CN116555061A - 一种酿酒酵母突变体及其在制备酒精中的应用 - Google Patents
一种酿酒酵母突变体及其在制备酒精中的应用 Download PDFInfo
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Abstract
本发明公开一种酿酒酵母突变体及其在制备酒精中的应用。本发明提供了Hsp82蛋白突变体为将Hsp82蛋白对应于序列2所示的氨基酸序列第485位残基进行突变，得到具有相同活性的蛋白。本发明提供的Hsp82蛋白S485A酿酒酵母突变菌株，不能利用乙醇作为碳源，可以减少在培养基营养不足时酵母细胞利用乙醇进行氧化磷酸化供能的生物学过程以提高乙醇产量。
Description
技术领域
本发明属于基因工程领域，涉及一种酿酒酵母突变体及其在制备酒精中的应用。
背景技术
酿酒酵母又称为面包酵母(baking yeast)或者出芽酵母(budding yeast)，属于真菌界真菌门子囊菌纲酵母目酵母菌科酵母菌属。酿酒酵母是一种常见的单细胞真核生物，通常为球形或者椭圆形，被广泛应用于制作馒头、面包等食品及酿酒。在营养丰富的环境中，酿酒酵母会将培养基中的发酵类碳源(主要是葡萄糖或者蔗糖)通过糖酵解途径转化为乙醇并为自身提供能量。当营养物质不足并且供氧充足的情况下，酿酒酵母会对培养基中的乙醇再次利用，通过氧化磷酸化的途径生成CO2和水并获得能量。而在工业酒精生产过程中，酵母细胞利用乙醇进行氧化磷酸化的生物学过程会大大降低酒精的产量。
ATP生产和乙醇合成的生物学过程是相耦合的，Steffen Klamt实验室通过在酿酒酵母中异源表达大肠杆菌F1-ATPase基因的αβγ亚基，提高ATP酶活性，减少ATP的产生来提高乙醇产量(与对照菌株相比增加了10％)。
也有研究人员通过双敲除ADH2基因(乙醇脱氢酶Ⅱ)和ALD6基因(乙醛脱氢酶Ⅵ)，分别阻断催化乙醇生成乙醛的过程和降低乙醇到乙酸的代谢流来提高乙醇的产量。通过气相色谱检测乙醇含量，与ADH2基因缺失菌株(YS2-Δadh2)和出发菌株(YS2)相比，双基因敲除菌株最高乙醇产量分别提高了6.9％和12.5％。
现有技术中主要采用了基因敲除，降低酿酒酵母代谢过程中某基因的表达量或过表达外源ATP酶降解ATP来提高乙醇产量的技术手段。其中，基因敲除手段往往改变单一基因表达量无法达到预期效果，需要在单敲的基础上进行双敲甚至三敲来提高效果，而且受限于酵母细胞同源重组的效率，因此筛选工作量较为繁重。而在酿酒酵母细胞中过表达外源ATP酶的方法，同样也需要较为复杂的遗传操作手段，而且过表达ATP酶，加快细胞内ATP的消耗会对酵母生物量和生长速率有显著的影响，降低了菌株生长速度和单位体积乙醇生产率。为了克服这一问题，需要将菌株生长和乙醇生产的过程分开。
因此，寻找一种新的提高乙醇产量的方法为研究热点。
发明内容
本发明的一个目的是提供一种不能够利用乙醇作为唯一碳源的酿酒酵母突变体在工业上生产酒精。
本发明提供的重组菌在生产乙醇或提高乙醇产量中的应用；
所述重组菌，为将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变，且该蛋白其他氨基酸残基不变，得到的菌株；
所述Hsp82蛋白，为如下A或B或C：
A)所述的蛋白为序列2所示的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白。
上述应用中，所述将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变为将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变为不发生磷酸化的氨基酸残基。
上述应用中，所述不发生磷酸化的氨基酸残基为丙氨酸。
本发明还提供了Hsp82蛋白突变体或其编码基因或含有所述编码基因的表达盒或重组载体或上述重组菌的用途。
本发明提供了Hsp82蛋白突变体或其编码基因或含有所述编码基因的表达盒或重组载体在生产乙醇或提高乙醇产量或制备生产乙醇或提高乙醇产量重组菌中的应用；
所述Hsp82蛋白突变体，为如下A或B或C：
A)所述的蛋白为将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变，且该蛋白其他氨基酸残基不变，得到具有相同活性的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白。
上述应用中，所述将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为将将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为不发生磷酸化的氨基酸残基。
上述不发生磷酸化的氨基酸残基为丙氨酸。
本发明还提供了如下应用：
使酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸去磷酸化的物质，在生产乙醇或提高乙醇产量或制备高产乙醇中的应用；
或，使酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸突变的物质，在生产乙醇或提高乙醇产量或制备高产乙醇中的应用。
上述应用中，所述物质为用于将酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸发生点突变的引物或试剂。
本发明还有一个目的是提供一种生产乙醇的方法。
本发明提供的方法，包括如下步骤：发酵上述第一个目的的重组菌，得到乙醇。
上述发酵中不能够利用乙醇作为唯一碳源，可以采用碳源为葡萄糖、甘露糖、半乳糖或棉子糖。
上述发酵到132h以上，与野生型重组菌相比，乙醇产量仍然稳定。
本发明还提供了一种Hsp82蛋白突变体。
本发明提供的Hsp82蛋白突变体，为如下A或B或C：
A)所述的蛋白为将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为丙氨酸，得到具有相同活性的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白。
含有上述Hsp82蛋白突变体的编码基因或含有所述编码基因的表达盒或重组载体也是本发明保护的范围。
本发明的实验证明，本发明提供的Hsp82蛋白S485A酿酒酵母突变菌株，不能利用乙醇作为碳源，可以减少在培养基营养不足时酵母细胞利用乙醇进行氧化磷酸化供能的生物学过程以提高乙醇产量；而且突变菌株在营养充足和不足的情况下，并不影响菌株的生长状态，在实际生产应用中具有优异表现。
附图说明
图1为WT和S485A菌株生长曲线。
图2为WT和S485A菌株生成乙醇的浓度。
图3为WT和S485A菌株对发酵碳源的利用。
图4为WT和S485A菌株对非发酵碳源的利用。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
序列1为野生型HSP82基因。
序列2为野生型HSP82蛋白。
序列3为用于同源重组构建重组菌GFP-HSP82的片段，序列3第1-534位和第7872-8352位为侧翼序列，第541-1254位为GFP基因，第1261-3390位为HSP82基因。
序列4为用于同源重组构建重组菌GFP-HSP82S485A的片段，序列4第1-534位和第7872-8352位为侧翼序列，第541-1254位为GFP基因，第1261-3390位为HSP82S485A基因。
W303-1A菌株记载在如下文献中：Han J,Li Q,McCullough L,Kettelkamp C,Formosa T,Zhang Z.Ubiquitylation of FACT by the Cullin-E3 ligase Rtt101connects FACT to DNA replication.Genes Dev.2010；(14):1485-1490.。
实施例1、突变体菌株的制备
一、构建HSP82突变基因
1、克隆HSP82基因
野生型HSP82基因的核酸序列为序列1所示，该DNA序列在NCBI Genebank上的登录号为855836，野生型HSP82基因编码野生型Hsp82蛋白(分子伴侣)，野生型Hsp82蛋白的氨基酸序列为序列2所示。
在酵母整合质粒CIp10载体的基础上设计特异性引物，构建靶基因原位标记的酵母整合质粒，具体如下：
以野生型菌株W303-1A基因组为模板，分别以HSP82-F和HSP82-R、Hsp82AB475-F和HSP82AB1003-R、HSP82CD3125-F和HSP82CD3605-R扩增得到HSP82基因(2130bp)、HSP82基因上游侧翼序列(529bp)和HSP82基因下游侧翼序列(481bp)。
以HSP82基因上游侧翼序列和HSP82基因下游侧翼序列为模板，以HSP82CD3125-F和HSP82AB1003-R为引物扩增得到融合侧翼序列(1016bp)。
CIp10-HSP82质粒为将融合侧翼序列(序列3第1-534、BamHI酶切位点、序列3第7872-8352位)替换CIp10载体(记载在如下文献中Xuan Shang,Guang Cao,Han Gao,Melinda Li,Guanzu Peng,Yanqiu Ji,Yansong Zhang,Wenzhe Zhang,Wanjie Li,FeiDou.A Single Site Phosphorylation on Hsp82 Ensures Cell Survival duringStarvation in Saccharomyces cerevisiae.Journal of Molecular Biology,Volume432,Issue 21,2020,Pages 5809-5824.)的KpnI和XhoI酶切位点间的片段，且将HSP82基因(序列1或序列3第1261-3390位)替换CIp10载体中ClaI和PstI酶切位点间的片段得到的质粒。
以CIp10-Met3pt-GFP质粒为模板，以GFP-F和GFP-R扩增得到GFP基因(714bp)。
CIp10-GFP-HSP82质粒为将GFP基因(序列3第541-1254位)替换CIp10-HSP82质粒的XhoI和ClaI酶切位点间的片段得到的质粒。
上述构建质粒使用引物：
HSP82-F:ATATCGATATGGCTAGTGAAACTTTTG
HSP82-R:ATCTGCAGATATCATTGGAGAAGTGTTC
HSP82AB475-F:GAACACTTCTCCAATGATATGGATCCATTTGGAAGCTTGATGACAG
HSP82AB1003-R:ATCTCGAGCATATCTTTGCGTGTTTGTTTG
HSP82CD3125-F:ATGGTACCGATTAGGTAGAACATCATGG
HSP82CD3605-R:GGATCCATATCATTGGAGAAGTGTTC
GFP-F:CCGCTCGAGATGTCTAAAGGTGAAGAATT
GFP-R:CCATCGATTTTGTACAATTCATCCATAC
CIp10-GFP-HSP82质粒用BamHI(该位点位于两段融合侧翼CD和AB之间)酶切，得到线性化质粒CIp10-GFP-HSP82。
2、定点突变S485A
参考定点突变试剂盒(KOD-Plus-Mutagenesis Kit，TOYOBO,SMK-101)的方法，设计点突变引物，在CIp10-GFP-HSP82质粒的基础上定点突变HSP82基因Ser485位构建S485A突变质粒CIp10-GFP-HSP82S485A，即将HSP82基因表达蛋白第485位点的Ser丝氨酸定点突变为Ala丙氨酸。
具体方法如下：以CIp10-GFP-HSP82质粒为模板，HSP82S485A-F和HSP82-R引物进行PCR扩增，得到突变质粒CIp10-GFP-HSP82S485A。
使用引物：
HSP82S485A-F:GCTCCATTTTTGGATGCCTTG
HSP82-R:CTTTTCGACAGCCTTTAGAGAT
突变质粒CIp10-GFP-HSP82S485A与CIp10-GFP-HSP82质粒唯一区别，就是将CIp10-GFP-HSP82质粒中的HSP82基因替换为HSP82S485A基因；
突变质粒CIp10-GFP-HSP82S485A含有GFP-HSP82S485A；该GFP-HSP82S485A的核苷酸序列为序列4第541-3390位，其中序列4第1261-3390位为HSP82S485A基因，序列4第541-1254位为GFP。
HSP82S485A基因为将HSP82基因序列1第1453位的T碱基突变为G碱基，得到的突变基因。
HSP82S485A基因编码HSP82S485A蛋白，HSP82S485A蛋白为将Hsp82蛋白序列2第485位的丝氨酸突变为丙氨酸，得到的突变蛋白。
突变质粒CIp10-GFP-HSP82S485A用BamHI(该位点位于两段融合侧翼CD和AB两段之间)酶切，得到线性化质粒CIp10-GFP-HSP82S485A。
HSP82S485A为将HSP82蛋白中能够被磷酸化的丝氨酸S突变成丙氨酸，实现HSP82第485位氨基酸残基去除磷酸化的目的。
二、构建S485A突变菌株
1、质粒转化酿酒酵母W303-1A菌株
通过醋酸锂转化法来构建酵母菌株，主要步骤如下：
收集对数期酵母细胞W303-1A菌株(1OD),3500g，离心3min,收集细胞弃上清。加入适量的双蒸水洗涤后3500g,离心3min，收集细胞弃上清。再加入1mL 0.1M醋酸锂水溶液重悬菌体，转移至2mL的EP管中17000g高速离心5s弃上清。依次加入如下试剂制备转化混合液：240μL无菌的50％(质量体积百分含量，g:ml)PEG 3350，36μL 1M的醋酸锂水溶液，5μL10mg/mL单链鲑鱼精DNA(Promega，D181B)，70μL水和2μg上述一制备的线性化的突变质粒CIp10-GFP-HSP82S485A DNA。用涡旋混匀仪混匀后置于30℃培养箱孵育1h。42℃水浴20min，6000rpm离心1min，弃上清。加入600μL无菌水，轻柔的吹打混匀，取出200μL涂布在相应的营养筛选平板(SD-Ura，即6.7g/L YNB(Solarbio，Y8040)、0.77g/L Ura DO Supplement(Clontech，630416)、20g/L葡萄糖、20g/L琼脂)将平板置于30℃培养箱培养3天，得到长有转化突变质粒CIp10-GFP-HSP82S485A的重组菌平板和长有转化质粒CIp10-GFP-HSP82的重组菌平板。
2、基因组水平鉴定
将筛选平板上的转化突变质粒CIp10-GFP-HSP82S485A的重组菌单克隆挑和转化质粒CIp10-GFP-HSP82的重组菌至含有相应的选择性液体培养基(SD-Ura，即6.7g/L YNB(Solarbio，Y8040)、0.77g/L Ura DO Supplement(Clontech，630416)、20g/L葡萄糖(Solarbio,G8150)、20g/L琼脂(Sangon Biotech,A600010))的试管中，将其置于30℃，250rpm摇床中过夜培养。取约0.1OD600的细胞悬液，3500g离心3min，弃上清，加入100μL0.2M的醋酸锂水溶液和5μL 20％(质量体积比)的SDS水溶液，上下颠倒混匀，置于70℃水浴锅中孵育15min。加入300μL 96％乙醇水溶液洗涤一次，15000g离心3min后弃上清。加入500μL 70％乙醇水溶液洗涤一次，15000g离心3min后弃上清。风干沉淀，加入适量双蒸水溶解沉淀，即为酵母基因组。
分别以上述不同酵母基因组为模板，用如下鉴定引物进行PCR扩增,得到约840bp的为阳性克隆。
鉴定引物：
HSP82AB-F:GCCTATCGTATTACAATTCG
GFP58-R:TTGTGACCATTAACATCACC
将来源于转化突变质粒CIp10-GFP-HSP82S485A的重组菌的阳性克隆命名为重组菌GFP-HSP82S485A，该重组菌为将序列4所示的含有GFP-HSP82S485A的片段通过同源重组替换酿酒酵母W303-1A基因组中的HSP82基因。其中序列4第1-534及7872-8352位为侧翼序列，第541-1254位为GFP基因，第1261-3390位为HSP82S485A基因；
采用同样的方法将线性化的CIp10-GFP-HSP82质粒转入酿酒酵母W303-1A菌株，得到重组菌GFP-HSP82，该重组菌为将序列3所示的含有GFP-HSP82的片段通过同源重组替换酿酒酵母W303-1A基因组中的HSP82基因。其中序列3第1-534及7872-8352位为侧翼序列，第541-1254位为GFP基因，第1261-3390位为HSP82基因。
实施例2、重组菌GFP-HSP82S485A在生产乙醇中的应用
一、GFP-HSP82S485A菌株的基本表型
1、酵母细胞GFP-HSP82S485A菌株生长曲线测定
将实施例1制备的酵母菌株重组菌GFP-HSP82S485A和重组菌GFP-HSP82分别接种至5mL YPD液体培养基(10g/L酵母提取物(Oxoid,LP0021)、20g/L蛋白胨(Sangon Biotech,A505247)、20g/L葡萄糖(Solarbio,G8150))中，30℃,250rpm过夜培养。第二天测定过夜培养物浓度OD600，以终OD600＝0.01转接至新鲜的10mL YPD液体培养基中连续培养132h，132h期间定时取样测定OD600并记录。实验结束后依据OD600值绘制生长曲线。
结果如图1所示，利用双尾T-test对时间点两菌株的差异进行显著性分析，*表示GFP-HSP82(WT)和GFP-HSP82S485A(S485A)菌株之间的差异，“*”p<0.05；“**”p<0.01；“***”p<0.001。)，可以看出，重组菌GFP-HSP82S485A在不同时间点的生长速度均明显慢于重组菌GFP-HSP82，且没有明显的双相转换期，生长到24h后生长速度基本维持不变达到平台期。
以野生菌W303-1A为对照，重组菌GFP-HSP82和野生菌W303-1A的结果无显著差异。
2、酵母细胞GFP-HSP82S485A培养中乙醇浓度测定
在营养丰富条件下，酵母细胞会利用葡萄糖进行有氧糖酵解生产乙醇和ATP供细胞快速增殖。在测菌株生长速率的同时，收集了不同生长时期培养物的上清检测，重组菌GFP-HSP82和重组菌GFP-HSP82S485A生成乙醇的浓度，具体如下：
在上述1定时取样测生长曲线的同时，取不同生长时间点的菌液100μL，3500g,3min离心取上清，分装于新的EP管，用封口膜封好存放于-80℃。待收集完一批样品后，按照乙醇检测试剂盒(EnzyChromTM Ethanol Assay Kit,ECET-100；BioAssay Systems,Cat.ECET-100)要求测定乙醇浓度(1vol％＝170mM)。
结果如图2所示，将重组菌GFP-HSP82(WT)和重组菌GFP-HSP82S485A(S485A)过夜培养物终OD600＝0.01转接至新鲜的10mL YPD液体培养基中连续培养132h，期间定时取酵母培养物上清测定乙醇浓度，1vol％＝170mM。同时利用双尾T-test对时间点两菌株的差异进行显著性分析，*表示WT菌株和S485A菌株之间的差异，“**”p<0.01；“***”p<0.001。)，结果显示：在0-24h时，重组菌GFP-HSP82(WT)和重组菌GFP-HSP82S485A(S485A)快速产生乙醇，而且生成的速率接近。培养至60h时，重组菌GFP-HSP82(WT)产生乙醇的浓度开始下降，酵母细胞利用乙醇进行氧化磷酸化以维持细胞生长，在培养至132h时乙醇浓度已低于0.5vol％。而重组菌GFP-HSP82S485A(S485A)在培养至60h时产生乙醇的浓度未达到峰值仍在上升，72h时乙醇浓度达到峰值2vol％(相当于340mM)。在84h后略有下降，这有可能是由于培养时间较长，乙醇挥发所导致的下降。
重组菌GFP-HSP82和野生菌W303-1A的结果无显著差异。
与重组菌GFP-HSP82相比，推测重组菌GFP-HSP82S485A菌株由于无法利用乙醇作为碳源，因此，在生长过程中会大量积累乙醇，具有工业化生产乙醇的潜力。
下面的实验会进一步证实。
二、GFP-HSP82S485A菌株对不同碳源的利用
将重组菌GFP-HSP82和重组菌GFP-HSP82S485A过夜培养物终OD600＝0.01转接至新鲜的10mL YPD液体培养基中连续培养10h至对数期早期，3500g离心3min时间收集细胞，用双蒸水洗涤2次后分别转接至对应的10mL发酵碳源液体培养基和非发酵碳源液体培养基连续培养12h，期间定时取酵母培养物测定OD600来反映菌株的生长速率。
发酵碳源液体培养基:1％酵母提取物(Oxoid,LP0021)、2％蛋白胨(SangonBiotech,A505247)和2％不同发酵碳源或2.36％棉子糖(Solarbio,D8180))，余量为水；百分比均为质量百分含量g:ml；其中，2％不同发酵碳源为2％葡萄糖(Solarbio,G8150)、2％甘露糖(Solarbio,G8370)、2％半乳糖(Solarbio,D8310)；
非发酵碳源液体培养基：1％酵母提取物，2％蛋白胨和2％不同非发酵碳源，余量为水；百分比均为质量百分含量g:ml；其中，2％不同非发酵碳源为2％乙醇(SangonBiotech,A500737)或2％甘油(西陇化工股份有限公司,12801801)；
结果如图3和图4所示，其中，WT为重组菌GFP-HSP82，S485A为重组菌GFP-HSP82S485A；可以看出，重组菌GFP-HSP82可以利用发酵碳源葡萄糖、甘露糖和半乳糖，以及非发酵碳源乙醇和甘油进行生长；而重组菌GFP-HSP82S485A可以较好的利用发酵碳源葡萄糖和甘露糖，但在以发酵碳源半乳糖、棉子糖和非发酵碳源乙醇和甘油为唯一碳源的条件下不能较好的生长，无法有效的利用这些碳源为细胞供能。
上述结果证明了与重组菌GFP-HSP82相比，重组菌GFP-HSP82S485A无法利用乙醇作为碳源，使其在生长过程中会大量积累乙醇。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110>北京师范大学
<120> 一种酿酒酵母突变体及其在制备酒精中的应用
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gacgaagtta atgaaagaat tggtaggggt accatcttga ggttattctt gaaagatgac 1800
caattggagt acttggaaga aaagagaata aaggaagtta tcaagagaca ttctgagttc 1860
gtggcctacc caatccaatt agtcgtcacc aaggaagttg aaaaggaagt tccaattcca 1920
gaagaagaaa agaaagacga ggaaaagaag gatgaggaaa agaaggatga agacgacaag 1980
aaaccaaaat tggaagaagt cgatgaagaa gaggaaaaga agccaaagac gaaaaaagtt 2040
aaagaagaag ttcaagagat agaagaacta aacaagacta agcctttgtg gactagaaac 2100
ccatctgata tcactcaaga agaatacaat gctttctata agtctatttc aaacgactgg 2160
gaagacccat tgtacgttaa gcatttctcc gttgaaggtc aattggaatt tagagctatc 2220
ttattcattc caaagagagc accattcgac ttgtttgaga gtaaaaagaa gaagaataat 2280
atcaagttgt acgttcgtcg tgttttcatc actgatgaag ctgaagactt gattccagag 2340
tggttatctt tcgtcaaggg tgttgttgac tctgaggatt taccattgaa tttgtccaga 2400
gaaatgttac aacaaaataa gatcatgaag gttattagaa agaacattgt caaaaagttg 2460
attgaagcct tcaacgaaat tgctgaagac tctgaacaat ttgaaaagtt ctactcggct 2520
ttctccaaaa atatcaagtt gggtgtacat gaagataccc aaaacagggc tgctttggct 2580
aagttgttac gttacaactc taccaagtcc gtagatgagt tgacttcctt aactgattac 2640
gttaccagaa tgccagaaca ccaaaagaac atctactaca tcactggtga atctctaaag 2700
gctgtcgaaa agtctccatt tttggatgcc ttgaaggcta aaaacttcga ggttttgttc 2760
ttgaccgacc caattgatga atacgccttc actcaattga aggaattcga aggtaaaact 2820
ttggttgaca ttactaaaga tttcgaattg gaagaaactg acgaagaaaa agctgaaaga 2880
gagaaggaga tcaaagaata tgaaccattg accaaggcct tgaaagaaat tttgggtgac 2940
caagtggaga aagttgttgt ttcttacaaa ttgttggatg ccccagctgc tatcagaact 3000
ggtcaatttg gttggtctgc taacatggaa agaatcatga aggctcaagc cttgagagac 3060
tcttccatgt cctcctacat gtcttccaag aagactttcg aaatttctcc aaaatctcca 3120
attatcaagg aattgaaaaa gagagttgac gaaggtggtg ctcaagacaa gactgtcaag 3180
gacttgacta agttattata tgaaactgct ttgttgactt ccggcttcag tttggacgaa 3240
ccaacttcct ttgcatcaag aattaacaga ttgatctctt tgggtttgaa cattgatgag 3300
gatgaagaaa cagagactgc tccagaagca tccaccgcag ctccggttga agaggttcca 3360
gctgacaccg aaatggaaga ggtagattag ctgcagctaa attgtaagcg ttaatatttt 3420
gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 3480
cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 3540
ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 3600
ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 3660
gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 3720
aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 3780
gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 3840
gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 3900
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 3960
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc 4020
gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc gcggccgact 4080
tggccaagcc tagatcccga ctaataggaa ttgatttgga tggtataaac ggaaacaaaa 4140
aaaagagctg gtactacttt ctttaaaatt attttattat ttgattttat ttaatagtat 4200
atattatatt ttgaacgtag attattttgt tgaaagttgc tgtagtgcca ttgattcgta 4260
acactaattc tgtattagtc attcctcttg tttgatagta tccaaaaaaa cggctatttt 4320
tttgcaatct tatttcctgc atattataca gataacataa tgaaagaaaa aatctttttt 4380
tttgttcttc aatgatgatt tcaaccattc ttttaaacat tgatcaattc ctgagcaaca 4440
accccataca cactggttta tataccgccc cttttacagt tgaagaaaga aatagaaata 4500
gaaatagcaa acaaaagata tgacagtcaa cactaagacc tatagtgaga gagcagaaac 4560
tcatgcctca ccagtagcac agcgattatt tcgattaatg gaactgaaga aaaccaattt 4620
atgtgcatca attgacgttg ataccactaa ggaattcctt gaattaattg ataaattagg 4680
tccttatgta tgcttaatca agactcatat tgatataatc aatgattttt cctatgaatc 4740
cactattgaa ccattattag aactttcacg taaacatcaa tttatgattt ttgaagatag 4800
aaaatttgct gatattggta ataccgtaaa gaaacaatat attggtggag tttataaaat 4860
tagtagttgg gcagatatta ccaatgctca tggtgtcact gggaatggag tggttgaagg 4920
attaaaacag ggagctaaag aaaccaccac caaccaagag ccaagagggt tattgatgtt 4980
agctgaatta tcatcagtgg gatcattagc atatggagaa tattctcaaa aaactgttga 5040
aattgctaaa tccgataagg aatttgttat tggatttatt gcccaacgtg atatgggtgg 5100
ccaagaagaa ggatttgatt ggcttattat gacacctgga gttggattag atgataaagg 5160
tgatggatta ggacaacaat atagaactgt tgatgaagtt gttagcactg gaactgatat 5220
tatcattgtt ggtagaggat tgtttggtaa aggaagagat ccagatattg aaggtaaaag 5280
gtatagaaat gctggttgga atgcttattt gaaaaagact ggccaattat aaatgtgaag 5340
ggggagattt tcactttatt agatttgtat atatgtagaa taaataaata aataagttaa 5400
ataaataatt aaataagggt ggtaattatt actatttaca atcaaaggtg gtccttctag 5460
aactagtgga tctgttgatc aaagttttac caacatttct gttttcaaaa gtggttggag 5520
ctttgatgtc aaaccaatct tttctggtga atgggtcaac gaccttcttt tttaatcctt 5580
tctttccttt ggacaatctc ttgtttttac cgacagccat gttgtacttg agttggatct 5640
acgcgtgaat tcgatatcgg tacccagctt ttgttccctt tagtgagggt taattgcgcg 5700
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 5760
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5820
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 5880
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 5940
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6000
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6060
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6120
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6180
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6240
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6300
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6360
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6420
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6480
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6540
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6600
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6660
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6720
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6780
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6840
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 6900
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 6960
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7020
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7080
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7140
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7200
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7260
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7320
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7380
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7440
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 7500
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7560
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7620
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7680
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 7740
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 7800
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 7860
gccacggtac cgattaggta gaacatcatg gccttgaata ggttataaac aaaacataat 7920
ataacgtata ggtattcgaa tgaataaata agtatgtaaa tagggcatct gcatggaaat 7980
aactgggtaa aacattacaa tgtatttttt tagagttttc actattccga cgcgtctagt 8040
gtgtcataca aaacgcgaag aatctgaaat cgtgaaatat acttaagaat ataactgaaa 8100
tagcttaagc ataaatggac tttactgcga agataaaggg tatgttgaaa ctcatctgct 8160
ttcattatgc gtccaactaa ttaacattgc atattttcgt atactatttt tttgtgtaga 8220
gctcgaacga gaactctcag agacgagtga ctataagacc ttgcagaaga agaccataag 8280
tcttcgaagt gagctgaata ctctgtcaca ttctttgaca tcttatgaaa aagaacactt 8340
ctccaatgat atggatcc 8358
<210> 4
<211> 8358
<212> DNA
<213> Artificial sequence
<400> 4
gatccatttg gaagcttgat gacagaatat tttaaaatat aacttagctt gcgtgtgcgt 60
atttgtattc ttcgtggtta cgatatgaac caacgtgcaa gcgtgtgata tatcacattc 120
cggagggtgt caccccccct ctctcaacac agtaatccat aaaccagttt tacatacacg 180
taaaaaagaa caggaataaa gcttaatcgg attattaact catacgcttg tcacatattg 240
ttcgaacaat tctggttctt tcgagtttcg cagaactttt tgaatttttc ttttttttct 300
agaacgccgt ggaagaaaaa cacgcgcatg gttttatgag cggttaattc tcatcttaat 360
accaaccagg tccttccgcc accccctaaa acatataaat atgcagctta tcccttcaat 420
tcttaacatc tgtgacctcc tcatttcttc ccgctgtatt agagttcaag aaatcatacc 480
tgatagaaaa tagagtccta taaacaaaag cacaaacaaa cacgcaaaga tatgctcgag 540
atgtctaaag gtgaagaatt attcactggt gttgtcccaa ttttggttga attagatggt 600
gatgttaatg gtcacaaatt ttctgtctcc ggtgaaggtg aaggtgatgc tacttacggt 660
aaattgacct taaaatttat ttgtactact ggtaaattgc cagttccatg gccaacctta 720
gtcactactt tcggttatgg tgttcaatgt tttgctagat acccagatca tatgaaacaa 780
catgactttt tcaagtctgc catgccagaa ggttatgttc aagaaagaac tatttttttc 840
aaagatgacg gtaactacaa gaccagagct gaagtcaagt ttgaaggtga taccttagtt 900
aatagaatcg aattaaaagg tattgatttt aaagaagatg gtaacatttt aggtcacaaa 960
ttggaataca actataactc tcacaatgtt tacatcatgg ctgacaaaca aaagaatggt 1020
atcaaagtta acttcaaaat tagacacaac attgaagatg gttctgttca attagctgac 1080
cattatcaac aaaatactcc aattggtgat ggtccagtct tgttaccaga caaccattac 1140
ttatccactc aatctgcctt atccaaagat ccaaacgaaa agagagacca catggtcttg 1200
ttagaatttg ttactgctgc tggtattacc catggtatgg atgaattgta caaaatcgat 1260
atggctagtg aaacttttga atttcaagct gaaattactc agttgatgag tttgatcatc 1320
aacaccgtct attctaacaa ggaaattttc ttgagagaac tgatatctaa tgcctcggat 1380
gcgttggata aaattagata caaatctttg tctgatccaa agcaattgga aacagaacca 1440
gatctcttta ttagaatcac tccaaagcca gagcaaaaag ttttggaaat cagagattct 1500
ggtattggta tgaccaaggc tgaattgatt aataacttgg gtaccattgc caagtctggt 1560
accaaagcct tcatggaagc tctatctgct ggtgccgatg tatccatgat tggtcaattc 1620
ggtgttggtt tttactcttt attcttagtt gccgacagag ttcaggttat ttcaaagagc 1680
aacgacgacg aacaatacat ctgggaatcc aacgctggtg gttctttcac tgttactcta 1740
gacgaagtta atgaaagaat tggtaggggt accatcttga ggttattctt gaaagatgac 1800
caattggagt acttggaaga aaagagaata aaggaagtta tcaagagaca ttctgagttc 1860
gtggcctacc caatccaatt agtcgtcacc aaggaagttg aaaaggaagt tccaattcca 1920
gaagaagaaa agaaagacga ggaaaagaag gatgaggaaa agaaggatga agacgacaag 1980
aaaccaaaat tggaagaagt cgatgaagaa gaggaaaaga agccaaagac gaaaaaagtt 2040
aaagaagaag ttcaagagat agaagaacta aacaagacta agcctttgtg gactagaaac 2100
ccatctgata tcactcaaga agaatacaat gctttctata agtctatttc aaacgactgg 2160
gaagacccat tgtacgttaa gcatttctcc gttgaaggtc aattggaatt tagagctatc 2220
ttattcattc caaagagagc accattcgac ttgtttgaga gtaaaaagaa gaagaataat 2280
atcaagttgt acgttcgtcg tgttttcatc actgatgaag ctgaagactt gattccagag 2340
tggttatctt tcgtcaaggg tgttgttgac tctgaggatt taccattgaa tttgtccaga 2400
gaaatgttac aacaaaataa gatcatgaag gttattagaa agaacattgt caaaaagttg 2460
attgaagcct tcaacgaaat tgctgaagac tctgaacaat ttgaaaagtt ctactcggct 2520
ttctccaaaa atatcaagtt gggtgtacat gaagataccc aaaacagggc tgctttggct 2580
aagttgttac gttacaactc taccaagtcc gtagatgagt tgacttcctt aactgattac 2640
gttaccagaa tgccagaaca ccaaaagaac atctactaca tcactggtga atctctaaag 2700
gctgtcgaaa aggctccatt tttggatgcc ttgaaggcta aaaacttcga ggttttgttc 2760
ttgaccgacc caattgatga atacgccttc actcaattga aggaattcga aggtaaaact 2820
ttggttgaca ttactaaaga tttcgaattg gaagaaactg acgaagaaaa agctgaaaga 2880
gagaaggaga tcaaagaata tgaaccattg accaaggcct tgaaagaaat tttgggtgac 2940
caagtggaga aagttgttgt ttcttacaaa ttgttggatg ccccagctgc tatcagaact 3000
ggtcaatttg gttggtctgc taacatggaa agaatcatga aggctcaagc cttgagagac 3060
tcttccatgt cctcctacat gtcttccaag aagactttcg aaatttctcc aaaatctcca 3120
attatcaagg aattgaaaaa gagagttgac gaaggtggtg ctcaagacaa gactgtcaag 3180
gacttgacta agttattata tgaaactgct ttgttgactt ccggcttcag tttggacgaa 3240
ccaacttcct ttgcatcaag aattaacaga ttgatctctt tgggtttgaa cattgatgag 3300
gatgaagaaa cagagactgc tccagaagca tccaccgcag ctccggttga agaggttcca 3360
gctgacaccg aaatggaaga ggtagattag ctgcagctaa attgtaagcg ttaatatttt 3420
gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 3480
cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 3540
ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 3600
ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 3660
gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 3720
aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 3780
gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 3840
gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 3900
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 3960
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc 4020
gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc gcggccgact 4080
tggccaagcc tagatcccga ctaataggaa ttgatttgga tggtataaac ggaaacaaaa 4140
aaaagagctg gtactacttt ctttaaaatt attttattat ttgattttat ttaatagtat 4200
atattatatt ttgaacgtag attattttgt tgaaagttgc tgtagtgcca ttgattcgta 4260
acactaattc tgtattagtc attcctcttg tttgatagta tccaaaaaaa cggctatttt 4320
tttgcaatct tatttcctgc atattataca gataacataa tgaaagaaaa aatctttttt 4380
tttgttcttc aatgatgatt tcaaccattc ttttaaacat tgatcaattc ctgagcaaca 4440
accccataca cactggttta tataccgccc cttttacagt tgaagaaaga aatagaaata 4500
gaaatagcaa acaaaagata tgacagtcaa cactaagacc tatagtgaga gagcagaaac 4560
tcatgcctca ccagtagcac agcgattatt tcgattaatg gaactgaaga aaaccaattt 4620
atgtgcatca attgacgttg ataccactaa ggaattcctt gaattaattg ataaattagg 4680
tccttatgta tgcttaatca agactcatat tgatataatc aatgattttt cctatgaatc 4740
cactattgaa ccattattag aactttcacg taaacatcaa tttatgattt ttgaagatag 4800
aaaatttgct gatattggta ataccgtaaa gaaacaatat attggtggag tttataaaat 4860
tagtagttgg gcagatatta ccaatgctca tggtgtcact gggaatggag tggttgaagg 4920
attaaaacag ggagctaaag aaaccaccac caaccaagag ccaagagggt tattgatgtt 4980
agctgaatta tcatcagtgg gatcattagc atatggagaa tattctcaaa aaactgttga 5040
aattgctaaa tccgataagg aatttgttat tggatttatt gcccaacgtg atatgggtgg 5100
ccaagaagaa ggatttgatt ggcttattat gacacctgga gttggattag atgataaagg 5160
tgatggatta ggacaacaat atagaactgt tgatgaagtt gttagcactg gaactgatat 5220
tatcattgtt ggtagaggat tgtttggtaa aggaagagat ccagatattg aaggtaaaag 5280
gtatagaaat gctggttgga atgcttattt gaaaaagact ggccaattat aaatgtgaag 5340
ggggagattt tcactttatt agatttgtat atatgtagaa taaataaata aataagttaa 5400
ataaataatt aaataagggt ggtaattatt actatttaca atcaaaggtg gtccttctag 5460
aactagtgga tctgttgatc aaagttttac caacatttct gttttcaaaa gtggttggag 5520
ctttgatgtc aaaccaatct tttctggtga atgggtcaac gaccttcttt tttaatcctt 5580
tctttccttt ggacaatctc ttgtttttac cgacagccat gttgtacttg agttggatct 5640
acgcgtgaat tcgatatcgg tacccagctt ttgttccctt tagtgagggt taattgcgcg 5700
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 5760
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5820
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 5880
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 5940
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6000
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6060
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6120
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6180
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6240
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6300
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6360
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6420
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6480
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6540
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6600
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6660
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6720
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6780
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6840
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 6900
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 6960
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7020
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7080
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7140
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7200
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7260
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7320
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7380
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7440
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 7500
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7560
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7620
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7680
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 7740
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 7800
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 7860
gccacggtac cgattaggta gaacatcatg gccttgaata ggttataaac aaaacataat 7920
ataacgtata ggtattcgaa tgaataaata agtatgtaaa tagggcatct gcatggaaat 7980
aactgggtaa aacattacaa tgtatttttt tagagttttc actattccga cgcgtctagt 8040
gtgtcataca aaacgcgaag aatctgaaat cgtgaaatat acttaagaat ataactgaaa 8100
tagcttaagc ataaatggac tttactgcga agataaaggg tatgttgaaa ctcatctgct 8160
ttcattatgc gtccaactaa ttaacattgc atattttcgt atactatttt tttgtgtaga 8220
gctcgaacga gaactctcag agacgagtga ctataagacc ttgcagaaga agaccataag 8280
tcttcgaagt gagctgaata ctctgtcaca ttctttgaca tcttatgaaa aagaacactt 8340
ctccaatgat atggatcc 8358
Claims (10)
1.重组菌在生产乙醇或提高乙醇产量中的应用；
所述重组菌，为将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变，且该蛋白其他氨基酸残基不变，得到的菌株；
所述Hsp82蛋白，为如下A或B或C：
A)所述的蛋白为序列2所示的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白。
2.根据权利要求1所述的应用，其特征在于：
所述将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变为将酿酒酵母基因组中的Hsp82基因编码的蛋白第485位或对应于该蛋白第485位的丝氨酸突变为不发生磷酸化的氨基酸残基。
3.根据权利要求2所述的应用，其特征在于：所述不发生磷酸化的氨基酸残基为丙氨酸。
4.Hsp82蛋白突变体或其编码基因或含有所述编码基因的表达盒或重组载体在生产乙醇或提高乙醇产量或制备生产乙醇或提高乙醇产量重组菌中的应用；
所述Hsp82蛋白突变体，为如下A或B或C：
A)所述的蛋白为将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变，且该蛋白其他氨基酸残基不变，得到具有相同活性的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白。
5.根据权利要求4所述的应用，其特征在于：
所述将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为将将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为不发生磷酸化的氨基酸残基。
6.根据权利要求5所述的应用，其特征在于：所述不发生磷酸化的氨基酸残基为丙氨酸。
7.使酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸去磷酸化的物质，在生产乙醇或提高乙醇产量或制备高产乙醇重组菌中的应用；
或，使酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸突变的物质，在生产乙醇或提高乙醇产量或制备高产乙醇重组菌中的应用。
8.根据权要求7所述的应用，其特征在于：
所述物质为用于将酿酒酵母基因组中的Hsp82基因编码的蛋白的第485位或对应于该蛋白第485位的丝氨酸发生点突变的引物或试剂。
9.一种生产乙醇的方法，包括如下步骤：发酵权利要求1-3任一中所述的重组菌，得到乙醇。
10.一种Hsp82蛋白突变体，为如下A或B或C：
A)所述的蛋白为将Hsp82蛋白对应于序列2所示的氨基酸序列第485位丝氨酸突变为丙氨酸，得到具有相同活性的蛋白；
B)所述的蛋白为将A所示蛋白的氨基酸序列末端添加标签序列且具有相同活性的由A衍生的蛋白；
C)所述的蛋白为与A)或B)具有至少95％，98％，99％的序列一致性的蛋白；
或，所述的Hsp82蛋白突变体的编码基因或含有所述编码基因的表达盒或重组载体。
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