacetylase genes ofSaccharomyces cerevisiae [J]. Yeast,1997,13( 4) : 327- 336.[43] PAMMER M,BRIZA P,ELLINGER A,et al. DIT101( CSD2,CAL1) ,a cell cycle ‐ regulated yeast gene re-quired for synthesis of chitin in cell walls and chitosan inspore walls [J]. Yeast,1992,8( 12) : 1 089 - 1099.
[44] SANZ M,TRILLA O A,DURAN N,et al. Control ofchitin synthesis through Shc1p,a functional homologue ofChs4p specifically induced during sporulation [J]. Mo-lecular microbiology,2002,43( 5) : 1 183 - 1 195.
[45] NEUWALD A F. Barth syndrome may be due to an acyl-transferase deficiency [J]. Current Biology, 1997,7( 8) : R462 - R6.
[46] BRIZA P,ELLINGER A,WINKLER G,et al. Charac-terization of a DL-dityrosine-containing macromoleculefrom yeast ascospore walls [J]. Journal of BiologicalChemistry,1990,265( 25) : 15 118 - 15 123.
[47] BRIZA P,KALCHHAUSER H,PITTENAUER E,et al.N,N' Bisformyl Dityrosine is an in vivo Precursor of theYeast Ascospore Wall [J]. European journal of biochem-istry,1996,239( 1) : 124 - 131.
[48] BRIZA P,ECKERSTORFER M,BREITENBACH M.The sporulation-specific enzymes encoded by the DIT1and DIT2 genes catalyze a two-step reaction leading to asoluble LL-dityrosine-containing precursor of the yeastspore wall [J]. Proceedings of the National Academy ofSciences,1994,91( 10) : 4 524 - 4 528.
[49] FELDER T,BOGENGRUBER E,TENREIRO S,et al.Dtr1p,a multidrug resistance transporter of the major fa-cilitator superfamily,plays an essential role in spore wallmaturation in Saccharomyces cerevisiae [J]. Eukaryoticcell,2002,1( 5) : 799 - 810.
[50] 李毅。 酿酒酵母孢子固定化酶催化 D-葡萄糖合成 D-阿洛酮糖的研究 [J]. 2015.
[51] CHOW C K,PALECEK S P. Enzyme encapsulation inpermeabilized Saccharomyces cerevisiae cells [J]. Bio-technology progress,2004,20( 2) : 449 - 456.
[52] ROMERO C,SANCHEZ S,MANJON S,et al. Optimi-zation of the pectinesterase / endo-D-polygalacturonase co-immobilization process [J]. Enzyme and microbial tech-nology,1989,11( 12) : 837 - 843.
[53] MEN Y,ZHU Y,ZENG Y,et al. Co-expression of d-glucose isomerase and d-psicose 3-epimerase: develop-ment of an efficient one-step production of d-psicose [J].Enzyme and microbial technology,2014,64: 1 - 5.
[54] MASARU S,MITSUTAKA Y. A new enzymatic serumcreatinine measurement based on an endogenous creatine-eliminating system [J]. Clinica chimica acta,1984,143( 2) : 147 -155.
[55] FOSSATI P,PRENCIPE L,BERTI G. Enzymic creati-nine assay: a new colorimetric method based on hydrogenperoxide measurement [J]. Clinical chemistry,1983,29( 8) : 1 494 -1 496.
[56] COLUCCIO A E,RODRIGUEZ R K,KERNAN M J,etal. The yeast spore wall enables spores to survive passagethrough the digestive tract of Drosophila [J]. PLoS One,2008,3( 8) : e2873.